Immunology and Cell Biology (1994) 72, 69-74

Stimulation of gastrointestinal antibody to Shiga toxin byorogastric immunization in mice

MARK A. SUCKOW,' DAVID F. KEREN," J. EDWARD BROWN^ andGERALD T, KEUSCH"*

' Unit for Laboratory Animal Medicine, University of Michigan Medical School, ^Warde Medical Laboratory, AnnArbor. Michigan, '^Division of Geographic Medicine and Infectious Diseases, Department of Medicine. New EnglandMedical Center and Tufts University School of Medicine. Boston, Massachusetts. USA and ^ Armed Forces Institute ofMedical Sciences. Bangkok, Thailand

Summary Shiga toxin (ST) is a protein toxin o^ Shigella dysenteriae type 1, a causative agent of severediarrhoea and dysentery. In this report we describe the gastrointestinal secretory antibody response ofmice following orogastric immunization with ST. Gastrointestinal secretions were sampled by a gas-trointestinal lavage technique weekly for 5 weeks after initial immunization. Assay of lavage samples byELISA showed that mice vaccinated orogastrically with various doses of ST developed gastrointestinalantibody to ST in a dose-dependent manner. Serum anti-ST activity developed by 5 weeks after initialimmunization. The ability of ST to act as a mucosal immune adjuvant was investigated by coadminis-tration of ST and keyhole limpet haemocyanin. In contrast to cholera toxin, a potent adjuvant, ST didnot demonstrate adjuvant activity. The mouse gastrointestinal lavage model could be useful for furtheranalysis of the cellular basis of ST immunogenicity.

Key words: cholera toxin, mice, Shiga, Shigella dysenteriae.

Introduction

The mucosal surfaces of the body, especially thoselining the gastrointestinal, respiratory and genito-urinary tracts, are continuously exposed to a widevariety of potentially pathogenic agents including vi-ruses, bacteria, protozoa and toxins. In response,plasma cells in the mucosa are directed to synthesizeand secrete antigen specific antibody.' At mucosal sur-faces, the predominant antibody isotype is secretoryIgA, while IgG predominates in serum. In either case,antibody can bind to and may inhibit harmful activityof encountered pathogenic agents.

Shiga toxin (ST) is a protein product of Shigelladysenteriae type 1, a causative agent of severe diar-rhoea and dysentery.'"^ In addition, ST has been impli-cated in the development of haemolytic uraemic syn-drome.'*^'" It is believed that ST may induce disease bydamaging vascular endothelium.^"^

Although the exact role of ST in clinical diseaseremains undefined, the toxic effects of ST have been

Correspondence: M. A. Suckow. Laboratory Animal Program,Purdue University, South Campus Courts-D, West Lafayette,Indiana 47907, USA.

Received 12 January 1993; accepted 20 May 1993.

described in several experimental models. The toxin islethal for rabbits and mice when given paren-terally,'^" results in fluid accumulation in acutelyligated rabbit ileal loops,'" '^ and is cytotoxic for vari-ous cell lines including human vascular endothelialcells,''^•'''"'^ ST appears to act by binding of a prote-olytically generated subunit to the 60S ribosome withsubsequent inhibition of protein synthesis and celldeath.' '

ST is structurally similar to cholera toxin (CT), apowerful adjuvant for the mucosal immune system, inthat both are multimeric proteins composed of five Bsubunits which presumably bind to susceptible cellsand a single A subunit believed responsible for gener-ating a toxic subunit following endocytosis,'^'^

Systemic humoral immunity to ST is readily stimu-lated and immune sera from humans and rabbits canneutralize in vitro activity of the toxin."'^•-' The protec-tive value of serum anti-ST antibody is questionable asdemonstrated by failure of high circulating anti-STtitres to protect Rhesus monkeys against clinicalshigellosis when orally challenged with live Shigelladysenteriae.'^^ Although these studies clearly describethe serum antibody response to ST, there is a lack ofdata describing local immunity in the gastrointestinal

70 M. A. Suckow et al.

tract. Studies in rabbits have shown that, like CT, STstimulates a powerful antigen specific secretory IgAresponse when administered directly into Thiry-Vellaileal loops.'- Furthermore, anti-ST secretory IgA in-hibits HeLa cell cytotoxicity and fluid accumulation inacutely ligated rabbit ileal segments.'^

Precise definition of the cellular basis for stimu-lation of the gastrointestinal antibody response to SThas been hampered by the expense and effort of usingthe rabbit Thiry-Vella ileal loop model, the lack ofinexpensive inbred lines of rabbits, and the lack ofreadily available monoclonal antibodies to rabbit lym-phocyte antigens. In this regard, an easily manipulatedmouse model to study the mucosai immune responseto ST would be useful. Described here is the applica-tion of a mouse orogastric lavage technique to investi-gate dose sensitivity of the gastrointestinal anti-STsecretory IgA response, development of this responseover time, and studies to determine if ST, like CT, canexert an adjuvant effect for a weak mucosal proteinantigen, keyhole limpett haemocyanin (KLH)."'

Materials and methods

Immunogens

CT was obtained from List Biological LaboratoriesInc. (Campbell, CA, USA). Purified ST used for immu-nization of mice was prepared by affinity column chro-matography as described in detail elsewhere."^ PurifiedST used as antigen in ELISA assays was prepared fromthe ammonium sulfate precipitate of culture lysatesfrom S. dysenteriae by using a combination of ion-exchange chromatography, gel filtration chromatogra-phy and preparative isoelectric focusing.'^

Animals

Eight to twelve week old C57B1/6J female mice fromthe Jackson Laboratory (Bar Harbor, ME, USA) wereused in all animal studies. All mice were specific patho-gen free and were housed under conditions suitable tomaintenance of a constant microbiologic status. Use ofthese animals was approved by the institutional animalcare and use committee.

Immunizations

To examine the dose sensitivity of the gastrointestinalsecretory IgA response to ST, groups of five mice weredosed orogastrically with 0.01,0.10, 1.0, 10.0, 25.0 or50.0 \i% of ST in 0.5 mLof 0.2 mol/L NaHCO_, via a 22

gauge stainless steel feeding needle at days 0, 7, 14 and21.

To examine the adjuvant activity of ST, groups offive mice were dosed orogastrically as described abovewith 0.5 mL of 0.2 mol/L NaHCO, solutions contain-ing 5 mg KLH (Sigma Chemical Co., St Louis. MO,USA), 5 mg KLH and lO^gCT, 5 mg KLH and 10ngST, 10 |ig CT or 10 g ST. In addition, one cohort wasdosed with only 0.5 mL of 0.2 mol/L NaHCO3.

Collection of gastric lavage samples

Gastrointestinal contents were sampled prior to immu-nization and then weekly for 5 weeks following initialimmunization by a previously described orogastric la-vage technique."^ Brieffy, mice were orogastricallydosed via a 22 gauge stainless steel feeding needle fourtimes, 15 min apart, with 0.5 mL of a hyperosmoticsolution consisting of 0.025 mol/L NaCl, 0.040 mol/LNa.SOj, 0.01 mol/L KCl, 0.002 mol/L NaHCO, and0.0485 mol/L polyethylene glycol. Thirty minutes afterthe last lavage, each mouse received 0.5 mLof lactatedRinger's solution subcutaneously and 1,0 mL of 0.1%(w/v) pilocarpine intraperitoneally. Intestinal se-cretions were collected over the next 60 min in 3 mL of0.1% (w/v) soybean trypsin inhibitor. The sampleswere diluted to 6 mL with PBS and centrifuged at600^? for 10 min to remove gross faecal debris. Thirtymicrolitres of 17% (w/v) phenylmethylsulfonylfluoride(PMSF) in 95% (v/v) ethanol was added to the super-natant prior to centrifuging at 27 000^ for 20 min at4°C. Next, 1% sodium azide (w/v) and PMSF wereadded to the resulting supematant. After incubating15 min at room temperature, 50 iL of fetal calfserum/mL of sample were added and the samples werefrozen at - 2 0 ^ .

Collection of serum samples

Blood samples were collected by retro-orbital bleedtngof mice under methoxyfluorane anaesthesia (Meto-fane. Pitman-Moore, Washington Crossing. NJ, USA)prior to immunization and 5 weeks after initial immu-nization. Sera were stored at - 2O'C.

Enzyme-linked immunosorbent assay

Sera and gastrointestinal lavage samples were assayedin duplicate for anti-KLH, and anti-ST IgA, IgG andIgM via an ELISA technique. Polystyrene microdi-lution wells were coated with either K.LH (1.0 |ag/well)in 200 mmol/L carbonate buffer (pH 9.6), or pure ST

Mucosal immunity to Shiga toxin in mice 71

{25 ng/well) in carbonate buffer. Following overnightincubation, the plates were washed three times withPBS, pH 7.4, containing 0.1% (v/v) Tween 20, andpost-coated with 3% (w/v) bovine serum albumin(Sigma Chemical Co., St Louis. MO. USA). Followingremoval of unbound protein by washing with PBS/Tween. the plates were stored at 4°C for up to 2 weeks.Protein concentrations of gastrointestinal lavage andserum samples were measured by the Bradford methodand standardized by dilution with PBS. Immediatelyprior to addition of serum or intestinal secretions, theantigen solutions were removed and the wells wereagain washed with PBS/Tween 20. Samples to be as-sayed were diluted in this buffer (1:2 for intestinallavage samples and 1 : 10 for sera), added to wells(100|iL/weU) and incubated for 12 h. Following thisincubation, the wells were washed with the buffer andincubated overnight at 4 X with 1 : 1000 (v/v) sol-utions containing either alkaline phosphatase conju-gated rabbit anti-mouse IgG, alkaline phosphataseconjugated rabbit anti-mouse IgM or alkaline phos-phatase conjugated goat anti-mouse IgA (Zymed Inc..South San Francisco, CA, USA) in PBS/Tween 20.

Monospecificity of IgG and IgM antisera was con-firmed with ELISA. Slight anti-IgM activity in the anti-IgA reagent was adsorbed out by adding 1 : 2000 (v/v)mouse IgM (Sigma Chemical, St Louis, MO, USA).For anti-KLH assays, plates underwent an additionalwash with buffer and the substrate reaction was carriedout with 0.1 mL of p-nitrophenyl phosphate (I mg/mL) in carbonate buffer at a constant temperature of2 5 ^ . The kinetics of the enzyme-substrate reactionwere measured to 100 min on a Titertek MultiscanMicroELISA Reader (Flow Laboratories Inc.. McLean,VA, USA) at an optical density of 405 nm (OD 405).For the anti-ST assay, an ELISA Amplification System(Bethesda Research Laboratories, Gaithersburg, MD,USA) was used in which plates underwent an initialreaction with nicotinamide adenine dinucleotide phos-phate (NADPH) followed by reaction with iodoni-trotetrazolium violet (INT-violet). Final incubationwith substrate was carried out at a constant tempera-ture of 25X. Optical density at 490 nm (OD 490) ofthe reaction was measured 30 min after addition ofsubstrate.

Statistical analysis

Means and standard errors of ELISA absorbancevalues were compared using the Wilcoxon rank sumtest. Statistical significance was reached whenP<0.05.

Results

Mucosal immune response to ST

Mice inoculated orogastrically with ST mounted astrong dose-dependent intestinal anti-ST secretory IgAresponse (Fig. 1). Two weeks following initial inocu-lation, mice dosed with 1.0-25 ng of ST began to de-monstrate significantly elevated anti-ST sIgA titreswhich continued to increase through the fourth andfifth weeks.

A weak anti-ST IgG response was detected in theintestinal secretions (data not shown), however anti-STIgM activity was not found. No blood was grosslyvisible in any of the samples. Control mice dosed withNaHCO3 did not develop significant anti-ST antibodyactivity (data not shown).

Four of five animals dosed with 50 iig of ST diedwithin 2 days after the initial immunization, presum-ably due to toxic effects of ST. The single survivinganimal from this cohort had a weak anti-ST response.

Serum antibody response to orally administered ST

Five weeks following orogastric administration of0.1 ng or greater doses of ST. serum anti-ST IgG ac-tivity increased markedly (Fig. 2). This activity wassignificantly greater than pre-immunization anti-ST ac-tivity in groups dosed with i .0 [ig or more of ST. Im-munization with at least 1.0 (ig of ST also stimulatedweak serum IgM and IgA anti-ST activity (data not

1.2 1

1,0 -

E 0.8 -

0 ,6 -

0,4 -

0,2 -

0,0

II 1

iI I

I

1

1

0 1 2 3 4 5

Time after immunization (weeks)

Fig. 1. Gastrointestinal anti-ST IgA activity for 5 weeks fol-lowing initial immunization (week 0). Dose groups are0.01 ^gST(B),o.io^gST(Ea), l.OngST(a). lo.and 25.0 ^g ST (D).

72 M. A. Suckow et al.

0 5Time after immunizalion (weeks)

Fig. 2. Serum anti-ST IgG activity prior to immunizationand at the time of killing (5 weeks after initial immunization).Dose groups are 0.01 ig ST (•), O.lO^g ST (M), LO^tg ST{H), 10.0 ngST (ED) and 25.0 ngST(n) .

1.00-1

0,75-

0.50-

0.25 -

0.001 2 3 4 5

Time atler immunizalion [weeks)

Fig.3. Gastrointestinal anti-KLH I&A activity through 5weeks following initial immunization (week 0). Dose groupsare NaHCOj (•). KLH (^), KLH + ST (B), KLH + CT(Q). and KLH + ST + CT (D).

shown). Mice dosed with 0.01 ^g of ST did not mount aserum anti-ST response. Interestingly, mice dosed with25 jig of ST mounted a significantly weaker serum IgGanti-ST response than those dosed with 10 jig.

Role ofSTas a mucosal immune adjuvant

Having established ST as a strong mucosal immuno-gen in the mouse, we examined whether ST sharedwith CT the ability to act as a mucosal immuneadjuvant. In this regard, mice were dosed with KLH,a weak mucosal immunogen. alone or in combinationwith ST or CT. As expected, mice dosed with KLHand CT developed significantly elevated anti-KLHsIgA activity by the third week after initial inocu-lation. This response experienced further significantincreases during the fourth and fifth weeks (Fig. 3).Mice dosed with KLH, CT and ST developed anti-KLH sIgA activity which did not significantly differfrom mice dosed with KLH and CT. In contrast, micedosed with NaHCO3, CT, ST or KLH alone or KLHin combination with ST did not demonstrate signifi-cantly elevated anti-KLH sIgA activity over thecourse of the study. Similarly, anti-KLH gastrointesti-nal IgG activity was significantly enhanced in micedosed with KLH and CT, but not in mice dosed withKLH alone or in combination with ST (data notshown). These results indicate that ST lacks the abil-ity to act as an adjuvant for the mucosal immuneresponse to KLH.

Discussion

ST is likely an important vinalence factor of Shigelladysenteriae, the causative agent of human dysentery.Like CT, a powerful mucosal antigen and adjuvant,ST elicits a strong secretory IgA response to itselffollowing administration into rabbit Thiry-Vella ilealloops.'^"^'^^ The purpose of the present study was todevelop a mouse model in which to study gastrointes-tinal immunity to ST and to examine the time courseof the anti-ST gastrointestinal antibody response fol-lowing orogastric immunization.

Anti-ST intestinal IgA and IgG activity developedin a dose-dependent manner in response to orogastricimmunization with ST. Previous studies conducted inthe rabbit chronic Thiry-Vella ileal loop model foundthat anti-ST sIgA activity is detectable by ELISA bythe first week following direct intraloop immunizationand increases after the second and third weekly doses.We found that in the mouse intestinal lavage modelsimilar activity is detectable by the second weekfollowing orogastdc immunization with ST. It is inter-esting that IgG directed against ST was found ingastrointestinal lavage samples following immuniz-ation. Although no blood was grossly observable inthese samples, it is possible that small amounts ofundetected blood were present. Alternatively, pilo-carpine could result in transepithelial transport ofserum components such as IgG, since cholinergicagents have been shown to cause transient opening oftight junctions in the gut.~^

Mucosal immunity to Shiga toxin in mice 73

Interestingly, mice dosed with 25 ng of ST had lowerserum anti-ST IgG activity than mice immunized with10.0 |ig ST. A possible explanation might be the devel-opment of systemic tolerance to ST. Systemic tolerancefollowing orogastric exposure has been described forseveral protein antigens.~^"^' For many antigens,larger doses result in a greater and longer-lasting stateof tolerance.

The mouse intestinal lavage model holds severaladvantages over the rabbit Thiry-Vella ileal loopmodel for the study of the mucosal antibody responseto ST. First, greater numbers of animals can bestudied using the mouse model, since it is less expen-sive and cumbersome than the surgically manipulatedrabbit model. Second, the availability and use ofinbred strains of mice diminish individual variation,due to genetic heterogeneity, in response to antigens.Third, commercial monoclonal antibodies to specificmouse T-cell antigens and cytokines are readily avail-able and will permit a thorough investigation of thecellular basis of ST immunogenicity. Fourth, the oralroute of antigen administration more closely mimicsthe natural route of antigen exposure than exposurethrough a surgically created, isolated intestinal loopsystem.

Previous investigators have described the ability ofCT to enhance the mucosal antibody response to avariety of unrelated antigens.^^-*^ The exact mechan-ism by which CT exerts this effect is unknown.Although several studies have suggested that adjuvan-ticity occurs through a cAMP independent pathway,other evidence suggests that this effect may be medi-ated through elevation of intracellular cAMP. -*" * Inthe present study, ST was unable to enhance themucosal antibody response to a weak, coadminis-tered, protein antigen (i.e. KLH). These results showthat although ST is similar to CT in that it is both astrong immunogen and a multimeric enteric bacterialtoxin, it may not be an adjuvant for the mucosalimmune system. Further studies will be needed todetermine if ST demonstrates adjuvancy for antigensother than KLH.

In conclusion, ST is a potent oral immunogen inmice, but lacks adjuvanticity. Although the preciserole of ST in clinical disease remains largely specu-lative, the antigenicity of ST following orogastricimmunization warrants further study to examine thepotential usefulness of ST in immunization strategiesto protect against Shigella-associaXed disease. Estab-lishment of the mouse as an animal model in which tostudy gastrointestinal immunity to ST will permit amore detailed examination of the cellular basis of thisresponse.

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