lNF ORMATION Tb USERS

This manusaipt has been nproduced fmm the miaofwm master. UMI films the

text directly from the original or copy submitted. Thus, some thesis and

dissertation copies are in typewriter face, Mile mers may be fmm any type of

cornputer printer.

The qwlity of this nproduction is âepenâont upon the qurlity d the copy

submitîed. Broken or indistinct print, coloced or poor quality illustrations and

photographs, print bkdthmugh, substanâard margins, and improper alignment

can adversely affect reproduction.

In the unlikely event that the auüior ad not send UMI a complete manuscript and

there are missing pages, Cties8 will be noted. AISO. if unauüiorited copyright

material had to be mmoved, a note will indicate the deletion.

Ovenize materials (e.0.. maps, drawings, &arts) are reproduced by monhg

the original, beginning at the upper left-hand corner and continuing frwn left to

fight in equal sections wiîh m a l overlaps.

Photographs induded in the original manusaipt have been reproduœd

xerographicaily in this copy. Higher qwl-Q 6' x 9. black and white photographie

pcints are availirble for any phaographs or illusbations appearing in this copy for

an additional charge. Contact UMI diractly to order.

Bell & Howell Information and Leaming 300 North teeb Road, Ann Amr, MI 481061346 USA

DIETARY PROTEIN DEFICIENCY MODIFIES

SYSTEMIC AND GUT-ASSOCIATED lMMUNE

RESPONSES IN MICE INFECTED WITH

HELJGMOSOMOIDES POLYGYRUS (NEMATODA)

REBECCA YAT LOO ING

Institute of Parasitology & S c h d of Dietetics and Human Nutrition

McGiil University, Montreal Quebec, Canada

A thesis submitted to the Faculty of Graduate Studies and Research in partial fuVilment of the reqoirements for a joint degree of Master of

Science Crom the Institute of Parasitology & S c h d of Dietetics and Human Nutrition

@ Rebecca Y.L. h g October 1998

National Library Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibliographie Sewices services bibliographiques

395 Wellington Street 395. rue Wellington Ottawa ON K1A ON4 Ottawa ON K I A ON4 Canada Canada

The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant ii la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, dismbute or sel1 reproduire, prêter, distribuer ou copies of this thesis in microforni, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de

reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts ffom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

ABSTRACT

Protein deficiency may increase susceptibility to gastrointestinal (GI) parasitic

infections. possibly as a result of impaired systemic andor intestinal effector responses

induced by downreguhtion of Th2 cytokines andor upreguiation of Th1 cytokines. To test

this hypothesis. femaie B ALBlc mice (n= 1 lldiet) were fed a control(24%), marginal (7%).

or deficient (3%) protein diet and given a challenge infection with the GI nemûtode,

Heligmsomoides polygyrus. The 3% mice had higher wom burdens at 1.2 and 4 weeks

post-challenge uifectio n (pci). 10 wer increases in serum IgE, reduced intestinal eosino philia,

and depressed mucosd mast ceii proliferation and activation at 1 to 2 weeks pci To

determine wkther these suppressed effector responses in the 3% rnice were usociated with

altered spleen and mesenteric lymph node (MLN) cytokine profiles. ceh were restimuiated

in vitro with parasite mtigen and cytokine concentrations were measured. Deficient MLN

ceUs secreted significantly less IL4 and more IFN-y at 1-2 weeks pci thui did control

MLN ceB. Deficient spleen cek &O secreted more I M - y at 2 weeks pci compared with

convol spleen cells. From RT-PCR analyses, the 3% rnice &O had lower IL-4 mRNA

expression in spleen and MLN at 1-2 weeks pci Our study supports the hypothesis that

protein deficiency exacerbates the sudval of a GI nematode parasite by decreasing IL-4

(Th.2) and increasing IFN-y ml) emly in the infection, leading to reduced gut and

systemic Th2 effector responses.

La carence benta i re protidique a démontri! son influence à la prédisposition aux

infections parasitaires gûstrointestinales chez ia souris. Une relation possiblement due à une

d&&ioration de l'action des effecteurs gbnéraw et/ou intestinaux provoqude par une

régdation à la baisse des cytokines Th2 et/ou d'une régulation B la hausse des cytokines '

Thl. Pour tester cette hypotMse, des souris BALBIc (n=l8/di&e) ont tt6 nourri selon trois

types de dietes protidiques diffttrentes: un groupe ternoin (24%)' marghd (7%) et deticient

(3%). De plus, ces souris &aient infestées par le nematode Heligmosomoides polygyms

selon un schéma de premi2re infection suivie d'une réinfection. Le 3 8 groupe avait un

taux d'infection plus elevé à 1,2 et 4 semaines après la réinfection. les augmentations des

taux d'IgE les plus bas dans le serurn, une 6osinopM&nie intestinale, ainsi qu'une

prolifhtion et une activation reduites de mastocytes de h muqueuse à L et 2 semaines

post-réinfection. Pour déterminer si cette diminution de i'activite des effecteurs d m le

3% groupe est le résultat d'un profde dt6rr5 de la rate et des cytokines du GLM, des

cellules ont et6 stirnuEes in vitro avec de l'antigène de parasite. Les cellules du GLM du

groupe déficient ont sécréters beaucoup moins d'IL-4 et d'IFN-y durant les 2 semaines

suivant la réinfection que le groupe de cellules du GLM du groupe ternoin. Les cellules de

la rate ont eues aussi sdcrétées plus d'IFN-y durant les 2 semaines aprés la réinfection en

comparaison aux cellules du groupe tdmoin. D'après des analyses au PCR. le groupe DP

n'exprimait pas aussi bien IL4 ARN messager de k rate et du GLM B 1 ou 2 semaines

post-réinfection. Nos dsultats sou tiennent l'hypo these que la carence protidique exacerbe

la survie d'un dmatode gastrointestiniil en diminuant IL-4 (Th2) et en augmentant CFN-y

(Thl), qui m8nent une diminution de l'activitd des effecteurs Th2 intestinaux et g6n6nux.

ACKNOWLEDGEMENTS

1 thank my supervisors, Dr. Marilyn Scott and Dr. Kristine Koski for their insights,

guidance and expertise which were instrumental to the success of this research project. My

work hm k e n greatly facilitated by their continuous support. enthusiasm and ability to

embrace new concepts and directions. Most irnportantly, I am grateful to them for giving

me the unique opportunity to study both pansitology and nutrition despite my lack of

previous experience in either discipline. 1 also thank Dr. Linda Wykes for her contribution

to my understanding of protein metabolism. I thank McGiU University for the Max Stern

Recruitment Fellowship and the Christine Gagnon Mernorial Scholuship. I also am

gratefûl to rny supervisors for their fmancial support in the last months of my studies.

1 gratefully acknowledge the contributions of my kbmates toward my resevch

project. My deepest appreciation to Fananeh Jalili for her worm counts and assistance

with the infections and mouse harvesting. 1 thank Dr. Zhong Su for performing the

histological work and RT-PCR experiments and for s h h g his immense expertise on

pansite immunology. I will remember fondly the many hugh and beers we shmd

together and especially our late night conversations in the lab. By helping me feed the mice

and enter data, Mike Clark engaged me in lively discussions as weii as spued me many

hours of shvery and monotony. 1 am indebted to Dr. Hai Ning Shi and Sarah Conly for

teaching me the ce1 culture and ELISA procedures. 1 th& Christine Gagnon for her

many words of wisdorn and encouragement and for inspiring in dl of us an appreciation of

science and Ne.

1 th& Gordon Bingham for his tireless and impeccable care of my mice and the

animal facilities. My thousand th& to Shawn Mohammed and Darren Campbell for

helping me troubleshoot problems with the imrnunological assays and for our afternoon

coffee breaks. 1 am gratehil to Christhne Trudeau for her wterfbl French translation of

my absuact. 1 thank Dr. K Chadee, Dr. G. Ruben, Dr. C. Tanner, Dr. J. Smith, Dr. M.

Stevenson. and Dr. S. Mahanty for their ~sistuice and advice during my studies. Also, 1

thYiL dI the students and techniciam at P d t o l o g y and Nutrition for shûring theu

resources and for their fiiendship which Lifted my spirits on the long, tough resevch days.

Thank you Dr. CC. Miny for seeing me through the years it took to me to become

an independent person and competent scientist. Above ail, thank you for showing me the

many wondehl things to iive for beyond acdemics and for your invaluable friendship.

I thank my f d y for their unwavering moral and hancial support which has

sustabled me throughout my academic endeavors. I dedicate this thesis to my parents who

selflessly sacrificed their own professional and personal lives to give their children a better

Me full of endles posibilities. I cm only hope that m y accomplûhments are worthy of

their past hardships. pride and enduring belief in my abilities.

Listly but imrnensely, 1 also dedicate this thesis to J.P. Paiement. Thuik you for

everythmg that you are and more. Elephant shoe.

TABLE OF CONTENTS

TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . v

LISTOFFIGURES ................................................... viii

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

THESIS OFFICE STATEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

STATEMENT OF CONTRIBUTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . xü

CHAPTER II. LITERATURE REVZEW . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . 5

1. Host Immunity to Nematode Parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.1 Th1 Versus Th2 Response Phenotype as Medhted by Il-4 and IFN-y . . . . . 7 1.2 Th1 Versus Th2 Ceil Differentirition .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.3 Roleofni2Cytokines . . . . . . . . .. .. .. . . .. .. . . .. . . . . . . ... .. . . . . 12 1.4 Roie of Th1 Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 1.5 Overview of Gut-Associated Mucosal Immune Responses . . . . . . . . . . . . 16

2. The Heligmosomoides polygym-mouse Mode1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 2.1 Host Immunity to Primyy Infection with H. polygyrus . . . . . . . . . . . . . . . 2 1 2.2 Systemic Versus Gut Mucosai Immunity to Primary Mection with H.

polygyrus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 2.3 Host lmmunity to Chdknge Infection with H. po lygyw . . . . . . . . . . . . . 30

3. Effect of Protein Dekiency on HeImtoth Infections . . . . . . . . . . . . . . . . . . . . . . . . 34 3.1 Effect of Protein Dekiency on Primaxy Infection with H. polygynrr . . . . . 34 3.2 Effect of Protein Defkiency on Challenge Mection with H. polygynrr . . . 36

4. Effect of Protein Deficiency on Systemic CeiI-Mediated and Humorai Immunity . . 38

TABLE OF CONTENTS continued

5 . Effect of Rotein Dekiency on Gut Mucosal Immunity ..................... 42

6 . Effect of Protein Deficiency on Immune Responses to Intestinal Nematodes Including ..................................................... H.polygyms 45

....................................................... . 8 References 49

CHAPTER iII . STIJDY OBJECTIVES ................................... 69

CHAPTER IV . DIETARY PROTEIN DEFICIENCY PROLONGS S U R W A L OF A GASTROINTESTINAL PARASITE IN MTCE BY SUPPRESSING TH2 MMUNITY ......................................................... 70

INTRODUCTION ............................................... 72

.................................... MATERIALS AND METHODS 74 ........................................ Experimental Design 74

Experimental Protocol ..................................... - 7 4 Mice .................................................... 76 Diets ................................................... 77 Parasite and Parasite Antigen ................................. 77 Puasite Wonn Burdens ..................................... 79

....................................... Nutritionai Mesures 79 ELISAs for IgE. parasite-specinc IgGl and MMC protease- 1 ......... 79

..................... Intestinai Mast Cell and Eosinophil Histology 81 Spleen Ceii and Mesenteric Lymph Node CeIl Prepmtion ........... 81 Production of Th2 (IL.4. IL.5. IL- 10) and Th1 (IFN-y) Cytokines ..... 81 Cytokine mRNA Expression by Semi-Quantitative RT-PCR .......... 83

......................................... Statisticd Analyses 84

..................................................... RESULTS 85 ......... ............................ Nutritional Outcornes .. 85

Parasite Outcornes ..... .. .................................. 87 ................................... Senun EEector Responses 87

Intestinal Effector Respoases ................................. 95 Systemic and Local Cytokine Responses to Parasite Antigen in vivo .... 98 Systemic and Local Cytokine mRNA Expression ................. 102

TABLE OF CONTENTS continued

DISCUSSION ................................................. 105

ACKNOWL, EDGEMENTS ....................................... I l 8

REFERENCES ................................................ 119

CHAPTER IV . GENERAL DISCUSSION ................................ 125

APPENDICES ...................................................... 128

APPENDIX A Resuits from Two-Way ANOVA on EEects of Diet. Tirne . & Diet by Time Meraction .............................................. 128

APPENDIX B Results from One-Way ANOVA on Main Effect of Diet ............ 131

APPENDIX C

........... Results from One-Way ANOVA on Main Effect of Time 136

vii

LIST OF FIGURES

CHAPTER 11, LITERATURE REVIEW

Figure 1: Th2 And Th1 Immune Response Phenotypes .......................... 6

Figure 2: DBerentiation of CD4+ T CeUs into Thl or Th2 ........................ 8

Figure 3: Mucosal Immunocpe Tnfnf ..................................... 18

CHAPTER III . DIETARY PROTEIN DEFICIENCY M O D E S TH2 IMMUNE RESPONSES IN MICE TO A GASTROINTESTINAL PARASITIC INFECTION WITH HELIGMOSOMOIDES POLYGYR US (NEMATODA)

Figure 1: Schematic Representation of Experimentai Protocol .................... 75

Figure 2: Effects of Dietary Protein Deficiency and Tirne on W o m Burdens ......... 88

... Figure 3: Effects of Dietary Protein Deficiency and Time on S e m Anu'body Lrvels 90

............ Figure 4: Effects of Dietary Protein Deficiency on Eosinophdh in Blood 93

Figure 5: Effects of Dietary Protein Deficiency and Time on MMC Counts. Intestinal Eosinophil Numbers. and MMCP- 1 Concentration ..................... 96

Figure 6: Effects of Dietary Protein Deficiency on ni2 and Thl Production .......... 99

.... Figure 7: Effects of Dietary Protein Deficiency on Th2 and Thl Cytokine mRNA 103

LIST OF TABLES

CHAPTER II. LITERATURE REVIEW

Tabie 1: Kinetics ofIn2 Cytokine and Effector Production Post-prim;isr and Post-chdlenge .......................... Infection with H. p o l y g y w in BALBk Mice 28

CHAPTER III. DIETARY PROTEIN DEFICIENCY MODIFTES TH2 IMMUNE RESPONSES IN MICE TO A GASTROWSTINAL PARASITIC INFECTION VATH HELJGMOSOMOIDES POLYGYRUS (NEMATODA)

.......................... Table 1: Nutrient Composition of Experirnental Diets 78

Table 2: The Effect of Protein Deficiency on Nutritional Parameters of Challenged Mice KüledonDay28pci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 6

THESIS OFFICE STATEMENT

In accordance with the reguiations of the Faculty of Graduate Studies and Research of

McGiIl University, the following statement is included in this thesis:

"Candidates have the option of including, as part of the thesis. the text of a paper(s)

submitted or to be submitted for publication. or the cleuly-duplicated text o h published

paper(s). These texts must be bound as an integral part of the thesis.

' n i e thesis must stiU conforrn to ail other requirements of the "Guidelines for

Thesis Preparation". The thesis must include: A Table of Contents. an abstract in English

and French, an introduction which clevly States the rationale and objectives of the study, a

comprehensive review of the litenture. a final conclusion and surnrnary and a thorough

bibliognphy or referencc üst.

Additional material must be provided where appropriate (e.g. in appendices) and in

sufficient detail to allow a clev and precise judgernent to be made of the importance and

oiginality of the resevch reported in the thesis.

"In case of mmuscripts CO-authored by the candidate and others. the candidate is

required to make an expücit statement in the thesis as to who contributeci to such

wodc and to what extent. Supervison must attest to the accuncy of such statements at

the doctoral oral defense. Since the task of the examiners is made more dinicult in these

cases, it is in the candidate's b a t intrrest to make perkctly clear the responsibilities of dl

the authors of the CO-authored papers. Under no circunirtances can a CO-author of any

component of such a thesis serve as an examiner for that thesis."

STATEMENT OF AUTHORSHIP

AU experimental work in this thesis was performed by Rebecca Y.L. Ing with the

exceptions of the wom counts, histological evaiuations, and RT-PCR assays. Dr. Zhong

Su performed the histology and RT-PCR experiments. This thesis was written by Rebecca

Y.L. hg. Dr. M.E. Scott and Dr. KG. Koski served as research/thesis supenrisors,

critiqued data presentation and andyses, and pre-edited aü written material. The authors

gratefitiiy acknowledge Fananeh .JaU for her work in counting the w o m burdens.

LIST OF ABBREVXATIONS

ADCC

ANOVA

MC

BSA

BUN

CM1

ConA

DNA

DTH

GALT

GM-CSF

HBSS

HEV

HPRT

IE

E L

IL

m-Y r, L I

LP

LPS

mAB

rnRNA

MLN

MHC

MMC

antibody-dependent cellular cytotoxicity

andysis of variance

antigen presenting ceii

bovine serum dbumin

blood urea nitrogen

concanavalin A

deoxyribonucleic acid

dehyed type hypersensitivity

gut-associated lymphoid tissue

granulocyte-macrophage CO lony stimuiating factor

Hank's Balanced Salt Solution

high endothelid venule

hypoxanthine phosphoribosyluansferase

intraepithelium

intraepithelial lymphocytes

interferon-gamma

third-stage W m

fourth-stage lmae

lipo polysaccharide

monoclonal mtibody

messenger riionucleic acid

major histocompatibüity complex

mucosd mut ceU

LIST OF ABBREVIATIONS continued

MMCP- I

NK

PBS

pci

PHA

pi

P P ~ PWM

RNA

RT-PCR

SRBC

Th

Th1

Th2

TCR

TNF-a

mucosd m u t cell protease- l

naturai Mer

phosphate bugered saline

post-challenge section

phyto hemagglu tinin

post-infection

post-prirnary infection

pokeweed mitogen

ribonudeic acid

reverse transcriptase - polyrnerase chah reriction

sheep red blood ceil

T helper

T helper type 1

T helper type 2

T cell receptor

tumour necrosis factor - alpha

CHAPTER 1 INTRODUCTION

Protein malnutrition and helminth infections of the human gastrointestind tract are

among the most cornmon causes of morbidity and mortality worldwide (WHO. 1987). The

coexistence of enteric helminth infections with dietary deficiencies, particularly in

populations of devdoping couniries, has been weU-documented in epidemiological and

clinid studies (Crompton, 1986). Although the prevailing socioeconornic and

environmental conditions (e. g. poor sanitation, inadequate preventive health care) may

predispose the individual to both malnutrition and parasitic infection, the complex

bidirectiond interactions between the two conditions may influence disease patterns and the

efficacy of nutritional repletion or anti-puasitic interventions (Bundy & Golden, 1987).

Malnutrition is known to depress immunocompetence in humans and experimentai animals

(Good & Lorenz, 1992; Kuvibidih et ai., 1993), leadhg to increased suscepuiility CO

parasitic infections. Aiternatively, intestinal parasites rnay influence host defenses ( tg.

villous atrophy and damage to the mucosai epithelial integrity) which in tum disrupt the

uptake and utilization of dietary nutrients (Upadhyay et al., 1985; Solomons, 1993).

Pansitic infections are also implicated in human w u @ and nutrient loss associated with

protein deficiency due to expenditure of resources for imrnunological processes rather thm

for tissue spthesis, to excessive intesthai blood loss and to the lealcage of p h m a proteins

across the damaged gut epitheiial barrier (Lunn & Northrop-CIewes, 1993). Thus, host

immune function may be one mechankm underlying the mutually aggravating interactions

of mdnuuition and helminth infections. Given the possible reiationships in the

malnuuition-uifection-immunity complex, this project was h e d at determining the effect

of protein mainuvition on host immune response to intestinal nematode parasites.

Protein deficiency, in conjunction with inadequate energy intake, h a k e n

associated with profound growth retardation (stunting). metabolic dysfunctions. increased

child mortality, and greater prevaience of intestinal parasitic mfections (WHO, 1987;

Latham, 1990; Kuvii idf et al, 1993; Nesheim, 1993). Epidemiological studies have

suggested a strong association between protein malnutrition and immune function

(Chandra, 1983). however, the evaluation of causal rehtionships in the human setting is

cornpiicated by the presence of CO-existing confounding variables which make it dificult to

establish whether the impaired immune function is a direct result of protein deficiency or

whether it is secondary to the presence of concomitant infection, unrehted diseases, other

nutritional deficiencies, or environmental factors. Despite the i n t ~ s i c interprerative

problerns in epidemiologic data, the conclusions from human studies have k e n supported

by experiments showing that protein deficiency depressed various indices of immune

function in uninfected mimals (e.g. Conzen & Janeway, 1988; Larnont et al., 1988; McGee

& McMurruy, 1988; Woodward et aL, 1995) and increased the transmission d o r

s u ~ v a l of Nippostrongylus brasiliensis. Trichuris muris , and Heligmosornoides polygyrus

in rodents (Duncombe et al.. 1981; Slater, 1988; Keyrner & Tarlton, 1991; Michael &

Bundy. 1992a). However. the majority of these studies either did not involve parvitized

hosts when investigating the rob of protein nutrition in host irnrnunocompetence or did not

examine imrnunologicai rnechanisrns relevant to the control of helminthiasis. Therefore,

rnuch is unknown about the effect of protein malnutrition in host-pansite interactions.

Functional immunity to nematode parasites depends on specific cytokine

production by type 2, T helper cells (Th2) which induce and mediate antibody-dependent

effector responses involving IgE. IgG 1, mucosd mast ce& (MMC) and eosinophils

(MOMO y & E ~ q u e z , 1992; Nredhm & Lillywhite, 1994). Yet. ody a few studies have

investigated the effect of dietary protein deficiency on Th2-type host immune responses to

intestinal nematode parasites (Shter & Keymer, 1988; Boulay et al.. 1998) and therefore it

is currently unclear whether protein deficiency potenthtes intestinal parasitic inf'ections by

impairing the development and maintenance of specific T ceU-dependent humorai immune

responses. In addition. the severe Ieveis of protein restriction (e.g. 0.5%) used in studies on

protein malnutrition and immune function (q. Woodward et al., 1992a & 1992b) limit the

generahbility of the results to human populations in which müd to moderate malnutrition

is much more prevhnt than profound nutrient deficiencies (Kuvibidh et al., 1993). Few

researchen have evaluated comprehensively the relative impact of mild to moderate protein

deficiency on protective immunity to nematode parasites m a dynamic host-parasite system.

Also, there is Utle information regardhg the interactions of host immunity and pro tein

malnutrition during secondary pansitic infections despite accumukted evidence that the

magnitude md efficacy of immune responses are innuenced by the frequency of antigen

exposure. In experiments using repeated or challenge inlection protocols that stimulate

protective immunity in normally immunocompetent hosu, pro tein deficiency (2-346) hu

k e n shown to prolong H. po lygym s u ~ v a l and to impair the production of secondary

total IgG 1 and eosinophil responses (Slater & Keymer. 1986; Keymer & Tarlton, 199 1;

Bouiay et al.. 1998). To date. the imrnunologicd parameters examined in these studies and

in most previous research on host immunity to nematode parasites hm ken confined to the

effector mechanisms in systemic cornputmenu such as the penpherd circuiation and

spleen. As a result, we know less about the nutritional deterrninants of gut mucosal

immunity despite reports that the gut mucosa hm one of the highest protein turnover rates

of ail tissues in the body (Nakshabendi et al., 1995) and is potenMy very sensitive to

changes in host nutritional status (Wykes et al., 1996). Research on gut mucosai immunity

is essential to the understanding of host mûlnutrition-parade interactions because the gut

and its associated lymphoid tissues comprise the fvst site of contact between host immune

de fenses and invasive intestinal nemntodes. Recent studies have s ho wn thrt the expression

of Th2 immunity in intestinal lymphoid tissues may determine the s u ~ v a l of H. polygyrus

during prirnary infection in mice (Behnke et a l , 1993; Fuiklemm et al., 1997; Wahid et ai..

1994). The putative importance of gut mucosal immunity in the resolution of nernatode

infection and its potentiai for nutritiond modulation are persuasive reasons for evaluathg

intestinal as weli as systemic immune processes in ordet to M y ascertain the mech3nisms

underlying the rektionships between protein deficiency and intestinal parasitism

The overall objective of this research project w u to detennine the systemic and

intestinal Th2 immune responses by which protein deficiency may influence the duration

and severity of an intestinai nematode infection. To better understand the cornplex

interrelationships of protein deficiency. host immunity and intesthaî parasitism, this study

integnted ail three conditions using an experimentd H. poIygyncs-mouse system as a mode1

for gastrointestmal helminthiasis and expression of Th2-type immune responses. Mice were

fed varying ievels of dietuy protein and subsequently subjected to a challenge infection

pro toc01 that has k e n shown to stimulate acquired resistance to secondary infections with

polygyms in well-nourished hosts (Behnke & Robinson. 1985). We hypothesized that

pro tein deficiency would reduce the magnitude of intestinal a d o r systemic cytokine and

effector responses duMg challenge infection with H. polygyms. Also. we expected that the

level of diet-induced impairment of host pro tective Unmunity would be proportional to the

severity of protein restriction such that a Iow protein diet (e.g. 3%) would exacerbate

parasite survival by inhiiiting acquired imrnunity to a greater extent th;ui would a

margindy deficient diet (e.g. 7%). We had anticipated that a certain degree of

physiological adaptation to maintain homeostasis and hinctionality might occur with mild to

moderate malnutrition but that there would be a threshold of nutritional deficiency beyond

which imrnunocompetence would be notably impaired. Assuming that gut mucosal

immunity is important for the rapid termination of a nematode infection and for prirning

systemic irnmunity. we postulated that protein deficiency would cause a more profound

impairment of acquired immunity at the intestinal ievel than that at the systemic leveL

Fidy. we speculated that protein deficiency would impact cytokine-medisited effector

mechanisrns to vuyhg intensities at different phases of a nematode infection. To determine

this possible effect of protein deficiency on the kinetics of Th2 immune responses, we

conducted a tirne course study of various effector and cytokine outcomes zt multipk time

points afier prirnafy and challenge kifection with H. polygynrs. Addresshg these

hypotheses would dvance out understanding of the mechaniîrns of parasite survhal in

protein malnourished populations and thereby help d e b e immunoIogicaI or nutritional

targets for therapeutic interventions.

CHAPTER 11 LITERATURE REVIEW

1. HOST IMMUNITY TO NEMATODE PARASITES

In general, immune responses in mice to foreign antigens involve two categories of

T helper (Th) cek: type 1 Th ("M) c e k are responsible for celi-mediated immunity (CMI)

and type 2 Th (TU) cek mediate antibody-dependent immunity. Each Th ceii response

phenotype is characterized by distinct patterns of cytokine and d u n e effector production

that correhte weil with their irnmunologic specincity (see Figure 1). Presentation of

parasite-denved antigens by antigen-presenting celis (APC) to CD4' T c e h induces the

dinerentiation of Th ce& to the Th2 functional phenotype. Th2 ceUs secrete interleukin

(IL,)-4, L-5. IL-9 and IL-10 which promote the prolifention and activation oCTh2-

associated effectors such as IgE- or IgG1-secreting plasma ceiis, eosinophils. and mucosal

m a t ce& (MMC). These efkctors are features of IgE-mediated host defense mechanisms

against many helminth parasites (Cox & Liew, 1992). Altematively, Th1 ceUs produce L-2

and IFN-y and interact with APCs to synthesize IL-12. These Thl-associated cytokines

are important in macrophage activity and isotype selection for IgG2a. IgGZb and IgG3,

which mediate antibody-dependent celluhr cytotoxicity (ADCC) and complement activity

against bacteridor viral infections (Locksley, 1994). BothTh cell types secrete IL-3,

RIF-a, and GM-CSF (Mosmann & Sad, 1996). The pohrked patterns of cytokine and

immune ceil production correspond to activated effector Th phenotypes generated during

immune response to a particuiar antigen but are no t observed in nive CD4+ T ce& or even

long-tem. quiescent memory ce& (Mosmann & Sad, 1996; Bunce & Bell, 1997).

Th2 lmmune Responses To Nematode Parasites IgE-Medieted Humoral Responses:

Plasma Cells IgE lgG1

Th2 Cytokines: ILS, 119, IL10

Eosinophilia

a MMC Proliferation

Th1 Immune Res~onses To Bacteria and Viruses Cell-Mediated tmmunity:

IL-1 2 and IFNy Macrophage Activity

lgG2a, lgG2b & 1963

Figure 1 : Th2 and Th1 lmmune Response Phenotypes

1.1 Th1 Venus Th2 Response Phenotrpe as Mediated by Ii-4 and IFN-y

The characteristic cytokine products of T'hl and Th2 cells are mutudy mtagonistic

for the differentiation and activity of effectors belonging to the reciprocal phenotype (see

Figure 2). Hence, L-4 and in some circumstances 5 1 0 inhibit cytokine synthesis by Th1

cells and macrophage hinction (Kopf et ai., 1993; Lagoo et d., 1994; Mosmann & Sad,

1996) whereas IFN-y suppresses the production of ThL-type effectors and cytokines (King

et aL, 1993; Urban et ai., 1993). This cross-regulation oCTh ceU polarization and immune

responses may explain the svong bias towvds Th2 responses during nematode infections

and why the alteration of Th2 response patterns by IFN-y or other Th1 cytokines (e.g. IL-

12) reverses host resistance to nematode infection (see UrSm et al.. 1993; Fiiemm et aï.,

1994). In humans infected with helminth parasites, serum IgE and IgGl be ls are directly

proportional to parasite-induced IL4 production and inversely related to IFN-y synthesis

(King et al., 1993; King & Nutman, 1993) whereas IgE-medhted eosinophil and mast ce1

activities are associateci with eflective resolution of helminth infections (Men & Maizels.

1996). Experimentai evidence to support the view thrt development of a Th2 cytokine

response stimuiates strong resistance to nematode puasites hm ken denved f?om studies

that measured the cytokine profile of cells isohted from tissues of genetically resistant

animais (Wahid et al., 1994). conducted adoptive tramfer of protection with immune c e h

known to secrete Th2 cpokines in vino (Korenaga et of.. 1989; Ramaswamy et al..

1994a). andyzed knockout animais (Kopf et al., 1993), used neutralizllig antibodies to Th2

cytokines ( C o h a n et al., 1989; Urbm et al.. 199 1 b), and andyzed the effects of different

cytokines on the NI vitro development of Thl and Th2 celi hes (e.g. Maggi et ai-. 1992).

Collectively, observations from these studies suggest that hnctional immunity to helrninths

involves Th2-type cytokines and effectors and that the reciprocal amounu of parasite-

elicited IL4 and LFN-y produced determine the magnitude of Th2 irnrnunity raised against

the nematode pansites.

11-1 2 IL4 O

1FN-y Q t

Humoral CelCMediated lmmunity (viruses, bacteria)

Immunity (Nematodes)

Figure 2: Differentiation of CD4+ T helper cells into Th1 or Th2 type: cross=regulation by 11-4 and IFNy. Adapated from Mosmann & Sad, 1996

1.2 Th1 Versus Th2 Ce11 Differentiation

The source and p ~ c i p a i early events affecting Th 1Khî cell dinerentiation

ultimately influence the type, speed and magnitude of the host immune response to

nematode parasites. Naive CD4' T cells and even some subsets of rnemory Th cells are

uncommitted and have the potentid to secrete cytokines belonging to both phenotypes.

When stimulated by antigen presented on APCs, these Th0 ceh secrete primarily IL-2 as

weU as smdl amounts of other cytokines, including IL-12, IFN-y and IL-4. There is

agreement that IL- 12 and IFN-y direct differentiation to a Th1 phenotype (Seder et al.,

1991; Bradley et al., 1996) and IL-4 drives dif'ferentiation to a Th2 phenotype (Le Gros et

al.. 1990; S wain et al., 1990). Several studies suggest that IL- 12 is denved from

macrophages and IFN-y is secreted by NK cells in response to IL12 or by naive CD4' T

ceik independently of APC or IL- 12 (S wain. 1988). The initial source of IL-4 and factors

required for the genention of IL-4-producing precursors are less weil-dehed. Classical

APCs (e.g. macrophages, dendritic ce&) are not known to produce IL-4 while the

proportion of IL-éproducing T ceUs among naive T ceil populations is very smdi (Swah eet

al., 1988; Le Gros et al., 1990). In fact. naive CD4+ T celis fkeshly isoiated from murine

spleen do not secrete detectabk quantities of IL-4 until afier the cells are primed and

cestimulated with C o d or antibodies to the TCR (Swain et ai., 1988) or ifexogenous IL4

is added to the priming culture (Le Gros et al., 1990; Seder e t al., 1992). Mast cells and

eosinophils cm produce IL-4 (Srder et al.. 199 1; Moqbel et al.. 1995; Ndcajima et aL,

1996) and provide ceil contact signals required for IgE production by B ceils (Gauchat et

al., 1993). but these ceiis are not abundant in lymphoid tissues where T ce1 priming and

daerentiation occur upon fkst exposure to the mtigen. L-4 is ako produced by a minor

subpopulation of T cells. CD3TD4'NKl. l* T cells (Yoshimoto & Paul, 1994) but how

these celis become activated to secrete IL-4 is not known.

SimiIarly. the driving force for initial Th2 priming during nematode infection has not

been clevly elucidated. IL-4 mRNA expression duMg prirnary idection in mice with

Heligmosomoides polygyrus is restricted to CD4'. TCRdw ce&. occurs relatively Iate

compared to other Th2 cytokines, and is not preceded by elevations in IL-2 mRNA (Svetic

et al., 1993) as observed in some Th ceil transitions from Th0 to Th2 expression. Also. NK

T ceils with the markers, CD6L-low. CD44-high, CD45RB-low, fiom mice geneticdy

deficient in I L 4 receptor retain their capacity to produce IL4 durhg Nippostrongylus

brasiliensis infection (No ben-Trauth et al., 1997). suggesting diat the induction of Th2-

type responses to nernatode pansites does not require evly IL-4 or signaiing through the

IL-4 receptor. Conside~g that production of IL-4 by mast ceh, eosinophils or CD4' T

cells in response to nernatode parasites appeus to be a Lte event without an initial transient

Th0 phase dominated by IL-2 synthesis. other cytokines or cell types may be hvolved in

initial Th2 dinerentiation. A recent study has s h o w that IL-6 produced by APCs can

vigger the Th2 pathway by inducing naive CD4+ T cells to produce IL-4 which in turn

would act as an autocrine factor to potentiate the upreguktion of IL-4 gene expression

while inhibithg IFN-y production (Rincon et al.. 1997). The CD4' T ceUs are then

polarized to a Th2 phenotype using their own source of IL-4 or IL-4 secreted by

sirnultaneously activoted basophiis and eosinophils. It is possible that these IL-6-dependent

events leading to IL-dproducing Th2 tek occur during responses to nematode parasites

because Svetic et al. (1993) have reponed that non-T ceUs (CD4XD8') are iikely the evly

source for elevated expression of IL-3, IL-5 and IL-9 mRNA and C M + T ce& becorne the

hter source of these cytokines including IL-4. Although IL-6 was not analyzed by Svetic et

al. (1993) or other rehted studies on helminth immunology, it may be a patentid initial

factor iduencing the choice between a Th1 or Th2 response to nematode parasites.

The presence of an appropriate pattern of cytokine production is essentiai for the

development of beneficiai immune responses to intestinal helminths. Even though the

initial sources of IL4 remah uncenain, it is weii recognized that IL-4 is a crucial r e m t o r

of the cornmitment of CD4+ T ceUs to the production of I L 4 The inhibition of early IL4

production ushg antibodies to I L 4 and the IL-4 receptor is sufficient to promote the

dinerentiation of Th1 ceils by increasising IL-2 and IFN-y production by naive CD4+ T cells

(Seder et al., 1992; Tanaka et al., 1993). Consistent with these data. mice deficient in

Stat6 gene (the signal transducer and transcriptionai activator of IL-4) and infected with N.

brarilienris secreted lower amounts of Th2 cytokines (Takeda et aL . 19%) and showed

decreased production of IgE but increased levels of IgG2a. IgG2b and IgG3 which are

dependent on IFN-y (Shmioda et al., 1996). Moreover, blocking of IL4 induced the

development of memory effector Th 1 ceUs (Bradley et al. , 1995). Unlike IL-Cproducing

T cells which require priming and restimulation, naive Th cells secrete IL2 and IFN-y upon

fist antigenic stimulation even in absence of IL- 12 (Bradley et al., 1996). Taken together.

these hdings Mply that the pattern of Th1 cytokines appeus to be the default phenotype

in naive mice as weii as in nematode-infected rnice when IL-4 synthesis or activity is

resuicted. M o . it is clear that IL-4 is an absolute requirement for Th2 cell dinerentiation

and resultant Th2 immune responses because it selectively promotes the expansion of Th2-

committed cells via positive feedback and/or suppresses IFN- y synthesis. Multiple sources

of IL,-4 including CD4' T cek, APC. mast tek. eosinophils and their costirnulatory

molecules (e.g. signaling between B7.1 or B7.2 on B cells and CD28 or C'T'LA-4 on T

ceh; expression of CD40 ligand on activated Th ce&) are involved in induchg the

differentiation of TM cells and subsequent production of IL-4 (Lu et ai., 1994; Lu et al*.

1996; Greenwdd et al*. 1997). A selective impairment of these ceh a d o r of their contact

signais may dom-reguiate ni2 response andor concomitantly up-regulate Th1 responses,

resulting in an inappropriate or inadequate host immune response to nematode parasites.

Because immune effectors are controlled by T ceii-derived cytokines, the foUowing

pangraphs wül review briefly the imrnunologic roles of the major Th2 and Th1 cytokines.

It is apparent that each cytokine has pkiotropic effects on multiple types of lymphoid cek

and that there is mucb duplication of function mongst different cytokines.

1.3 Role of Th2 Cytokines

IL4 is involved in the activation and maintenance of most Th2 effector responses.

IL4 produced by Th2 ceh evly after antigenic stimulation drives ail subsequent immune

responses toward the Th2 phenotype by acting as an autocrine growth factor for

differentiated Th2 ceils (Mosmann 8 Sad, 1996). enhancing the production of other Th2

cytokines (Kopf et al.. 1993), and inhibiting IFN-y production by Th1 cells (Tanaka et al..

1993). Mice deficient in IL-4 receptor displayed markedly diminished IgE, I f i l , IL-5, and

IL- 10 responses to N. brmiliensis Uection (No ben-Trauth et al., 1997). indicating that

induction of Th2 responses to helminths is dependent upon signaling through the IL-4

receptor. iL-4 promotes isotypc: switching of membrane IgA-positive and IgM-positive B

ceiis to IgE- and IgGl-expressing c e b (Finkelman et ai.. 1988 b; Zhang et al.. 199 1). As

IgM4 B ceb are frequently found in peripherd blood, the IgM to IgE switch may account

for much of circukting IgE after nematode infection (Zhang et aL, 1991). Also. IL-4-

mediated switch of IgA to IgE-expressing B c e b may be important for protective mucosai

immunity given the abundance of IgA+ B ceils in gut lyrnphoid tissues such as Peyer's

patches (Cebn & Shroff. 1994) and the association of IgE with anthelrninthic resistance.

Katona et al. (1991) demonstrated that antibodies to the IL-4 protein and recepior

compietely inhiaited secondary IgE responses to H. polygynrr but did not affect the ability

of IgE-expressing B cek to daerentiate into IgE-secreting ce&. Taken together, these

data suggest two important aspects of IL-dinduced imrnunoglobulin responses: 1) IL-4 is

required by virgin B ceh to express the IgE and IgG 1 isotype but may not be necessuy for

the tenninal dinerentiation of these cells into IgE- and IgG1-secreting plasma cek; and 2)

IgE and IgG 1 responses generated in rnice chailenged with H. polygyrus are not derived

from memory B c e h that were primed to express IgE and IgGl during the initiai exposure

but from IgA* or IgM* B cells that were induced by IL4 to switch to IgE and IgG 1

expression during secondw exposure. IgE and IgG 1 in tum, when crossed-iinked by

pansite-denved antigen. trigger the degranuiation of mast cells and eosinophüs (Gounni et

oL, 1994; Chen & Enerback, 1994 & 1996). Aiso, IL-4 has been shown to enhance L-5-

mediated chernotaxis of eosinophils and to induce MMC hyperphia dong with I L 3 and

stem cell factor (Madden et al., 199 1 ; Rennick et al., 1995). IL-4 up-regulates the

expression of endothelial adhesion molecules (e.g.VCAM-1) on intestinal endothelial c e h

and therefore may be important in the reguhtion of effector ceU traficking and horning of

lymphocytes, eosinophils and basophils to intestinal mucosal sites (Chin et al., 199 1;

Haradken et al.. 1996; Schleimer et al., 1992). Similarly, IL-4 treatment in vivo has been

shown to enhance the uptake of injected IgE into the gut mucosa and lumen following

infection with Trichinella spirulis, possibly by upregulating the affuiity of 1% receptors on

MMC and eosinophils present in the infected intestinal tissues (Ramaswarny et al., 1994).

In addition to promoting the expansion and recruitment of Th2 effectors. IL-4 rnay be

cniciril for effector ceil activation and function. IL-4 stirnuiates and promotes IgE binding

to m a t cek and IgE-induced degrmuhion of mut ce& when sensitized by antigen.

possibly via control of mut ceii FcrRI expression (Banks & Coleman. 1996; Tom et al..

1996). Lastly, preliminary experiments by Urban et al. (1996) suggest that IL-4 amplines

smooth muscle contraction in the smail intestine through mast cell and leukoviene D4-

dependent mechvllsrns which could in tum expedite wom expulsion from the intestine.

The main activities of IL-5 are to promote the growth and terminal differentiation of

eosinophils, to attract eosinophils to sites of inflrimmation. and to enhance IgE- or IgG 1-

induced eosinophil degranulation (Co f i a n et al.. 1989; Kita et al.. 1992). These activities

are complemented by L-4 (e.g. via IgE or IgG switching and eosinophil recruitment) and

by IL- 10 (e.g. via IgG 1 switching). IL-5 is secreted by Th2 cek, basophils and eosinophils

afier IgE- and IgG1-induced activation. thus providing an autocrine pathway for locd

eosinophil activation (Dubucquoi et al., 1994). The production of I L 5 in T and NK ceils is

augmented by IL4 and inhîbited by IL- 12 (Warren et al*, 1995).

Preiiminq data suggest that IL-9 potenthtes IL-4induced synthesis of IgE and

IgGl by LPS-prirned or antigen-stimuiated B lymphocytes (Dugas et ul. 1993; Petit-Frere

et al., 1993), optimizes mucosal mastocytosis synergisticany with IL3 and I L 4 (Behnke et

aL, 1993; Huitner et al., 1990). and stimulates the uanscription of genes coding for mast

cell granules such as mouse mast cell protease-1 (MMCP-1) which is secreted specitically

by intestinal MMC (Eklund et ai., 1993). The role of IL-9 in mediating protective

immunity to nematode parasites has not ken studied as extensively as that of other Th2

cytokines, however, a recent paper has demonstrated that IL9 mRNA is expressed early

during Trichuris m r i s infection and its elevation Nt vivo is associated with enhanced

intesthal mastocposis, production of IgE and IgG1, and loss of the parasite from the

intestine (Faulkner et al., 1998). Collectively, these observations indicate thrt IL-9

contribu tes to host resistance to helminths by promoting IgE-dependent MMC responses.

A recognized roh OPIL-IO in mediiiting Th2 responses is its inhibition of Th1 ceil

differentiation via suppression of IFNy production by Thl-cornrnitted celis. IL-10 does

not act directly on Th1 ceils but impairs the capacity of splenic and peritoneal macrophages

to stimulate Th1 cytokine synthesis by Thl cells (Fiorentino et al., 1991). Ding et al.

(1993) showed that this effect is partly due to the ability of IL40 to suppress the activity of

B7.1 (CD80) costimukory molecule on macrophages while de Waal et al. (199 1) found

that IL40 down-regulated the expression of clûss II MHC on APCs that interact with

antigen-activated Thl c e k to induce cytokine synthesis. IL-10 contributes to mucosai

mastocytosis by promoting the growth of mast cell progenitors and expansion of later

colonies in the presence of I L 3 and IL-4 (I3eh.de et al., 1993; Thornpson-Snipes et of.,

199 1). Similar to the effect of IL-9, E- 10 induces the transcription of genes for MMCP- 1

and other serine proteases secreted by MMCs of nematode-infected mice (Ghildyal et al.,

1992 & 1993). IL-10, dong with IL4 and stem cell factor, induces the synthesis of

histamine in MMC (Bissonette et al., 1995). h o , IL40 may promote isotype switching to

IgG4 in humans (Lagoo et al., 1994); although human IgG4 is homologous to muMe

IgG 1, this particular function of IL- 10 ho not been shown with murine B lymphocytes.

IL-3 is a cytokine secreted by Tho. Th1 and ni2 tells (Mosmann & Sad, 1996). Ir

is a multi-colony stimulating factor for severai haematopoietic ceils and thek progenitors in

vitro. Tt-3 is an essentiai cofactor for the growth and maturation of MMC dong with I L 4

and stem ceil factor (Madden et al., 1991; Re-k et al., 1995) and its administration into

nematode-infected mice induces strong mucosal mastocytosis and successful expulsion of

the nematode Snongyloides ratti (Abe & Nawa, 1988; Abe et al., 1993).

1.4 Role of Th1 Cytokines

IFN-y is an important Th1 cytokine that serves primdy to activate natunl killer

ceh and mononuclear phagocytes (e-g. macrophages and neuuophils) which are

responsible for cytotoxic activity againsr tumor celis and microbes (Mosmann & Sad,

1996). Research suggests that IFN-y inhibits or dehys protective immunity to nematodes

by suppressing the growth and expansion of Th2 ce& via a dom-regdation of IL-4

(Maggi et ni., 1992; Urban et al., 1992), inhibithg production of IgGl (Fielman et ai.,

1988a), suppressing the ability of IL4 to elicit IgE switching and dinerentiation (King et

al.. 1993; Uchikawa et al. 1994). blocking eosinophüia (Urban et ai., 1993), decreasing

the TM-a-dependent cytotoxicity of MMC (Bissonette et al.. 1995). and delaying IgE

response and MMC hyperplasia Li N. brudiensis-infected rats (Urban et al.. 1993). These

inhibitory effects of IFN-y on Th2 effector responses have ken associated with prolonged

s u ~ v a l of intestinal nematode parasites such as N. brasiiiensis and T. muris in rodents

(Lirban et al., 1993; Eise et a l , 1994).

L I 2 is secreted by macrophages and B lymphocytes and potenthtes Thl

phenotypic expression by enhancing priming for IFN-y production by naive CD4' T ceUs

(Seder et al., 1993) and naturd killer ceils (Chan et al.. 199 1 ) . IL- 12 also induces Th1

dserentiation &er antigenic stimuhtion by enhncing IFN-y spthesis and inhibiting the

development of IL-4-producing Th c e h (Myietti et al.. 1993; Windhagen et al.. 1996),

even in the absence of the costirnuhtory receptor CD28 on T ceh (Chu et al., 1997). The

presence o f IL- 12 in vivo has k e n shown to reduce the suppressing effecu of IL,-4 on

IFN-y production by naive CD4+ T cells (Seder et al., 1993). These anti-Th2 effects of IL-

1 2 have ais0 been dernonstrated in vivo: IL- 1 2 inhibits IgE S ynthesis and corres ponding IL-

4 production by lymphocytes isolated from helminth-Sected patients (King et al.. 1995)

whüe administration of K- 1 2 to N. brasiliensis-infected rnice prolo nged w o m s u ~ v d ,

stimulated IFN-y production. inhibited IL-4. IL-5 and IL-9 mRNA expression, and

prevented increases in nematode-induced IgE. MMC and eosinophü responses ( F i e h a n

et aL, 1997).

1.5 Overview of Gut-Associatecl Mucosal Immune Responses

Much of Our knowledge about specinc immune responses to nematode infection is

based on analyses of antibody concentrations fiom blood and cytokine production by

splenocytes. The underlying assumptions in these studies are that peripheral levels of

effectors or cytokines refiect intestinal IeveB and that protective T cells exert their principal

hinction by releasing Th2 cytokines in gut mucosa where the parasite resides and

reproduces. The fxst assumption was deemed invalid by hdings of a study showing that

serum IgE concentration in T. spiralis-infected mice represented only 0.2 to 1.0% of total

amount of IgE produced at the peak of intestinal antibody response and that the vast

rnajority of IgE produced in vivo is directed to the small intestine and does not enter the

bloodstream (Negarao-Correa et al.. 1996). Therefore, to understand the bill capacity of

host resistance to intestind parasites, we need to e x d e immune responses occurring in

gut mucosai tissues in addition to those in systemic compartments.

The srnail intestine contains speciaüzed lymphoid tissues that are categorized h t o

two anatornically and hinctionally distinct cornpytments: 1) the affierent hb consists of

aggregated lymphoid ceils (Peyer's potches). isolated lymphatic follicies and mesenteric

lymph nodes (MLN), where APCs present antigen to naive T lymphocytes (Bogen et al.,

199 1; Laissue et al., 1993; Regoli et aL, 1994) and 2) the eKerent h b c o n t h the

inuaepithelium (IE) and lamina pro pria (LP) of the intestinal rnicrovilli, where antigen-

specifk effectors such as IgE, IgG1, MMC and eosinophils mediate their anthelminthic

reactions (Beagley & Elson, 1992). Recently, Saito et al. (1998) desctibed a new intestind

lymphoid cornpartment (cryptopatches) Iocated under the layer of epithelid cells lining the

gut which consisted of immature and dEerentiated intraepithew lymphocytes (EL).

Although progenitors of al l immune cells originate nom bone m m w , Saito et ai. (1998)

found that T ceii precursors in cryptopatches were capable of maturing in the gut without

passing through the thymus and thus provide evidence that the intestinal mucosa contains a

population of thymus-independent mature T cells bat may interact directly wïth enteric

antigem. Cokctively, these immunologicai compartments are kno wn as gut-associated

lymphoid tissues (GALT) which help ensure mucosal integrity and comprise the fkst line of

host protection against ingested antigens and intestinal pathogens. The function of the gut

mucosa includes nonspecitic defenses provided by the epithekl bYrier and mucus as weli

as specinc immune responses medited by gut-associated cytokines and effectors.

The continuous. nonrandom migration of lymphocytes between mucosal lymphoid

tissues and the bloodstream is an essential process in the development of immune responses

at the appropriate sites of infection anywhere in the body. Naive lymphocytes fkst interact

with antigens in the Peyer's patches and isolated lymphoid follicles and then hrther

dinerenthte and mature in the germinal centers of the lymphoid follicles (Guy-Grand et ai.,

1978). Thereaftet. the antigen-primed c e h Ieave these inductive sites of GALT via

mucosai lymphatics. coiIect in the thoracic duct and enter the peripheral circulation (see

Figure 3). From the blood, the lymphocytes migrate to systernic lymphoid organs such 3s

the spleen and peripheral lyrnph nodes where the lymphocytes continue to mature and

proliferate to become effector cells (e.g. secrete cytokines) andor memory ce& (Salmi &

JaIkanen, 1991). Early studies on lymphocyte rectculation proposed a common mucosal

immune system in which lymphocytes mignte freely among the gut, peripheral lymph

nodes. genitd tissues and the lung (McDermott & Bienestock. 1979). However, ment

papers suggest that lymphocytes of lung and gut ongin have distinct recirculation routes

and express different homing adhesion receptors that bind to high endothehl venules

(HEV) lining the mucosai lymphoid tissues (Joel& Chmana, 1985; Abitonbi et al., 1996).

For example. activated and memory gut-drrived lymphocytes preferentidly extravasate

nom the blood to intestinal effector sites by expressing integrin a4P7 and Lselection,

adhesion receptors which are much less expressed by lymphocytes recirculating through the

lung (Schweighoffer et al., 1993; Abitonbi et ai., 1996). niese homing molecules are

receptors for mucosal vascular addressin cell adhesion molecule (MAdCAM)-I whifh is a

vascular addressin found selectively on endothelia of the p t and associated lymphoid

tissues (Nakache et al.. 1989; Berlin et a l . 1993; Roberts 8 Kilshaw. 1993). Thus. most of

the differenthted lymphocytes that enter the effector sites of the GALT (LP, E) are likely

to have had prior contact and specinc activation with antigens located in the gut mucos&

Inductive Sites: Antigen Presentation

Effector Sites: IE and LP

+ Lymphatics Lymphatics

C Systemic MLN -W Thoracic - Circulation

Duct Spleen

PLN

6 Antigen Figure 3: Mucosal lmmunocyte Traff ic @ B ceii

\ IgE or IgGl

In addition to the intestinal migration of lymphocytes, MMC progenitors, basophils,

and circulating eosinophüs are attracted by chernotaxis induced by gut-associated cytokines

to the IE and LP where they mature and degranulate after cross-linkllig of their FceRI

receptors by IgE ruid pansite antigens (Pewce et al.. 1982; Smith & Weis, 1996). MMC

in the gut mucosa or MLN are histochernicdy and functionally dBerent Erom masr cells of

the peritoneal cavity ûnd connective tissues (Shanahan et al., 1986; Befùs et al., 1987) and

are the predomuiant type of mast cells that proliferates in response to parasitic infections of

the gastrointestind tract (Arizono et a l , 1993 & 1994). Infection with N. brusiliemis

induces migration of MMC precursors from peripherd blood to the intestine (Kasugai et

al., 1995), development of MMC cells located Ni situ (Miller & Jarrett, 1971), and release

of serine proteases that may alter intestinai mucosal pemerbility (King & Miller, 1984;

Wasthg et a l , 1997). Mucosal mastocytosis is dependent on T cell-denved IL-3, IL-4 and

IL10 (Haig et ai., 1982; Madden er al., 199 1; ReMick et al., 1995). In tuni. MMC

produce Th2 cytokines (e-g. IL-4) and provide contact signals for IgE synthesis which help

augment local Th2 reactions (PIaut et al., 1989; Seder et al., 1991; Gauchat et al., 1993).

Cytokines such as IL-4, IFN-y and TNF-a rnay enhance the adhesiveness of

endothew ligands on HEV ceils for lymphocytes (ThornhiU et ai., 1990 & 199 1; Chin et

QL, 1991). Previous studies have docurnented the synthesis of Th2 cytokines by

lymphocytes derived fiom Peyer's patches and MLN (Svetic et aL. 1993; Wahid et al.,

1994) as weii as the GALT-specinc production a d o r uptake of Th2 effectors early f ier

nematode iruection (Ramciswmy et al., 1994; UcMcawa et aL, 1994; Wahid et al., 1994).

Cytokine gene expression in GALT and transport of IgE into intestinal lumen occur within

24 hours after primary T. spirulis infection (Ramaswuny et al., 1994; Svetic et ai., 1993)

while cytokine secretion by MLN ceUs, mucosai mastocytosis, and IgE-mediated MMC

degranulation occur by 5 to 10 days of N. braifiensis and H. polygynrs infection (Arizono

et al., 1994; W W et aL, 1994; Chen & Enerback, 1996, 1996). Th2 cytokine leveis in the

intestinal lymph of T. spiralis-infected mice were found to accuntely r e k t the responses

in the gut mucosr. leding to the conclusion that the most important source of Iocd

cytokines duMg early infection is the mucosai ce& residing in the gut and not the draîning

lymph nodes (Ramaswamy et ai.. 1996). Similarly, others have observed that the mjonty

of cytokines produced in MLN, which receive both activated CD4+ T ceUs that were prirned

in Peyer's patches and naive lymphocytes circuhting from systemic cornputmenu, are

produced by cells denved Rom intestinal lyrnphoid tissues (Ho & Massouh, 1997).

Moreover, lymphocytes in the MLN were found to be the most potent source of growth-

stimulating activity for MMC hyperplasia during N. brmiliensis infection compared to

those in spleen or peripheral lymph nodes (Haig et a!. . 1984). These results imply that

cytokine production is inithted locdly at intestinal sites of infection and that these gut-

associated cytokines induce chernotactic recmitment of lymphocyte and effector

progenitors to IE or LP where they mature and dzerentiate into antigen-specific cefl

popuiations. Furthemore. the migrational pattern of mucosd lymphocytes from the gut to

systemic sites suggest that irnmunological mediators produced in GALT are important in

priming and perpetuating host responses in systernic compartments.

A further indication that immune responses in the mucosa are unique is the large

percentage of T c e k with the y 8 receptor. This y 6 popdation, which constitutes only 5%

of circuhting T cells (the majority carry the ap receptor), makes up approxirnately 508 of

the T lymphocytes in the gut mucosa of mice (Kaufmm. 1996). The role of y6 T cek in

Th2- type responses to nematode parasites has not k e n clevly elucidated. Most y6 ceils

are not CD4' T cells which have been shown to be important for protective immunity to

nemûtode parasites (Urbui et al. 199 la; Koyama et ai., 1994) and y 6 ceh do not

recognize MHC-associated peptides. (Raulet. 1989). However, there is evidence that y6

T celis mediate Th2 immune responses by providing 3 contact signal for B cell isotype

switching to IgE production in the presence of IL4 (Gascan et al., 1992; Wen et ai.. 1996)

and by preferentidy secreting Th2 cytokines spontaneously (Hiroi et al., 1995) or during

infection with N. brusiliensis in mice (Femck et ai., 1995). CD4' y8 T ceUs are rare but

may proMerate during infection in the gut and help drive the development of Th2 clones in

vitro (Wen et al., 1998). The selective expression of Th2 cytokines by y6 T cells and the*

substantial accumulation in the gut mucosa of mice suggest that y8 T ceIls play an

important role in the induction of Th2 response to intestinal nematode parasites.

2. THE HEUGMOSOMOIDES POLYGYRUS-MOUSE MODEL

Heligmosomoides polygynrs (= Nematosp iroides dubius) is a trichostrongyloid

pansite of murine rodents and has been proposed as a experirnentd mode1 for human

hookworm infection (B~tlet t & Ball, 1972) and other nematode infections of veterinary

importance (Monro y & E ~ q u e z , 1992). The chronicity of primary infection, the capacity

for acquired immunity to secondary infections, and the similvity of epidemiological

patterns between laboratory-rnaintained and free-living populations (Scott & Tanguay,

1994) make the H. polygyrus-mouse system a suitable mode1 for studying chronic

gastrointestinal helmintbis in humans.

The life cycle of H. polygyrus involves both free-living and parasitic stages and

occurs predomuiately in the small intestine of its murine host. As described by Bryant

(1973), parasite eggs are passed in the feces of the infected host. hatch within

approxirnately 36 hours to become the fûst h a 1 stage (L,), which then undergoes two

moults to form the ensheathed non-feeding infective third lamal stage (LJ. Ingested L-,

exsheath in the lumen of the stomacch and migrate to the srnail intestine where they

penetrate the serosal musculature and mature to fom the fourth larvai stage ( L,). Within

7-8 days post-infection (pi), the pre-adults migrate out of intestinal mucosa and emerge into

the intestinai lumen. Adult worms migrate preferentiaily to the anterior duodenum where

they mature, mate and produce eggs. Signincant levels of egg output are observed within

12- 14 days pi (Bryant, 1973) and an average of 1200- l5OO eggs per fende worm per day

are released in the feces of mice infected once with 100 infective L, (Kerboeuf. 1982).

2.1 Host Immunity to Primary Infection with H. polygyrus

Prirnary infections are chronicdiy mriuitained for 4 to 10 months depending on the

rnouse svain (Lawrence & Pritchard, 1994; Wahid et al., 1989). Apart from nuvitional

factors, parasite establishment, survival and fecundity duMg primary infection are

influenced by multiple externai variables such as host population density. level of exposure,

genetic background. and host immune response phenotype (Behnke & Wahid, 1991;

Kerboeuf, 1985; Keyrner & Tarlton, 1991; Scott, 1990). Even in mouse strains which are

able to reject primary infection with H. polygyms. the process of worrn expulsion is

protracted over several weeks with considerable variation among individual animais

(Behnke & Robinson, 1985). Primary H. p o l y g y m infections in most murine hosts are

long-lived despite the ability for gene expression and production of Th2 cytokines (Svetic et

al. 1993) and the presence of pansitic-specinc effector responses such as IgGl (Wahid &

Behnke, 1993). niis chronic survival of H. polygyms during primvy infection is believed

to be related to the immunosuppressing excretory-secretory proteases of adult w o m

(Monroy et af., 1989). These substances released by the adult worm have been shom to

suppress the activity of host generated immune ce&. to resuict intestinal peristûlsis and

mucus secretion, and to neutralize cytotoxic substances such as free oxygen radicals

produced by mucosal mut cells (MMC) or eosinophils (Dehhwi et al., 1987; Dehlawi &

Wakelin. 1988; Monroy & Enriquez, 1992; Pritchard et al., 1994; Reed et al., 1988; Smith

& Bryant, 1986). Aithough these proteolytic and antioxidmt proteases may moduiate host

immune responses, the overaii contribution of these immunosuppressing activities of the

parasite to the chronicity of primary infection has not been dennitively resolved.

Prirnvy infection with H. polygynrs induces marked enlugement and increased

celiulafity of the spleen and MW (Parker & Inchley, 1990a). Prelllninvy work by Ali and

Behnke (1985) suggested that the intensity and npidity of the prolifentive response in

secondary lymphoid organs are related to the inherent capacity of the mouse strain to expel

the Worms. In support of these hduigs. Parker and Inchley (1990b) reported that an

increase in the proportion of B lymphocytes in MLN (taken as a cmde measure of active

humoral immunity) was greatest and most prolonged in modente to high responder strswis

(BALBk and NIH mice) compared to slow responders (CBA mice). T ceN proüferation

was delayed in cornparison with changes in B cell freguency, while total T ceII numbers and

the ratio of T helper to T suppressor ceb were not ditrerent among the zhm strains.

MaxïmaX hyperplasia of lymphoid organs and peak B lymphocyre cefluiarity occmed within

7-12 days post-infection and coincideci with the appeuance of L, in the gut mucosa (Parker

& Inchley, 1990a & 1990b). These results support other observations that L, stimulate a

strong antibody-dependent inflmmatory and granulomatous response in intestinal tissues of

responder suains (Penttila & Jenkin, 1983; Monro y & EMquez, 1992).

Although primary polygyms infection in mice induces inmtration of innYnmatory

mediators (e.g. neuuophils and eosinophils) and parallel increases in serum IgE and IgG1,

mucosal mastocytosis is limited early d e r infection and noticeably absent once adults have

estabhhed in the anterior duodenurn (Dehlawi & Wakelin. 1988; Reed et al., 1988). The

suppression of mucosal mastocytosis by adult H. polygyms is not due to invinsic defects of

rnast cells because mastocposis can be stimulated by concomitant T. spiralis infection

(Dehhwi & Wakelti, 1988) but is associated with the selective dom-regulation of IL-9

and IL40 production in MLN (Brhnke et al., 1993). Although IL-3 and L-4 are

necessary for helminth-kiduced mucosal mastocytosis (Madden et al., 1991), both IL-9 and

IL40 have k e n shown to be important for optimal MMC proMeration and production of

MMC-derived granule proteases (Ghildyal et al., 1992 & 1993; Hultner et al., 1990;

Rennick et al., 1995). In contrast. other researchers have found high numbers of intestinal

MMC in mice infected with H. polygyruî compmd with that of naive mice (Urban et a l ,

1995) but this nematode-induced mucosal mastocytosis appears only after, and not before,

pronounced worm expulsion (Wahid et al., 1995). It has ken mggested that knpaired IgE-

or IgG1-induced activation of mat cek, and thus the reduced degranulation and release of

granule proteins, may be a more important reason for the prolonged worm s u ~ v a l during

p r i m q infection than the hufficient enlugement of MMC in GALT (Wahid et al., 1994;

Woodbury et al., 1984). Thus, the chronicity of primary infection may result from not only

immunosuppressive secretions from the parasite but also, and perhaps more imponmtiy,

from parasite-induced d ysreguia tion of Th2- type cytokine and effector responses.

It is controvenial as to whether IgGl, IgE, and eosinophil responses are beneficid

to the host infected once with H. polygynrs. Pritchard et al. (1983) reported that puritied

IgGl Crom immune semm of rnice infected with kt polygynrr caused severe stunting of

w o m and promoted adherence of peritoneal exudate ceUs to the surface of L, and adult

w o m in vitro. However, a Iater in vivo study by Wahid et al. (1993) showed t h t

parasite-specific IgGl responses did not correlate with the rate of worm expulsion and

were margindy elevated in rnice with heavier wonn burdens. Moreover, the inhibition of

IL4 and its receptor by neutralizing antibodies increved wom burdens in primary H.

polygyrus idection without concurrent aiterations in IgG 1 responses ( F i e h a n et al..

1988b; Katona et al.. 199 1). Later studies showed that adult woms induced mass

production of nonspecific IgG l (Robinson & Gustad. 1995) that could be bound with

proteins on the surface ofH polygynrs (Robinson et ai.. 1997). This was interpreted by

these authors as evidence of an attempt by the p m i t e to mask specinc antigens from

recognition and interaction with host immune celis. Other researchers have also questioned

the hinctional role of Th2 effectors during primvy H. polygyms infection. The

neutralization of secreted IgE by an anti-IgE antibody fded to promote wonn s u ~ v a l

although injection of anti-IL4 mtibody, which suppressed semm IgE concentrations,

resulted in increased worm burdens and fecundity (Urban et al., 199 Lb). IL4 treatment

elllninated an established primary H. polygyrus infection in immunocompetent mice but was

not associated with changes in eosinophü or serum IgE levek (Urban et al., 1995).

Furthemore, injection of an anti-IL-5 antibody suppressed the development of eosinophilia

but had no effect on worm numbers or egg production (Urban et al.. 1991b). In contnst to

these results, Wahid et al. (1994) found that the ability of mice to expel adult H. polygyrus

d u ~ g primary infection was directly reiated to their potential to secrete Th2 cytokines and

to produce prominent and sustained IgG1, IgE, ûnd MMC responses. Thus, an increase in

parasite-induced production of Th2-type effectors reflects a capacity for specific

immunological responsiveness but the precise functions of these Th2 effectors in mediating

host immunity to H. polygynrs infection remah poorly dehed. The inhiiition of a single

parameter of Th2 defense mechanism without a subsequent detectable eEect on worm

expulsion or fecundity does not n e c e s s ~ y prove that these effectors are not hinctiondly

active or important in the host immune response to nematode parasites. but suggests that

there may be some redundancy of host defenses which has evolved to prevent the selection

of parasites that are resistant to any one of these defenses (as reviewed by Fieiman et a[-,

1997). To this end, the host may have developed a complex set of defense mechanisms that

cokctiveIy, through their interactions and compensatory responses. act to effectivdy resist

infections with nematode puasites. However, for reasons that remain poorly understood.

this putative network of cooperative host effector responses fails to provide adequate

protection against primary infections with H. polygynis. In summuy, low immunologicd

responsiveness and chronic primary infection with H. polygyms appear to be related to

geneticaily detemiined host susceptibility to pansite-induced immunomodul;ition, dthough

the critical protective mechanisrns thnt are suppressed are not clearly defined.

Despite the equivocal evidence for the benefit of Th2-type effector mechitnisms. the

production of Th2-type cytokines is thought to be crucial for the resolution of H. polygyms

Section Injection of anti-IL4 or anti-IL-4 receptor antiiodies into rnice idected with H.

polygyrus increased worm survival and fecundity in mke (Urban et al.. 1991b). The

importance of IL-4 to protection a g h t prirnary infection was confirmed by a later study

which found that treatment of H. polygyms-inocuhted mice with exogenous purûied IL4

signincantly lirnited puasite s u ~ v a l and completely suppressed fecundity (Urban et al..

1995). Interestingly, the effector mech;uiisms by which IL-4 tenninated the primary

infection were uncleu because leveis of MMC. IgE, and eosinophils did not dBer between

IL-4-treated and untrerted rnice. The independent ability of increased IL-4 concentrations

in vivo to selec tively enhance these Th2 e ffec tor responses in the absence of altered

concentrations of other Th2 cytokines is no t known. The observation that IL-4 treatment

inhibited adult worm s u ~ v d to a greater extent than h a 1 establishment led Urbm et al.

(1995) to specuiate that IL-4 rnay activate nonspecitic immune defenses such as intestinal

smooth muscle contraction. mucus secretion, or peristalsis which in tum rnay help facilitate

worm expulsion f?om the s m d intestine. Recently, preliMnary data suggest that IL4 rnay

increase gut spasticity. s m d intestinai permeability, and decrease fluid absorption in the

s m d intestine (Urbui et ai.. 1996). These changes in smooth muscle contractility and

intestinal fluid dynamics may faciltate expulsion of H. polygy~us during primary Section

by decreasing parasite anachment to the intestinal viIli (thus limiting parasite establishment)

ancilor by testricihg access of worms to their food source within mucosai tissues.

Alternatively, IL-4 treatment rnay have initiated immune-mediated responses bat damrged

the lame or young adult Worms and thereby reduced their potentid for chronic suMvaL

2.2 Systemic Versus Gut Mucosal Immunity to Rimary Infection 16th H. poiygyms

Aside from the analysis of mucosai rnastocytosis, much of our knowledge on

immune responses to H. polygyrus ha been based on serum levels oETh2-associated

antibodies (e.g. IgE and IgG 1) or cytokine production (TL-4, IL-5. IL9 or IL- 10) in the

spleen. Studies examining the specifc advantages of Th2 effector mechanisms such as IgE-

mediated immune reactions have produced inconsistent results perhaps because they have

not examined these Th2 immune responses at the usual site of infection in GALT. It

appears logical that induction of Th2 cytokine and effector ceU production also occurs in

intestinal mucosal tissues where the nematode piirasite first interacts with the host and

spends the majority of its life cycle. However. ody recently bas there k e n concentnted

research on the role and relative importance of gut mucosd immunity in the resolution of

intestinal nematode infections.

Parker & Inchley (1990a) reponed that the pcak cellular proliferative response to

H. polygyms infection occurred eulier in MLN than in spleen (Parker & Inchley 19903,

thus suggesting that the k s t Ipphocytic response to an intestinal nematode is restricted to

infected enteric regions. Unfortunately, the authors did not determine whether this eulier

cellular proliferative response in MLN resulted in a more rapid omet of functionai immunity

at the gut kveL To mess tirne-dependent cornpartmentalization of Th2 immune responses

to H. polygyrus, Svetic et al. (1993) studied Th2 cytokine gene expression in spleen and

GALT (MLN and Peyer's patch) within 12 days after primary Section The ruthors found

that IL-3. IL-4, IL-5, and IL9 mRNA expression as measured by a RT-PCR assay was

markedly elevated in the Peyer's patch and MLN ceils within 2 days pi and remained above

u ~ e c i e d levels for up to 12 days afier infection. In contrasr. the gene expression for

these Th2 cytokines remained unchmged in splenic c e h diroughout the experirnentd

period. These results suggested that the early hinctional immune response to H. polygynrs

as measured by mRNA activîty w u locaiized at intestinai sites of infection CurrentIy, no

study has c o n h e d whether the higher Th2 cytokine mRNA expression in the gut during

EL polygym infection is accornpanied b y equivalent increases in pro tein secretion although

gene expression usuaiiy correlates well with protein synthesis. Table 1 shows a compilation

of data on the kinetics and site-specifcity of Th2 cytokine and effector responses following

prllnary Section with H. polygynrs. The principal conclusion to note from this table and

the above studies is that Th2 cytokine gene expression and secretion in GALT usually

precede the production of Th2 cytokines in the spleen as weU as the onset and peak of Th2

effector responses in the peripheral circulation.

Because wom outcomes were not assessed by Parker & Inchley (1990a) or by

Svetic et al. (1993), it is unclear whether this earlier omet of ceiiuiar and cytokine

prouerative responses in GALT i s critical for protective irnmunity to the nematode

parasite. In one study that examined the correlation between Th2 immune responses in

GALT and worm burden of a primary response, Wahid et al. (1994) detemiined whether

Th2 cytokine secretion in MLN as weil as MMC responses difZered between fast and slow

responder mouse s trah. The fast responder suain (SAR mice) expelled over 961 of the

Worms by 8 weeks pi compared to the slow responder strain (CBA mice) which

experienced no signincuit worm Ioss throughout the experimental penod. The MLN of

SAR mice secreted consistently higher amounts of L-3, IL-4, and IL-9 over the course of

infection compared to MLN of CBA rnice. Moreover, significantly higher MMC counts

and senirn concentrations of MMCP-I (used as a systernic marker of mucosal activation

ancUor matocytosis) were found in SAR rnice versus the CI3 A mice. Thus, suMvaI of the

parasite and ultimately the chronicity of infection may depend on the intensity and

persistence of Th2 cytokines and effector responses ar the intestinal kveL The relative

importance of gut mucosal versus systemic immunity to infection outcomes such as worm

s u ~ v a i has not been studied in Our experimental H. polygynrs-mouse modeL

Table 1: Kinedcs of Th2 Cytokine and Effetor Production Post-primary (PH) and Post-challenge (PCI) Infection with H. pofygyms in BALBlc mice (unless othenvise indicated).

The Post-infectio n PPI PCI CytokineEffector Response Tissue Location

Onset of IL-3, a-5, IL-9 gene expression PP1 Marginal increase in IL4 gene expression MLNL

1-2 days Sustained L 3 , IL-9 gene expression PP & MLN'

2-4 days

4-6 days

6-8 days

Omet of IL4 gene expression PP & MLN' Onset of I L 3 & IL-4 secretion (SAR mice) MLN'

3-5 days Omet of eosino philia (NIH mice) Whole Blood3

Peak IL-5 gene expression Peak IL-3 gene expression

PP' PP & MLN'

4-6 days Onset of total IgE response Serum4* ' Onsei of total I Q response (NIH mice) Semrn3

5-7 days Peak eosinophilia Whole Blood3* Onset of parasite-specifk IgG 1 (NIH mice) Senimg

Sustained IL-5 gene expression MLN ' Peak IL-9 secretior (SAR mice) MLF Muginai increase in IL- 10 secretion MLN2 Onset of MMCP- I increase Semm2 Onset of total IgG 1 response (NIH mice) Serurn3

7 days Peak total IgG l response Serum6

8-9 days Peak total IgE response Sem4* ' Deche of eosinoph (NIH mice) Whole B100d3

7-14 days Onset of to tai IgE and to ta1 IgG 1 response Serum4*

continued PP = Peyer's patches MLN = Mesenteric lymph nodes

Table 1 : continued

T h e Post-infection PP PC CytokindEffector Response Tissue Location

12 days Peak MMC numbers (SAR. CBA mice) Smdl IntestineZ Peak IL-5 gene expression MLNL

10-15 days Peak pansite-specific IgG l response serurns

14-15 days

16 days

20-21 days

24-30 days

Peak eosinophilia Whok Blood6 Peak total & specitic IgGl response serumb

14-2 1 days Peak total & specitic IgGl response Serum6* ' Peak serum IgE response Serum63

Peak MMCP-1 leveis (SAR. CBA rnice) Semmz

Second peak of IL-9 secretion M N Decline of IL-4 secretion (SAR/CB A rnice) MLN2 Peak total IgE response Senim4

Dccline of MMC numbers (SAR mice) S m d Intes the' Decline of MMCP- 1 leyels (CBA mice) Serum'

MLN = Mesenteric lyrnph nodes T

' Svetic et ai., 1993 * W;ihid et al., 1994 ' Shter & Keymer, 1988 Urban et al., 199 1 a ' Wahid & Behnke, 1993 Boulay et al., 1998

'Urban et al., 1991b ' Katona et al.. 1991

Wahid & Behnke. 1992

2.3 Host Imminity to Challenge Infeftion with H. polygyrus

The hast is capable of developing effective resistance against secondary exposures

to H. polygyrus following the removal of adult worms from a primary infection by an

anthelmintic-abbreviated regirne (Behnke & Wakeh, 1977; Behnke & Robinson. 1985).

oral inocuiation with irndiated b a e (Slater & Keymer. 1988). or other immunizing

methods (Urban et al., 199 la). The rapidity and intensity of acquired immunity achieved

upon chdenge infection depend on the dose of the immunking inoculum, the genetic

susceptibility of the host, and the infectivity of the larvae ( Jacobson et al., 1982; Ali &

Behnke, 1985; Behnke & Robinson. 1985; Dobson et al.. 1985; Enriquez et al., 1988).

Protective immunity to challenge infection with H. polygyms is demonstrated by reduced

won site, depressed fecundity. and more rapid expulsion of adult wonns (as reviewed by

Monroy & E ~ q u e z , 1992). The L, and L, IYvae of H. polygyms are believed to be potent

inducers of protective immunity in responder strains of mice (Monroy & Enriquez. 1992;

Wahid & Behnke, 1992). In most murine straiils, p r i m q H. polygyms infections, which

are terminated before the parasites mature to adulthood by drug treatment or irradiation.

induce high levels of acquired resistance to iarval challenge infections (Behnke et al.. 1983;

Behnke & Robinson. 1985; Wahid & Behnke, 1992). The strong and npid acquired

imrnunity to secondary lyval exposures fo flowing an inmuniMg regime is thought to

overcome any inherent immunomoduIatory stntegies employed by the nematode puasites

(Monro y & Enriquez, 1992). However. develo pment of ihis pro tective immunity depends

on the stage of the parasite used in the irnmuniPng pmcedures because no protection wûs

evident in mice when adult w o m were used for the initial sensitization. when the primyy

infection was terminated after the appearance of adults worms in the intestinal lumen, and

when the mice were chatiienged with adult worms (Behnke et al.. 1983; Enriquez et al..

1988; Jacobson et al., 1982; Pleass & Bianco, 1994). The appevance of increased effector

responses upon h a 1 challenge of previously immunized hosts suggests that immun0 Io gical

processes primed during primuy exposure are important in the effective resolution of

secondary infections (as reviewed by Monroy & Enriquez, 1992).

Immunological memory is characterized by accelerated speed, increased size and

persistence, dl of which are observed during secondary immune responses to challenge or

repeated idections with nematode parasites. Immunocornpetent hosts are able to develop

strong resistance ta challenge infection with H. polygyrw as evidenced by the dramatic

reduction in worm survival, wonn size, and fecundity shortly after secondvy exposure

(Behnke & Wakelin, 1977; Behnke & Robinson, 1985). This rapid and intense temination

of secondary H. polygynrs infections is thought to result from the mobilization of protective

Th2 immune responses that had been primed during previous mtigenic exposure. Acquired

immunity to challenge infection following an anthelminthic-abbreviiited primary infection is

CD4' T celi dependent and involves Th2 effector responses (Urban et ai., 1991a). Indeed,

senun IgGl rose ten-fold from resting levels in mice repeatedly immunized with immune

mouse serurn (Williams & Behnke, 1983) and the-fold in mice challenged with the

pansite compared to mice given only a single inocuiation (Urban et al., 199 la). Passive

tramfer of pr imvy infection senirn containhg pansite-specinc IgGl did not protect

unprimed mice from H. polygyrw (Williams & Behnke, 1982) whereas multiple Section

sera were capable of transferting immunity to naive recipients (Behnke & Parish. 1979).

Also. concentrations of semm IgE and numbers of MMC were significantly higher in

chaiienged mice relative to primary infection controls (Dehlawi & Wakelin, 1988; Urban et

al., 199 la). Consistent with the concept that secondary exposwe to polygyncs in

previously immunized mice stimuhtes npid activation of memory Th2 immunity, Table 1

shows that the onset and peak of IgE, IgG 1 and eosinophil responses in the peripheral

circulation of challenged mice generdy occurs 2 days to a week earlier than that foUowing

primary infection. Unfortunately, there are no studies evduating specitic cytokine profles

during challenge infection and therefore it is u n c o ~ e d as to whether an ekvated

secretion of Th2 cytokines is responsibk for the apparent potent activation of Th2-type

effector responses in mice chdienged with nematode parasites. However, this cytokine and

eEector interaction in the gut is highly probable considering that cytokines are the primary

factors that induce and reguiate effector responses. A cytokine inhriition study by Urbm et

al. (199 1 b) provided some indirect evidence that Th2 cytokines are important for the

resolution of secondary infections. Injection of an anti-IL4 antibody suppressed

secondary IgE responses and was associated with increasised worm counts and fecundity on

day 48 pci Interestingly, the role of IL4 and associated eosinophil response in acquired

imrnunity to H. p o l y g y m is not clear because neutrdimtion of IL-5 blocked nematode-

induced eosinophilia but did not affect worm burden or fecundity in chaiienged mice.

The relative contribution of naive and rnemory effector c e h to immune responses

observed during challenge infection is not cleu. Memory Th ceUs are not as stringent in

their rcquirements for IL-4 or costirnulatory signais (CD28/CTLA4 and B7.1tB7.2

interactions) during activation and effector activity compared to naive Th ceUs (Gause et

aL. 1995; Huang et aL. 1997). It is possible that the priming of Th2 cytokines during

prirnary infection rnay be a more criticai immunologicai event than secondvy responses for

the development of protective imrnunity to nematode parasites. However, some studies

have suggested that memory CD4+ T ceh are unstable, depend on the persistence of

antigen, and may reven to a resting, quiescent form that is phenotypicdy indistinguishable

from naive CD4' T c e h (Bunce & Bell. 1997; Bell et al.. 1998). IL-4 was found to be

crucial for maximal capacity for Th2 cytokine secretion by memory Th effector c e h that

came from a heterogeneous pool of resting memory CD4* T ceUs (Bradley et al.. 1995)

and therefore may be important in potentiating previousiy comrnitted active T ce& as weii

as in reactivating memory revenant T cek. In addition. Katona et al. (1991) and

F i e h a n et al. (1 997) reported that IgE responses in secondary infections with H.

polygynrs were derived mostly from naive or uncommitted B cells rather thm fiom IgE-

committed memory B ceils produceci during initiai nematode infection. These preliminary

results must be verined by experirnents on other Th2 effectors to provide precise

description of the immune popuktions and reguliitory factors that are activated during

challenge infection.

In sumrnuy, functional immunity to H. polygyms appears to be a Th2-dependent

process because it involves specific cytokines such as IL-4 and effectors such as IgE-

dependent eosinophils and MMC. The magnitude and eficacy of Th2-mediated immune

mechanisrns may d s e r according to the infection protocol used with the greatest level of

protection seen d e r secondary exposure. Preliminary data point to the importance of Th2

immunity in GALT which may play a d e in promothg the O bserved increases in serurn

Th2 effector responses. Given that immunocompetent hosts are able to mount a strong and

effective acquired immunity upon subsequent infections, it would be important to determine

the magnitude. kinetics and companmentdhtion of Th2 immune responses during

challenge infection and to mess whether these characteristics are altered by nutritionai

deficiencies such as protein malnutrition.

3. EFFECT OF PROTEIN DEFICIENCY ON HELMINTH INFECTIONS

Pro tein deficiency is generdy synergistic with bo th naturd and experimental

helminth infections and thereby results in an interaction that is more seriously harmhl than

would be expected Erom the occurrence of either condition alone (Scrimshaw et al., 1968).

Protein malnutrition and intestinal parasitism share similar geogrrphicd distribution patterns

with the sarne individuais experiencing bo th diseae States simultaneously throug hout their

lifetime. For exampie, a study of rurd Nigerim children found that protein intakes were

inversely proportional to the number of CO-existing intestinai parasitic infections

(Rosenberg & Bowmm, 1984). Additiondy. Trichuris egg counts in children iiving in an

urbm slum region of Indonesia were correlated with stunting. a physiological indicator of

chronic protein calorie malnutrition (Hadju et ai., 1995. These data supports the concept

that malnourished children living in helminth endernic areas are more heavily parasitized

than theY webnourished counterparts. However. given the multi-dimensionai nature of

malnutrition-parasite interactions and the interpretative pro blems of epidemiological data, it

is difncult to ascertain which condition was the preceding or causative factor.

3.1 Effect of Pmtein Deficiency on Pnmrry Infection with H. polygyms

ControUed experirnents are more conducive to studying the nature of putative

casual rehtionships between protein mdnutrition and intestinal parasitic Uuection. These

systems atternpt to reconsvuct the natunl transmission of nematode parasites o c c u ~ g in

human settings by establishg a cohon of susceptible animals inhabithg an arer of endemic

helminth infection. Shter (1988) housed N c e in large, unrestricted cages which provided

free access to a 2% or 16% protein diet. An initialcohort of mke w u infected with H.

polygyrus, and at 5 week intervals, subsequent cohorts of young uninfected rnice were

introduced into the enclosures whüe previous cohorts were removed. The rate at which the

uninf'ted mice acquired the parasites was higher in the low protein colony, thus îndicaiing

th t protein deficiency may enhance the rate of pansite transmission within a mobile

population of hosts. The prevalence of infection arnong protein-deficient mice was higher

compared with that of the welI-nourished mice and may be related to the observed increase

in s u ~ v a i of aduit womis in these deficient animals over the experhental period. These

results show that, under naturd infection conditions. protein deficiency may potentiate the

prevalence and intensity of a nematode parasitic infection.

Evidence to support these fkdings using controiled p h a r y infection protocois is

inconclusive. An euly study by Bawden (1969) showed that a marginal protein diet (white

bread with 12.3% crude protein) produced greater establishment of H. polygym in mice.

signincant stunting of female adult w o m . and more widespread distribution of nematodes

dong the srnall intestine relative to the nomal protein diet (pelleted concentrate with

25.2% protein). Interestingly, protein restriction had no effect on the long-term expulsion

rate of nematodes but w u associated with decreased fecundity at 28 days post-infection.

Bawden suggested that the retarded growth and dtered distribution of femde w o m

recovered in the malnourished hosu adversely affected their reproductive capacity. Given

that H. polygym w o m tend to mate and produce eggs in the antenor duodenum, the

10 nger migration route induced by pro tein restriction rnay have subsequently impeded their

usud reproductive processes. It is dficult to interpret these results due to the intermediate

level of protein restriction and the use of diets with difTerent macronutrient constituents.

These uncontrolled d i e t q factors may have been responsible for the absence of nutritional

impact on chronic worm survivd Using purifed synthetic diets, Bnilsford and Mapes

(1987) reported that a more severe restriction of dietvy protein (2% casein) did not

significmtiy increase wom establishment or s u ~ v a l despite inhibiting body weight gain

and producing overt hypoalbuminaemiû. during primuy infection. These authors found that

worm burdens in control(204b protein) and protein-deficient (2% pro tein) animais

remained equivalent at 3-4 weeks post infection. Previous studies in our laboratory found

that marginal (7%) and low (3%) protein diets resulted in greater worm burdens at day 29

post-primary infection (Boulay et al.. 1998). These results dEer fiom those of Bnilsford

& Mapes (1987) perhps because Boulay et al. (1998) found unusudly low parasite

numbers in the control diet group (rather than larger wonn burdens in the deficient mice)

which may have kad to signûicant differences in worm sumival between dietxy groups.

It is possible that a primary infection protocol in which laboratory anima are

exposed only once to the nematode parasite may not be a sensitive experimental system to

evduate the influence of dietary protein on outcornes of H. polygyn<s infection. P N n q

infections with H. p o l y g y m are usually chronic in othenvise immunocompetent murine

hosts. suggesting that the parasite has the inherent capacity to down-regulate or prevent

protective host immune responses (Behnke et al., 1992; Dehhwi & Wakelin, 1988; Smith

& Bryant, 1986). Assuming that secondary infections nonnally stimulate development of

protective immunity in weU-nourished immunocompetent hosts (Monroy & Enriquez

1992) ami that protein deficiency impairs irnmunological processes, a repeated or challenge

infection protocol mny help to maximize the chances of detecting any possible effects of

dietary pro tein deficiency on intestinal helminth popuiation dynamics.

3.2 E f k t of Protein Deficiency on Challenge Infection with H. polygym

Experimental infection studies show that the level of dietvy protein influences the

extent to which the host cm develop protective irnrnunity to repeated or secondvy

infections. Slater and Keyrner (1986) fed mice 2% or 8% protein diets and repeatedly

infected animais with vvying doses of infective k a e of H. polygyms every 2 weeks for a

period of 12 weeks. Mice fed the 2% protein diet accumulated worms in direct proportion

to the intensity of exposure throughout the experiment whereas mice fed the 896 protein

diet showed partial resistance to repeated infection at higher doses. Also. net egg

production was increased in rnice fed the Iow protein diet due to both higher per capita egg

production of femde w o m and hewier worm burdens. The authors attributed the

increased s u ~ v a i and fecundity of w o m in the mainourished mice to either a diet-induced

inhibition of acquired immunity to reinfection or to increued sensitivîty to parasite-induced

irnmunosuppression. Both explanations rely on the assumption that protein deficiency

impairs optimal irnmunocompetence. Consistent with the results obtained by Skiter and

Keymer (1986). Keymer and TuIton (1991) observed higher wonn burdens in repeatedly

infected mice fed 3% protein diet compared with those fed 16% protein diet. These

authors also used a challenge infection protocol in which the primary infection was

abbreMated by an anthelmintic dmg and the mice were then reinfected once with 100

infective bac. At two weeks post-challenge infection, mice fed the low protein diet had

more w o m thm their weU-nourished counterparts, suggesting that pro tein deficiency

impairs development of irnrnunological memory normally primed by previous exposure to

infectious pathogens. Using a chailenge infection protocol. Shter & Keymer (1988)

recovered significantly more w o m in &male NIH mice fed diets containhg 2% protein

compared with 16% protein at 21 days post-challenge infection. In contrat to restlts of

their previous study (Shter & Keyrner, 1986) which had ernployed a repeated infection

protocol, Slater & Keymer (1988) found that per capita fecundity of a femde worrn in mice

challenged with larval H. pofygyms was not altered signüicantly by the low protein diet. It

is unknown whether repeated infection protocois elicit different immunological responses

than challenge infection protocols and whether this rnay be responsible for these confiicting

effecu of protein deficiency on worm fecundity. Bouhy et al., (1998) reported that o 3%

protein diet increased total egg output compared to a 24% protein diet throughout four

weeks of challenge infection with H. polygyrus. Taken together, protein restriction

impaired the capacity for acquired host resistmce to secondary infection as indicated by the

increased worm burdens in previously immunized protein-deficient mice.

In sumrnary, Gndings from controiled labo ratory and experimentai epiderniology

studies demonstrate that the overd protein nutriture of the host affects the population

dynamics of a nematode parasite. Although many researchen have attributed the increased

survivai and fecundity of nematodes in protein-deficient hosu to impaired host imrnunity,

only a few studies directly examined Th2 immune responses after both nematode infection

and protein deprivation (e.g. Boulay et al., 1998). Inasmuch as host immune defenses are

important in mediating susceptibility to parasitic infection, the immunologic mechanisms by

which protein deficiency rnay exacerbate intestinal parasitism deserve in-depth

investigation. To this end, the foilowing sections discuss in d e t d some of the literature on

protein malnutrition and host immunity.

4. EFFECT OF PROTEIN DEFICIENCY ON SYSTEMIC CELL-MEDIA'IED AND HUMORAL IMMUNITY

The predorninant approach of previous literature on interactions of malnutrition

and immunity has k e n to examine diet-induced structural and hnctional changes in

systemic lymphoid tissues and theu associated immune ce1 populations. The thymus and

bone rnarrow are essential tissues for the production of lymphocyte, monocyte and mast

celi pro genito rs, ho wever, the y are no t specitically designed to facilitate immune responses

upon antigenic stimulation. Initiation of rnost primary immune responses to blood-borne

pathogens including antigen presentation, activation of B and T lymphocytes, and the

production of antibodies and T ceU-derived cytokines occurs mainly in the spleen. Antigen-

specific immune effectors (e.g. imrnunoglobulins and differenthted T cells) produced in the

spleen enter the systernic circulation to migrate to sites of infection and are detectable in the

peripheral blood. Thus, the term systemic immunity used in this paper wili apply to

nonspecific and specitic immune responses in penpherai sites such as the bloodstreun,

thymus. or spleen.

Dietary protein deficiency has been shown to adversely affect the morphology of

systemic lymphoid orgus, particukly in the irnrnediate postweaning period which

represents a crucial stage of development and maturation of the lymphomyeloid system

(Speu & Edehan, 1974). Thymic and splenic atrophy is chmcteristic of protein-energy

malnutrition in children and young kboratory animais (Chuidra, 1983). Bell et al. (1976)

found significant organ weight loss, decreved ceil yields, and stnicturally disorganized cells

in the thymus and spleen of w e d h g mice fed a 4% protein diet for four weeks cornpmd

to mice fed a 2 8 4 protein diet. Given the importance of the thymus and spleen in the

production and maturation of T lymphocytes, respectively, a likely consequence of

lymphoid organ involution is decreased numbers or altered proportions of T lymphocyte

subpopulations. Pro tein-deficient rats and patients with kwashiorkor have lower counts of

total cucuiating or splenic T lymphocytes (Abbott et al., 1986; Bises et aL, 1987;

SIo bodivlik et al., 1984) whiIe pro tein-rnalnourished adults also had lower percentages of

CD3+. CD4+, and CD8' T cells in blood (Abbott et aL , 1986). Contradicting these fjndings,

Woodward and Miller (1991) and Woodward et al. (1995) reported rhat a very low protein

diet (0.5% protein) increased the percentages of CD4' and CD8+ ce& reiative to totd ceU

numbers in spleens of weanluig mice. The biological significance of these proportional

increases is unclear considering that the total cell population was drastically reduced (96-

998) by the severe protein deficiency. Some researchers found greater proportion of

cytotoxic/suppressor (CD8+) T cells relative to that of T helperfinducer (CD4+) c e h in

pro tein-deficient anirnals (Nimmanwudipong et aL. 1992) while others reported that the

ratio oFCD4+ to CD8+ celis did not v u y from control values in protein mainourished

animals or humans (Abbott et al., 1986; Bises et al.. 1987; Woodward & Miller. 1991).

Therefore, it is equivocal as to whether an altered ratio of helper to suppressor T ceUs

contributes to the Th cell-dependent immunodepression observed in protein malmurished

animals (Woodward & M e r , 1991). A recent paper examining the effects of protein

deficiency in experimental pulmonary tuberculosis in guiner pigs reponed that pmtein

deprivation (10% ovalbumin versus 30%) did not affect the proportions of ap T c e h

(CD4' and CD$+) in the spleens of naive animais (Mainali & McMurray, 1998). When

mycobacterial infection was superimposed on the protein deficiency, there was a d m &

Ioss of ap T ce& Erom the spleen ûnd a large decrease in the CD4+/CD8+ ratio in lymph

nodes due to a disproportionate increase of the CD8' subset at the expense of CD4+ T ceiis.

These results imply that analysis of immune cells in uninfected animais may not necessdy

reflect induced responses to antigenic stimuhtion. Lower numbers of CD4' ceh would

have O bvious implications for the capacity of the host io respond effectively to nematode

parasites because CD4+ T cells control the development and activation of B ceUs and

effectors via secretion of cytokines or direct cellular interactions. Interestingly, Woodward

et of. (1995) observed higher numbers of CD4+ and CDS* cells expressing the CD45RA'

surface market of quiescent or hyporesponsive T cek relative to ceils with the CD45RA'

marker of memory-phenotype T ceIlse These results provide the first indication that protein

deficiency decreases the number of responsive T ceîl subsets in the absence of exposure to

an extemal antigen The exact mechuiisms by which protein d e e n c y contnïlutes to

depressed activation and maturation of T ceiis remain to be elucidated.

Recent data point to the involution and dysfunction of specinc thymic

compartments, namely the thymic epithelium which promotes functional dserentiation of T

lymphocytes through the release of thymulin and other hormonal factors, as contribuhg

factors in the decrease in T lymphocyte nurnbers seen in protein depleted subpcts (Wade et

aL. 1988; Woodward et al., 1992a). Administration of thymuh by Parent et al. (1994) to

malnourished children with marasmus andor kwashiorkor increased the percentages of

CD3'. CD4+ and CD8' and decreased the proportion of immature T lymphocytes (CD 1 a) in

their penpherai blood, Also, thyrnulin is reputed to exert a positive influence on

prolifentive capacity of peripheral T ce&. Thus. the thymic and splenic atrophy induced by

protein malnutrition in we&gs may result in depressed maturation and prolifierative

capacity of specific T lymphocytes. Unfortunately, none of these studies e x d e d the

hinctional hpücations ofdecreased or altered T ceii subpopuktions during infection in

mainourished mixnds or humans.

In addition to dtered nurnbers of specific types of T lymphocyte. protein deficiency

may have detrimental effects on the efiïcacy of CMI responses to Mtogenic or antigenic

stimulation. Deitch et al. (1992) found that splenic lymphocytes from mice fed a nearly

protein-kee diet (0.03% protein) for 7, 14 or 21 days had progressively lower biastogenic

responses to two T ceil mitogens (PHA and ConA) compmd to basehe levels. The lower

mitogen responsiveness of T lymphocytes from mice fed a low protein diet is a crude

indicator of depressed nonspecitic CM1 but does not necessarily refiect altered T cell

responses to speciiïc antigens. Lamont es al. (1988) showed that the vansfer of ovdbumin-

primed lymphocytes fiom donor mice fed a 24% protein diet to the footpads of naive mice

fed 4% protein die< resulted in a depressed cutaneous delayed type hypersensitivity @TH)

response (a characteristic feature of CMI) whereas a positive DTH response w u elicited in

footpads of naive mice fed 24% protein. Using a graft rejection assay. Larnont et al.

(1988) found that splenic T cells derived Crom rnice fed 4% protein induced significant

splenornegaly in webnourished recipients. The authors interpreted these resuits as evidence

that protein deficiency did not impair the functional capacity oCT cells perse but rather it

altered other ceU-mediated immune responses (e.g. innltratioa of leukocytes to site of

antigen challenge or T cekdependent activation of macrophages) which are necessary for a

positive DTH response. Thus. protein deficiency may decrease not only the proliferative

capacity of T lymphocytes but dso impaircd T cell-dependent immune processes and

interfered with celluiar interactions required for effective CM1 responses.

Although thymic involution and su bsequent alterations in T lymphocyte numbers are

frequent rndestations of protein deficiency, evidence is inconclusive as to whether CMI is

more seriously impaired by malnutrition than is thymus-dependent humord imrnunity.

Severe protein restriction (0.5% protein) induced a greater reduction in splenic B

lymphocyte numbers than in T lymphocyte numbers (Woodward & Miller, 1991).

However, changes in lymphocyte numbers do not necessarily correlate with altered

hnctiond lymphocyte responsiveness. Woodward et al. (1 W b ) found that protein

denciency impaired DTH to sherp red blood cells (SRBC) but had no effect on the serum

titres of anti-SRBC antibodies. Human studies consistently report depressed DTH

responses, lower mitogen-stimulated proliferative capacity of T lymphocytes, and lower

numbers of circulating T lymphocytes but presemed B cell function and

hyperimmunoglobuiinemia in chiidren with protein malnutrition (Chandra. 1983; Ozkm et

aL, 1993; Punilo et al.. 1976). In support of these fmdings, Deitch et al. (1992) reported

that severe protein deficiency (0.03% protein) decreased biastogenesis of spknic

lymphocytes to T cell mitogens (PHA and C o d ) to a greater extent than that to a B celi

mitogen (PWM). Although these mitogens do not elicit exclusive proliferation of either T

or B lymphocytes, these results do imply that competence of CM1 rather than of humorai

immunity is more sensitive to changes in dietary protein IeveL In sumrnary, protein

deficiency causes structural and functional defects in systemic lymphoid organs and

peripheral lymphocytes which are required for the induction and propagation of CM1

ac tivities and T cell-de pendent hum0 rd respo mes.

5. EFFECT OF PROTELN DEFICIENCY ON GUT MUCOSAL IMMUNITY

The functional relevance of systemic immune respoases to intestinal heiminths is not

clear because infections with these parasites are predominantly localized in enteric tissues.

Given that the gut c o n t a speciaüzed secondary lymphoid tissues that can react directly

with antigens present in intestinal mucosa or lumen. it is of cruchi importance to examine

the consequences of protein deficiency on gut mucosal immunity to nematode parasites.

The impact of protein deficiency on gut mucosd immunity cm be tirst observed by changes

in the gross morphological chancteristics of GALT. Bell et al. (1976) reported that dietvy

protein restriction (4% protein) induced atrophy of MLN of we;uiling mice as seen by the

reduction in organ s k and loss of mature B cells over the four week feeding penod.

However. the authors found chat the tissue loss and structurai defects in the protein

deficient mice were less pronounced in MLN than those in the thymus and spleen.

Simiiiirly, Lochmilier et al. ( 1992) reported no signifcant differences in the number and

average total weight of Peyer's patches in the srnail intestine among weanling rats fed 4%

and 16% protein diets for 6 weeks. When expressed relative to body weight. Peyer's

patches were 84 to 95% larger in the protein-deficient rats compared to the wehourished

rats. indicûting that the low protein diet crused a greater decrease in total body weight than

in the weight of Peyer's patches. In contrast. Woodward and Miller (1991) and Deitch et

al. (1992) found that severe protein rndnuuition equaily depressed splenic and M W orgm

weight and lymphocyte pools. The CD4/CD8 ratio and percentage of CD4' or CDS' ceh

in MLN were not affected by feeding a very low protein for 3 weeks (Lee & Woodward.

1996). Taken together, these studies indicate bat gut lymphoid tissues mîy be more

resistant to gross organ stunting induced by moderate protein restriction than peripheral

lymphoid organs or body mus in growing animais. However, the very severe ievels of

protein restriction used in bo th studies (0.5% and 0.03% pro tein, respectively) and the use

of nYve animais preclude my applicable conclusions to be drawn Erom these 6ndings

toward nematode infections in protein-depkted humans. Regardless of the relative extent

of gross structural changes to intestinal lymphoid tissues. there is considerable evidence that

protein deficiency induces morphological abnomalities in the intestinal villi (e.g. crypt

hypoplasia) md increves pemeability of the gut mucosal bvrier (Curnmins et al.. 1987a &

1987b; Deitch et al., 1992). Diet-induced villus atrophy and a disrupted mucosal barrier

integrity may enable parasites to penctnte and establish in gut mucosal tissues and thus

could be more dethentai to host resistance thm gross changes in lyrnphoid organs and

lymphocyte numbers. Moreover, an increasingly penneable gut barrier may ailow leakage

of p h m a proteins and nutrients which may further compromise gut-associated Unmunity.

In addition io the O bserved morpho lo gical defects, reseuch has documented

irnpaired functional capacity of gut-associated immunity in pro tein-restricted animals.

B lastogenic responsiveness of lymphocytes from M W and Peyer's patches to T cell

mitogens (PHA and ConA) was depressed over t h e in mice fed 0.03% protein diet (Deitch

et al., 1992). Although B cell responsiveness to mitogen is rehtively retained in protein-

deficient mice (Deitch et al.. 1992). protein restricted diets (2 to 4% protein) have k e n

associated with reduced intestinal IgA concentrations (Lim et al., 198 1; McGee &

McMurray, 1988; Sullivan et al.. 1993). impriired dinerentiation of IgA' precursors in

Peyer's patches ta a mature IgA-expressing B ceil phenotype (Lopez & Roux . 1989). and

dimùiished ability of gut lymphocytes to mediate mtibody-dependent ceil-mediated

cytotoxicity ngainst SRBC (Lim et al.. 1981). Recent reports show that the reduced

intestinal I ~ A concentrations during protein-energy malnuvition may not result fiom

impaired IgA synthesis but more Likely fiom decreased transport of IgA precursors to the

intestinal epithelium (Ha et aL. 1996; Ha & Woodward, 1997). The devance of these

defecu in gut-associated IgA and rnitogen-stimulated proliferative responses to helminth

section is not cleu. dthough decreases in the intestinal IgA+ B cell pool mriy lower the

potential for nemntode-elicited production of IgE- and IgGl-expressing B cek in GALT.

Another mechanism by which protein deficiency may contribute to impaired host

immunity to intestinal parasites is altention of the homing patterns of mucosal lymphocytes.

McDermott et al. (1982) transferred radiohbded MLN lymphobhts prepared from well-

nourished donor rats to recipients with or without protein-calorie malnutrition and found

that radioactivity in small and h g e intestines did not differ among normal or mahourished

rats. In conuast, radiolabekd MLN lymphobhu fiom malnourished donors inje!cted into

well-fed recipients resulted in decreased radioactivity recovered in s m d and large

intestines. The authors concluded h3t protein-calorie mdnutrition did not alter the

capacity of the gut to take up mucosd lymphocytes but did impair the selective intestinal

locûliz;ition of GALT-derived lymphobiasts. Although these preliminq data do not

provide any information regarding molecuIar or ceiiular mechmkms, the authors specuiated

that protein deficiency may have modified the function mdlor structure of lymphocyte

horning receptors that mediate lymphocyte binding to the endothelium of HEV in gut

mucosd tissues. Composition of the diets and the degree of deprivation induced in the

recipient rats were not specifed and therefore these results are diffcult to hterpret and to

generalize to systems involving both rnûlnutrition and intesthai parasitic infection.

6. EFFECT OF PROTEIN DEFICIENCY ON IMMUNE RESPONSES TO INTESTINAL NEMATODES INCLUDmG H. POLYGYRUS

Given that T helper lymphocytes are cnicial for antibody responses. depressed T

lymphocyte numbers, impaired functiond capacity of CM1 responses. and defects in homing

of gut-derived T lymphocytes have important implications for Th2-mediated humorai

immunity to intestinal nematode pansites. It is premature. however, to conclude that these

changes o bserved in blood and lymphoid organs of naive subjects interfere with host

immunologicd responsiveness to helminths, especidly considering that the impact of

protein deficiency on host protection depend on the proliferative and functional capacity of

Th2 cells. As discussed previously, changes in humoral responses are not chuacteristic of

protein malnutrition in uninfected humans. Malnourished children have k e n reported to

have elevated antibody titres in blood (Ozkan et al.. 1993) and elevated totd IgE ievek

(Abbassy et al.. 1974) while other studies found no relationship between malnutrition and

IgE levels (Punïio et al., 1976). However. these results on total antibody levels do not

reflect antigen-specific responses during protein malnutrition. Hagel et al. (1995) reponed

that Ascaris-infected cliildren h d increased tord serum IgE but also lower anti-Ascaris IgE

concenuations. This stud y sho wed that mainu trition may enhance polyclonal IgE synthesis

induced by the parasite and in tum dirninish the ability to mount specifk anti-parasite IgE

responses that are important in host resistmce to helminthic infections.

In another study thet is relevant to anthelmirithic immunity, Rickard and Lagunoff

(1989) found that a 0.5% protein diet reduced the nurnber of IgE receptors on peritoned

mat ceb of naive rats but did not affect the nurnber of mut c e k nor the histamine content

per ceL The authors suggested two possibüities for these results: 1) protein deficiency is

more detrimental to IgE receptor synthesis than to synthesis of pro teins necessary for m u t

ceiî or histamine production andor 2) decreased levels of circulating IgE down-regulated

the expression of mast ceiî receptors for IgE. The kst explmation requires ihat pro tein

malnutrition does not cause generalized hypoimmunoglobuluiemia. however, the htter is

eqiially plausible in individu& whose puasite-specinc IgE synthesis is impaired by protein

restriction (Hagel et aL, 1995). Interestingly. Chen & Enerback (1994 & 1996) found that

the density of FccRI receptors and binding of IgE to FceRI on peritoneal m u t ce& of

naive or N. brasiliensis-uifected rats were independent of T lymphocytes as demonstrated

by the lack of differences in these panmeters between athymic and euthymic rats. In

athymic rats which lack T lymphocytes. IgE synthesis may be induced by Th2 cytokines and

cell contact signals rebased from mut ceUs and basophüs (Gauchat et al., 1993; Huek et

a l , 1995). However, the IgE-mediated histamine reiease by mut c e k in athyrnic rats was

signincantly lower compared with that of the normal rats despite similar leveis of semm 1%

and IgE receptors. Thus, interaction with T ceils and their medktors may be necessary for

activation and degranulation of mast cells. The potential for protein deficiency to impair

the synthesis of IgE receptor and the secretory capacity of MMC by depressing Th2

proiif'ntion and cytokine production rem& to be investigated.

In one study that exiunined gut-associated immunological outcomes of protein

deficiency during nematode infection, Cummins et al. (1987a & 1987b) found that a 66

protein diet venus a 208 protein diet impaired worm expulsion and decreased the number

of intestinal MMC but did not affect semm leveh of rat mucosal mu t ceIî protease II

(released upon MMC degranulation) in rats infected once with N. brasiliensis. Thus.

increased worm s u ~ v a l in the protein-deficient rats rnay be attributed to depressed MMC

proliferation rather than to defective mast ceil activation. It is possible that the rehtively

rmld level of pro tein deficiency (6% protein ) was no t conducive to determining the impact

of protein malnutrition on histamine releasibility of mast ceh. Also. protease

concentrations are an indirect meuure of MMC activation and may be infiuenced by the

rate of protease turnover in blood. Given that cytokine production w u not measured in

these studies, it is unknown whether protein deficiency down-reguiates the growth and

maturation of MMC by decreasing the production and release of cytokines from Th2 and

other immune ceils. These hdings of diet-relnted impairment of intestinal immunity have

obvious implications for the capacity for tesistance to nematodes in mahourished hosu.

Surprisingly, there are few studies that have examined whether protein d e f i n c y

potentiates chronicity of infection with nematode puasites by impairing Th2 immune

responses. A low pro tek diet (4%) increased the establishment. survival and egg output of

T. muris in mice (Michael & Bundy. 1991) compared with a 16% protein diet. but this was

not associated with defects in humoral immunity as indicated by the normal to elevated

serurn titres of pansite-specific IgGl and IgA in the protein deficient group (Michael &

Bundy, 1992a & 1992b). These fhdings were interpreted by the researchers as evidence

that protein deficiency is detrimentai to irnmunological processes such as CMI or intestinal

barrier function but has little impact on systernic antibody-mediated responses. Studies

using the mouse-H. polygyms system have produced more conclusive data showing the

detrimental effecr of protein malnutrition on Th2 irnmunity during nematode infection.

Slater and Keymer (1988) fed mice a 2% or 16% protein diet and then chalienged the

animals with H. polygym. The worm burdens at day 21 post-challenge infection were

significantly higher in mice fed the low protein diet cornpared to those recovered in the

protein sufficient mice. The blood eosinophil response was signincantly deiayed and

reduced in the protein deficient rnice over the chdlenge infection period. The rate of

increase in parasite-specific IgG response was signüicantly reduced in the protein deficient

mice while absolute concentrations in IgG did not dSer between the diet groups. It is

unciear whether this slower omet of IgG response may account for the lower level of

acquired protection observed in these animais because changes in total IgG response may

not necessarily correspond with that of subset IgG 1 response which is known to be mediate

protective immunity against H. polygynrr. Consistent with these hdings by Shter and

Keymer (1988). p u t experirnents in Our Lbontory showed that mice fed a low (3%)

protein diet had significantly higher w o m burdens and higher net egg production at 4

weeks post-chalIenge infection compued to mice fed 24% protein (Boulay et al., 1998).

The hck of functional resistance to challenge infection in these pro tein-deficient rnice was

related to their lower periphenl eosinophiüa and lower senun total IgGl concentrations

observed throughout the c W n g e infection process. Considering that Shter and Keymer

(1988) and Bouiay et aL (1998) did not memure production of other Th2 immune effectors

such as IgE, intestinal eosinophils. MMC and specific Th2 cytokines. there are still major

gaps in out understanding of the s pecific immunologicai mechanisms underlying the

interrelationship between protein deficiency and challenge infection with H. polygyms. In

particular, we know very Little about how pro tein deficiency aEects the development of

protective irnmunity to secondary exposures to H. polygyncr in GALT. It is obvious from

the limited scope of current ïiterature that much more research on protein deficiency and

specific Th2 immunity to helminth infections is needed. Our study integrated dl t h e

conditions (ie. protein deficiency, intestinal nematode infection, and host irnmunity) in a

single experirnentai host-pansite system with particular focus on them systernic and

intestinal immun010 gical mechIlliiSms mediating the interac rions between pro tein

malnutrition and intestinal parasitism.

REFERENCES

Abbott, W.C., Tayek, J.A., Bistrian, B A , Maki, T., Ainsley, B.M., Reid, L.A., & Blackburn, G.L. (1986). The effect of nutritional support on T-lymphocyte subpopulations in protein calorie malnutrition. i. Am. Cokge Nutr. 5: 577-584.

Abe, Te & Nawa, Y. (1988). Wonn expulsion and mucosal mst ceil response induced by repetitive iL-3 administration in Strongyloides rani-infected nude rnice. Immunology 63: 181-185.

Abe, Te, Sugaya, He, Ishida, K., Khan, W.L, Tasàemir, I., & Yoshimura, K. (1993). Intestinal protection against Strongyloides roM' and mastocytosis induced by administration of interkukin-3 in mice. Immunology 80: 1 16- 12 1.

Abitorabi, M.A., Mackay, CR., Jerome, E.H., Osorio, O., Butcher, E.C., & Erie, D.J. (1996). DSerential expression of horning molecules on recùcuhting lymphocytes from

sheep gut. peripheral. and lung lymph. J. ImmunoL 156: 3 1 1 1-3 1 17.

Allen, J.E. & Maizels, RM. (1996). Immunology of human helminth infection. Int. Arch AUergy & IrnmunoL 109: 3- 10.

Ali, N.M.H. & Behnke, J.M. (1985). Observations on the gross changes in the secondary lymphoid organs of mice Uifected with Nematospiroides dubius. J . Helminth. 59: 167-174.

Arizono, Na, Kasugai, Ta, Yamada, M., Okada, M., Morimoto, M., Tei, H., Newlands, G.F.J., Miller, H.R.P., & Kitamura, Y. (1993). Infection of Nipposnongylus brusiliemis induces development of mucosal-type but not comective tissue-type m a t cells in gene tically mat ceii-deficient Ws/Ws nts. Blood 81:2572-257 8.

Arizono, Ne, Yamada, M., Tegoshi, Ta, Okada, M., Uchikawa, R, & Matsuda, S. (1994). Mucosal m u t ceîi proliferation folIowing normal and heterotopic îdections of the nernatode Nippostrongyius brasiliensis in nu. APMIS 102: 589-596.

Abbassy, AS., Badd ECDin, M.K., Hassan, A L , Aref, G.H., Hammad, S.A., El- Araby, LI., & Badd ECDin, A.A. (1974). S tudies of ceU-mediated immunity and akrgy in protein energy malnutrition. II. Immedkte hypersensitivity. J. Trop. Med. & Hyg. 77: 18-21.

Banks, E.M.S., & Coleman, LW. (1996). A comparative study of peritoneal mut cells fiom mutant IL4 deficient and normal mice: evidence that IL4 is no t essential for mast ce1 development but enhances secretion via control of IgE binding and passive sensitization. Cytokine 8: 190-196.

Bartiett, A. & Bdl, P.A. J. (1972). Nematospiroides dubius in the mouse as a possible model of endemic human hookworm infection. Am. Trop. Med. P~asitoL 66: 129- 134.

Bawden, RJ. (1969). Some effects of the diet of mice on Nematospiroides dubius (Nematoda). Parasitology 59: 203-2 13.

Beagley, &W. & Elson, C O . (1992). Ceh and cytokines in mucosal immunity and inflammation. GastroenteroL Clin. N.A 21 : 347-365.

Befus, A.D., Dyck, No, Goodacre, R., & Bienenstock, J. (1987). Mast ceUs fiom the human intestinal lamina propria. Isoiation. hisrochernical subtypes and functional characterization. J. Imrnunol. 138: 2604-26 10.

Behnke, J.M. & Wakeün, D. (1977). Nematospiroides dubius: stimulation of acquked imrnunity in inbted strains of mice. J. Helminthol 51: 167-176.

Behnke, J.M. & Parish, HA. (1979). Expuision of Nemotospiroides dubius nom the intestine of mice treated with immune serum. Parasite Imrnunol 1: 13.

Behnke, J.M.9 Hannah, J., & Pritchard, D.I. (1983). Nemurospiroides dubius in the mouse: evidence that adult w o m depress the expression of homologous immunity. Parasite Immuno 1. 5: 397-408.

Behnke, J.M. & Robinson, M. (1985). Genetic control of imrnunity to Nematospiroides dubius: a 9-day anthelminthic abbreviated inununiMg regime which separates weak md strong responder strains of mice. Parasite Immun01 7: 235-253.

Behnke, J.M. & Wahid, RN. (1991). Immunological relationships during primary infection with Heligmsomoides polygynis (Nematospiroides dubius): H-2 linked genes determine w o m survival. Parasitolo gy 103: 157- 164.

Behnke, J.M., Cnbaj, W., & Wakeün, D. (1992). Susceptibility of adult Heligmsomoides polygyms to intestinal infiammatory responses induced heterologous infection. Int. J. PmsitoL 22: 75-86.

Blnke, JeMo, W W , F.N.9 Gren&, RK., Else, KJo, Ben-Smith, A.W., & Goyd, P.K. (1993). Immunological relationships during primary infection with Heligmosomoides polygyrics (Nema~ospiroides drtbius) : do wnreg ulation of speciûc cytokine secretio n (IL-9 and IL-IO) correlates with poor mastocytosis and chronic swvival of adult w o m . Parasite ImmunoL 15: 415-421.

Bell, E.R, Sparshott, SoM., & Bunce, C. (1998). CD4+ T-cell memory, CD45R subsets and the penistence of antigen - r unifjing concept. ImmunoL Today 19: 60-64.

Bell, RG.9 Hazell, L.A., & Mce, P. (1976). Muence of dietary protein restriction on immune cornpetence II. Effect on lymphoid tissue. C h Exp. ImmunoL 26: 3 14-326.

Berlin, Cm, Berg, ILLw, Briskin, M. J., Andrew, D.P., Kilîhaw, P.J., Holzmann, B., Weisman, LL., Hamann, A., & Butcher, E.C. (1993). a4P7 integrin mediates Iymp hoc yte binding to the mucosal vascular addressin MadCAM- 1. Cell74: 1 8 5.

Bises, Ge, Mengheri, E., & Goetad, S. (1987). T-lymphocyte subsets in zinc deficient and in protein mahourished rats. Nutr. Rep. Int. 36: 1371-1378.

Bissonette, E.Y., Chin, B., & Befw, A.D. (1995). Interferons dinerentially regdate histamine and TNF-cr in rat intestinal mucosal mast ceiis. Immunology 86: 12-17.

Bogen, S.A., Weinberg, DaS., & Abbas, AwKo (1991). Histologic analysis of T lymphocyte activation in reactive lymph nodes. J. Immunol. 147: 1537- 154 1.

Boulay, M., Scott, M.E.9 Conly, S.& Stevenson, M.M., & Koski, K.G. (1998). Dietvy pro tein and zinc restrictions independently modw a Helig msomoides polygyrus (Nematoda) infection in mice. Puasitology 116: 449-462.

Bradley, LM*, Yoshimoto, K., & Swoin, S.L. (1995). The cytokines IL-4. EN-y, and L 1 2 regulate the development of subsets of mernory effector helper T celis in vitro. J. ImmunoL 155: 1713-1724.

Bradley, Lw, M., Dalton, DeK., & Croft, M. (1996). A direct role for IFN-y in reguhtion of Th1 cell development. J. IrnmunoL 157: 1350-1358.

Brailslord, T J. & Mapes, C. Je (1987). Cornparisons of Heligmosomoides polygyrus primary infiection in protein-deficient and well-nourished mice. P~asitology 95: 3 1 1-32 1.

Bryant, V. (1973). The life cycle of Nematuspiroides dubius. Baylis 1926 (Nematoda: Heligmosomidae). J. Helminthol 47: 263-268.

Bunce, C. & Bell, E.B. (1997). CD45RC isoforms dehe two types of CD4 memory T cek. one of which depends on persisting antigen. J. Exp. Med. 185(4): 767-776.

Bundy, DeAmPm and Golden, M.H.N. (1987). The impact of host nutrition on gastrointestinal helminth populations. Pansitology 95: 623-635.

Cebm, JJ. & S h d , K.E. (1994). Peyefs patches as inductive sites for IgA cornmitment. In: (Eds. P.L. Ogn, W. Stroker, J. Mesteckey. et al.), Handbook of Mucosd Immunology. New York: Academic Press Inc.

Chan, S.H., Penissia, B., Gupta, J.W.9 Kobyashi, M., Pospisil, M., Young, H.A., Wolf, S.F., Young, D., Clark, S.C., & Tdnichieri, G. (1991). Induction of interferon y production by natural killer ceil stimulatory factor: characterhtion of the responder celis and synergy with other inducers. J. Exp. Med. 173: 869.

Chandra, R.K. (1983). Nutrition. irnmunity. and infection: present knowledge and hture directions. The Lmcet 1: 688-69 1.

Chen, XJ. & Enerback, L. (1994). IgE receptors, IgE content and secretory response of mast cells in athyrnic rats. Immunology 83: 595-600.

Chen, X J. & Enerback, L. (1996). Immune response to a Nippostrongylus brasiliensis Lifection in athymic and euthymic rats: sudace expression of IgE receptors. IgE occupancy and secretory ability of mast cells. Int. Arch. Allergy & IrnmunoL 109: 250-257.

Chin, Y.H., Cai, J.-P., & Xu, Xe-M. (199 1). Tissue-specific homing receptor rnediates lymphocyte adhesion to cytokine-stimulated lymph node high endothehl venule ceb. Immunology 74: 478-483.

Chu, NA, DeBenedette, M.A., Stiernholm, J.N., Barber, B.H., & Watts, T.H. (1997). Role of IL,- 12 and 41BB ligand in cytokine production by CD28+ and CD28- T ceils. I. ImmunoL 158: 3081-3089.

Coffman, R.L., Seymour, B.W., Hudak, S., Jackson, J., & Rennidr, D. (1989). Antibody to IL-5 inhibits heiminth-induced eosinophilia in rnice. Science 245: 308-3 10.

Conzen, S.D. & Janeway, C.A. (1988). Defective mtigen presentation in chronically protein-deprived mice. Irnmunology 63: 683-689.

Cox, F.E.G. & Liew, E.Y. (1992). T-ceU subsets and cytokines in pyasitic infiections. ParasitoL Today 8: 371-375.

Crompton, D.W.T. (1986). Nutritional aspects of infection. Trans. R Soc. Trop. Med. & Hyg. 80: 697-705.

Cumniins, A.G., Kenny, A.L., Duncombe, V.M., Boün, T.D., & Davis, A.E. (1987a). The effect of protein deficiency on systemic reiease of rat mucosd mast ceii protease II during Nipposnongylus brusiliensis infection and foilowing systemic anaphyW. IrnmunoL CeU BioL 65( Pt 4): 357-363.

Cummins, A.G., Duncombe, V.M., Boiin, T.D., Davis, A.E., & Yong, J. (1987b). The respoase of the s d intestine of the pro tein-deficient rat to infection with Nippostrongylus brmiIiensis. fnt. J. ParasitoL 17: 1445- 1450.

Dehlawi, M.S., Wakelin, Dm, & Behnke. (1987). Suppression of mucosal mastocposis by infection with the intestinal nematode Nemutospiroides dubius. Pansite Immun01 9: 187- 194.

Dehlawi, M.S. & Wakeün, D. (1988). Suppression of mucosal mastocposis by Nematospiroides dubiur results from an adult wom-mediated effect upon host lymphocytes. Parasite ImmunoL 16: 85-95.

Mtch, E.A., Xu, Dm, Spdan, RD., & Berg, RD. (1992). Protein malnutrition done and in combination with endotolcin impairs systemic and gut-associated imrnunity. P E N 16: 25-3 1.

Ding, Lm, Linsley, P.S., Huang, Lm-Y., Germain, R.N., & Shevach, E.M. (1993). IL10 inhibits macrophage costimulatory activity by selectively inhibithg the up-regulation of B7 expression. J. IrnmunoL 151: 1224.

Dobson, C., Sitepu, P., & Brindley, P.J. (1985). Muence of primary infection on the population dynamis of Nematospiroides dubius after challenge infections in rnice. Int. J. ParasitoL 15: 353-359.

Dubucquoi, S., Desreumaux, P., Janin, A., Klein, O., Coldman, M., Tavernier, Je, Capron, A., & Capron, M. (1994). Interleukin 5 synthesis by eosinophüs: association with granules and immunoglobulin-dependent secretion. J. Exp. Med. 179: 703-709.

Dugas, B., Renauld, J.C., Pene, J., Bonnefoy, J.Y., Peti-Frere, C., Braquet, P., Bousquet, J., Van Snick, J., & Menaa-Huerta, J.M. (1993). Interleukin-9 potenthtes the interleukin-4-induced irnmunoglo bulin (QG, IgM and IgE) production by nomai human B lymphocytes. Eur. J. ImmunoL 23: 1687- 1692.

Duncombe, V.M., Boün, T.D., Davis, A.E., & Kelly, J.D. (198 1). Dehyed expulsion of the nematode Nippmtrongylus brusiliensis fiom rats on a low protein diet: the role of a bone marrow derived component. Am. J. Clin. Nutr. 34: 400-403.

Eklund, K.K., Ghildyal, N., Austen, K.F., & Stevens, R*L. (1993). Induction by L-9 and suppression by L-3 and IL-4 of the be l s of chromosome lbderived transcripts that encode late-expressed mouse mast ce1 proteases. J. ImmunoL 151: 4266-4274.

Else, KJ., Finkelman, F.D., Maliszewski, CR, & Grencis, RK. (1994). Cytokine- mediated regulation of chronic intestinal helminth infec tioa J. Exp. Med. 179: 347-35 1.

Enriqucz, FJ., Cypess, RH*, & W-nt, D.L. (1988). Influence of mimunizing dose and presence or absence of adult wonns on the development of resistance to Nematospiroides dubius challenge infections of mice. J. ParasitoL 74: 409-4 14.

Faulkner, Hm, Renauld, J.-Ca, Van Snlck, j., & Grenas, RK. (1998). Interieukin-9 enhances resistance to the intestinal nematode Tn'churis muris. fnfect. Immun. 66: 3832- 3840.

Femck, D.A., Schrenzel., M.D., Mulvania, T., Hsieh, B., Ferlin, W.G., & Lepper, Ha (1995). Differential production of interferon-y and interleukin-4 in response to Thl- and Th2-stimulating pathogens by y6 T cells in vivo. Nature 373: 255-257.

Finkelman, F.D., Katona, LM., Momann, Tek , & Coflman, R*L. (1988a). IFN-y regdates the iso types of Ig secreted during in vivo humoral immune responses. J. Immun01 lm: 1022- 1027.

Finkelman, F.D., Katona, EM., Urban, J.F., Holmes, J., Ohara, J., Tung, AS., vG Sample, J., & Paul, W.E. (1988b). IL4 is required to generate and sustain in vivo IgE responses. J. ImrnunoL 141: 233XU41.

Finkleman, F.D., Madden, K.B., Cheever, A.W., Katona, LM., Morris, S.C., Gately, M.K., Hubbard, B.R, Gause, W.C., & Urban, J.F. (1994). EfTects of interkukin 12 on immune responses and host protection in mice infected with intestinal nematode parasites. J. Exp. Med. 179: 1563-1572.

Finkelman, F.D., Shea-Donohue, Tm, Goldhill, J., Sullivan, C.A., Morris, S.C., Madden, K.B., Cause, W.C., & Urban, J.F. (1997). Cytokine regulation of host defense against pansitic gastrointestinal nematodes. Annu. Rev. IrnmunoL 15: 505-533.

Fiorentino, D.F., Zlothnik, A., Vieira, P., Mosmmn, T.R., Howard, M., Moore, K.W., & OpGama, A. (1991). IL40 acts on the mtigen-presenting ceii to inhibit cytokine production by Th1 ce&. J. ImmunoL 146: 3444-345 1.

Flo, J. & Massouh, E. (1997). Age-rehted changes of naive and memory CD4 rat lymphocyte subsets in mucosal and systernic lymphoid organs. Dev. & Comp. IrnmunoL 21: 443-53

Gascan, H., Aveisa, G.G., Gauchat, J.F., Van Vlasselae, P., Ronurolo, M.G., Yssel, Hm, Kehry, M., Spits, Ha, & De Vries, JE. (1992). Membranes of activated CD4+ T cells expressing T c d receptor (TCR) alpha beta or TCR gamma delta induce IgE synthesis by human B ceb m the presence of interleukin-4. Eur. J. ImmunoL 22: 1 133- 1141.

Gauchat, J.-Fa, Aenchoz, S., Mazzel, Ga, Aubry, &P., Brumer, T., Blasey, H., Life, P., Talabot, Dm, Fiores-Romo, Lm, Thompson, J., Kishi, K., Butterfield, J., Dahinden, C., & Bonnefoy, &Y. (1993). Induction of human IgE synthesis in B cells by mast cells and basophihi. Nature 365: 340-343.

Gaw, W.C., Urban, J.F., Linsley, P., & Liu, P. (1995). Role of B7 signalling in the differentiation of naive CD4+ T cells to effector interleukin-4-producing T helper cells. ImmunoL Res. 14: 176-188.

Ghidyal, N., McNeil, H.P., Stechschulte, S., Austen, K.F., Silberstein, D., Gurish, M.F., Somerville, L.L., & Stevens, EL. (1992). IL-10 induces transcription of the gene for mouse mast cell protease-l, a serine protease preferentially expressed in rnucosal mast cells of Trichinella spiralis-infected mice. J. ImmunoL 149: 2123-2129.

Ghidyal, N., Friend, D.S., Nicodemus, C.F., Austen, K.F., & Stevens, RL. (1993). Reverslfile expression of mouse mast cell protease 2 mRNA and protein in cultured mast cells exposed to IL-10. J. ImmunoL 151: 3206-3214.

Good, RA. & Lorenz, E. (1 992). Nutrition and cellulv immunity. Int. J. ImmunopharmacoL 14: 36 1-366.

Gounni, A.S., Larnkhloued, B., Ochalal, K., Tanaka, T., Delaporte, E., Capmn, A., Kinet, J.-P., & Capmn, M. (1994). High-affinity IgE receptor on eosinop hils is involved in defence against parasites. Nature 367: 183- 186.

Greenwald, R J., Lu, P., Hdvomn, M. J., Zhou, X., Chen, S.-J., Madden, K.B., Pemn, PJ., Moms, S.C., Finkelman, F.D., Peach, R., Linsley, P.S., Urban, J.F., & Gaw, W.C. (1997). Effects of blocking B7-1 and B7-2 interactions during a type 2 in vivo immune responses. J. ImmunoL 158: 4088-4096.

Guy-Grand, D., Griscelll, C., & Vassalli, P. (1978). The mouse gut T lymphocyte: 3

novel type of T celL Nature, origin, and traffic in mice in normal and graft-versus-host conditions. J. Exp. Med. 148: 1661- 1677.

Ha, C.-L., Paulino-Radne, L.E., & Woodward, B.D. (1996). Expylsion of the humord effkctor cell compartment of both systemic and mucosal immune systems in a weanling murine model which duplicates critical features of human proteinenergy malnutrition. Br. I. NUU. 75: 445-460.

Ha, C.-L. & Woodward, B. ( 1997). Reduction in the quantity of the polymeric immunoglobulin receptor is sufficient to account for the low concentration of intestinal secretory immunoglobulin A in a weanling mouse model of wasting pro tein-energy malnutrition. J. Nutr. 127: 427-435,

Hagel, I., Lynch, N.R., Di Prisco, M.C., Sanchez, J., 8 Perez, M. (1995). Nutritional status and the IgE response against Ascuris lumbricoides in children from a tropical slum. Trans. R Soc. Trop. Med. & Hyg. 89: 562-565.

Haig, D.M., McKee, T.A., Jarrett, E.E.E., Woodbury, R, & Miller, HoRP. (1982). Generation of mucosd mast ceUs is stirnulated in vitro by factors derived from T cells of helminth-infected rats, Nature 300: 188- 190.

Haig, D.M., Jarrett, E.E.E., & Tas, J. (1984). In vitro studies on mast ceU prolifention in Nipposrrongylus brasiliensis infection. Immunology 51: 643-65 1.

Haradlsen, G., Kvale, De, Lien, B., Farstad, LN., & Brandbaeg, P. (1996). Cytokine- regulated expression of E-selectin, interceiiulv adhesion molecule- 1 (ICAM-1). and vascular ceU adhesion molecule- 1 (VCAM- 1) in human intestinal microvascuiar endo thelial ceh. J. IrnmunoL 156: 258-2565,

Hiroi, Ta, Fujhashi, K., McGhee, J.R., & Kiyono, H. (1995). Polarued Th2 cytokine expression by both mucosal y6 and ap T ceb. Eur. J. ImmunoL 25: 2743-2751.

Huang, H., Hu-Li, Je, Chen, H., Ben-Sasson, S.Z., & Paul, W.E. (1997). IL,-4 md L- 13 production in dinerentiated T helper type 2 ce& is not IL,-4 dependent. J. ImmunoL 159: 373 1-3738.

Hueh, C., Germann, Tm, Goedert, S., Hoehn, P., Koelsch, S., Hultner, L., Palm, Na, Rude, E., & Schmitt, E. (1995). Co-activation of naive CD4' T cells and bone marrow- derived mast results in the development of Th2 ceh. Int. h u n o L 4: 525-532.

Hultner, L., Druez, & C., Mwller, J., (1990). Mast cell growth enhancing activity (MEA) is stnicturdly rekted and functionûlly identicai to the novel mouse T ceIl growth factor P40/TCGFIII (Interleukin 9). Eur. J. ImmunoL 20: 14 13- 14 16.

Jacobsen, RH., Brooks, B.O., & Cypess, RH. (1982). Irnmunity to Nematospiroides dubius: parasite stages responsible for md subject to resistmce in high responder (LAFl/J) mice. J. ParasitoL 68: 1053-1058.

Joe!, DoD.9 & Chanana, A.D. (1985). Cornparison of pulmonary and intestinal lymphocyte migrationid patterns in sheep. Ann. NY Acad. Sci. 459: 56.

Kasugai, Te, Tei, H., Oknda, M., Hirota, S., Morimoto, M., Yamada, M., Nakama, A., Arizono, N., & Etmura, Y. (1995). Infection with Nippostrongylus brcrsiliensis induces invasion of mast cefl precursors from peripherai blood to small intestine. Blood 85: 1334-1340.

Katona, LM., Urban, J.Frn, Kmg, S.S., Paul, W.& & Finkelmnn, F.D. (1991). IL4 requirements for the generation of secondq in vivo IgE responses. J. h u n o L 146: 42154221.

Kaubann, S.H. (1996). Gamrnddelta and other unconventional T lymphocytes: what do they see and what do they do? Proc. NatL Acad. Sci. USA 93: 2272.

Kerboeuf, Ds (1 982). Egg output of Heligmosomoides polygyrus (Nematospiroides dubius) in mice infected once ody. Ann. Recherches Veterinaires 13: 69-78.

Kerboeuf, Dm (1985). The effect of host density on the establishment and fecundity of Heligmosomoides polygynrs. Parasitology 91 : 177- 183.

Keymer, A.E. & Tariton, A.E. (1991). The population dynvnics of acquired immunity to Heligmosomuides polygyrur in the laboratory mouse: strain. diet and exposure. Parasitology 103: 121-126.

King, CL., Low, C C , & Nutman, T.B. (1993). IgE production in human helminth infection: ceciprocal interrelationship between IL-4 and IFN-y. J. ImrnunoL 150: 1873- 1880.

King, C.L. & Nutman, T.B. (1993). IgE and IgG subclass reguhtion by IL4 and IFN-y in human helminth infections: assessrnent by B ceil precursor frequencies. J. ImmunoL 151: 458-465.

King, CL., Hakirni, J., Shab, M.T., & Medhat, A. (1995). IL-12 reguiation of parasite antigen-dnven IgE production in human helminth infections. J. ImmunoL 155: 454-46 1.

King, S. J., & Miller, H.RP. (1984). Anaphylactic release of mucosai m u t ceU pro tease and its relationship to gut permeability in Nipposnongylus-primed nu. Immunology 51: 653-660.

Kita, Hm, Weiler, D.A., Abu-Ghazaleh, R., Sandemn, CJ., & Gleich, G.J. (1992). Release of granule proteins from eosinophils cultured with IL-5. J. Imrnunol. 149: 629- 635.

Kopf, M., Le Gros, G., Bachmmn, M., Lamen, M.C., Bluethmann, H., & Kahler, G. (1993). Disruption of the murine ïL-4 gene blocks Th2 cytokine responses. Nature 362: 245-248.

Kocenaga, M., Wang, CH., Bell, RG., Zhu, Dm, & Ahmad, A. (1989). Intestinal immunity to Trichinella spiralis is a propeny of 0x8' 0x22 T helper ceh that are generated in the intestine. Immunology 66: 588-594.

Koyama, K., Tamauchi, Hm, & Ito, Y. (1994). The role ofCD4+ and CD8+ T ceils in protective immunity to the murine nematode parasite Tkhuris muris. Parasite ImmunoL 17: 161-165.

Kuvibidila, S., Yu, L., Ode, Dm, & W d e r , RP. (1993). The immune response in protein-energy malnutrition and single nutrient deficiencies. In: (Ed. D.M. Klurfeld), Human Nutrition - A Comprehensive Treatise. Volume 8: Nutrition and Immunology. New York: Plenum Press.

Lagoo, AS., Eldridge, J.H., Lagoo-Deenadaylan, S., Black, C.A., Ridwan, B.U., Hardy, K. J., McGhee, J.R, & Beagley, K. W. (1994). Peyer's patch CD8+ memory T ce& secrete T helper type 1 and type 2 cytokines and provide help for immunog10buli.n secretion, Eur. J. Immun01 24: 3087-3092.

Laissue, J.A., Chappuis, B.B., Miller, C., Reubi, J.C., & Gebbers, JO-0. (1993). The intestinal immune system and its relation to disease. Digestive Diseases 11: 298-3 12.

Lamont., A.G., Gordon, M., & Ferguson, A. (1988). T lymphocyte fùnction in protein deprived mice. Clin. Exp. Imrnunol. 72: 1 13- 1 17.

Latham, M.C. (1990). Protein-energy rnahutrition - its epidemiology and conuol. J. Environ PathoL ToxicoL & OncoL (JEPTO) 10: 168-180.

Lawrence, C.E. & Pritchard, D.L. (1994). Immune response pro f i s in responsive and nonresponsive mouse strains infected with Heiigmosomoides polygyrus. Int. J. PansitoL 24: 487-494.

Lee, W.-H. & Woodward, B.DO (1996). The CD4KD8 ratio in the blood does not reflect the response of this index in secondvy lymphoid organs of weanling mice in models of protein-energy malnutrition known to depress thymus-dependent irnmunity. J. Nutr. 126: 849-859.

Le Gros, R, Ben-Sasson, S.Z, Seder, R., Finkelman, F.D. & Paul, W.E. (1990). Generation of interleub 4 (IL-4)-producing cek in vivo and in vitro: IL2 and IL-4 are required for in vitro genention of IL-4 producing cells. J. Exp. Med. 172: 921-929.

Lim, T.S., Messiha, N., & Watson, RR. (198 1). Immune components of the intestinal mucosae of ageing and protein deficient mice. Immunology 41: 401-407,

Locksley, RM. (1994). Th2 ce&: help for helminths. J. Exp. Med. 179: 1405-1407.

Lochrniller, RL., Vestey, M.R., & Nash, Da (1992). Gut-associated lymphoid tissue in the Cotton rat (Sigrnodon hispidus) and its response to protein restriction. J. Wiidlife

Lopez, M. del C. & Roug M.E. (1989). Irnpaired dinerentiation of IgA-B ce1 precursors in the Peyer's patches of protein depieted rats. Dev. & Cornp. ImmunoL 253-262.

Lu, P., di Zhou, Xe, Chen, S.& Mwnnan, M., Morris, S.C., Finkelman, RD., Linsley, P., Urban, J.F., & Gause, W.C. (1994). CTLA-4 ligands are required to induce an in vivo interleukin 4 response to a gastrointesthd nematode parasite. J. Exp. Med. 180: 693-698.

Lu, P., Urban, J.F., di Zhou, X., Chen, S.- J., Madden, K., Mooman, M., Npyen, Ha, Morris, S C , Finkelman, F.D., & Gause, W.C. (1996). CD40-mediated stimulation contributes to lymphocyte proliferation. antibody production, e o s i n o p ~ and mastocytosis during an in vivo type 2 response. but is not required for T cell IL-4 production. J. ImmunoL 156: 3327-3333.

Lunn, PG. & Northrop-Clewes, C.A. (1993). Symposium on 'Parasitism and protein and energy metabolism in man and animais. Proc. Nutr. Soc. 52: 101-1 11.

Madden, K.B., Urban, J.F., Ziltener, HmJ*, Schrader, J.W., Finkelman, F.D., & Katona, LM. (199 1). Antibodies to L-3 and IL-4 suppress helminth-indoced intestinal mastocytosis. J. Irnmunol. 147: 1367-139 1.

Maggi, E., Parronchi, P., Manetti, Ra, Simonelli, CD, Piccinni, M..P., Rugiu, ES., De Cadi, M., Rica, M., & Romagnani, S. (1992). Reciprocal reguhtory effects of IFN-y and IL-4 on the in vitro development of human Th 1 and Th2 clones. J. ImmunoL 148: 2142-2147.

Maindi, ES. & McMurray, D.N. (1998). Protein deficiency induces aiterations in the distribution of T-ce1 su bsets in experimental puhonary tu bercuiosis. Infect. Immun. 66: 927-93 1.

Manetti, R, Parronchi, P., GiudiP, MG., Picrinni, M., Ma&, ED, Trinchieri, G., & Romagnani, S. (1993). Naturd killer ceii stimuhtory factor (interleukin 12 (IL-12) induces T helper type (Th1)-specific immune responses and inhibits the development of L- 4-produchg Th ce&. J. Exp. Med. 177: 1199.

McDermott, M.R & Bienenstock, J. (1979). Evidence for a cornmon mucosd immunologie system. Migration of B immunoblasts into intestinal. respiratory, and genitd tissues. J. ImmunoL 122: 1892.

McDemott, MeRa, Mark, DA., Beliis, A.D., Baîiga, B.S., & Suskhd, RM. (1982). Irnpakd intestinal locaiization of mesenteric lymphobhts associated with vitamin A deficiency and pro teindorie malnutrition. Immun010 gy 45: 1-5.

McGee, D.W. & McMumy, D.N. (1988). The effect of protein malnutrition on the IgA immune nsponse in rnice. Immunology 63: 25-29.

Michael, E. & Bundy, D.A.P. (1991). The effect of the protein content of CBNCa mouse diet on the population dynamics of Trichuris m r i s (Nematoda) in primary infection. Parasitology 103: 403-4 1 1.

Michael, E. & Bundy, D.A.P. (19923). Nutrition, immunity and helminth infection: effect of dietary pro tein on the dynamics of the primary antibody response to Trichuris m r i s (Nematoda) in CB AICa mice. Parasite ImmunoL 14: 169- 183.

Michael, E., & Bundy, D.A.P. (199%). Protein content of CBA/Ca mouse diet: relationship with host antibody responses and the population dynamics of Tnchuris muris (Nematoda) in repeated infection. Parasitology 105: 139- 150.

Miller, H.R.P. & Jarrett, W.F.H. (197 1). Immune reactions in mucous membranes. 1. Intestinal mat ceil response during heiminth expulsion in the rat. Imrnunology 20: 277- 288.

Monroy, FaGa, Adam, J.H., East, L J., & Dobson, C. (1989). Excretmy-secretory antigens from adult Nematospiroides dubius. Immunol. Ceil Bi01 67: 1 15- 120.

Monroy, F.G. & Enriquez, FJ. (1992). Heligmsomoides polygyrus: a mode1 for chronic gastrointestind helminthiasis. PansitoL Today 8: 49-54

Moqbel, Ra, Ying, S., Barkans, J., Newman, T.M., Kirnmitt, P., Wakeün, M., Taborda-Barata, Lm, Meng, Q., Comgrn, C a J., Durham, SR, & Kay, A.B. (1995). Identification of messenger RNA for IL-4 in human eosinophils with granule localkation and releue of the translated producr. J. Immunol. 155: 4939-4947.

Mosmann, T.R & Sad, S. (1996). The expanding universe of T-ceil subsets: TM, Th2 and more. Immunol. Today 17: 138- 146.

Nakache, M., Berg, EL., Streeter, P.Ra, & Butcher, E.C. (1989). The mucosal vascuiar addressin is a tissue-specific endothelid cell adhesion molecule for circuhting lymphocytes. Nature 337: 179.

Nakajima, Ha, Gleich, G.J., & Kits, He (1996). Constitutive production of IL-4 and IL- 10 and siimuked production of L-8 by normal peripherd blood eosinophils. J. IrnmunoL 156: 4859-4866.

NaMabendi, LM., Obeidat, W., Russell, RL, Downie, S., Smith, K., & Rennie, MJ* (1995). Gut mucosal protein synthesis meuured using intravenous and intragastric delivery of stable tracer amho acids. Am. J. PhysioL 269: E9960E999.

Needham, C.S. & Lillywhite, J.E. (1994). Immunoepidemiology of intestinal h e h t h i c infections. 2. Immunological correlates with pattern of Trichuris uifection Trans. R. Soc. Trop. Med. & Hyg. 88: 262-264.

Negrao-Coma, D., Adams, L.S., & Bell, R.G. (1996). intestinal transport and catabolism of IgE. J. ImmunoL 157: 4037-4044.

Nesheim, M.C. (1993). Human nutrition needs and p d t i c infections. P~asitology 107 (Suppl): S7-S 1 8.

Nimmanwudipong, T., Cheadle, W.G., Appel, S.H., & Polk, H.C. (1992). Effect of protein malnutrition and irnmunomodulation on immune ceil populations. I. Surg. Res. 52: 233-238.

Noben-Trauth, N., Shultz, L.D., Brombacher, F., Urban, J.F., Gu, H., & Paul, W.E. (1997). An interleukin 4 (IL-4)-independent pathwûy for CD4+ T cell IL-4 production is revealed in IL-4 receptor-deficient mice. Proc. NatL Acad. Sci USA 94: 10838-10843.

Ozkan, Hm, Logun, N., Sasmaz, E., Abacroglu, H., Okuyan, M., & Cevik, N. (1993). Nutrition. immunity and Uifections: T lymphocyte subpopuhtions in protein-energy malnutrition. J. Trop. Ped. 39: 257-260.

Parent, G., Chevaiier, P., Zalles, L., Sevilla, IL, Bustos, M., Dhenin, J.M., & Jambon, B. ( N94). In vitro lymphocyte-differentiating effects of thyrnulin (Zn-FTS) on lymphocyte subpopulations of severely malnourished children. Am. J. Clin. Nutr. 60: 274-8.

Parker, SJ. & Inchley, C. J. (1990a). E d y lymphocytic responses to Heligmosomuides polygym infections in rnice. J. HelminthoL 64: 35-45.

Parker, SJ. & Inchley, C. J. (1990b). Heligmosomoides polygyncs: infiuence of infection on lymphocyte subpopulations in mouse mesenteric lymph nodes. Exp. ParasitoL 71: 249-258.

Pearce, F.L., Befus, A.D., Gauldie, J., & Bienenstock, Je (1982). Mucosal mast cea. II. Effects of anti-alergic cornpounds on histamine secretion by isohted intestinal mast celis. J. ImmunoL 12%: 248 1-2486.

Penttila, I.A., Ey, P.L., & Jenkin, C.R (1983). Adherence of murine peripheral blood eosinophils and neutrophils to the different parasitic stages of Nematospiroides dubiur. Aust. J. Exp. BioL Med. Sci. 61: 617-627.

Petit-Frem, Cm, Dugas, B., Braquet, P., & Mena-Huerta, J.M. (1993). Interkukin-9 potentiates the interleukin+hduced IgE and IgGl rekase fiom murine B lymphocytes. Immunology 79: 146-151.

Plaut, M., Pierce, J.H., Watson, Cm J., Hanley-Hyde, J., Nordan, R.P., & Paul, W.E. (1989). Mast cell lines produce lymphokines in response to cross-linkage of FcERI or to calcium ionophores. Nature 339: 64-67.

Pleass, R. J. & Bianco, A.E. (1994). The role of adult w o m in suppressing function protective imrnunity to Heligmosomoides po lygym bakeri challenge infections. Parasite ImmunoL 16: 619-628.

Pritchard, D.I., William, D.J.L., Behnke, J.M., & Lee, T.D.G. (1983). The role of IgG l hyperg;unmaglobulinaemia in immunity to the gastrointestinal nematode Nema tospiroides dubius. The immunochemical purification. antigen-s pecincit y and in vivo anti-parasite effect of IgG 1 from immune semm. Immunology 49: 353-365.

Pritchard, D.L, Lawrence, C.E., Appleby, P., Gibb, LA., & Glover, K. (1994). Immunosuppressive proteins secreted by the gastrointestinal nematode parasite Heligmosomoides polygyrus. Int. J. ParasitoL 24: 495-500.

Purtilo, D.T., Riggs, RS., Evans, R., & Nedie, RCw (1976). Humoral immunity of parasitized. mainourished chüdren. Am. J. Trop. Med. & Hyg. 25: 229-232.

Ramaswamy, K., Goodman, RE., & Bell, R.G. (1994a). Cytokine profile of protective yiti -Trichinella spiralis CD4' 0x22' and non-protective CD4+ OX22+ thoracic duct ceils in rats: secretion of IL-4 done does not determine protective capacity. Parasite Imrnunol. 16: 435-445.

Rarnaswamy, K., Hakirni, J., & Bell, R.G. (1994b). Evidence for an interleukin-4- inducible immunoglobulin E uptake and transport mechuiism in the intestine. J. Exp. Mcd. TM: 1793-1803.

Ramaswamy, Kw, Negrao-Coma, D., & Bell, R (1996). Local intestinal immune responses to infections with Trichinella spiralis. J. ImmunoL 156: 4328-4337.

Raulet, D.H. (1989). The structure, Function. and molecul;ir genetics of the gammddelta T ce1 receptor. Annu. Rev. ImmunoL 7: 175.

Reed, N.D., Dehlawi, M.S., & Wakelin, D. (1988). Medium conditioned by spleen cek of Nematospiroides dubius-infected mice does not support development of cultured m a t ceUs. Int. Arch. AUergy & AppL Immun01 85: 1 13- 1 15.

Regoii, M., Borghesi, C., BerteIli, E., & Nicoletti, C. (1994). A morphological study of the lymphocyte trattic in Peyer's patches d'ter an in vivo antigen stimulation. Anat. Rec. 239: 47-54.

Rennidc, De, Hunte, B., Hollmd, Ge, & Thompson-Snipes, L. (1995). Cofactors are essential for stem ceil factor-dependent growth and maturation of mast ceil progenitors: comparative effects of interleukin-3 (IL-3). IL-4. IL-10. and fibroblasts. Blood 85: 57-65.

Rickard, ADLm & Lapnotr, Dm (1989). Protein malnutrition: effect on rat peritoned mast cell number, histamine content. and IgE receptors. Am. J. C h . Nutr. 49: 641-645.

Rincon, M., Anguita, J., Nakamura, Te, Fikrig, E., & Fiavell, RA. (1997). Interleukin a ) - 6 directs the differentiation of IL-4-producing CD4+ T cells. J. Exp. Med. 185: 46 1 - 469.

Roberts, K. & Kilshaw, P. JD (1993). The mucosal T cell integrin aM290P7 recognizes a ligand on mucosai epitheiid ce1 lines. Eur. J. ImmunoL 23: 1630.

Robinson, M. & Gustrid, T+R. (1995). In vitro stimulation of naive mouse lymphocytes by H e l i g m s o m i d e s polygyms adult worm antigens induces the production of IgG 1. Parasite IrnmunoL 18: 87-93.

Robinson, M., Gustad, TDRD, & Meinhamit, S. (1997). Non-specific binding of mouse IgGl to Heligmsomoides polygyrus: parasite homogenate cm riffinity purify mouse monoclonal antibodies. Parasitology 114: 70-84.

Rosenberg, I.H. & Bownman, B*B. (1984). Impact of intestinal parasites on digestive fiinction in humans. Fed. Proc. 43: 246-250.

Saito, Ho, Kanamori, Y., Takemori, T., Nariuchi, Hm, Kubota, E.9 Takahashi- Iwanaga, Iwanaga, Tm, & Ishikawa, Ha (1998). Genention of intestinal ceh f?om progenitors residing in gut cryptoprtches. Science 280: 275-278.

Salmi, M. & Jalkanen, S. (1992). Regdation of lymphocyte tmftïc to mucosa-associated lymphatic tissues. GastroenteroL Clin. N.A 20: 495-510.

Schleimer, R.P, Sterbinsky, S.A., Kaiser, J., Bickel, C.A., Klunk, D.A., Tomioka, K., Newman, W., Lusrinskas, F.W., Gimbmne, MrnA.9 Mcïntyre, B.W., & Bochner, B.S. (1992). IL4 induces adherence of human eosinophils and basophüs but not neutrophils to endo theliurn. Association with expression of VCAM- 1. J. ImrnunoL 148: 1086- lO92.

Schweighoffer, Ta, Tanaka, Y., Tidswell, M., Ede, DJa, Horgan, KJ., Luce, G.E., L m v i t s , A o L , Buck, Dm, & Shaw, S. (1993). Selective expression of integrin a4P7 on a subset of human CD4' memory T cek with hallmarks of gut-uophism. J. Immun01 151:717.

Scott, M.E. (1990). An experimental theoreticai study of the dyndcs of a mouse- nematode (Heligmsomoides polygynis) interaction Parasitology 101: 75-92.

Scott, M.E. & Tanpay, G.V. (1994). Heligmosomoides polygyrus: a iaboratory mode1 for direct Me cycle nematodes of humans and livestock. In: (Eds. M.E. Scott & G. Smith), Pansitic and Xnfectious Diseases. pp. 279-300. New York: Academic Press hc.

Scrimshaw, N.S., Taylor, C.E., & Gordon, J.E. (1968). Interactions of Nuvition and Infection. WHO Monognph Series No. 57: Geneva,

Seder, RA., Plaut, M., Barvieri, S., Urban, J., Finkelmm, F.D., & Paul, W.E. (1991). Mouse splenic and bone marrow celi populations hat express high aEfÏnity Fc epsilon receptors and produce interleukin 4 are highly enriched in basophils. Proc. NatL Acad. Sci USA 88: 2835-2839.

Seder, RA., Paul, W.E., Davis, M.M., Fazekas, D.S., & Groth, B. (1992). The presence of interleukin-4 duMg in vitro priming determines the lymphokine producing potential of CD4+ T cells from T ceii receptor transgenic mice. J. Exp. Med. 176: 109 1.

Seder, RA., Gdnel l i , R, Sher, A., & Paul, W.E. (1993). IL- 12 acts directîy on CD4+ T ceils to enhance priming for IFN-y production and diminish IL-4 inhibition of such priming. Proc. Nati. Acad. Sci USA 90: 1018840192.

Shanahan, F., Lee, T.D.G., Denburg, J.A., Bienenstock, J., & Befus, A.D. (1986). Functional chuacterization of mast cells generated in vivo fiom the rnesentenc lymph node of rats infected with Nippostrongylus brasiliensis. Immunology 57: 455-459.

Shirnoda, K., van Deursen, J., Snagster, M.Y., Sarawar, S.R., C~rson, RT., Tripp, RA., Chu, C., Quelle, RW., Nosaka, T., Vignali, D.A.A., Doherty, P.C., Grosveld, DG., Paul, W.& & Dile, J.N. (1996). Lack of IL-4-induced Th2 response and IgE clas switching in rnice with disrupted Stst6 gene. Nature 380: 630-633.

Slater, A.F.G. (1988). The influence of dietvy protein on the experimental epiderniology of Heiigmosomoides polygyrus (Nematoda) in the iabontory mouse. Proc. R. Soc. Lond. B 234: 239-254.

Slater, A.F.G. & Keymer, A.E. (1986). Heligmosomides polygyw (Nematoda): the influence of dietvy protein on the dynamics of repeated infection. Proc. R Soc. Lond. B 229: 69-83.

Slater, A.F.G. & Keymer, A.E. (1988). The influence of protein deficiency on immunity to Heligmosomoides polygyrur (Nematoda) in Mce. Parasite ImmunoL 10: 507-522.

Slobodianik,N.H.,Cos~nsky,RC., Langini,S.H., Roux, M.E., Rio,M.E.,& Sanahuja, J.C. (1984). Effect of severe protein deficiency on surface and intracelluiar markers of growing rats' lymphoid organs. Nuu. Rep. Int 29: 957-964.

Smith, N.C. & Bryant, C. (1986). The role of host generated free radicals in helminih infections: Nippostrongylus brdiensis and Nemutospiroides dubius compared. Int. J. ParasitoL 16: 6 17-622.

Smith, T J. & Weis, J.H. (1996). Mucosai T cells and mast cells share cornmon adhesion receptors. ImmunoL Today 17: 60-63.

Solomons, N.W. (1993). Pathways to the impairment of human nutritional status by gastrointestind pathogens. Parasitology 107 (Suppl): S 19433.

Spear, P. & Edelman, G.M. (1974). Maturation of the humoral immune response in mice. i. Exp. Med. 139: 249.

Sulüvan, D.A., Vaerman, J.-P., & Soo, C. (1993). Influence of severe protein malnutrition on rat lacrimal. salivary and gastrointestind immune expression duMg development. adulthood and ageing. Immunology 78: 308-317.

Svetic, A., Madden, K.B., di Zhou, X., Lu, P., Katona, LM., Finkelman, F.D.9 Urban, J.F., & Cause, W.C. (1 993). A prirnary intestinal helrninthic infection rapidly induces a gut-associated elevation of Th2-associated cytokines and IL-3. J. Imrnunoi. 150: 3434.

Swain, S.L., McKenPe, D.T., Weinberg, A.D., & Hancock, W. (1988). Characterization of T helper 1 and 2 ceU subsets in normal mice: Helper T cells responsible for IL-4 and IL-5 production are present as precursors that require priming before they develop into lymphokine-secreting ceils. J. ImmunoL 141: 344503455.

Swain, S.L., Weinberg, A.D., Engüsh, M., & Huston, G. (1990). IL-4 directs the development o f Th2-W<e helper effectors. J. IrnmunoL 145: 3796-3806.

Takeda, K., Tmaka, Tm, Shi, W., Matsumoto, M., Minami, M., Kashiwamura, S.L, Nakanishi, K., Yoshida, N., Kishimotom, Tq & Akira, S. (1996). Essential role of Stat6 in IL-4 s ignahg Nature 380: 627-630.

Tanaka, T., Hu-Li, J., Seer, R.A., de, S., Groth, B.F., & Paul, W.E. (1993). Interleukin 4 suppresses interleukinZ and interferon gamma production by naive T ceUs stimuhted by accessory cell-dependent receptor engagement. Proc. Nat. Acad. Sci USA 90: 5914.

Thompson-Snipes, L., Mar, V., Bond, M.W., Mosrnann, T.R., Moore, K.W., & Rennick, D. (1991). Interleukm 10: a novel stimuhtory factor for mast ceb and their progenitors. J. Exp. Med. 173: 507.

Thomhill, M.&, Kyan-Aung, U., & Haskard, D.0. (1990). IL4 increases human endotheiiai ceïï adhesiveness for T ceiis but not for neutrophils. J. ImmunoL 144: 3060.

Thomhill, M.H., Welücome, S.M., Mahiouz, D.L., Lanchbury, J.S.S., Kyan-Aung, U., & Haskard, D.O. (1991). Tumor necrosis factor combines with IL-4 and IFN-y to selectively enhance endothelid ceU adhesiveness for T cells. J. Immunol 146: 592-598.

TON, Ha, Ra, C., Nonyama, S., Suniki, K., Yata, Je-Ia, & Nakahata, T. (1996). Induction of the high-dfhity IgE receptor (FcrRJ) on human mast cells by IL-4. Int. ImmunoL 8: 1367-1373.

Uchikawa, Ra, Yamada, M., Matsuda, S., K u d a , A., & Anzono, N. (1994). IgE antibody production is associated with suppressed interferon-y levels in mesentenc lymph nodes of rats infected with the nematode Nipposnongylus brmiliensis. hrnunology 82: 427-432,

Upadhyay, P., Ganply, N.K., Mahajan, R.C., & Walia, BeNaSm (1985). Intesthai uptake of nutrients in normal and mahourished animais infected with Giardia lamblia. Digestion 32: 243-248.

Urban, J.F., Katona, LM., & Finkelman, F.D. (1991a). Heligrnusomides p o l y g y w : CD4+ but not CDS+ T ceb regulate the IgE response and protective imrnunity in mice. Exp. ParasitoL 73: 500-5 1 1.

Urban, J.F., Katona, LM., Paul, W.E., & Finkelman, F.D. (199 1 b). Interleukin 4 is important in protective immunity to a gastrointestinal nematode infection in mice. Proc. Natl. Acad, Sci USA 88: 35 13-34 1%

Urban, J.F., Madden, K.B., Svetic, A., Cheever, A., Trotta, P.P., Cause, W.C., Katona, LM., & Finkelman, F.D. (1992). The importance of Th2 cytokines in pro tective immunity to nematodes. ImmunoL Rev. 127: 205-2 19.

Urban, J.F., Madden, KWBa, Cheever, A.W., Trotta, P.P., Katona, LM., & Finkelman, F.D. (1993). IFN inhibits inflanmatory responses and protective immunity in mice infected with the nernatode parasite, Nipposnongylus brdiensis . I. Immunol. 151: 7086- 7094.

Urban, J.F., Maliszewski, C.R., Madden, K.B., Katona, LM., & Finkelman, FaDe (1995). L-4 treatment can cure established gastrointestinal nematode infections in imrnunocompetent and Unmunodeficient mice. J. ImrnunoL 154: 4675-4684.

Urban, J.F., Fayer, R a , Sulüvan, C., Gofdhill, J., Shea-Donohue, T., Madden, K., Morris, S.C., Katona, L, Cause, W., Ruff, MM., Mansfield, L.S., & Finkelman, F.D. (1996). Local Th1 and Th2 responses to parasitic infection in the intestine: reguhtion by IFN-gamma and 1 . 4 . Vet. ImmunoL & ImmunopathoL 54: 337-344.

de Waal Malefijt, R a , Haanen, Je, Spits, H., Roncarolo, M.-Ga, teVelde, A., Figddor, C., Johnston, K., Kaastelein, Ra, Yssel, He, & deVries, J.E. (1991). Seiection of a humm T helper type 1-lüce T cell subset by mycobacterh. J. Exp. Med. 174: 9 15.

Wade, S., Parent, Ge, Bleiberg-Daniel, F., Maire, B., Fall, M., Schneider, Dm, Le Moullac, B., & Dardenne, M. (1 988). Thymulin (Zn-FïS) activiîy in pro tein-energy malnutrition: new evidence for interaction between malnutrition and infection on thymic function. Am. J. Clin. Nutr. 47: 305- t 1.

Wahid, F.N., Robinson, M., Behnke, J.M. (1989). Irnmunological relationships during primary infection with Heligmosomides polygynrs (Nemutospiroides dubius): expulsion of adult worms from fast-responder syngenetic and inbred strains of mice. P&tology 98: 459-469.

Wahid, F.N. & Behnke, J.M. (1992). Stimuli for acquired resistmce to Heligmsomides polygyms from intestinal tissue resident L3 and LA larvae. Int. J. P~ûsitoL 22: 699-7 10.

Wahid, F.N. & Behnke, J.M. (1993). Immunologicd relationships during primary infection with Heligmusomoides polygyrus (Nemutospiroides dubius): parasite-specifc IgGl antibody responses and primvy response phenotype. Parasite Irnrnunol 15: 401- 413.

Wahid, F.N., Behnke, J.M., Grenas, R.K., Else, KJ., & Ben-Smith, A.W. (1994). Immun010 gical relatio nships during p h y y infection with Heligmosomoides polygyms: Th2 cytokines and primary response phenotype. P~asitology 108: 46 1-47 1.

Warren, &S., Kinnear, B.F., Philüps, J.H., & Lanier, L.L. (1995). Production of IL-5 by human NK ceils and regulation of IL-5 secretion by IL-4. IL- 10. and IL- 12. J. ImrnunoL 154: 51445152.

Wastllng, J.M., Scudamore, C.L., Thomton, E.M., Newlands, G.F.J., & Miller, H.RP. (1997). Constitutive expression of mouse m u t ceil protese-1 in normai BALBk mice and its up-regulation duMg intestinal nematode infection. Immunology 90: 308-3 13.

Wen, L., Pao, W., Wong, ES., Peng, Q., C d , J., Zheng, B., Kelsoe, G., Owen, J. Je, & Hayday, A.C. (1996). Germinal center formation immunogIobulin ciass switching and autoantibody production driven by "non ap" T ce&. J. Exp. Med. 183: 227 1.

Wen, L., Barber, D.F., Pao, W., Song, F.S., Owen, MJ., B Hayday, A. (1998). Primary y6 ce1 clones can be dehed phenotypicdy and functionally as T h l m tek and illustrate the association of CD4 with Th2 differentiation. J. ImmunoL 160: 1965-1974.

Wodd Health Organization (WHO). (1987). Prevention and Control of Intestinal Parasitic Infections. Geneva: World Health O r g h t i o n , Technical Report Series, no. 749.

Wilüams, D J.L. & Behnke, J.M. (1982). Host protective antibodies and semm immunoglobulin isotypes in mice chronically infected or repeatedly immunized with the nematode parasite Nemutospiroides dubiiis. Immunology 48: 37-47.

Windhagen, A., Anderson, D.E., Carrizosa, A., Williams, RE., & Hafler, D.A. (1996). IL- 12 induces human T ce& secreting IL- IO with IFNy. J. ImmunoL 157: 1 12%

W d b u y , RG., Miller, H.R.P., Huntley, J.F., Newlands, G.F.J., Palliser, A.C., & Wakeün, Dm (1984). Mucosal mast ceh are hinctiondy active during spontaneous expulsion of intestinal nemntode infections in nt. Nature 312: 450-452.

Woodward, B.D. & Miller, R.G. (1991). Depression of thymus-dependent immunity in wasting protein-energy miilnutrition does not depend on an altered ratio of helper (CD47 to suppressor (CDg*) T ceh or on a disproportionately iarge atrophy of the T-ceii relative to the B-ceil pool Am. J. Clin. Nutr. 53: 329-335.

Woodward, B., Dwivedi, A., & Nmgpd, A. (1992a). Mechanisms of thymic epitheliai involution in weanüng pro tein-energy mainutrition. Nutr. Res. 12: 1253- 1264.

Woodward, B.D., Woods, J.W., & Crouch, D.A. (1992b). Direct evidence that primvy acquired ceil-mediated immunity is Iess resistant thm is primary thymus-dependent humoral immunity to the depressive Suence of wasting protein-energy malnutrition in weaniing mice, Am. J. Clin, Nutr. 55: 1 180- I 185.

Woodward, B.D., Bezuison, K.D., Hillyer, L.M., & Lee, W.-H. (1995). The CD45RA' (quiescent) celluiar phenotype is overabunduit reiative to the CD45W phenotype within the involuted splenic T ceil popuhtion of weanling mice subjected tow wasting protein-energy malnutrition. J. Nutr. 125: 247 1-2482.

Wykes, L.J., Fiorotto, M., Bumn, DG., Del Rosario, M., Frazer, ME., Pond, W.C., & Jaboor, F. (1996). Chronic low protein intake reduces tissue protein synthesis in a pig mode1 of pro tein malnutrition. J. Nutr. 126: 148 1- 1488.

Yoshimoto, T. & Paul, W.E. (1 994). CD4*M<l. 1+ T ceUs prornp tly produce interleukin- 4 in response to in vivo ch;illenge with mti-CD3. J. Exp. Med. 179: 1285-1295.

Zhang, X., Werner-Fom, Ce, Tang, H., Bmuswers, Ne, Bonnefoy, J.Y., & Zubkr, RH* (1991). IL4dependent IgE switch in membrane IgA-positive humaa B cells. J. IinmunoL 147: 3001-3004.

CHAPTER III STUDY OBJECTIVES

Previous studies have reported independentiy that protein deficiency increases the

prevalence and intensity of experimental nemtode infections and impairs systemic and gut

mucosal immune huiction in naive animais. However, very few studies have examkied the

interactions between all three conditions (Le. protein deficiency, nematode infection and

host irnmuniiy) in the s m e host-parasite system. Inasmuch as host resistance to challenge

H. polygyrus infection depends on intense. npid and sustained Th2 immune responses. it is

important to examine whether the capacity for acquired immunity to intestinal nematodes is

adversely modified by protein malnutrition. The overd purpose of this study was to

detennine whether dietvy protein deficiency potentiates a chdenge Uifection in rnice with

the gastrointestinal nematode, H. polygyrus, by impairing the magnitude. site-specificity

and kinetics of secondary cytokine and effector responses. A the course study of serum

IgE. parasitic-specific IgG1, peripheral and intestinal eosinophils, MMC, MMCP-1, and

cytokine production in spleen and M W was performed from days 3 to 28 pci These

immune responses were &O measured in naive rnice yidlor afker primary section to

compare the ievel of protective immunity acquired during challenge infection.

The k s t specinc objective of this study was to determine the effects of dietary

protein deficiency on systemic md intestinal Th2 immune responses to polygyms

Mection as indicated by the level of serum effecton (eosinophils, IgE, parasite-specinc

IgG l), the production of spleen cytokines (lL-4, IL-5. IL- 10, IFN-y ), the proliferation of

gut mucosd effectors (MMC, intesthai eosinophüs, MMCP- I), and the synthesis of gut-

associated cytokines in MLN. Secondly, we wanted to ascertain whether the seventy of

dietary protein restriction (7% versus 3%) would afTect the extent of impaired Th2

immunity and parasite survivd. The third objective was to compare the impact of protein

deficiency on systemic versus intestinal Th2 immune responses to validate our assumption

that gut mucosal immunity may be more suscepuile to protein mdnutritioa Fiidy, we

intended to characterize and contrast the kinetics of systemic and local Th2 immune

responses during graded leveis of protein dekiency in mice challenged with polygynrs.

PROTEIN DEFICIENCY PROLONGS SURVIVAL OF A GASTROINTESTINAL

PARASITE IN MICE BY SUPPRESSING TH2 IMMUMTY

Rebecca Ing12, Zhong Su', Marilyn E. Scott', Kristine G. Kosk?

'Institute of Parasitology and 'School of Dietetics and Humm Nuuition.

McGill University (Macdonald Campus)

21.1 11 Lakeshore Road, Ste-Anne de Bellevue, Quebec H9X 3V9. Canada

Corresponding Author:

Dr. KG. Koski

School of Dietetics and Human Nutrition

Macdonald Campus, McGilI University

21.1 11 Lakeshore Road

Ste-Anne de Bellevue, Quebec H9X 3V9, Canada

Telephone: 5 14-398-7845

Fsix: 5 14-398-7739

Email: koski@agndiahmcgülca

ABSTRACT

Protein deficiency has been shown to increase susceptibüity to gastrointestind (GI)

parasitic infections in mice, possibly as a result of impaired systemic and intestinal effiector

responses induced by the dom-regulation of Th2 cytokine d o r up-regdation of Th l

cytokine. To test this hypothesis, B ALBlc mice (n= 18/diet/time point) were fed a control

(24%), marginal (7%). or deficient (3%) protein diet and given a challenge infection with

the nematode, Heligmosomides polygyrus. The protein deficient (PD) rnice had higher

food intake per gram of body weight, lower body weight gain, reduced BUN concentration,

and depressed reiative spleen and mesentenc lymph node (MLN) weights compared to the

control and marginal mice. The PD mice had higher worm burdens at 1,2 and 4 weeks

post-change infection (pci), suggesting chronic absence of a fbnctional immune response.

Indeed, the PD rnice h d lower increues in serum IgE, reduced penpheral and intestinal

eosinophilia, and depressed MMC pmliferation and activation at 1 to 2 weeks pci

Parasitic-specinc IgG 1 was not affected by protein deficiency. To determine whether these

suppressed effector responses in the PD rnice may have resulted in piut from aitered spleen

and MLN cytokine pronles, c eh were stimulated Ni vino with parasite antigen and

cytokine concentration in the culture supematmts was measured by sandwich ELISA. The

deficient MLN ceh secreted signincantly las IL4 and more EN-y within 2 weeks pci

than did control MLN cek. The deficient spleen ce& &O secreted more IFNy at 2 weeks

pci compared with control spleen cells. There were no significuit dserences in IL-5 or IL-

10 secretion by MW or spleen c e h among the dietuy groups. Frorn RT-PCR mûlyses,

the PD mice also had lower IL-4 rnRNA expression in spleen and MLN at 1-2 weeks pci

In summary, protein deficiency has detrimental type- and site-specifk effects on cytokine

and effector responses at euly pci time points critical for expulsion of H. polygyrur. Our

data support the hypothesis that protein deficiency exacerbates the suniival of a GI

nernatode parasite by decreasing IL-4 (TM) and increasing IFN-y ml), leading to

reduced gut and systemic Th2 effecto r respo mes.

INTRODUCTION

Protein malnutrition and gastrointestind nematode infections are chronic diseases

that frequently CO-exist in human populations of developing countries (Crompton, 1986).

Acquired resistance to intestinal parasitisrn depends on the magnitude and rapidity of Th2

cytokine (e.g. E-4, IL-5. IL-10) and effector responses ( e o s h o p ~ . IgE, wtocytosis) as

well as the concomitant absence of Th1 responses such as IFN-y and macrophage activity

(Urban et al., 1993; Monroy & E ~ q u e z . 1992; Urbm et ai., 199 1). Although protein

deficiency increases susceptibility to nematode parasites during repeated and challenge

infections (Shter & Keymer, 1988; Keymer & Tarlton. 1991), it rernains unclev whether

protein deficiency potenthtes intestinai parasitic infections by hpaVing the development

and maintenance of specific Th2-dependent immune responses in penpheral andior

intestinal tissues. Earlier studies showed that higher worm burdens in mice fed low protein

dieu were associated with depressed blood eosinophilia and lower titres of cuculating

parasite-speciflc IgG (Slater & Keymer, 1988; Bouhy et al.. 1998). However, it is

unknown whether milder protein restriction has the same effect on Th2 immunity and

whether the irnpaired effector responses resulted from diet-induced perturbations in

cytokine synthesis. Additionally, lit tle Uifomation exists on the CO nsequences of pro tein

deficiency on host protective immunity in gut-associated Iymphoid tissues (GALT) even

though immune responses in GALT comprise the k s t line of defense ag;iinst intestinal

parasites and may influence the appropriateness, proiiferative magnitude and traffïcking of

systemic responses. The rate of protein turnover in gut mucosal tissues is 25-30 times

greater than that observed for muscle (Nakshabendi et al.. 1995) and is potentisilly very

sensitive to changes in host protein status (Wykes et al., 1996).

Our labontory has been involved in studies on the interactions between acquired

immunity, parasitic infection, and nutritional deficiencies in mice infected with a

gastrointestinal mouse nematode, Heiigmosomoides polygynrr. P r i m q infections with H.

polygyncs are usually chronic but immunocompetent hosts are capable of developing

resistance to challenge infections. The capacity to resist secondary infections and the

intesthal site of infection make the polygyrus-mouse system a suitable mode1 for

studying the Muence of nutritionai factors on acquired immunity and gut mucosal immune

responses during gastrointestinal heùninthiasis. Previous experimenu in our labontory

have shown that protein deficiency uicreased worm burdens, decreased serum IgGl and

lowered eosinophilia in mice chdienged with H. p o f y g y w (Boulay et al.. 1998). Gut-

associated Th2 effector responses and cytokine production in a protein deficient host

chaknged with polygyncs have no t k e n chuacterized. The overd objective of this

study was to determine the effects of graded levels of protein deficiency on the magnitude,

site-specincity and time course of Th2 immune responses during challenge infection with H.

pofygynrs. We postuhted that pro tein deficiency prolongs parasite s u ~ v a l by impairhg

host cytokine respo mes in gut-associated ando r systemic lymphoid tissues that normaily

help to resolve a secondary H. polygyms infection. The reduced Th2 cytokine responses

andlor increased T'hl cytokine synthesis may subsequently lead to decreased concentrations

of Th2 effectors in intestinal and systemic sites. Assuming that gut mucosal immunity is

more susceptible thui systemic immunity to protein malnutrition and given its putative role

in priming and directing host resistance, we expected protein deficiency to cause a more

profound impairment of intestinai irnmunity than of systemic responses. Also, we expected

that a low protein diet (3%) would inhibit acquired immunity, and in tum increase

parasitisrn, to a greater extent than would a marginally deficient diet (7%). FimaDy, we

speculated that these effects would diner in intensity at dinerent phases of infection given

that the most intense stimulus for acquired immunity is elicited by tissue-resident Wae at

1-2 weeks post-challenge infection (Wahid & Behnke, 1992).

Our results show that protein deficiency promoted suxvival oEH. p o l y g y w by

altering cytokine and effector responses in a selective mamer depending on the tissue

cornpartment, the level of restriction and the time point of infection. In pyticulu. the host

responses that normaily help to resolve challenge Uifections with H. pofygynrs (increased

IL-4, decreased IFN-y, eievated IgE, mucosal rnastocytosis, MMC activation) were

pro foundiy impaired by pro tein deficienc y. These data support the hypo thesis that pro tein

denciency prolongs sunRvaï of n nematode parasite by decreasing gut-associated I L 4

production and Th2 effector responses in the gut and peripherd circulation.

MATERIALS & METHODS

Experimental Design

Mice were fed a diet that was sufficient (24%). marginal (7%). or deficient (3%) in

protein and given prirnary or chûllenge uifections with H. polygynrs. Penpheral

eosinophih was measured in tail veh blood using the Unopette Test (#5877 Fiher

Scientinc, Montreal. PQ). Worms in smali intestines of infected mice were counted at 6

and 28 days post-primuy infection (ppi) and at 6, 14.2 1 and 28 days post-challenge

infection (pci). Cardiac blood w u separated into serum and plasma sampks and stored at

-80°C in Microtaher tubes (Becton Dickinson, Rutherford, NJ. USA) for iater antibody and

mucosal mast ceU pro tease- 1 (MMCP- 1) analyses. Intestinal tissues were O btained for

histological counting of mucosal mast cells (MMC) and intestinai eosinophils. At 3. 6, 14

and 28 days pci, spleen and rnesentenc lymph nodes (MW) ceii suspensions were

prepared, and their cytokine production and gene expression were determined in vitro.

Expenmental Protocol

The experiment was conducted using an aithelmintic-abbreviated immunizing

protocol that hm been shown to stimulate protective imrnunity against a challenge infection

with H. polygynis (Behnke & Robinson, 1985). niree weeks after feeding with

experimentd diets, mice (6/diet group/time point) were intubated ordy with 100 third-

stage Iarvae (L,, of H. polygynrs (day O ppi); 12 rnice per diet group remained uninfected

(Figure 1). Six days &er primary infection, 6 mice per diet group were iciIIed to connmi the

Uifectivity of L, (P6 rnice). On days 9 and 14 ppi, the remaining infected rnice were trerted

oraily with pyruitel pamoate (Pfker Canada Inc.. Kirkland, Quebec, Canada, dose of 172

mgkg body weight) to remove the adult parasites fkom the intestine. Six mice per dietary

group were LiUed CO ensure efficac y of the dmg ueatment which required an absence of

w o m in the intestine. One week foilowing the second anthelmintic treatment ( d g 2 1

ppi), mice (6ldiet group/time point) were reinfected with 100 L, of H. poiygyn*r (challenge

cohort).

Challenge Cobort - -21 D DO D6 D 9 D l 4 D 21 1 Posi-primary Infection (ppi)

DO D 3 D 6 D 9 D 14 D 21 D 28 Post-challenge Infection (M)

Feeûing differenr diets 100 L, P6 Drug h g 100 L, C3 C6 0 Cl4 C21 C28 Primary Challenge Infeciion In feciion

-21 r) DO D 28 - Posi-primary Infection (ppi)

Feeding different diets 100 L, Primary Infection

Feeding different die& Nai ve mirols

Figure 1: Schematic representation of the experimental protocol and various infection groups. The numbers on top of each line represent tirne course (in days) of the infection protocol. The values below the iine represent the procedures perfomed ai the correspondhg day and infection groups killed at each t h e point.

Two groups of control mice were included in the experiment. One group of previously

uninfected mice (6 per diet group) were given a primary infection with 100 L, at the tirne of

challenge infection and killed at 28 days ppi (P28 mice). These primary infection mice were

used to compare the degree of protection acquired duMg the challenge infection protocoL

A second cohon of mice (6 per diet gmup) remained uninfected throughout the

experimental penod to serve as naive infection controls and were kiüed on day 28 pci

(naive controls). Mice given the challenge infection were H e d at 3, 6.9, 14, 2 1 and 28

days pci and are referred to as C3, C6, Cg, C 14. C2 1 and C28 Mce. respectively.

Mice

Three week old femaie B ALBlc rnice (Charles River, St. Constant, PQ, Canada)

were acchatized to a 14h light-l0h duk cycle in a temperature-controlled (22-25'C)

animai room for 3 days while fed the protein-sufficient diet. Mice were then weighed and

randomly docated to one of the 3 dietary groups. AU experimental diets were offered ad

libitum in plastic Mouse Powder Feeders (Lab Products Inc., Montreal, PQ) specifically

designed to minimize food spihge. Distilled water was rvaiiable to ail animais ad libinim.

Mice were housed individudly in Nalgene cages (Flher Sckntific, Montreai, PQ) with

stainless steel covers, filter tops, and heat-treated hardwood bedding. AU procedures were

approved by the McGill Animai Care Committee according to the Canadian Council on

Animal Care (1984).

Diets

Mice were fed a semi-pudied, biotin-fortified diet with spray-dried egg-white soiids

as the sole source of protein (Bouhy et al., 1998). The 2446 (control) protein level is

considered adequate and not excessive for the hboratory mouse (NRC, 1995). The

protein-restncted diets contained either 7% (marginal) or 3% (deficient) protein and were

made isoenergetic to the 24% protein diet by substitution of protein with equivalent

amounts of comtatch on a weight buis (Table 1). AU other nuvients were identical

among the three diets and were included at levels sufflciently above the NRC (1995) mouse

requirernents to ensure that a 30% reduction in food intake as a result of protein restriction

or infection would not generate any other nuvient deficiencies in the marginal or deficient

protein groups. Biotin was added at 18 times the NRC recommended level to counteract

the biotin-chehting effects of avidin in the egg-white solids.

Parasite and parasite an Agen

Infective third-stage Irirvae (b) of H. polygyms were obtained by culturing the

feces of stock CD1 mice (Charles River) on moist fdter paper for 7 days. The cultured

l ame were suspended in distilled water (approlamately la0 Y20 pl) and adrninistered by

o n l gavage to mice using a Gilson pipetmm (Mandel ScientSc, Guelph. Ontario). The

accuracy of the dose and viabiîity of the iarvae was estimated by counting the number of

live b a e per 20 p1 after every 15-20 intubations.

Parasite antigen was prepared using fourth stage lyvae (LJ of H. p o l y g y m

obtained fiom s m d intestines of mice infected with L, 6-7 days previously. The coiîected

L, were washed with PBS and sonicnted on ice for 10-15 min. The homogenate was

centrifuged at 1500 g at 4°C for 1 hr. The recovered supernatant was sterjlized using a

0.22 pm Acrodisc (Gehui Sciences, AM Arbor, MI, USA) and stored at -20°C. Pro tein

concentration of the antigen homogenate was determined ushg a dye-binding protein assay

(Bio-Rad, Mississauga, Ontario, Canada) with BSA as the standard.

Table 1: Nutrient composition of experimental diets.'

1ngredient2 Pro tein sufficient Pro tein marginal Protein deficient (24%) (7%) (346)

Egg white 240

Corn oil 80

Corn starch 296

Glucose 296

Alphacel 30

Vitiunin mix3 12

Mineral mix' 46

l Ingredients are g/kg diet

ICN Biochemicals. Division of ICN Biocfiemiuis, Inc.

V i m i n mix (mgkg diet) as formulnted with X N Biahemicals & Anachernia Science (Anachemin

Canadû, ïnc.); niacin 50.0; d-calcium panthothenate 32.0: riboflcivin 24.0; pyridoxine bydrochloride 24;

thivnin hydrocbloride 16.0; folic acid 2.0; d-biotin 3.6; cymocobalunin 0.05; a-tocopheryi aceote (1 .O

iU/mg) 96.0; menaquinone 9.0: vitamin 4 (400.W iUlg) 7.5; viiamin A acetate ( 5 0 0 . 0 i ü g ) 8.0;

butylated hydroxytoluene 100.0; choline chIoride 4000.0; alphacel3748.5.

' Minerai mix (gkg diet) as farmulated with ICN Biochemicals. Anachemia Science, Sigma Chernicals

(Sigma Canada), & Fisha Chemids (Fisher Scientific Canada); diuiaum phosphate 27.16; potassium

bicarbaiate 10.25; soâium chloride 2.54; magnesium sulphate 4.95; chromium potassium sulphnte

0.0384; cupric carbonate 0.0157; potassium iodate 0.01; ferrous sulpbate 0.2489; manganous carbonate

0.1883; zinc carbonate 0.115; sodium selenite 0.0004; sodium moiybdate 0.00045; potassium fluoride

0.0099; citric acid 0.48;

Parasite wonn burdens

The numbers of L, parasites were counted at 6 days after primary or challenge

uifection. The nurnbers of adult worrns in chdienged rnice were determined at days 14.2 1

and 28 pci and in the primary group at day 28 post-infection. L, pansites embedded in the

intestinal mucosa were counted from the smaii intestine that was pressed between two g h s

slides. Parasite s u ~ v a l was indicated by the number of w o m recovered from the entire

srnail intestine at necropsy.

Nutritional measures

Mouse body weight and food intake were measured every 3-4 days. Body weight

gain was calculated by subuacting the initiai body weight taken prior to randornkition to

the experirnental dietary groups from the hal body weight obtained at necropsy. The

weighu of spleen, thymus and MLN are expressed relative to the h a 1 body weight. The

concentration of albumin in plasma simples was osayed by dye-binding of bromcresol

green to the protein (No. 63 1. Sigma. St-Louis. MO) as rneasured with the Abbott VP

Sumper S ystem Discrete Analyzer (Abbo tt Diagnostic, Mississauga, Ontario). Blood urea

nitrogen @UN) concentration was detemhed by coupled enzyme reactions (No. 67-UV.

Sigma) as measured with the Abbott Analyzer.

ELISAs for IgE, parasite-specific Ig GI and MMC proteme-I

Total semm IgE and MMCP-1 were rneasured by two-site ELISA Immulon II

phtes (Dynatech Laboratories Inc., Chantilly, VA) were coated with purined rat mti-mouse

IgE mAb (021 11D clone R35-72, PharMingen, San Diego, CA) at 2 pgrd and incubated

ovemight at 4°C. Plates were blocked with 1% BSA in PBS for 2 hrs at room tempenture

to imnmiize nonspecinc binâing in weUs. Serinl dilutions of purified mouse IgE standard

mAb (03 121D, PharMingen) and diluted sera were added to the welk and incubated

oveniight at 4°C. Biotinylated, streptavidin-horseradish peroxidase conjugated rat anti-IgE

mAb (02122D clone R35-92, PharMingen) at 2 pglml was used as a detecting antibody-

Avidk- pro xidase conjug ate (Cedariane Labontories Limited, Homb y, ON) was used as

the secondary hyer and the reaction w as visualized b y addition of 2.2'-azino-(3-ethyl-

bennhiazoline-6-sulphonic acid) (ABTS; Sigma) and read at 405 nrn after 25 min with an

ELISA microplate reader (Dynatech MR5000. Dynatech Laboratories). IgE concentrations

were deterrnined 6rom standard curves and are expressed as mean p g / d î SE.

For measurement of semm MMCP-1. Immulon ï f plates (Dynatech Laboratories)

were coated with sheep mti-MMCP-1 capture antibody (Moredun Scientif'c Ltd.. Peniciuk.

Scotland) at 2 pglml and incubated for 24 hr at 4°C. Purified mouse MMCP-1 isolated

fkom mouse srnaii intestine was used as the standard (Moredun Scientinc). M e r overnight

incubation at 4°C with standards and diiuted serurn sampks. rabbit anti-MMCP- l mAb

(Moredun Scientinc) conjugated to sueptavidin-horseradish peroxidase was added as the

detecting antibody. The reaction was visuaijzed by the substrate. orthophenylene diamine

(OPD. Sigma) and read at 450 m with ELISA reader after 25 min incubation. MMCP-1

concentrations are derived Erom MMCP-1 standard curves and expressed as nglml I SE.

Parasite-speciûc IgGl in sera was deterrnined by direct ELISA Immulon II phtes

(Dynatech Laboratories) were coated with L, antigen homogenate (2.5 pg/rnl). prepared as

above. in PBS and incubated ovemight at 4OC. Piates were blocked with 1% BSA in PBS

for 2-3 hr at room temperature. Serum samples and interna1 convols from uninfected and

infected mice diluted 1:100 in PBS with 1% BSA were incubated in weUs at 4°C overnight.

Biotinyhted, streptavidin-horsendish peroxidase conjugnted rat anti-mouse IgGl mAb

(PharMingen) was used as the detecting antibody. Avidh-peroxidase conjugate (Ceduhe

Limited) was dded and plates were developed with ABTS substrate solution for 12 min.

The reaction was read at 405 nm with an ELISA reder. The results are reported as mean

optical density (OD) I SE.

Intestinal mus? cell and eo~hophil histology

Segments of small intestine (approximately 0.5 cm long, 10 cm fkom pyloms) were

h e d in Camoy's fixative (for MMC) or in 4% paraformaldehyde at 4°C overnight (for

intestinai eosinophils). The h e d tissue was dehydrated and embedded in paranin following

standard histological procedure. To count mast cek, sections (5-6 prn thick) were stained

with Aician Blue, counterstained with Sakanine 0, and the aumber is expressed as mem

per 20 villus crypt units (v.c.u.). Eosinophils in intestinal mucosal sections (5-6 pm thick)

were stained with 0.5% Chromotrope 2R and are expressed as mem counts per V.C.U.

Spleen ceil and mesenteric iymph node ceil preparation

Spleens and MLN were weighed. teased apart with needles. and then passed

aseptically through a 60 mesh stainless steel screen (Sigma). Red blood ceiIs were lysed by

osmotic shock with cold W C 1 lysing bufEer solution. Membrane debris was removed

through repeated washings with RPMI 1640 supplemented with 1096 FBS and

centrihigation at 469 g for 10 min at 4°C. Ceil viability was deterrnined by trypan blue

exclusion (0.1% Trypan Blue in PBS; Gibco) and was dways greater thm 90%. The ceh

were resuspended in complete medium consisting of RPMI 1640 (Gibco, Burlington,

Ontario), 10% heat-inactivated FBS (Gibco), 20rnM HEPES buffer (Sigma), 200 Ulm1

penicillin (Sigma), 200 p ghd streptomycin sulfate (Sigma), 25 p g/d gentamich (Gibco),

and 50 pM 2-mercaptoethanol diluted in Hank's balanced salt solution (Gibco). The stock

concentration was adjusted to 10 x 106 ceWrnL

Production of Th2 (IL-4, IL-S and IL- I O) and ni I (IFN- y) cytokines

Spleen and MLN ceil suspensions were plated in complete medium at a hai

concentration of 5 x 106 ceWmL Aliquots of 1.0 ml were incubated in 24-weîI Hat-bottom

ceil cuhure pktes (Costar, Charlotte, NC) with L, antigen (7.5 pg proteWml) prepared as

above. This concentration of parasite antigen was determined in a separate experiment to

be the lowest concentration required to stimulate maximal cytokine secretion by infected

s p k n cek. CeIl cultures were incubated at 37OC in 5% CO2 aunosphere with 90%

humidity. Supernatants were recovered afler 48 hr incubation by centrifugation of the

suspensions at 300 x g for 10 min. The supematants were stored at -80°C until they were

assayed for IL-4, IL-5, IL- 10 and IM-y. A subset of the cultured ce& (3 per diet, second

block) were lysed with Trizol (Gibco) for mRNA analysis.

Cytokine concentrations in the supematants were measured by two-site sandwich

ELISA that involved the following paired antibodies: purified rat anti-mouse IL4 mAb

1 1B 11 (18 MD. PharMingen. San Diego, CA) and biotinylated rat anti-mouse IL-4 mAb

BVD6-24G2 (180429, PhYMingen); purified rat anti-mouse IL-5 mAb TRFK-5 (18051D,

PharMingen) and biotinylated rat anti-mouse IL-5 rnAb TRFK-4 (18062D, PharMingen);

purined rat anti-mouse IL- 10 mAb JES5-2A5 (1 8 141D, PhYMingen) and biothyhted rat

anti-mouse IL-10 mAb SxC-1 (18 152D, PharMingen); and purified rat anti-mouse IFN-y

mAb W6A2 (1 8 18 1D. PharMingen) and biotinylated rat anti-mouse IFN-y mAb XMG 1.2

(18 112D, PhrirMingen). Irnmulon II piates (Dynatech Laboratories) were coated with the

capture antibody ai 2 p g h l in O. 1 M Na&PO, (pH 9.0) and incubated ovemight at 4°C.

Seriai dilutions of cytokine standards and supernatants were added and incubated ovemight

at 4°C. Biotinylated anti-cytokine detection antibodies were added at 1 pg/d and

s~ptavidin-horseradish peroladase conjugate (Cedariane Laboratories Limited, Homby,

ON, Canada) was used as the secondvy hyer. The reaction was visudized with the ABTS

(Sigma). The concentration of cytokine was cdcuhted fiom standard curves generated

fiom known concentrations of recombinant (r) murine I L 4 (1 923 1 V, PharMingen), rIL-5

(19241V, PhitfMingen), rIL-10 (19281V, PhluMingen), and rIM-y (19301T.

PhuMingen).

Cytokine mUNA expression by semi-quantitative RT-PCR

Cytokine mRNA Ievels in cultured spleen and MLN cells were quantitated by RT-

PCR assay. RNase free plastics and water were used throughout the assay. Concentration

of the extracted RNA was determined spectrophotometrically. The RNA was suspended in

water, heated to 65°C for 10 min and reverse tramcriid in a 65 PI final volume containing

6.5 pl of a 10 mM mU of four deoxynucleotide triphosphates (m), 13 pl of 5X RT

buffer, 22. pl of O.1M dithiothreital, 0.26 p1 of RNase inhibitor (40 UIpI), 0.52 p1 Oligo dT

(0.5 pg/pI), 0.65 p l of Moloney Murine Leukemh Virus (MMLV) reverse transcriptase

(200 Ulpl) (Gibco), 36.27 pl distilled water, and 5.2 pl total RNA (0.3 pgfpl). The mix

was incubated at 37'C for 1 hr and then heated to 95°C for 5 min. A 10-pl aliquot of the

RT reaction was used in a 100 p1 PCR reaction which contained 0.5 p1 of Taq DNA

polymeme (Gibco), 10 pl of 10x PCR buffer, 0.2 pl of 10 m M d m . 4 11 of 50 rnM

MgCl, 1 pl of each appropriate forwvd and reverse primers (50 pdpl). The sequence

primers for IL-4, IL-5, IL10 and IFN-y and the ceference gene, hypoxanthine phosphori-

bosyltransferase (HPRT) were selected as descnid by Svetic et al. (1993). The samples

were subjected to 29 cycles as foilows: 1) 94OC for 0.5 min; 2) 56°C for 1 min; and 3) 72°C

for 2 min. A final extension step was performed by heating to 72°C for 8 min. The nurnber

of PCR cycles and the total RNA input in the RT reaction were determined in a preliminary

experiment to ensure that the amplification was well below the saturation under this assg

condition. After PCR amplitication. a 10 p1 diquoi w u mixed with 2 pl loading buffer and

ekctrophoresed on a 1.2% agrose gel containing ethidium b r o d e (0.25 pg/ml). The

resulting DNA bands in the gel were photographed using a Polaroid camera Quantitation

of cytokine mRNA was accomplished by densitomeuic scanning of the photograph ushg

Macintosh Photoshop and NIH Image 160 software. HPRT mRNA levels did not dZer

signincantly among dietary groups (FI, 4 4 7 ; p=0.49), infection times (F,, e l .77;

p=û.17), or tissues (F,,,,=û.O6; p=0.81). The mRNA Ievel for each cytokine w u

nomialued to HPRT and expressed as ratio to HPRT mRNA for each sample.

Statistical analyses

nie experimental protocol was repeated 3 times over a penod of 8 months. The

results for nutritional, parasite, antibody and histologicd outcornes were obtained from 3

blocks. The cytokine concentration data were from the second and third blocks while the

mRNA expression data were obtained from a subset (n=3/diet/time point) in the second

block. Results are reported as means SE. Bmktt's test was used to detennine

homogeneity of variance among groups to be compmd. When the variances of the

cornpuison groups were non-homogenous (PcO.OS), analyses of eosinophils, IgE, MMCP-

1, histological counts, cytokine were perfomed with logarithmic transfonned data.

To assess the overall effects of diet and tirne, data on wom burdens. mtibody titres.

histological counts, MMCP- 1 concentration, cytokine production and cytokine mRNA

expression were pooled by infection protocol (primary or challenge) and each was anaiyzed

for the effects of diet, tirne and diet by time using a two-way analysis of variance

(ANOVA). Interactions between diet and tirne were reponed only if signincant. Repeated

mesures ANOVA was used to assess effects of diet and iime on peripheral eosinophils.

One-way ANOVA was used to detect the effects of diet on nutritional parmeters of

chdenged mice killed on day 28 and on parasite and irnmunological data of naive mice.

Although our study was designed to examine the overail effects of diet and tirne

during the entire course of the prirnary or challenge infection (as assessed using two-way

ANOVA), we ako studied the efTect of diet at each post-infection tirne point because we

expected dietuy modulation of host immune response to vary in magnitude at different

stages of the infection. To thiS end, the w o m burdens, antibody titres, histologicd counts,

MMCP-1 concentration. and cytokine data were analyzed for the effect of diet at each post-

~ e c t i o n time point with a one-way ANOVA foliowed bypost hoc p-e cornpuisons

(Tuicey) when diet w u signiticant. Only results from pusr-hoc cornparisons are reported.

The analyses were perfomed using SAS (SAS Institute I~c., Cary, NC) and considered

sipifkant at P c 0.05.

Num*tional Outcornes

Table 2 summarizes the nutritional outcomes of a representative group. challenged

mice killed on day 28 pci Although challenged rnice fed the 3% protein diet ate the same

amount of food curnulatively, and hence calories, as mice fed the 24% protein diet, they

gzined significantly less body weight. Thus. the 3% protein diet induced protein but not

energy deficiency that Frequentiy accompanies dietary protein restriction. In fact. the 3%

protein group ate significmtly more food per gram body weight compared to the other 2

dietvy groups. However, ihis increased relûtive energy intake did not support normal

growth as s h o w by the 71% lower body weight gain of protein deficient mice compared to

the control mice. In contrast to the 3% group, the marginal protein (7%) group ate

signincantly more food overd and as a result gained comparable weight as the 24% group.

Protein restriction at both the 3% and 7% level signincantly decreased rehtive

weight of mesentenc lymph nodes (MLN). The 3% protein diet also signincantiy lowered

relative spleen weight. Thymus weight was not affected by protein restriction. Dietary

protein deficiency modified biochemical indices of protein status: plasma albumin

concentration was decreased by the 3% protein diet only when compared with the 7%

group, but neither groups differed from the 24% mice. However, blood urei nivogen

(BUN) concentration showed a dose-dependent response to protein level in the diet.

Parasite Outcornes

The hinctional significance of protein deficiency on immunity is shown in Figure 2

which presents the worm burdens of mice fed the experirnental dieu and given either a

primq or challenge Section wiih H. polygyrus. Previously irnmunized mice fed the 24%

and 7% protein diets were capable of expeiling over 95% of their parasites by day 28 pci

compared to their primary infection counterparts who eliminated only 56-60s of the

infective dose. In contrut. worm burdens were signincmtly higher in mice fed the 3 8

pro tein diet throughout the primary and chdenge infections, thus supporting the prernise

that protein deficiency impairs the development of protective immunity to H. po1ygyrusS

During challenge infection. protein deficiency increased both the establishment of H.

p o l y g y w at day 6 pci and the s u ~ v a l of aduit wonns at days 14-28 pci The wom

burdens of the 3% mice were also signincuitly higher cornpared to the 24% and 7% mice

during prirnvy infection. however, these were not as dnmatic as that observed during

challenge infection.

Serum Effector Responses

To assess the effects of protein deficîency on serum IgE responses, we measured

IgE concentration in nive mice, during primary infection and throughout the challenge

infection 3A). Serum levek of IgE were neady undetectable in uninfected rnice

and low in prirnary mice but increased dnmatically after chdenge infection. Serum IgE

levels in ail dietvy groups changed over the course of challenge infection (time effect,

Pcû.05): mice fed 24% and 7% protein diets h d noticeable peaks in serum IgE at days 6 to

9 pci whüe this elevation was blunted in rnice fed 3% protein. In al chaiienged mice. serum

IgE levels gradudy deched over tirne and by 28 days pci were simi7u to ievels observed

during primary Section. Mice fed 3% protein had signiacantly lower serum IgE levels

cornpared to mice fed 24% pro tein throughout the chdknge infection and on day 28 pp i

Serum IgE ievels in the 71 mice were simily to the 24% mice on days 3.6 and 14 pci.

were intermediate between the 24% and 3% groups on days 9 and 2 1 pci, and were as low

as the 3% group by day 28 pci, thus suggesting a possible dinerential response over time to

graded levels of dietary protein

Infection Groups

Figure 2

Figure 2. Effects of d i e t q protein deficiency and time on worm burdens of mice given r primary or challenge infection with H. polygynrs. Values are mean + SE. During primvy infection. the number of wonns was significantly higher in 3% mice compared to 24% and 7% mice (F,=13.35. Pd.0001) and was signXcmtly higher on day 6 ppi cornpared to day 28 ppi (F,*,=5.29, P=0.024). During challenge infection, the worm burdens were signincantly higher in 3% mice compared to 24% and 7% rnice (F,,,,=163.37, P=O.Oûû 1) and were signiticmtly ai3ected by time (F,,,,,=47.63, P=0.000 1). There were signincant diet and time interactions in primary infection (F,,=4.22. P=û.018) and in challenge infection (F6,1,=9.15, P=O.ûûûl), indicating that 24% and 7% rnice but not 3% mice eiiminated progressively more worms over thne. Dinerent letters at each time point represent significant dEerence between dietary groups ( P d . 0 5 ) based on posr-hoc pairwise cornparisons (Tukey).

ParasiteSpecif ic lgGl O.D. O O a --L

O in ô in P O

Serum IgE (uglml)

Figure 3. Effects of dietary protein deficiency and time on semm antibody bels. Values are + SE. Total semm IgE (A) and parasite-specitïc IgGl (B) were determllied in naive mice, primary irifection (days 6 and 28 ppi). and challenge infection (days 3.6.9. 14, 21. and 28 pci). A: In naive mice, there were no signiricant clifferences in serum IgE detected between the dietary groups (F,,,=O. 89, P=O.43). During primary infection. semm IgE was not affected by diet overaii (F,=2.43, Pd.094) but was signüicuitly higher on day 28 ppi compmd to day 6 ppi (F,,=254.5, P=0.0001). During challenge infection. serum IgE was signülcantly different arnong ail three diets (F,,,=44.3, W.0 1) and signincantiy dtered over t h e (F,,,,=17.67, P=0.0001). There were no signincant interactions of diet and t h e in serum IgE during primiuy or challenge Uifection. B: Parasite-specific IgG 1 concentration wu no t signincantiy affected by diet overd in naive rnice, primvy groups or challenge gro ups. Tirne significantiy afTected parasite-specinc IgG 1 levels among die tvy groups d u ~ g primary infection (F,,8,=583.18, P4.000 1) and during challenge infection (F5,,,=150.62, P=0.0001). For IgE, different letters represent signincant merence between dktq groups (Pd .05) based on post-hoc painvise cornparisons (Tukey) perfbrmed following signincant effect of diet in one-way ANOVAs at each time point.

Parasite-specinc IgGt responses were also measured in the different infection and

dietary groups (Figure 3B). Sirniliir to IgE responses. parasite-specific IgGl was not

detected in naive mice or evly during primary section but w u signincuitly elevated after

challenge infection. However, protein deficiency did not impair the synthesis of parasite-

specific IgGI at any tirne during the prirnary or challenge infection period. In ail dietary

groups, parasite-specific IgGl rose rapidly evly after chaUenge infection and peaked by day

9 pci (tirne effect. P<0.05) to levels greater thm that observed during primvy Uifection.

Higher parasite-specinc IgG 1 levels persisted in aii dietary groups after 4 weeks pci despite

the near absence of worms in the rnice fed 24% or 7% diets.

The patterns of peripheral eosinophil responses were deterrnined duMg p r i m ~ y and

challenge infection with H. polygyrus (Figure 4). Pro tein deficiency signincantly Io wered

the numbers of peripheral eosinophils throughout the primary infection (Figure 4A).

Although eosinophil counts appeued to be higher in the 24% mice compared to the 3%

mice on day 5 pci, the overd effect of diet on peripheral eosinophilia throughout challenge

infection was no t signincant (Figure 4B). At day O pci (on which the challenge mife

received theû second infection and the p r i m q mice received their fïrst infection), blood

eosinophil counts in previously immunized mice of the challenge group were higher than in

mice of the primary group. indicating the more intense secondary response characteristic of

acquired irnmunity. In the f h t 2 days afier challenge infection. circuiating eosinophil

numbers in t d vein blood decreased in di dietary groups. suggesting a preferenthi

recniitment of blood eosinophils to the parasite-infected intestine. This was followed, in

mice fed 24% or 7% protein. with a shvp increase in blood eosinophh during the fbst

week of infection which was double die eosinophilüi observed in prirnary mice fed either

diets. Blood eosinophil responses declined rapidly after day 5 pci in the chaknged mice

fed 24% or 7% protein possibly because of the decreasing number of w o m in the gut. In

contrast. elevated blood eosinophilia persisted in the protein sufficient mice throughout the

primary infection.

B Challenge Infection

Figure 4

2 5 13

Days Post-Infection

Figun 4. EfTects of dietary protein deficiency on eosinophüÿt in tail vein whoie blood during both primary (A) and challenge infection (B) with H. po lygyw . Values are î SE. A: During primary infec tien, perip henl eosinophili;i was signitfcuitly aEected by die t (F,=8.7 1, P = W û 1) and tirne (F6,,,4=47.99, W.000 1). B: During challenge infection, peripheral eosinophdia was significantiy aected by rime oniy (F,,,,,=6.87. Pd.000 1 ). There were no significmt interactions of diet and t h e during primtuy or challenge infection.

Intestinal Effector Responses

We counted the number of MMC and intestinai eosinophils in sections of intestinal

tissues obtained from nive rnice, primary mice. and chaIIenged rnice (Figure 5). In aii

challengecl mice. the numbers of intestinal MMC increased 5-fold by day 3 pci compared

with the uninfected levels and were consistently higher at ai l pci t h e points cornpared with

early p h v y mice (Figure 5A). Overali, the 3% protein diet significantly impaired MMC

proliferation during challenge infection but no t during primary infection. High numbers of

MMC in chailenged mice fed 24% or 7% protein die6 persisted throughout the infection

period (tirne effect. PsO.05) but dropped in the 396 group after day 9 pci (time effect,

PeO.05). Therefore, significant dserences in MMC numbers between 245 and 3% groups

were detected on day 9 pci and successively thereafier. Numbers of intestinal MMC were

lower in the 7% protein diet compared with the 24% group ody on day 9 pci and at other

pci time points were either sunilar to 244 mice or intermediate between the 24% and 3%

groups. Hence, the marginai protein mice maintained intermedite but adequate leveis of

MMC d u ~ g challenge Section. MMC counts were not statisticdly different among the

dietary groups in d e c t e d mice or duMg prirnary infection.

Proiein de ficienc y also significuitly prevented the increase of intestinal eosino phils

during challenge infection but did not af5ect numbers in naive or primary mice (Figure SB).

In spite of the fluctuation in the numbers of intestinal eosinophils after challenge inlection.

they showed m overall dose-response to the dietary protein leveL Compared to the

uwected level. a doubiing of intestinal eosinophil numbers was apparent within 2 weeks

pci only in mice fed 24% protein. Marked increases in intestinai eosinophilia were not

obserwd in the chaîienged rnice fed 7% or 3% protein compared to their naive

counterpans, and therefore. gut eosinophils fded to prouerate after challenge infection in

these protein restricted groups. Due to the considenble variability in counts obtained at

each pci time point (e-g. dûys 3 and 9 pci), intestinai eosinophil bels did not change

signincantiy over tirne in any of the dietary groups (ticne effect, A0.05). It is important to

note that high numbers of gut eosinophiis were found in 24% mice on day 3 pci despite Iow

numbers of circulating eosinoplds obsemed very eariy afier challenge infection (Figure 4).

Naive

Naive P6 P28 C3 C6 Cl4 C28

Infection Groups Figure 5

Figure 5. Effects of dietvy protein deficiency and t h e on the numbers of MMC per 20 vcu (A), numbers of intestinal eosinophils per vcu (B), and the secretory capacity of MMC as measured by MMCP- 1 concentration (C) in naive mice, p M i q mice (6 and 28 days ppi), and challenge mice (3.6.9, 14.21 and 28 days pci). Values are * SE. A: In naive mice, the numbers of MMC were not signiticantly atrected by diet. During primary infection. the numbers of MMC were not significantly different among dietary groups but were signincmrly altered over the (F,e7O. 19, P=0.000 1). DuMg challenge infection, the nuben of MMC were signüifantly lower in 3% mice compared to 24% and 7% rnice (F,,=21.69, Ps0.0001) and signincantly affected by tirne (F,,,=4.09, P d . 0 0 3 ) . B: Numbers of intestinal eosinophiis were no t significantly affected by diet overail in naive or prirnary rnice. During primary infection, intestinai eosinophih was signincantly highet on day 28 ppi compared to day 6 ppi (F,,=7.02, P4.014). During challenge infection, the numbers of intestinal eosinophüs were signincantly dinerent among di dietary groups (F,,=29.6 1, P=ûAlûû 1) and signincuitly different over time (F,,=3.67, P=0.0059). C: S e m MMCP-I concentration was not significantly affected by diet in naive mice. During primary infection. semm MMCP-1 levels were not significantiy affected by diet or time but there was a signincant diet and time interaction (F,,=3.84, P4.028). During challenge infection, MMCP-1 concentration was not significmtly aected by diet overall but w u significantly affected by t h e (F,,,=44.59, P=O.ûûû 1). For MMC, gut eosinophils and MMCP-1, the different ietters represent signincuit difference between dietary groups (P4.05) based on post-hoc pairwise cornparisons (Tukey) foIiowing significant effect of diet in one-way ANOVAs at each t h e point.

To determine the secretory capacity of MMC duMg nematode infection in mice

with v-g degrees of protein deficiency, we measured the concentration of s e m

MMCP-1, a protease expressed predominantly in intestinal MMC (Figure SC). Protein

deficiency did not alter MMCP-1 ievek in uninfected rnice, prirnary mice or in challenged

mice. Although there were no overd diet effects during chailenge infection, we found that

protein deficiency significantly inhibited the increase of MMCP- 1 secretion at days 6 and 14

pci ( P d . 0 5 ) . The time course of MMCP- 1 response parallels that of semm IgE such that

MMCP-1 concentration in the protein suficient mice were higher after challenge infection

compared with unllifected or prirnary mice. In ail dietary groups, peak MMCP-1

concentration occurred on day 14 pci (tirne effect, P<0.05) which occurred &ter peak

MMC numbers in the gut mucosa (in 244 mice on day 9 pci) and peak bels of serum IgE

and parasite-specinc IgGl (day 9 pci).

Systemic and Local Cytokine Responses tu Parasite Antigen in vitro

To determine whether the impaired effector responses observed in the protein

deficient mice were reiated to diet-induced perturbations in cytokine production in local

a d o r systemic lymphoid tissues, we measured post-challenge Th2 cytokine (a-4, IL-5,

IL- IO) and Th 1 cytokine (IM-y) production by spleen and MLN ceik restimulated with

parasite antigen in vitro mgure 6). In general, Th2 cytokine secretion was higher in the

fnst week of primuy infection compared with the challenge infection (with the exception of

IL-5 production in spleen), indicating a strong priming of CD4* c e h to secrete Th2-type

cytokines. Also, the Th2 cytokine proNe durhg challenge infection was similu among the

3 dietuy groups in that the response was greater in MW cells than in spleen cells, peaked

by one week pci, and declined npidly thereder. Effecu of protein deficiency on Th2

cytokine production were detected only in IL,-4 secretion by MLN cells early after

challenge infection @gure 6A & 6B). The MLN ceik of the 7% protein group secreted

significantly Iess IL-4 at day 3 pci compared to their 2446 protein counterparts. S W l y at

day 6 pci, IL-4 production was significantly lower in MLN of mice fed 3% protein

compmd to mice fed 24% protein We also observed differences in mucosal IL-4

SPLEEN MLN

Figure 6 Infection Groups

Figure 6. Effects of dietary protein deficiency on Th2 cytokine (IL-4. IL-5. IL-10) and Th1 cytokine (EN-y) production in spleen (A, C. E. G) or MLN (B. D, F, H) ceUs in mice with either primary or challenge infection of H. polgynrs. Values are I SE. Spleen and MLN ceh from P6. C3, C6, Cl4 and C28 mice were cuitured, restimuhted with parasite antigen in vitro, and assayed for cytokine secretion using sandwich ELISA Values are mean nglml I SE. A and B: At day 6 ppi, IL-4 production was signincantly lower in spleen 01 3% mice compared to 24% mice (F,,=3.87, P=0.03 1) but not in MLN. During chdknge infection, IL-4 production in spleen was signincantly affected by tirne only (F, ,=6.55, P4.0004) and in MLN was signincuitly &ted by both diet overdi (~;,=6.08, W . 0 0 3 3 ) and tirne CF,,=14.06, P=0.0001). C and D: IL-5 secretion by spleen or MLN cells was not signiticantly dinerent among dietary groups at day 6 ppi and during c W n g e infection. Time signincantly aEected IL-5 production oniy in MLN ceiis duMg chdenge infection (F3,,,,=16.74, Pd.0001). E and F: IL-IO production by spleen or MLN ceUs wu not significantly affected by diet overd at day 6 ppi and throughout the challenge infec tioa During challenge infection. tirne significantly affected IL- 10 production in spleen (F, p l 3.4 1, Pa000 1) and in M W (F3,8,=9.97, P=û.0001). G and H: IFN- y production kas not signincantly atrected by diet overall at day 6 ppi or throughout challenge infection. During chailenge infection, time significantly affected IFN- y production in spleen (F3,,,=35.06. P=0.0001) and in MLN (F,,,=30.89. P=0.0001). For a i l cytokines produced in spleen or MLN, different letters represent significant differences between dietary groups (PcO.05) based on post-hoc p M e cornparison (Tukey) following significant enect of diet in one-way ANOVAs at each post-challenge time point.

production between the dietary groups at a t h e immediately prior to challenge section

(basal challenge levels). In these primed mice. the MLN cells of the 2 4 4 group produced

more IL4 (8.66 ng/d * 0.71. n=2) compared to 3% group (3.98 nglml I 0.96, n=2) while

the MLN of mice fed 7% pro tein secreted an intermediate level (5.16 nglml* 2.43). The

7% mice were capable of increasing IL-4 production by 6 days pc5, suggesting a capacity to

up-regdate IL-4 production at a post-infection tirne critical for maximal stimulation of

effector responses. By cornparison, the decreased production of IL-4 in MLN cells of m k e

fed 3% protein preceded the impairment of intestinal and systemic Th2 effector responses.

As shown on Fgure 6C to 6F, the protiles of IL5 and IL40 production in spleen and

MLN were s h h r to IL-4 responses but. unlike IL-4, were not signifïcantly affected by

protein deficiency overaii duMg chaüenge infection. Despite an overail nonsignifïcant

effect of diet, we found that L-5 secretion in MLN was higher in mice fed 3 4 protein

compared with either 24% or 7% groups at day 3 pci but this was not associated with

m k e d elevation of gut eosinophils.

As shown in Figure 6G & 6H. IFN-y secretion by both spleen and MLN ceh

restimuhted with parasite antigen in vitro increued signifïcuidy throughout the challenge

infection in a.li d i e t q groups (tirne effect, P d . 0 5 ) . except in spleens of mice fed 3%

protein (tirne effect, P>0.05) This higher IFN-y response in spleen and MLN over time

coincided with decreasing ieveis of IL-4 and IL10 in MLN and increasing worm loss in the

protein sficient mice. We did not detect an overall significant effect of diet on IFN-y

production during challenge infection period. However, at day 14 pci, mice fed the 3 2

protein secreted significantly more IFN-y compared to rnice fed 24% protein in spleen

(P=0.0003) and in M'LN ( P 4 . 0 2 3 ) . Taken together, protein deficiency did not alter the

inherent capacity of spleen or MLN ce& to synthesize ?FN-y but increased IFN-y

production at the onset of adult worm expulsion (day 14 pci) in a c h a n g e infection.

Systemic and Lucal Cytokine m W A Expression

To address the question of whether altered IL-4 and IFN-y profiles in protein

deficient mice resulted from diet-induced changes in cytokine mRNA expression, we

analyreci be l s of cytokine mRNA using semi-quantitative RT-PCR (Figure 7). The spleen

and MLN ceh of mice fed either 24% or 3% protein (n=3) were restimdated with parasite

antigen in vitro and then assayed for E-4, IL-5, IL10 and IFN-y mRNA In mice fed 3%

protein. IL4 mRNA expression in spleen and MLN cek fded to increase and remained

low throughout the chalienge infection (Figure 7A & 78). The dinerences between dietary

groups were statisticdy signincant on d g 14 pci in spleen celis and on days 6 and 14 pci in

MLN ce&. Also, IL40 mRNA expression in spleen and MLN was significantly lower in

mice fed 3% protein compared with their 24% protein counterpûru at day 14 pci (Figure

7E & 7F). The ievels of IL-5 (Figure 7C & 7D) and IFN-y (Figure 7G & 7H) rnRNA

expression were not significuitly affected by protein deficiency at any t h e point during

c hdenge infection.

Figure 7 Effecu of dietary protein deficiency and tirne on Th2 cytokine mRNA (IL-4, IL- 5, IL-10) a d ni1 cytokine mRNA (IFN-y) expression in spleen (A,C. E, G) or MLN (B, D. F, H) ceh in mice with challenge infection of H. p o l y g y m . Spleen and MLN cells from C3, C6. Cl4 and C28 mice were cultured and restimulated with parasite antigen in vitro. Cells fiom the supematants were lysed for total mRNA and mRNA expression of each cytokine was determined with semi-quantitative RT-PCR Values are expressed as the ratio of cytokine mRNA to HPRT mRNA for individual simples (mean î SE, n=3). A and B: IL4 mRNA levels in spleen and MLN were signincantly affected by diet (F,,=12.28. P=û.ûû 15) and the (F,,3,=6.43. P4.M) 17). C and D: No significant dinerences in IL-5 mRNA expression in spleen and MW were detected between the dietary groups or time points. E and F: IL,- 10 rnRNA leveis in spleen and MLN were signtncantly aEected by diet (F ,,,, =6.64, Pr0.015) and tirne (F,,p17.18, P4.0001). G and H: IFNy mRNA expression in spleen and MLN was no t significantly affected by diet or time. For ail cytokine mRNA levek in spleen or MLN, dserent letters represent signincant dinerences between dietvy groups (P4.05) based on post-hoc pYwise cornparisons (Tukey) foiiowing signincant effect of diet in one-way ANOVAs at each tirne point.

DISCUSSION

This study is the fist comprehensive investigation on the effects of dietary protein

deficiency on the magnitude and kinetics of Th2 immune responses in mice to a challenge

infection with a gastrointestinal nematode parasite, Heligmosomoides p o l y g y w . Contrary

to conventional belief, dietriry protein deficiency did not suppress aU immune processes but

instead exened dinerential site- and type-specific effects on cytokine and effector responses

to H. polygym infection To iuustrate this point, four important hdings are noted: 1)

Protein deficiency impaired the proliferation of gut eosinophils and mucosal mast cells

(MMC) but only duMg the evly phase of infection; 2) Pro tein deficiency decreased the

levels of circulating IgE but not of parasite-specifc IgGI; 3) Protein deficiency down-

regulated IL-4 production only in a gut Iymphoid tissue and did not ûffect secondary

responses of other Th2 cytokines; and 4) Protein deficiency up-reguhted levek of IFN-y at

2 weeks pci which is a cnticai period for expression of acquired immunity. Additiody,

the Ievel of d i e t q protein influenced the degree of protective immunity and parasite

s u ~ v a l duMg a challenge infection with H. polygynrs. Our results support the hypothesis

that protein deficiency prolongs sunival of a gastrointestinal nematode parasite by

decresing gut-associated IL4 (TM) and increuing IFN-y (Thi) early in the infection,

leading to reduced intestinal and sysremic Th2 enector responses.

Acquired immunity to secondary infections with H. polygyms is assochted with

strong and persistent Th2 effector responses (Monroy & Enriquez, 1992), although the

precise mechuiisms by which these Th2 effectors exen their protective effect have not ken

ciearly elucidated. Eosinophiiia is a typicai Th2 infiamatory response observed during

nematode parasitic infections but some researchers have suggested that host resistance to

H. polygyrus or Trichinella spirulis infections in mice is not dependant on eosiaophilis

Prevention of e o s i n o p h induced by the neutraüzation of IL4 antibody or deletion of the

IL-5 gene did not afliect worm survival or fecundity (Urban et al.. 199 1; Herndon & Kayes,

1992; Takamoto et aL, 1997). However, these authors measured eosiwphiiia in circulating

blood, an indicator bat does not necessvily refiect eosinophil responses in intesiinal sites of

Section which are more relevant to host immunity against enieric parasites (e.g. Svetic et

ai.. 1993; Negno-Coma et al., 1996). An example of the svong bias towards gut mucosal

localization of eosinophils during nematode infection is our hding of a transient drop in

blood eosinophils at day 2 pci that coincided with elevated numbers of intestinal eosinophils

in mice fed 24% protein. We interpret this pattern as evidence that eosinophüs are

trmported preferentiaiiy to the infec ted intestine hmedkely folIo wing c haiienge infection

until gut-associated lyrnphoid tissues are capable of producing high quantities of local

eosinophils. Similx to this reputed migrational pattern of eosinophils during polygy~us

challenge infection, others have observed that the uptake of IgE by intestinal ceUs is greatly

increased within 24 hours after T. spiralis Uifection (Rarnaswamy et al., 1994) and that the

vast rnajority of intestinal IgE (>99%) detected by 4 days post-infection is produced locdy

in the gut (Negrao-Correa et al.. 1996). These results suggest that high levels of immune

effectors in the gut are hinctiondy more important than peripherai quantities during

intestinal nematodiasis. Thus, Our fkding that intestinal but not peripherd eosinophilio was

inhibited by protein deficiency during challenge infection may be one factor contnbuting to

the prolonged worm s u ~ v a l in the protein deficient mice. However, the roie of

eosinophils in pro tective immunity to helminth infections has been chaUenged by studies

showing that murine eosinophils, unWte human eosinophüs, hck IgE receptors (the high-

S i t y FcdU and the low-afnnity FceRWCD23) but do express IgG receptors (de Andres

et al., 1997). Accordingly, the cross-Mage of IgG receptoa by pansite antigen may be

the main activation pathway of eosinophil degmuiation and oxidative burst. Thus, the

functional significance of reduced gut eosinophilia in mice fed 3% protein to parasite

expulsion remains uncenain because the synthesis of puasite-specinc IgGl (the IgG isotype

associated with Th2 responses to nematodes) after chdenge infection with H. p o l y g y m

was no t impaired by pro tein dekiency.

This lack of responsiveness of parasite-specifi~ IgGl to the detrimental effecu of

protein deficiency during primary or challenge infection with H. polygyrus corresponds

with previous results from Our laboratory (Bouhy et aL, 1998) and rnay be explained by

literature suggesting that the regdation of IgGl response does not require IL-4. Eatly

studies demonstmted that the synthesis and expansion of IgG1-secreting B cells is

dependent on CD4+ T cells and that IL-4 promo tes isotype switching of IgM+ B ce& to

membrane expression and antibody secretion of IgGl (Vitetta et al.. 1985; Coffmui et al..

1986). However. hter animai studies showed that IgGl responses can be induced duMg

H. polygynrs or Nippos~ongylus brasiliensis infections even in the absence of IL-4 activity

in vivo and the disruption of the murine IL-4 gene (FYike1rna.n et al.. 1988; Katona et al.,

1991; Kopf et al.. 1993). Furthemore. treatment with exogenous IL-4 increased the

transport and uptake of IgE but not of IgG1 into the intestines of T. spiralis-infected rats

(Ramaswany et al., 1994). Therefore, any disturbances in IL-4 production caused by

protein mainutrition as observed in Our study may not necessdy lead to subsequent

changes in IgGl responses during H. polygyms Section. Also, the observation that

protein deficiency signincantiy prolonged parasite s u ~ v d without impairing concomitant

IgGl responses indicate that we should reûppraise the immunologie d e of IgGl in

protective immunity to nematode parasites. Pritchard et ai. (1983) reported that purified

IgGl from immune sera of mice infected with H. polygyrus caused severe stunting of

woms and promoted adherence of pentoneal exudate celis to the surface of immature and

adult woms in vino. However. Wahid et aL (1993) later showed that parasite-specific

IgGl responses did not correlate with the rate of wonn expulsion and were rnargindy

elevated in mice with heavier worm burdens. Moreover, the inhibition of IL4 and its

receptor by neutraiizing antibodies prolonged worm survivaî in primary H. poiygyrus

infection without concunent impairment of IgG 1 responses (Finkelman et al.. 1988;

Katona et al., 199 1). The undtered parasite-specific IgGl levels observed in the heavüy

pivasitized protein deficient mice fûnher support the contention that IgGl may contribute

iess to host protection a g d t intestinal parasites than other Th2 effectoa in a c W n g e

infection.

MMC hyperpiasia is another prominent feature of Th2 effector responses to

intestinal helrninths including T. spiralis and N. brasiliensis (Woodbury et ai., 1984;

Dehlawi et al., 1987). The presence of adult polygyrur in the intestinal lumen is

associated with poor mastocytosis, chronic parasite s u ~ v a l , and inhibition of MMC

responses stimuhted by concomitant T. spirulis infections, indicating that adult H.

p o l y g y w exerts an immunosuppressive effect on MMC development and proliferation

(Dehlawi & Wakelin. 1988; Behnke et al., 1993). Moreover, spontaneous expulsion of H.

polygyrus in some resistmt strains of mice and elimination of N. brasiliensis in nu were

not accompanied by concurrent mucosal mastocytosis which had peaked afier maximal

worm loss (Woodbury et al., 1985; Dehhwi et al*, 1988; Wahid et al., 1994). However,

increased size and accelerated development of MMC responses have been observed during

challenge infections with FL polygyrus in which there was &O a reduction in the number of

penetnting b a e and expedited elhination of adult w o m (Dehhwi et al., 1987). thus

implying that MMC may contribute to acquired resistance by arresting h a 1 development

or darnaging young adult worms following b e n a l emergence. Indeed, we found that a

challenge infection with If. polygyms induced prominent and persistent MMC responses

that peaked (day 9 pci) prior to mvked eltnination of adult parasites (beginning on day 14

pci). The concept that secondq MMC responses are a critical mechanism of acquired

resistance is supported by Our finding that protein deficiency irnpûired the intestinai

prolûeration of MMC within 2 weeks pci but did not affect MMC numbers during prirnvy

Uifection in which more w o m were recovered thm after chdienge infection Funhermore.

the onset of depressed mucosai mastoc~tosis immediately preceded the loss of adult w o m

in the pro tein sutficient mice, thereby implying that MMC responses were elicited by larvae

tesident in the gut mucosa and that local MMC induced early after chdienge infection

directly phcipated in pansite expulsion in conjunction with IgE or IgG1.

IgE response is an essential component of acquired immunity to nematode pansites

and any increases in this antibody represent an induced response in helminth infections. We

found that serum IgE levels were low in uninfected mice and duMg primary infection but

hcreased substmtially after challenge infection- However, this nematode-induced eievation

in IgE was absent in the protein deficient mice which ais0 had lower MMC levels in the gut

mucosa. nie importance of IgE in mediating protective immunity to helminth parasites is

infened from studies showing that the neutraiization of IL-4 in vivo or inhibition of IL-4

receptor ameliorated semm IgE responses and increased worm numbers (Fieman et a l ,

1990; Urban et al.. 199 1). Moreover, mice with a mutation of the IgE gene have increased

burdens of Schistosoma monroni despite prominent elevations of antigen-specific IL-4-

secreting ceils (King et al., 1997). Binding of IgE to high atfinty receptors on mast ceh

and cross-linking by parasite antigen cause the degranulation of MMC and reiease of

inflanmatory mediators. Accordingly. an effective IgE-medhted immune response requires

not only sufficient Ieveis of MMC md associated IgE receptors but also abundant IgE

antibody at the appropriate sites of infection in order to enhance m a t ceil FceRI expression

and secretory capacity (Chen et al., 1994; Yamaguchi et ni., 1997). In our study, the t h e

courses of serum IgE response, MMC proliferation and parasite expulsion were tightly

correlated such that the onset of parasite expulsion in the protein sufficient mice (day 14

pci) occurred immediately following pedc serum IgE and intestinal MMC numbers (day 9

pni. Funhermore, the protein deficient mice h d higher w o n burdens throughout 4 weeks

of challenge infection as wel as consistently decreased serum IgE titres and lower numbers

of MMC in the gut mucosa Taken together, these results provide additionai evidence that

protective immunity to nematode parasites requires maximal induction of IgE production

and concurrent proliferation of MMC in situ or intestinai trafkking of mast ceii precursors.

Aithough semm IgE concentration was found to correlate poorly with intestinal IgE

ievels (Negrao-Coma et al., 1996) and with IgE occupancy or density of IgE receptors on

mast cells (Chen & Enerback, 1994 & 1996). Our results on the secretory capacity of MMC

iikely implicate a functional consequerice of lower serum IgE bels detected in the protein

deficient mice. Inasmuch as MMC activation md degranulation depend on expression of

IgE receptors and acquisition of IgE by the mast cell. the lower MMCP- 1 concentrations in

protein deficient mice at days 6 and 14 pci may be due to both decreased MMC numbers

and reduced levels of IgE in gut-associated lymphoid tissues. Lower serurn MMCP-I

levels likely represent the true extent of the immunosuppressive efEects of protein deficiency

on MMC secretory capacity given that the intestine is the major source of systernicdIy

secreted MMCP-1 and that semm levels correlate positively with concentrations in the

intestinal lumen of nematode-infected mice (Wastling et al.. 1997). There are &O

preliminary indications that MMCP-1 has a physiological role in puasite expulsion by

affecthg epithelial permeabüity and translocation of antibodies from blood to the gut lumen

(Wastling et a l , 1997). Lower IgE responses, depressed MMC numbers and impaired

MMC secretory activity were apparent by day 9 pci in mice fed 346 protein at a t h e point

that preceded the onset of marked parasite expulsion in immunocompetent mice fed 24%

protein. Although we did not measure MMCP-1 on day 9 pci, other studies have shown

hat MMC protease concentration in blood peaks slightly iater than maximal intestinal

mastocytosis in nematode-parasitized mice and rats (Woodbury et al., 1984; Wastiing et

aL, 1997). Thus, prolonged w o n survivd in rnice fed 3% protein may be attributed in part

to the impaired Ig E-dependent MMC pro Werative and secre tory responses O bserved evly

after challenge infection with H. polygynrs.

We investigated whether these impaired enector responses in protein deficient rnice

were reLzted to perturbations in Th2 cytokine production. It is currently accepted that IL-

4 is the critical cytokine directing ni2 differentiation and respowe phenotype d u ~ g

nematode iiifections (Mosmann & Sad, 1996). IL-4 promotes the dinerentiation of Th ceh

to the Th2 phenotype (Rincon et al., 1997). the generation of memory Th2 and B cells

(Bradley et ai.. 1995). isotype switching of B cells from IgA+ and IgW to I g F (Zhang et

al., 1991), the proliferation of IgE-comrnitted cells and differenthted MMC (Fïnkleman et

al., 1988; Katona et al., 1991), mast ceii FceRI expression (Tom et al., 1996). binding of

plasma IgE to intestinal ce& of T. spirulis-infected mice (Ranaswany et al.. 1994). and the

expulsion of established H. p o l y g y w in mice (Urban et ai.. 199 1). Our kdings b a t

protein deficiency decreased the capacity of MLN celis of H. polygynrs-infected mice to

secrete IL,-4 and to express IL-4 mRNA, depressed Th2 effector responses, and prolonged

parasite s u ~ v a l support previous reports (e.g. Urban et al.. 1991 & 1995) that IL-4 is a

crucial cytokine required for strong and persistent Th2 effector responses and npid

elhination of nematode parasites. In contrast to IL-4. protein deficiency did not impair IL-

S and IL10 production even though the 3% protein diet reduced IL40 mRNA ievels.

(Yamaguchi et al.. 1988; Coffman et al.. 1989; Behnke et al., 1993). This indicaies that

relaiively smdi amounts of IL-4 may be suEcknt for production of other Th2 cytokines

and that protein deficiency may a e c t gene expression without irnpairing translation of

mRNA to protein. In fact, IL-5 secretion by MLN was relatively higher in mice fed 3%

protein at day 3 pci, but the functiond signif~cance of this event is unclear because the up-

regulated IL5 response at this early tirne point was not associated with concomitant

elevation in gut eosinophilia, It is possible that IL-5 and IL- 10 are constitutively expressed

during nemat ode infection and their regdation is less responsive to exogenous factors.

Other researchers have questioned the hnctional impact of IL-5 and IL40 during

nematode infection The expression of E-10 mRNA does not correlate with the mRNA

levels of other Th2 cytokines in H. polygyms Mection (Svetic et al.. 1993) and IL12 cm

enhance the production of IL-10 by humm T ce& (Widhagen et al.. 1996). thus implying

that IL- LO may no t be a s tric tly Th2-resuic ted cytokine. h o , IL- IO gene expression is

stirnulated by IL42 trertment in mice during brusiliensis infection in which there was

l o prolonged w o m survival and reduced IgE, MMC and eosinophil responses (Fihicehan

et al., 1994). SimilarIy, Th2-type mechanisms of resistance to nematode parasites rnay be

independent of I L 4 as shown in experiments in which the neutraiization of endogenous IL-

5 fyled to S e c t parasite loads in T. spiral& H. polygynrs or brailiensis Sections even

though anti-IL4 antiiody prevented eosinophüia in blood and lung mucosa (Herndon &

Kayes, 1992; F i e m a n et al.. 1997). This selective impact of dietary protein deficiency on

host immune function has also been reported in studies on cytokine production m murine

tuberculosis (Chan et al.. 1996) and on neutrophü mobïiization during staphyIococcal

infection (Nwankwo et al., 1985). Taken together, the dinerential effects on ni2 cytokines

suggest protein deficiency impairs specinc components of Th2 cytokine response (a-4

production in intesthi lymphoid tissues) that are important for develo pment of acquired

immunity to H. polygyrus.

The significant effect of t h e on Th2 cytokine response profiles suggests that

kinetics of cytokine production during chdenge infection are important to the regulation of

host protection. Cytokines are likely most effective in promoting effector responses during

the first week of infection when H. polygynrs is in its tissue-dwehg b a l stage which is

h o wn to elicit the most intense expression of acquired immunity in mice (Wahid &

Behnke, 1992). Indeed. we found that Th2 cytokine concentrations in MW were higher

than in spleen and bat levels graduaily decline after 14 days pci Consistent with these

tirne-dependent effects of protein deficiency, Chan et al., (1996) detected depressed

cytokine mRNA expression in lungs of protein deficient mice only at early tirnes (cl4 days)

afier tuberculosis infection. In our study, the early intense response in local lymphoid tissue

refkcts the strong antigenic stimuli provided by the invasive and tissue-resideni I;trvae.

Once the worm matures and enters the pt lumen, there is much less antigenic stimulation

to gut-assochted lymphoid ceils for cytokine synthesis. Also, the presence of adult worms

in the anterior duodenum has been associated with suppression of mucosal mastocytosis

(Dehwi & Wakeh, 1988) and selective down-reguktion of IL-9 and IL-10 production

(Behnke et al., 1993), leding to the conclusion thot adult parasites are capable of

compromishg host immune responses. Taken together, these results suggest that acquired

mistance to H. po lygym requires elevated Th2 cytokine production in intestinal lymphoid

tissues within one week pci before adult parasites emerge into the gut lumen. During

challenge mfection with polygyrics, protein deficiency modified IL4 production by MLN

only during the first week pci in a selective manner depending on the seventy of restriction.

The MLN ceUs of marginal protein mice were able to rebound nom initiai impaired I L 4

synthesis (day 3 pci) whereas the MLN ce& of the protein deficient mice were unable to

secrete sufficient quantities of IL-4 at a slightly hter t h e point (day 6 pci). Tbis hter time

point rnay have been critical for stimuhtion of subsequent Th2 effector responses because

peak serum IgE and MMC responses occurred on day 9 pci Moreover, we found that

basal IL4 production by MLN of mice fed 24% protein was high prior to challenge

infection and remained elevated during the first week pci but that IL-4 secretion in MLN of

mice fed 3% protein was low at baseline and had f i d to increase after challenge infection.

Therefore, susthed elevations in IL-4 production in the early phase of challenge infection

rnay be equally or more important for priming of effector rnechuiisrns thui transient

increases at any specific pci tirne point.

It is imponant to note that Th2 cytokine production by spleen and MLN w u

prominent early after primvy infection as suniluly reported by Svetic et al. (1993) and Shi

et al. (1997). However, this intense primary cytokine response was no t associated with

subsequent mvked increases in IgE, eosinophila or mucosal mastocytosis which were

significantly higher afier chdenge infection. Secondary IL-4 and L- 10 cytokine responses

to H. polygym were relatively low CO mpared to primuy levels and elevated levels were

limited to the first week of chailenge infection. It is possibie that the priming or initiation of

Th2 cytokine synthesis rnay be a more important immunological event dian secondary

responses for the developrnent of B ceii or Th2 memory and phenotypic expression.

However, IL-4 produced during chaenge but not primary infection with H. polygynrr is

required to generate and sustain secondary IgE responses which denve mostly from naive

or uncomrnitted B ce& rather than from IgE-committed memory B cells produced during

prirnary infection (Fiinkelrnan et ai., 1990; Katona et ai.. 1991). Therefore, the impaired

production of gut-associated IL4 in protein deficient mice observed early in challenge

infection rnay be the determinhg factor responsible for the subsequent impaired IgE, MMC

and intesbd eosino p hi1 responses, and in tum for the prolonged wonn s u ~ v a i

The reciprocal amounts of IL-4 and IFN-y produced duMg challenge infection may

be an additional factor affecthg the magnitude of Th2 Unmunity raised against nematode

parasites. IL-4 and IFN-y are mutuaily antagonistic for Th ceii differentiation and effector

activity belonging to reciprocal Th ceil phenotypes. IL-4 inhibits cytokine spthesis by Th1

cek and impairs macrophage function (Kopf et al., 1993; Tan& et al., 1993; Lagoo et

al., 1994) whereas IFN-y suppresses the production of Th2 effectors and cytokine and

reverses host resistance to nematode parasites (Maggi et al., 1992; King et al., 1993; Urban

et ai.. 1993). Collectively, these data indicate that a high level of IL.-4 and the concomitant

Iow quantity of IFN-y ultimately influence the appropriateness. speed and magnitude of

Th2 immune responses raised against nemtode parasites. We found that protein deficiency

decreased gut-associated IL4 response (protein level and mRNA expression) and increased

both gut-assochted and systemic IFN-y production (protein level on day 14 pci). Although

we did not detect effects of diet on IFN-y gene expression. it is possible that protein

defkiency aitered other steps in the IFN-y synthesis pathway such as mRNA tnnsiation or

post-translational modification. The up-reguhted IM-y production duMg chaîienge

infection coincided with decreasing K-4 leveis in MLN and thus may be a by-product of

decreasing antigenic stimulation of Thtassochted responses within gut mucosal tissues.

Despite an overall nonsignincant effec t of diet on IFN-y throughout challenge Section. we

detected signincmtiy higher IFN-y production in spleen and MLN of mice fed 3% protein

compared to controk at day 14 pci During the initial 2 weeks pci, a criticai the for

developrnent of appropriate and sufficient Thî responses, the increased IFN-y coupled

with decreased IL-4 rnay have contnbuted to the lower Th2 enector responses in the

protein deficient mice. The mech;uiisrns by which protein deficiency could up-regdate

nonpro tective IFN-y and simultaneously dom-regulate protective IL4 are unknown but

these results suggest that malnutrition aiters the balance of cytokine responses that

r e m t e IgE-dependent effector mechanisms during challenge infection with H. polygyw.

The selective impact of protein malnutrition on host imrnunity was further

evidenced in the graded responses to varying levels of dietary proteh We found that 3%

proteh deficiency signincantly decreased body weight gain, rehtive spleen and MLN

weights and BUN concentration but h d no effect on piasrna aibumin concentration despite

a feeding period of 10 weeks. The absence of hypoalburninaemia in Our mice fed 7% or 3 1

protein is consistent with previous experiments in our labontory using similar diet and

infection protocols (Boulay et al., 1998) and with protein restriction in rabbits (Weidel et

of., 1994). Although biochemical indicators of pro tein deficiency typicdy have included

levels of transport proteins. urea nitrogen and hematocrit (Golden, 1982), plasma albumin

concentration has k e n regarded classically as an index of long-terni protein status

(McFuhe et al.. 1969). However, recent studies on pro tein deficiencies in pigs and

rabbits show that phma albumin concentration does not refiect albumin turnover nor

provide information on the mechanisms of how the phma albumin pool is maintained

(Jahoor et al., 1996). Several studies have reported that protein deficiency significmtly

reduces the fiactional rates of albumin synthesis and catabolism (Kirsch et al., 1968; Jahoor

et al., 1996), but the rate of dburnin turnover was sirniiar between control and protein

deficient animais (Weidel et al., 1994). These metabolic adaptations in aibumin turnover

rate m a y e x p h the unchanged plasma albumin concentration duMg protein deficiency

despite obvious impairment of growth and other physiological functions. Immune indices

are P sensitive parameter of nutritional status and help define the bc t iond consequences

of malnutrition by predicting morbidity and diseme outcornes (Chandra, 199 1). To this end,

Our results show ihat impaired immune fùnction occurs prior to detectable changes in

plasma aibumin concentration. Also, we cm suggest that the smakr body weight in the

protein deficient mice may have ennbled these mice to maintain plasma albumin homeostasis

at the expense of pro tein synthesis for other hinctionai capacities such as host

immunocompetence. Indeed, the suppressed Th2 immunity observed in H. polygytus-

inlected mice fed 3% protein confhned that we had created a functionaî protein deficiency

without concunent reductions in plasma albumin concentration.

Findy, this study is important because it suggests that a certain degree of

physiological adaptation occurs with marginal malnutrition but there is a threshold of

nutritional deficiency below which immune function is signincantly compromised. Mice fed

a marginal protein diet (78) were capable of eliminating rnost of their parasites by day 28

pci and showed intermediate but adequate b e l s of IgE, MMC nurnbers. and eosinophilia.

In contrast, a further decrease in dietary protein level caused obvious defects in host

protective immunity at both intestinal and peripheral b e l s which lead to increased parasite

s u ~ v a l . The74b protein group adopted to their marginal protein intake by eating more

food which resulted in these mice ingesting more energy than the control and deficient

groups. This was confmed by the higher plasma albumin concentration in the 7% mice

compared with the 3% group. Increased energy intake rnay spare endogenous protein for

utilkation in protein synthesis and allow nitrogen balaace to be achieved duMg Wied

protein intake (Waterlow, 1986). Indeed, mmy of the nutritional and irnmuno1ogical

outcornes of the 7% mice were not statisticdy different h m rhose of the 24% rnice,

suggesting that the higher energy consurnption compensated for the margind protein intake

and in tum help protect these 7% mice against reidection with H. polygynrs.

A comparison of Mmunological responses between the 7% and 3% protein groups

rnay heIp to clarify the signiacance of Th2 effector responses in protective imrnunity against

nematode parasites. Previous studies in which a single pumeter of Th2 defense

mechuiisms was inhibited by neuualipng antibodies (e.g. Urbm et al., 1991) or deletion o f

a cytokine-specific gene (e.g. Takmoto et d, 1997) were inconclusive as to whether Th2

eEectors such as IgE, MMC and eosinophüs were hinctionaiiy important in host resistance

to nematode parasites, but nised the possibility that there are some redundancies in host

immune hinction. Our study examining multiple parameters and tissue compuments of

host immunity suggests that Th2 effector and cytokine defenses act collectively andor

synergisticdy to promote acquired resistance to H. polygym. The rnarginaily resvicted

mice were capable of eliciting intermediate bels of Th2 effector responses b t were

apparently effective for rapid parasite expulsion In conuast, the 3% mice had lower gut-

associated IL-4, higher gut-associated and spkntc IFN-y. and consistently decreased IgE,

MMC and intesiind eosinophil responses during challenge infection which cumuiatively

may have resulted in theY higher parasite burdens.

In summary. Our study demonstrated that pro tein deficiency promo tes parasite

s u ~ v a l by dtering cytokine and effector responses in a seiective manner depending on the

specifc tissue cornpartment, the level of protein restriction and the time point of infection.

We found that protein deficiency w u more detrimental to IL-4 production in MLN than in

spleen during the early phase of a chdenge infection which m q have k e n responsible for

the subsequent decreased IgE, MMC and gut eosinophü responses. Based on Our results,

we suggest that protein deficiency exerts its adverse effecu at the following steps dong the

cascade of immunological responses to a Th2-induchg parasite. Adequate intake of

protein is necessary for mRNA expression and protein synthesis of TL-4 in GALT but does

not S e c t IL-5 or IL-10 protein production which appeared to be normal even in mice with

limited capacity to produce IL-4. This decreased IL-4 combined with increased IFN-y led

to reduced levels of IgE, MMC and gut eosinophils and in tum to prolonged parasite

survivai in the protein deficient mice. That intestinal MMC and eosinophil responses were

irnpaired in the 3 8 mice despite adequate production of IL-5 and IL40 suggests that

dietary protein affects cytokine-independent pathways in the cellular prolifention or

traficking of immune effectors. F m d y , the integration of protein deficiency into the Hp-

mouse system provided evidence thût, among the many cytokines and effectors elicited

during m Hp infection, the set of IL-Mependent, IgE-mediated MMC and eosinophil

responses in the gut mucosa is more cntical to host protection than IL-5, L- 10, peripherd

eosinophils or IgGI. Broadly, we conclude thrt dietvy protein deficiency predisposes

individu& to acquire higher levels of nematode uifection by suppressing specinc Th2

immunity at the intestinal kveL By contributhg to the understanding of the immunologicai

mechanisms of nematode parasite s u ~ v a l in protein-depleted popuiations, Our results

reved immunological targets and nutritional strategies that may help improve the naturd

abilities of the host immune system to protect against giutrointestinal nematode parasites.

This research was supported by the Naturd Sciences and Engineering Research Council of

Canada (NSERC) Collaborative Project Grant (0163762) and NSERC Research Grants

(A3624 and A3585). Research at the Institute oEParasitobgy is hnded in part by the

Fonds FCAR pour l'aide et le soutien à ia recherche, a Quebec provincial hnding agency.

The authors thank Famineh Jalili for her expert technical assistance.

REFERENCES

Behnke, J.M & Robinson, M. (198 5). Genetic control of immunity to Nematospiroides dubius: A 9-day anthelmintic abbrevhted immunizing regime which separates weak and strong responder strains of rnice. Parasite IrnmunoL 7: 235-253.

Behnke, J.M., Wahid, F.N., Grenas, R.K., Else, KJ., Ben-Smit, A.W. & Goyai, P.K. (1993). Immun010 gical relationships during primary infection with Heligmsomoides polygyrus (Nematospiroides dubius): downregulation of specinc cytokine secretion (n-9 and IL-IO) correhtes with poor mastocytosis and chronic s u ~ v a l of adult w o m . Parasite ImmunoL 15: 415-421,

Boulay, M., Scott, M.E., Conly, S.L., Stevenson, M.M. & Koski, K.G. (1998). Dietary protein and zinc restrictions independently rnodify a Heligmsomoides polygyrur (Nematoda) infection in mice. Parasitology 116: 449-462.

Bradley, L.M., Yoshimoto, K., & Swain, S.L. (1995). nie cytokines IL-4. IFN-y, and IL,-12 regulate the development of subsets of memory effector helper T cells in vitro. J. Immunol. 155: 17 13-1724.

Chan, J., Tian, Y., Tanaka, K.E., Tsang, M.S., Yu, K., Salgame, P., Carroll, Da, Kross, Y., Teitelbaum, R., & Bloom, B.R. (1996). Effects of protein cdorie malnutrition on tuberculosis in mice. Proc. Natl. Acad. Sci USA 93: 14857- 1486 1.

Chandra, R.K. (1991). Immunocompetence is a sensitive and hinctional barorneter of nutritional statu. Acta Pediatr. Scand. SuppL 374: 129-132.

Chen, X.J. & Enerback, L. (1994). IgE receptors, IgE content and secretory response of mast cells in athymic nts. Imrnunology 83: 595-600.

Chen, X.J. & Enerback, L. (1996). Immune response to a Nippostrongylus brariliensis infection in athymic and euthymic rau: surface expression of IgE recepton, IgE occupancy and secretory abiiity of mast cells. Int. Arch. Ailergy & Imrnunol. 109: 250- 257.

Cof'finan, RL., Ohara, J., Bond, M.W., Carty, J., Wotnik, A., & Paul, W.E. (1986). B ceii stimulatory factor4 enhances the IgE response of lipopolysacchuide-activated B ceUs. J. ImmunoL 136: 4538-4541.

Codlman, RL., Seymour, B.W.P., Hudak, S., Jackson, J., & Rennick, D. (1989). Antibody to interleukin-5 inhibits hekninth-induced eosinophilia in mice. Science 245: 308-3 10.

Crampton, D.W.T. (1986). Nutritional aspects of infection. Trans. R Soc. Trop. Med. & Hyg. 80: 697-705.

de Andres, B., Rakasz, E., Hagen, M., McCormik, ML., Mueller, A.L., Elliot, D., Metwail, A., Sandor, Britigan, B.E., Weinstock, J.V., & Lynch, RG. (1997). Lack of Fc-E receptors on murine eosinophils: implications for the functional significance of elevated IgE and eosinophils in pansitic infections. Blood 89: 382693836.

Dehfawi, M.S., Wakeün, Dm, & Behnke, J.M. (1987). Suppression of rnucosd mastocytosis by infection with the intestinal nematode Nematospiroides dubius. Parasite ImrnunoL 9: 187-194,

Dehlawi, M.S. & Wakelin, D. (1988). Suppression of mucosal mastocposis by Nemutospiroides dubius results from an adult wo rm-mediated effect upon hos t lymphocytes. Parasite ImmunoL 10: 85-95.

Finkelman, F.D., Katona, LM., Urban, J.F., Holmes, J., Ohara, J., Tung, A.S., vG Sample, J., & Paul, W.E. (1988). IL4 is required to generate ruid sustain in vivo IgE responses. J. Immunol. 141: 2335-234 1.

Finkelman, F.D., Holmes, J., Katona, LM., Urban, J.F., Beckmann, M.F., Park, L.S., Schooley, K.A., Cof'fman, R.L., Mosmann, T.R., & Paul, W.E. (1990). Lymphokine convol of in vivo immunoglobulin isotype selection. Annu. Rev. ImmunoL 8: 303-33.

Finkelman, F.D., Madden, K.B., Cheever, A.W., Katona, LM., Morris, S.C., Gately, M.K., Hubbard, B.R., Gause, W.C., & Urban, J.F. (1994). Effects of interleukin 12 on immune responses and host protection in M c e ~ e c t e d with intestinal nematode parasites. J. Exp. Med. 179: 1563-1572.

Finkelman, FaDe, Sheo-Donohue, T., GoldhilI, J., Sullivan, CA., Morris, S.C., Madden, &B., Gause, W.C., & Urbon, J.F. (1997). Cytokine regulation of host defense against parasitic g~uointestinal nematodes. Annu. Rev. Immunol. 15: 505-533.

Golden, MaHoNo (1982). Transport proteins as indices of protein status. Am. J. Clin. Nutr. 35: 1 159- 1 165.

Hemdon, F.J., & Kayes, S.G. (1992). Depletion of eosinophüs by anti-IL5 monoclond antibody treatment of mice infected with Trichinella spiralis does not alter parasite burden or irnmunologic resistance to reinfection J. ImmunoL 149: 364203647.

Jahoor, F., Bhattipmlu, S., Del Rosario, M., Bumn, D., Wykes, L., & Frazer, M. (1996). Chronic protein deficiency dEerentialIy atrects the kinetics of plasma proteins Pi young pigs. J. Nutr. 126: 1489-1495.

Katona, LM., Urban, J.F., Kang? S.S., Paul, W.E., & Finkelman, RD. (1991). IL-4 requirements for the generation of secondary in vivo IgE responses. J. Imrnunol. 146: 4215-4221.

Keymer, AwEm & Tarlton, A.E. (1991). The population dynmics of acquired immunity to Heligmosomoides polygyms in the laboratory mouse: suain, diet and exposure. P~asitology 103: 12 1- 126.

King, CL, Low, CC., & Nutman, T.B. (1993). IgE production in humm helminth infection: reciprocai intenelationship between IL-4 and IFN-y. J. Imrnunol. 150: 1873- 1880.

King, CmLm, Xianli, J., Malhoba, I., Liu, S., Mahmoud, A.A.F., & Oettgen, HwC. (1997). Mice with a targeted deletion of the IgE gene have increased worm burdens and reduced grnulornatous infiammation following primvy infection with Schistosoma mansoni. J. Immunot. 158: 294-300.

Kirsch, Rw, Frith, L., Black, E., & Hoffenberg, R. (1968). Regulation of albumin synthesis and catabolism by alteration of dietary protein. Nature 217: 578-579.

Kopf, M., Le Gros, G., Bachmann, M., Lamers, M.C., Bluethmnnn, H., & Kohler, Gw (1993). Disniption of the munne IL-4 gene blocks Th2 cytokine responses. Nature 362: 245-248.

Lagoo, A.S., Eldridge, J.H., Lagoo-Deenadaylan, S., Black, C.A., Ridwan, B.U., Hardy, K. J., McGhee, J.R., & Beagley, K. W. (1994). Peyer's patch CD8' memory T ceh secrete T helper type 1 and type 2 cytokines and provide help for irnrnunoglobuin secretion. Eur. J. IrnmunoL 24: 3087-3092.

Maggi, E., Paronchi, P., Manetti, R., Simonelü, C., Piceinni, Mrn-Pw, Rugiu, F.S., de Carli, M., Ricci, M., & Rornagnani, S. (1992). Reciprocal reguliitory effects of IFN-y a d IL-4 on the in vitro development of hurnan Th 1 and Th2 clones. J. IrnmunoL 148: 214292147.

McFarlane, H., Ogbeide, ML, Reddy, S., Adock, K. J., Adeshina, Hm, Gurney , J.M., Cwke, A., Taylor, G.O., & M o d e , J.A. (1969). Biochemical assessrnent of protein- calorie malnutrition. The Lancet 1: 392-394.

Monroy, F.G. & Endquez, F J. (1992). Heligmsomuides polygynrs: A mode1 for c hro nic gasuointestinai helrninthiasis. Parasito L Today 8: 49-53.

Mosmann, T.R 8 Sad, S. and more. ImmunoL Today

(1996). The expanding universe of T-cell subseu: Thl, Th2, 17: 138-146.

Nakshabendi, LM., Obeidat, W., Russell, RmL, Downle, S., Smith, K., & Rennie, M.J. (1995). Gut mucosai protein synthesis measured using htravenous and intragastric deiivery of stable tracer unino acids. Am. J. PhysioL 269: E996-E999.

Negrao-Correa, Dm, Adams, L.S., & Bell, RG. (1996). Intestinai transport and catabolism of IgE. J. Immunol. 157: 4037-4044.

National Research Council (NRC). (1995). Nutrient requirements of the mouse. In: Nuvient Requirements of Labontory Animals. Fourth Revised Edition. National Academy of Sciences, Washington, D.C.

Nwankwo, M.U., Schuit, K.E., & Glew, R.H. (1985). Effects oEmrtemd protein deprivation on the nutritional status and neutrophil function of suckling neonatai rats. J. Infect. Dis. 5 1 : 23-32.

Pawlowski, ZmSa (1984). Implications of parasite-nutrition interactions from a world perspective. Fed. Proc. 43: 256-260.

Pritchard, DaL, Williams, DDJ.Le, Behnke, J.M.9 & Lee, T.D.G. (1983). The role of IgGl hypergammaglobulinaemin in immunity to the gastrointestinal nematode Nematospiroides dubius. The immunochemicd purification. antigen-specificity and in vivo anti-parasite effect of IgGl from immune serum. Immunology 49: 353-365.

Ramaswarny, K., Hakimi, J., & Bell, R.G. (1994). Evidence for an interleukin-4- inducible immunoglobulin E uptake and transport mechanism in the intestine. J. Exp. Med. 18û: 1793-1803.

Rincon, M., Anguita, J., Nakamura, T., Fikrig, E., & Flavell, R.A. (1997). Interleukin (a)-6 directs the differentiation of Ldproducing CD4+ T cells. J. Exp. Med. 185: 46 1-469.

Shi, H.N., Koski, K.G., Stevenson, M.M., & Scott, M.E. (1 997). Zuic deficiency impairs T ceil function in mice during both primuy and challenge infection widi Heligmosomoides polygyrus (Nematoda). Parasite Imrnunol. 19: 363-373.

Slater, A.F.G. & Keyner, A.E. (1988). The influence of protein deficiency on immunity to Heligmosomoides polygyrur (Nematoda) in mice. Parasite ImmunoL IO: 507-522.

Svetic, A., Madden, K.B., di Zhou, X., Lu, P., Katona, LM., Finkelman, F.D., Urban, J.F., & Gause, W.C. (1993). A primary intestinai helminthic infection rapidly induces a gut-associated elevation of Th2-associated cytokines and IL-3. 1. Immunol. 150: 3434.

Takamoto, M., Ovington, K.S., Behm, C.A., Sugane, K., Young, LG., & Matthae, K.1. (1997). Eosinophilia, parasite burden and lung damage in Toxocara cmis infection in C57BY6 mice geneticdy deficient in IL-5. Immunology 90: 5 1 1-5 17.

Tanaka, T., Hu-Li, J., Seder, R.A., Fazekas de St. Groth, B., & Paul, W.E. (1993). 1nterleuk.i.n 4 suppresses interleukin 2 and interfemn y production by naive T ceils stimulated by accessory celi-dependent receptor engagement Roc. Natl. Acd. Sci. USA 90: 5914-5918.

Toru, Hm, Ra, C., Nonyama, S., Suzuki, K., Yata, J.-I., & Nakahata, T. (1996). Induction of the high-affinity IgE receptor (FceRI) on human mûst ceUs by IL4 Int. Immunol. 8: 1367- t 373.

Urban, J.F., Katona, LM., Paul, W.E., & Finkelman, FD. (199 1). Interleukin 4 is important in protective immunity to a gastrointestinal nematode infection in mice. Froc. Natl. Acad. Sci, USA 8%: 35 13-35 1%

Urban, J.F., Madden, &B., Cheever, A.W., Trotta, P.P., Katona, LM., & Finkelman, F.D. (1993). IFN inhibits infiunmûtory responses and protective immunity in mice infected with the nematode parasite, Nipposmnglyzu brusiliensis. J. Immunol. 151: 7086-7094.

Urban, J.F., Maliszewski, Cor., Madden, K.B., Katona, LM., & Finkelman, F.D. (1995). IL-4 treatment cm cure established gastrointestind nematode infections in irnrnunocompetent and immunodeficient rnice. J. ImrnunoL 154: 4675-4684.

Vitetta, ES., Ohara, J., Myea, C., Layton, J., Krammer, P.H., & Paul, W.E. (1985). Serological, biochemicai and functionai identity of B ceiI-stimulatory factor 1 and B ceii differentiation factor for IgG1. J. Exp. Med. 161 : 1726-3 1.

Wahid, FaNa & Behnke, J.M. (1992). Stimuli for acquired resistance to Heligmosomides polygyrus from intestinal tissue resident L3 and LA b a e . IntL J. Parasitoi. 22 (6): 699-7 10.

Wahid, F.N. & Behnke, J.M. (1993). Immunological relationships during primary infection with Heligmosomoides polygynrr (Nematospiroides dubius): parasite-specific IgG 1 antibody responses and primary response phenotype. Parasite Immunol. 15: 40 1 - 413.

Wahid, F.N., Behnke, J.M., Gnncis, RK., Else, KJ., & Ben-Smith, A.W. (1994). Immunological niationships during primary infection with Heligmosomoides polygyrus: Th2 cytokines and primary response phenotype. Parasitology 108: 46 1-471.

Wastüng, J.M., Scudamore, CL., Thornton, E.M., Newlands, G.F. J., & Miller, HmRPw (1997). Constitutive expression of mouse mast cell protease- l in nomal BALBlc mice and its up-regulation during intestinai nernatode infection. Immunoiogy 90: 308-3 13.

Waterlow, J.C. (1986). Metabolic adaptation to Iow intakes of energy and protein. Annu. Rev, Nutr. 6: 495-526.

Weidel, S.E., Smith, G., & Fleck, A. (1994). The effects of experimental malnutrition on albumin metabolism and distribution in mbbits. Br. J. Nutr. 72: 369-384.

Windhagen, A., Anderson, D.E., Camzosa, A., Wilüams, R.E., & Haflet, D.A. (1996). IL- 12 induces human T cells secreting IL- 10 with IFN-y . J. ImmunoL 157: 1 127- 1131.

Woodbury, R.G.9 Miller, H.R.P., Huntiey, J.F., Newlands, GwFwJa, Palüser, A.C. & Wakeiin, D. (1984). Mucosal mast ceils are hinctionally active during spontmeous expulsion of intestinal nematode infections in rat. Nature 312: 450-452.

Wykes, LJ., Fiorotto, M., Bumn, D.G., Del Rosario, M., Frazer, M.E., Pond, W.G., & Jahoor, F. (1996). Chronic low protein intake reduces tissue protein synthesis in a pig mode1 of protein malnutrition J. Nutr. 126: 148 1- 1488.

Yamaguchi, Y., Suda, T., Suda, J., Eguchi, M., Miura, Y., Harada, N., Tominaga, A., & Takatus, K. (1988). Pudied interleukin 5 supports the terminai differenthtion and proliferation of muMe eosinophilic precursors. J. Exp. Med. 167: 43-56.

Zhang, Xe, Werner-Favre, Cm, Tang, Ha, Brounvers, N., Bonnefoy, J.Y., & Zubler, RwHw (1991). IL-Uependent IgE switch in membrane IgA-positive human B celIs. J. Immunol, 147: 300 1-3004.

CHAPTER IV

GENERAL DISCUSSION

Protein malnutrition and gastrointestinai nematode infections are chronic diseases

thar are frequentiy endemic in populations of the developing world. Protein deficiency i s

associated with irnpaited host irnmunocompetence; however, the specific mechanisms by

which protein deficiency may influence the duration and severity of an intestinal nematode

infection are cornplex and poorly undentood. Inasmuch as host resistance to intestinai

nematode infections depends on the development and maintenance of Th2-type cytokine

and effector responses. we investigated whether protein deficiency exacerbates suMval of

a murine nematode puasite. Heligmsomoides polygym. by impairing parasite-induced

Th2 immunity in systemic a d o r intestinal cornpartments. Previous studies on the

interactions of protein deficiency, nematode infection and host immunity showed that

increased parasite s u ~ v a l was associated with depressed antibody (IgG, t o d IgGl) and

eosinophil responses. The effects of pmtein deficiency on secondary effector responses in

the GALT (e.g. MMC, intestinal eosinophilia) and on gut-associated or splenic cytokine

production have not been characterized. Our study w u designed to address these current

gaps in knowfedge as well as to asscss the temponl and comparmientalization aspects of

protein malnutrition and host immunity to nematode parasites. The major finding of rhis

study was that protein deficiency at the 3% level promoted parasite suMval in previously

immunized mice in association with altered cytokine responses to H. polygyrus (Le. up-

regulated Th l and down-regulated Th2) and impaired proliferation and activation of Th2

effectors within 2 weeks pci. Specifcaily. protein deficiency decreased gut-associated IL-

4 production, increased gut-associnted and systemic IFN-y synthesis, and inhibited

increases in intestinal and serum Th2 effector responses that were observed in weil-

nourished hosts during challenge infection. Interestingly, protein deficiency appeved to

exert differential site- and type-specîf?c effects on cytokine and effec tor responses to H.

po lygym challenge infection. For exarnple, protein deficiency inhibited the parasite-

elicited responses of intestinal eosinophils, IgE, MMC and I L 4 but did not signincmtly

depress peripherai eosinophilia, parasite-specifc IgGl synthesis, or production of other

Th2 cytokines (IL4 and IL-IO). Also, the intestinal cornpartment (gut eosinophils. IL-4

production in MLN) w u more dversely affected by protein resmction than systemic

responses in periphed circulation or spleen. The responses that were profoundly

irnpaired during protein malnutrition (n-41IFN-y balance, elevated IgE titres, mucosai

mastocytosis) are those known to be the most important markers associated with host

protective immunity to H. polygyrus. These impaired responses observed in the protein

deficient mice were detected by 3 days pci (e.g. IgE) until2 weeks pci (e-g. up-regulated

IFN-y), suggesting that functional Th2 immunity is most effective at terminating the

infection when activated at the e d y stage of challenge infection before the parasite

matures and migntes to the intestinal lumen. Once a worm reaches the gut lumen it is less

in contact with immune effectors in the gut mucosa and thus is capable of avoiding the

potentially dvnaging mediators of host immunity. Also. the level of protein restriction

Uifluenced the magnitude of host protection and parasite survival such that mice fed a

marginal protein diet were able to mobilize adaptive rnechanisms (e.g. increased total food

intake) which may have enabled them to mount an effective immune response and in tum

eliminate a challenge infection. However, a further decrease in dietary protein level from

7% to 3% caused profound defects in secondary Th2 immune responses as well as

significuitly prolonged parasite survival.

This study is the fint cornprehensive investigation of the interactions between

protein deficiency, Th2 immunity and nematode pansitic infection. and the results reveal

sevenl points for further investigation. Cytokine production in MLN likely reflects the

activities of lymphocytes in the Peyer's patches and cryptopatches of the gut mucosa that

are in more direct contact with parasite antigens. Considering that MLN receives

immunological components from both intestinal tissues and the periphenl bloodstream, a

definitive study of malnutrition-associated changes in cytokine production would require

measurement of cytokine production by lymphocytes in Peyer's patches or other epithelial

lymphoid tissues. Also, it would be important to determine the kinetics of cytokine

production dunng primary infection in order to ascertain whether primary cytokine

responses direct the magnitude and direction of secondary cytokine Rsponses and

resultant effector mechanisms. Moreover, the potential for imrnunornodulation by protein

malnutrition early after challenge infection (Le. 2 weeks pci) suggesu that nutritional

interventions (e.g. refeeding) or immunological therapy (e.g. I L 4 treaûnent) may be most

successful when implemented before or immediately following reinfection with nematode

parasites. Lady , the differentiai outcornes of the marginal protein group (7%) and the

protein deficient group (3%) indicate that total energy or cdoric intake may have an

important impact on host immunocompetence irrespective of, or in conjunction with, the

level of dietuy protein. The greater food intake by the marginal protein group which had

successfully terminated a challenge infection suggesu that one revonably cost-effective

and feasible stntegy to improve host resistmce to nematode parasites is to increase the

amount of energy consumption without a concomitant supplementation of dietvy protein.

Broadly, our study supports the premise that protein deficiency exacerbates the

suMvd of a gastrointestinal nematode pmsite by decreasing gut-associated L-4 (Th2)

and increasing systemic and gut-associated IFN-y (Thl) early in the challenge infection.

leading to reduced gut and systemic Th2 effector responses. These data contribute to Our

understanding of the immunologicd mechmisms of parasite survivai in proteindepleted

populations. Nso, the results reveal immunological mgeu and tissue compartments for

thenpeutic modulation in order to improve the nauiral abilities of the host immune system

to respond to intestinal nematode pansites. Cleuly. a consideration of the nutritionai

status in the population is essentid to the success of vaccination programs Ymed at

enabling npid and strong acquired immunity to secondary parasitic infections.

APPENDICES

APPENDIX A: RESULTS FROM TWO-WAY ANOVA ON EFFECTS OF DIET, TIME, & DIET BY TIME INTERACTION*

APPENDIX B: RESULTS FROM ONE-WAY ANOVA ON MAIN EFFECT OF DIET.

APPENDIX C: RESULTS FROM ONE-WAY ANOVA ON MAIN EFFECT OF TIME*

APPENDIX A: Results from two-way ANOVA analyses of diet, tirne and diet*time interaction main effects on panmeters measured postprimary infection BPI) or postchallenge infection (PCI). Numbea in parentheses are degrees of kedom.

Variable I Tinte

Worms - PPI

Worms - PCI

BI& Eosinophils - PPI BI& Eosinophils - PCI

S e m IgE - PPI

Semm IgE - PCI

Semm IgG1- PPI F (2.8 1) d .74 1 Pd.48 17

MMC - PPI

F (2.24) = 1 .O6 PPI GutEosinophiIs~ 1 M.3615

APPENDLX A continued. --

1 Variable 1 Die t Time

I IL-5 Spleui - PCI F (2, 109) = 2.41 I P=0.0948

I IL-5 MLN - PCI F (2, 101) = 0.02 1 P4.9839 1 P=0.0001 F (3, 101) = 16.74

1 iFN-y MLN - PCI F (2.92) = 0.35 I ~=0.7031

APPENDM B. Results nom one-way ANOVA analyses on the main effect of diet on parameters meaured at each postprirnary (PPI) and postchaknge (PCI) time point and in

1 Variable

1 Worm Burdens - P6

1 Worm Burdeos - C6

1 Worm Burdcns - Cl4

1 Worm Burdens - C28

1 Puasite-spedfic IgGl - P6

APPENDIX B continued.

1 Variable Die t

1 MMC - Cl4

1 MMC - C28

1 Gut Eosimphils - Naive

1 Gut Eosinaphils - P6

1 Gut Eosinophils - P28

1 Gut Eosinophils - C9 1 Gut Eosinophils - Cl4

I Gut Eosinophils - C21

Variable I

MMCP-1 - Naive

1 I L 4 Spleen - P6

MMCP- 1 - C6

MMCP- 1 - C 14

. - - . - - -

IL4 Spleen - C6 IF(2.29) = 1.91 id. 1665 1 - 1

Diet

F(2,6)=1.64 Pd.2705

Posthoc cornparisons J

F (2.47) = 4.37 P=O.O182

F (2.47) = 5.41 P=0.0077

1 IL4 Spleen - C6 1 F (2.30) = 0.39 P=0.6820 1 1

3% < 24%; 7%=345 & S M

1

3% c (24% = 7%)

IL4 Spleen - C 14 IL4 Spleen - C28

L

1 1L-5 Spleen - Cl4 1 F (2.27) = 1.59 Pd.2229 ( 1

F (2 .3 ) = 2.39 P=0.1126

F (2.20) = 0.18 P=0.8334

1 L i 0 Spleen - C6 1 F (2.30) = 3.14 P4.0576 1 1

1

IL-10 Spleen - P6 I

IL40 Spleen - C3

( ILI0 Spleen - Cl4 1 F (2.28) = 0.76 M.4765 1 1

F (2. 32) = 2.62 M.0880

F(2.25)=0.12 P=û.8856 -

I

APPENDIX B continued.

1 Variable 1 Diet 1 Posthoccomparisons

( m - y Spleen - P6 1 F (2. 32) = 1.29 P4.2897 ( 1 IFN-y Spleen - C3 IF(225)=1.15 Pd.3319 1

IL-4 MLN - C28

iFN-y Spleen - C 14

IFN-y Spleen - CS8

1 IL40 MLN - C3 1 F (2.22) = 0.70 M.5075 1

F (2.25) = 1 1.19 P=0.0003

F (2. 24) = 3.10 P=0.0633

IL-5 MLN - Cl4

IL-5 MLN - C28

1 IL40 MLN - C6 1 F (2.26) = 1.07 M.3564 1

(24% = 7%) < 3%

F ( 2.29) = 0.72 P4.4933

F (2, 22) = 2.1 1 Pa. 1452

1 L I 0 MW - C28 1 F (2. 10) = 3.22 M.0835 1

APPENDIX B continued.

1 Variable 1 Diet 1 PosthoccompPriso~ 1

1 IFN-y MLN - C3 1 F (2, 21) = 1.48 PG.2496 1 1 EN-y MLN - P6 F (2, 11) = 0.67 Pd.5330

i'

IFN-Y MLN - C6 v

IFN-y MLN - Cl4

1 Rdative Food Intake - C28 1 F (2.47) = 77.03 P=0.0001 1 1

Body Wei@ Gain - C28 Total Food btake - C28

1 Relative S p l m Weight - C28 ( F (2.39) = 7.27 P=û.0018 1 1

F (2, 22) = 0.29 P=0.7506

F(2,29)=4.31 P=0.0230

F (2,47) = 85.35 P=O.ûûûl

F (2,47) = 16.63 P=O.OUûl

1 Relative MLN Weight - C28 1 F (2, 32) = 8.57 k0.0007 1 1

24%<3%; 7% = 3% lk 24%

RelativeThymusWeight C28

Plasma Albumin Concentration - C28

- -

F(232)=0.21 P=û.8125

F (2,37) = 4.16 P4.234û 1

APPENDM C. Remlts from one-way N O V A analyses of thne on puameters measured within each dietary group (24%, 7%. 3%) postprimary infection (PPI) or postchdenge infection (PCI).

1 Variable 1 Time 1 1 Worm Burdens - 24% PPI 1 F (1.28) = 3.93 Pd.0573 1 1 Worm Burdens - 7% PPI 1 F (1,31) = 5.25 Pd.0289 1 1 Wonn Burdens - 3% PPI 1 F (1.33) = 2.37 Pd.1333 1 1 Worm Burdens - 24% PCI 1 F (3.49) = 22.29

1 W o m Burdens - 3% PCI I F ( ~ , s ~ ) = 1.31 P=û.2815 1

hem IgE - 24% PPI

( Senun IgE - 3 1 PPI 1 F (1, 31) = 78.53

1 Serurn IgE - 24% PCI

1 Se- IgE - 7% PCI 1 F (5.69) = 9.46 P~.OOOl 1

1 Parasite-speciâc IgG1-24% PPI 1 F (1,27) = 163.41 P4.0001 1

1 MMC - 7% PPI 1 F (1, 10) = 1.59 Pd.2392 1

Variable L

MMC - 24% PPI

1 MMC - 3% PPI 1 F(1,9) =OS1 Pd.4935 1

Time

F (1.9) = 5.09 P=0.0504

( Gut Eosinophils - 24% P H IF(^, 7) =0.00 P4.9645 1

I

MMC - 24% K I 1

1 Gut Eosinophils - 7% PPI

J

F (5.21) = 2.41 P4.07 1 1

1 Gut Eosinophils - 3% PPI 1 F (1,8) = 0.35 P4.5686 1 1 Gut EosinophiIs - 24% PCI 1 F (5.21) = 3.45 P4.1960 1 Gut Eosinophils - 7% PCI

-

IF&8)=1.40 P4.2708 1 1 Gut Eosinophils - 3 2 PCI 1 F (5, 19) = 0.54 P4.7471 1

1 MMCPl - 24% PPI 1 F (1. 19) = 13.47

1 MMCPl - 7% PPI 1 F (1, 18) = 0.14 P4.7136 1 1 MMCPl - 3% PPI 1 F(1, 13) ~ 0 . 2 4 W.6307 1

1 MMCPl - 7% PCI 1 F (3,58) = 19.85

I MMCPl - 3% PCI I F (3.66) = 10.23

APPENDIX C continued.

1 Variable 1 Time 1 1 I L 4 Spleen - 2 4 4 PCI ( F (3.35) = 6.85 P=O.OO09 1

-

1 IL4 Spleen - 7% PCI

1 IL-4 spleen - 3% PCI 1 F (3,34) = 1.35 P4.2747 1 1 IL-5 Spleen - 2446 PCI 1 F (3.37) = 0.18 P4.9073 1 1 IL5 Spleen - 7% PCI 1 F (3.35) = 0.67 Pd.5761 1 1 IL-5 spleen - 3% PCI 1 F (3.37) = 0.15 P4.9308 1 1 IL10 Spleen - 245 PCI 1 F (3.32) = 2.09 P=û.1205 1 1 IL-IO Spleen - 7% PCI

1 IL-10 spleen - 3% PCI 1 F (3.3 1) = 4.56 P==O.0093 1 1 IFN-y Spleen - 2446 PCI 1 F (3.35) = 22.97 P=O.ooOl 1 ( IFN-y spleen - 796 PCI 1 F (3,37) = 31.52 P=O.OOO~ 1

- . -. ..

1 IL-4 MLN - 24% PCI 1 F (3, 33) = 19.32 P=û.ûûûl 1 1 IL-4 MLN - 7 4 PCI 1 F (3,35) = 4.56

1 IL-5 MLN - 7% PCI 1 F (3. 36) = 6.03 P=û.0019 1

1 IL- 10 MLN - 24% PCI 1 F (3,31) = 5.50 P=0.0038 1

1 IFN-y MLN - 24% PCI 1 F (3,31) = 10.18