Cytokine profiles in metal-induced allergic contact

Cytokine responses in metal-induced allergic contact dermatitis: Relationship to in vivo responses and

implication for in vitro diagnosis

Jacob Taku Minang

Stockholm, 2005

Doctoral Thesis from the Department of Immunology, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

Cytokine responses in metal-induced allergic contact dermatitis: Relationship to in vivo responses and

implication for in vitro diagnosis

Jacob Taku Minang

Stockholm, 2005

ISBN 91-7155-158-1 pp 1-79 © Jacob Taku Minang PrintCenter, Stockholm University 2005 Stockholm 2005

“Without love, benevolence becomes egotism” -Dr. Martin Lurther King Jr. (1929-1968)

“People like you and I, though mortal of course like everyone else,

do not grow old no matter how long we live…[We] never cease to stand like

curious children before the great mystery into which we were born.”

- Albert Einstein (1879-1955): in a letter to Otto Juliusburger (1867-1952).

To my dear mum, Minang Margaret Atih

ORIGINAL PAPERS

This thesis is based on the following original papers, which are referred to in the text by their Roman numerals. Paper I.

Jacob T. Minang, Marita Troye-Blomberg, Lena Lundeberg and Niklas Ahlborg (2005).

Nickel elicits concomitant and correlated in vitro production of Th1-, Th2-type and regulatory

cytokines in subjects with contact allergy to nickel. Scand J Immunol. 62:289-296.

Paper II.

Jacob T. Minang, Iréne Areström, Bartek Zuber, Gun Jönsson, Marita Troye-Blomberg,

Niklas Ahlborg (2005). Regulatory effects of IL-10 on Th1- and Th2-type cytokines induced

in response to the contact allergen nickel. Submitted.

Paper III.

Jacob T. Minang, Iréne Areström, Marita Troye-Blomberg, Lena Lundeberg, Niklas Ahlborg

(2005). In vitro cytokine responses to metal ions in subjects with allergic contact dermatitis to

nickel, cobalt, palladium, chromium and gold. Manuscript.

Paper IV.

Jacob T. Minang, Niklas Ahlborg and Marita Troye-Blomberg (2003). A Simplified ELISpot

assay protocol used for detection of human interleukin-4, interleukin-13 and interferon-γ

production in response to the contact allergen nickel. Exogenous Dermatol. 2:306-313.

Reprints were made with permission from the publishers.

TABLE OF CONTENTS BRIEF OVERVIEW OF THE IMMUNE SYSTEM 7

The immune system 7 Innate immunity 7 Acquired immunity 8 T-lymphocyte subsets 8 The Th1/Th2 concept 9 T-regulatory cells (Tr1 and Tr2/Th3) 11 B lymphocytes and IgE class switching 12

HYPERSENSITIVITY/ALLERGIC REACTIONS 13 Classification of hypersentivity/allergic reactions 13 CONTACT DERMATITIS 14

Allergic contact dermatitis 14 Atopic contact dermatitis 16 Irritant contact dermatitis 17 Hapten immune system interaction in allergic contact dermatitis 17

RELATED BACKGROUND 20 Biochemistry of transition (heavy) metals 20 Transition metals and allergic contact dermatitis 20 Molecular and cellular mechanisms underlying metal-induced allergic contact dermatitis 22 Cross reactivity versus co-sensitisation in metal-induced allergic contact dermatitis 23 Cytokines investigated in this study 26 In vivo diagnosis of allergic contact dermatitis 28 In vitro diagnosis of allergic contact dermatitis 30 Cytokine detection assays 30

PRESENT STUDY 32 Objectives 32 Methodology 33

Study subjects 33 Measurement of cytokine levels in cell supernatants by ELISA (papers I, II and III) 34 Evaluation of the detection sensitivity of one-step and two-step reagents in capture ELISA (paper IV) 34 Enumeration of cytokine-producing cells by ELISpot 35 Phenotypic characterisation of Ni2+-specific cytokine producing cells using depletion experiments (paper II) 35 Statistical methods 37

Results and Discussion 38 Paper I 38 Paper II 42 Paper III 46 Paper IV 49

CONCLUSIONS AND PERSPECTIVES 53 FIGURE LEGENDS 54 ACKNOWLEDGEMENTS 58 REFERENCES 60 APPENDICES: PAPERS I-IV

ABBREVIATIONS

ACD Allergic contact dermatitis ALP Alkaline phosphatase APC Antigen-presenting cell Au1+/3+ Gold ions BcR B-cell receptor CD Cluster of differentiation CLA Cutaneous lymphocyte antigen Co2+ Cobalt ion Cr3+/6+ Chromium ions CTL Cytotoxic T-lymphocyte DC Dendritic cell DTH Delayed-type hypersensitivity ELISA Enzyme-linked immunosorbent assay ELISpot Enzyme-linked immunospot assay FACS Flourescent activated cell sorted FBS Fetal bovine serum FCS Fetal calf serum FSC Forward scatter GM-CSF Granulocyte monocyte colony stimulating factor IFN Interferon IL Interleukin KC Keratinocyte LC Langerhans cells LPS Lypopolysaccharide LTT Lymphocyte-transformation test mAbs Monoclonal antibodies MHC Major Histocompatibility Complex Ni2+ Nickel ion NK Natural killer cell PAMP Pathogen-associated molecular patterns PBMC Peripheral blood mononuclear cells PBS Phosphate buffered saline Pd2+ Palladium ion PHA Phytohaemagglutinin pNPP para-nitro-phenyl phosphate PPD Purified protein derivative PRR Pattern-recognition receptor PVDF Polyvinyldiflouride SA Strepavidin SSC Side scatter Tc T-cytotoxic cell TcR T-cell receptor TGF Transforming growth factor Th T-helper cell TLR Toll-like receptor TNF Tumour necrosis factor Tr T-regulatory cell TT Tetanus toxoid

Cytokine responses in metal-induced ACD

7

BRIEF OVERVIEW OF THE IMMUNE SYSTEM

The immune system

The human immune system is a truly amazing constellation of responses to attacks from

outside the body. It has many facets, a number of which can change to optimize the response

to these unwanted intrusions. The immune system has a series of dual natures, the most

important of which is self/non-self recognition. The others are: natural/adaptive

(innate/acquired), cell-mediated/humoral and primary/secondary immunity. Functional

integration of the immune system is accomplished mainly by cell-to-cell communication that

relies on cell adhesion molecules that act in concert with a number of small soluble

molecules. Every immune system cell is equipped to synthesize and release a variety of these

small molecules, mostly cytokines, which travel to other cells (both immune and non-

immune) and stimulate those cells to become either more active (up-regulated) or less active

(down-regulated).

Innate immunity

Innate immunity refers to antigen non-specific defense mechanisms that a host uses

immediately or within several hours after exposure to an antigen. This is the immunity one is

born with and is the initial response by which the body eliminates microbes and prevents

infection. Innate immune responses involve; anatomical barriers (skin and mucosa),

physiological barriers (temperature, pH), molecules (complement proteins, acute phase

proteins, antimicrobial peptides and cytokines), cells that release inflammatory mediators

(basophils, mast cells, and eosinophils), phagocytic cells (neutrophils, monocytes, and

macrophages) and natural killer cells (NK cells).

Cells of the innate immune system use proteins, pattern-recognition receptors referred to as

PRR’s (e.g Toll-Like Receptors; TLRs), encoded in an organism's germ line to detect

potentially dangerous substances. These cells are thought to recognise particular highly

conserved pathogen-associated molecular patterns or PAMP’s (e.g carbohydrate structures

such as lypopolysaccharides; LPS) present on many different microorganisms (reviewed in

Franc et al., 1999; Teixeira et al., 2002). New findings show that the innate immune system,

once thought to be the unnecessary vestigial tail of ancient antimicrobial systems that have

been made redundant by the evolution of acquired immunity, is the cornerstone of the body's

ability to fight infection. In many aspects, the innate immune response is also a prerequisite

for an efficient induction of the specific (acquired) immune response. Following activation the

Jacob Taku Minang

8

innate system induces key costimulatory molecules on antigen presenting cells (APCs), which

are essential for antigen-driven clonal expansion of T and B cells (Akira et al., 2001; Pasare

and Medzhitov, 2004; Hedges et al., 2005; Xu et al., 2005). Dendritic cells (DCs) activated

by innate stimuli and loaded with foreign antigen travel to regional lymph nodes to activate

the acquired-immune system. Subsequently, the activated acquired-immune cells move into

tissues, where the innate immune system sets-off the danger signal (Matzinger, 1994). The

chemokine system is an essential regulator of this dendritic cell and lymphocyte trafficking,

which is necessary to turn an innate immune response into an adaptive response (Luster,

2002).

Acquired immunity

Acquired (adaptive) immunity includes humoral immunity (antibody-mediated) and cell-

mediated immunity. Acquired immunity involves; APCs such as macrophages and DCs, the

activation and proliferation of antigen-specific T and B-lymphocytes and the production of

antibody molecules, cytotoxic T-lymphocytes (CTLs), and cytokines. T and B-lymphocytes

possess specific receptors that arise from complex gene recombination reactions giving rise to

a broad spectrum of antigen specificities that is a hallmark of the adaptive immune system.

The T-cell receptor (TcR) recognizes only epitopes on antigens processed by APCs and

presented in the context of the major histocompatibility complex (MHC) molecules (reviewed

in Sebzda et al., 1999; Inaba and Inaba, 2005). Self/non-self recognition is achieved by

having every cell displaying a marker based on the MHC complex (Doherty and Zinkernagel,

1975). Any cell not displaying this marker is treated as non-self and attacked (Oberg et al.,

2004). The B-cell receptor (BcR), in contrast can recognise specific epitopes on whole

antigens (Takata et al., 1995). The adaptive immune system has the capacity to “store”

instructions obtained from the innate immune system upon first encounter with a foreign body

by changing from a naïve to an appropriate memory phenotype (reviewed in José et al.,

1999). This property, that distinguishes the adaptive immune system from the innate, is

referred to as immunological memory.

T lymphocyte subsets

Immature T lymphocytes differentiate from pluripotent stem cells in the bone marrow and

migrate to the thymus where their maturation occurs. Surface expression of the cluster of

differentiation molecule, CD3, is a unique feature of T cells. T cells are further classified by

the type of TcR they express on their surface, having either α/β or γ/δ combination (Miescher


9

et al., 1988). The α/β TcR expressing T cells only recognize antigens as peptide fragments

presented as a complex with MHC antigens on APCs (Schwartz et al., 1976; Ball and Stastny,

1984, Braciale et al., 1987). There are two distinct subpopulations of T cells within the α/β

TcR expressing lymphocytes defined based on their surface expression of either CD4 or CD8

in combination with the accessory marker CD3 (de Gast et al., 1985). The CD4+ T cells,

designated T-helper (Th) cells, recognize peptides, which are bound to MHC class II

molecules. CD8+ T cells, referred to as T-cytotoxic (Tc) cells recognize antigens bound to

MHC class I molecules, displayed as peptide-MHC class I complexes on the surface of APCs

(Van Seventer et al., 1986). Antigen recognition by γ/δ TcR expressing T cells does not

require processing and is not MHC restricted (reviewed in Chien et al., 1996).

The Th1/Th2 concept

The Th1/Th2 hypothesis emerged in the late 1980s, stemming from observations in mice of

two distinct populations of CD4+ T-helper cells differing in cytokine secretion patterns and

other functions upon activation (Mosmann et al., 1986). The concept subsequently was

applied to human immunity (Mosmann and Coffman, 1989). Both the Th1 and Th2 subsets

are produced from a non-committed population of precursor (naïve) T cells (Lafaille, 1998).

The Th1/Th2 concept rests largely on a dichotomy of cytokine profiles, with Th1 cells

described as mainly IFN-γ, IL-2 and TNF-β producers while Th2 cells are IL-4, IL-5 and IL-

13 producers (McKenzie et al., 1993; Wang et al., 1994; Thomas and Kemeny, 1998).

However, as with other immune cells, the array of cytokines produced by the Th1 and Th2

cells varies greatly and is influenced by a large number of experimental variables, as well as

the danger from artefacts (Romagnani, G., 2000). The differences in cytokine profiles

between Th1 and Th2 responses described in earlier studies relied heavily on mice and

cultured cells. However, more recent research involving human subjects has proved much of

this earlier work to be highly simplistic or otherwise inaccurate (Reviewed in Dent, 2002).

Currently Th1 cells are referred to as cells that produce far more IFN-γ and IL-2 than do Th2

cells, while the Th2 cells produce far more IL-4 (and perhaps IL-5) than do the Th1 cells.

CD8+ T cells (Tc cells) have also been suggested to, depending on the type of antigen and

cytokine milieu, differentiate into cells which produce IFN-γ, but not IL-4 (Tc1), and cells

that produce IL-4, but not IFN-γ (Tc2) (Mosmann and Sad, 1996; Kemeny et al., 1999). A

third population of cells, Th0 and Tc0, has been described as lymphocytes exhibiting an

unrestricted profile of cytokines; i.e., they produce both Th1 and Th2-type cytokines

Jacob Taku Minang

10

(Firestein et al., 1989; reviewed in Bendelac and Schwartz, 1991; Torii et al., 2002). There

are two key features of the Th1/Th2 hypothesis; firstly, each cell subset produces cytokines

that serve as growth factors of that subset (autocrine effects) and secondly, cytokines

produced by the two subsets cross-regulate each other's development and activity either by

blocking polarised maturation of the opposite cell type or by blocking its receptor functions

(Abbas et al., 1996). Whereas commitment to the Th1 phenotype is reversible, that to the Th2

phenotype appears to be final, since efforts to reverse such differentiated cells have not been

successful (Rogge et al., 1997; Lafaille, 1998). Recently, it has been suggested that CD4+ T-

helper cells polarisation could be indicative of a more profound polarisation of the immune

system as a whole. That is, a kind of type 1/type 2 polarisation already begins with those cells

having the primary contact with antigens, including the DCs, monocytes and macrophages,

and other APCs (Moser and Murphy, 2000; Fujimura et al., 2004; Aktas et al., 2005). These

APCs likely polarise into type 1 and type 2 cells in response to the type of antigen experience,

and then subsequently bias the polarization of the T-helper population functionally

"downstream" from them.

The Th1-type cytokines

APCs secreting IL-12 upon uptake and processing of type-1 promoting (type-1 biased)

antigens promptly migrate to nearby lymph nodes. As this cytokine increases in concentration

it begins to influence naïve T cells to eventually become Th1 cells (Verhasselt et al., 1997;

Sparwasser et al., 1998; Hessle et al., 2000). NK cells also respond to the IL-12 environment

and proceed to release IFN-γ, which reinforces the APC's production of IL-12 and also helps

to drive the naïve T-cell commitment process (Hart et al., 2005; Walzer et al., 2005). As the T

cells attain maturity, Th1 cells also produce IFN-γ, which (together with the NK cells)

stimulates the APC and naïve T cells to polarise into more Th1 cells, in a self-reinforcing

"autocrine" loop. Th1-type cytokines are mainly produced in response to intracellular

pathogens, cell wall antigens or other smaller fragments of the organism, and trigger cell-

mediated immunity and/or production of opsonising antibodies (Table 1). Th1 cells are

hypothesised to lead the attack against viral and bacterial antigens through the classic

delayed-type hypersensitivity (DTH) reaction (Newport et al., 1996; Lienhardt et al., 2002).

Th1 cells are also important in the fight against cancer cells (Sato et al., 1998). On the

negative side, the Th1 pathway is often portrayed as being the more aggressive of the two,

and apparently, when it is over reactive, can generate organ-specific autoimmune diseases


11

(e.g., arthritis, multiple sclerosis, type 1 diabetes) (Tang et al., 1998; Pakala et al., 1997;

Schulze-Koops et al., 2001)

The Th2-type cytokines

The emergence of Th2 cells, like the Th1 cells, is also dependent on their cytokine

environment. Their maturation is likely initiated by the cytokine IL-6 (Croft and Swain, 1992;

Rincon et al., 1997) produced by APCs (monocytes and macrophages), endothelial cells,

fibroblasts and mast cells (Bradding et al., 1993; Derocq et al., 1994). IL-4 released by NK

cells, mast cells and eosinophils, also participates in driving Th2-cell maturation (Schmitt et

al., 1990; Marcinkiewicz and Chain, 1993). As the Th2 cells mature they also produce IL-4,

which together with the other participating cell types generate an autocrine loop to the naïve T

cells to make more Th2 cells (Chen et al., 2004) (Table 1). The Th2 pathway is thought to be

primarily involved in the triggering of IgE-mediated (immediate) hypersensitivity disorders,

including allergic asthma, eczema, hay fever, and urticaria (Singh et al., 1999) and also in

protection against helminthic infections (Coffman and von der Weid, 1997; reviewed in Dent,

2002).

T-regulatory cells (Tr1 and Tr2/Th3)

T-regulatory (Tr) cells were first described in the early 1970s based on their induction of

tolerance in other lymphocyte populations (Gershon and Kondo, 1970). However, failure to

define their exact phenotype led to controversy in the 1980s about their existence (Möller,

1988). Tr cells have recently become more prominent (Mason, 2001; McGuirk and Mills

2002; Chen et al., 2003) and have been shown to intervene and to block either Th1 or Th2

activities or both (reviewed in Kidd, 2003). CD4+CD25+ T lymphocytes have been suggested

to be the main cell population with immunoregulatory properties (Shevach, 2002; Nishimura

et al., 2004; Ruprecht et al., 2005). The CD4+CD25+ Tr cells comprise 5-10% of the total

peripheral T-cell pool (McGuirk and Mills, 2002) and constitutively express the CD25 marker

(IL-2Rα), already upon exit from the thymus (Sakaguchi et al., 1995). These cells are thus

referred to as “natural” Tr cells (Trn) (Table 1). Trn cells have been shown to possess a

contact-dependent cytokine-independent mechanism of immunosuppression (Fontenot et al.,

2003; Khattri et al., 2003). A second population of CD4+ Tr cells, generated in the periphery,

has been described (Baecher-Allan et al., 2001). This Tr subset has a cytokine-dependent

mechanism of action and is subdivided into Type 1 Tr (Tr1) and Type 2 Tr (Tr2) cells based

on their cytokine expression profiles. Tr1 cells secrete high levels of IL-10 and low-to-

Jacob Taku Minang

12

moderate levels of TGF-β, while Tr2 cells primarily secrete TGF-β (Groux et al., 1997;

Gorelik and Flavell, 2002). Tr2 cells were previously named T helper-3 (Th3) cells (Weiner,

2001) but later renamed as Tr2 cells to highlight their suppressor function (reviewed in

Horwitz et al., 2003). CD8+ T cells with regulatory properties have also been described

(Gilliet and Liu, 2002). Unlike CD8+ effector cells, CD8+ Tr cells lack cytotoxic activity and

produce IL-10 (Tr1) and/or TGF-β (Tr2) (Rich et al., 1995; Dhodapkar and Steinman, 2002).

Functional studies indicate that Tr1 cells and other Tr populations may help to terminate Th1-

related inflammatory responses to pathogens, tumors, and alloantigens (Fukaura et al., 1996;

Groux et al., 1997; reviewed in Gorelik and Flavell, 2002; Ruprecht et al., 2005).

B lymphocytes and IgE class switching

B-lymphocytes mature in the bone marrow and, unlike T lymphocytes, are able to recognise

soluble antigens without the need for processing (Takata et al., 1995). Antigen recognition by

naïve immunocompetent B cells is via surface expressed immunoglobulins mainly of the IgM

and IgD classes. Activated B cells differentiate into plasma or memory B cells in highly

organised compartments of secondary lymphode organs (i.e lymph nodes and spleen) during

which time, depending on the antigen and cytokine milieu, class switching occurs in the

immunoglobulin genes to give rise to other classes; IgG, IgE or IgA (reviewed in Coffman et

al., 1993) which, display the same antigen specificity but have different biological functions.

IgE has been shown to be the main mediator of type I allergies (see section on

‘Hypersensitivity/Allergic reactions’). The switch to the IgE antibody class is induced by IL-4

and IL-13 (Del Prete et al., 1988; Punnonen et al., 1993) (see section on ‘Cytokines

investigated in this study’). Measurement of total serum IgE antibody levels is used as a

marker of the atopic status of an individual although determination of allergen specific IgE is

considered a more reliable indicator of atopic allergy (Kasaian et al., 1995; Di Lorenzo et al.,

1997; Di Gioacchino et al., 2000; Jackola et al., 2004). IgE has also been shown to play a role

in the clearance of many parasitic infections (Coffman and von der Weid, 1997; reviewed in

Dent, 2002). There are a few reports of detection of nickel (Ni2+) specific IgE antibodies in

serum samples from some Ni2+ allergic subjects (Shirakawa et al., 1992; Estlander et al.,

1993); however, the relevance of these antibodies to the skin inflammatory response seen in

allergic contact dermatitis (ACD) is not known.


13

Table 1. General properties of CD4+ and CD8+ T regulatory and helper subsets

T cell subset

Predominant cytokine secreted

Response to antigena

Response to IL-2 or IL-15b

Regulatory/suppressor CD4+ CD25+ Trn None Unresponsive Proliferate CD4+ Tr1* IL-10 Unresponsive Proliferate CD4+ Tr2/Th3* TGF-β Unresponsive Proliferate CD8+ Tr1 or Tr2* IL-10 or TGF-β Hyporesponsive Proliferate

Helper/Cytotoxic CD4+ Th1 IL-2, IFN-γ Proliferate Proliferate CD4+ Th2 IL-4, IL-5c, IL-13 Proliferate Proliferate CD8+ Tc1 IFN-γ Proliferate Proliferate CD8+ Tc2 IL-4 Proliferate Proliferate *CD4+ or CD8+ Tr1 or Tr2 subsets can also display CD25 following activation. a, restimulation of T cells specific for ovalbumin or peptide 110–119 of influenza HA with the same antigen. b, both cytokines use the shared common cytokine receptor gamma chain (γc) and both function as T cell growth and survival factors. c, my addition. Culled from Horwitz et al., (2003).

HYPERSENSITIVITY/ALLERGIC REACTIONS

The term ‘allergy’ was first used by von Pirquet, to denote both host-protective and

potentially host-injurious immune responses (Bendiner, 1981). Today, hypersensitivity is

defined as an exaggerated reaction towards a stimulus, whereas allergy is a more specific

hypersensitivity reaction caused by an immunological sensitisation.

Classification of hypersentivity/allergic reactions

Hypersensitivity reactions are generally divided into four types depending on the effector

mechanism involved in the response (Reviewed in Rajan, 2003) (Table 2). The type I

hypersentivity response is very rapid (within minutes or hours of re-exposure to the allergen)

and is mainly IgE antibody mediated with allergen-specific IgE receptor bearing mast cells

and eosinophils playing a significant role (Siroux et al., 2004) (see also section below on

‘atopic contact dermatitis’). The clinical manifestation of type II and III hypersentivity

responses, just like the type I response, appear within a short time upon subsequent exposure

to the allergen but unlike the type I response, the IgG antibody class plays a major effector

role; either as opsonising antibodies promoting target cell lysis or in complexe with allergen

resulting in complement-mediated lysis of underlying epithelia (Skokowa et al., 2005). The

type IV response, also referred to as the DTH response, on the otherhand takes several days to

Jacob Taku Minang

14

appear upon re-exposure to the sensitising agent and is cell-mediated (Silvennoinen-Kassinen

et al., 1992; Shah et al., 1998; Grabbe and Schwarz, 1998).

The more important allergic disorders include bronchial asthma, perennial rhinoconjunctivitis

or seasonal rhinoconjunctivitis (hay fever), urticaria, extrinsic allergic alveolitis (farmer’s

lung, bird keeper’s lung), food allergies, acute generalised allergic reactions (anaphylactic

shock), allergic drug reactions, atopic dermatitis and contact dermatitis.

Table 2. The Gell-Coombs classification of hypersensitivity/allergic reactions

Gell-Coombs scheme

Characteristics Effector mechanism Manifestations

Type I IgE mediated Immediate reaction

IgE-dependent activation of mast cells and basophils (Th2-cell dependent)

Atopic allergy, anaphylaxis, hay fever, asthma

Type II Antibody mediated Rapid reaction

Antibodies to microbial antigens, C5a release Leukocyte infiltration

Cytotoxic reactions, thrombocytic purpura

Type III Antibody mediated Rapid reaction

Antibody-antigen complexes activating the complement system

Immune complex diseases, vasculitis

Type IV Cell mediated Delayed type reaction

T-cell mediated (type 1 T cells implicated)

Allergic contact dermatitis

Modified from Rajan, (2003).

CONTACT DERMATITIS

Contact dermatitis is defined as an inflammatory skin reaction subsequent to direct contact

with noxious agents in the environment. This phenomenon was first described as early as the

first century A.D. where some individuals were reported to experience severe itching when

cutting pine trees (reviewed in Rosenberg, 1955). Depending on the offending agent and/or

the mechanism of the reaction (immediate or delayed, allergic or non-allergic), three main

types of contact dermatitis reactions have been described viz; allergic contact dermatitis

(ACD), atopic contact dermatitis and irritant contact dermatitis.

Allergic contact dermatitis

ACD results from a specific immunological reaction (type IV allergy or DTH) (Grabbe and

Schwarz, 1998) and primarily affects the hands, head/face and feet in adults (Schnuch et al.,


15

1998; Shah et al., 1998; West et al., 1998). Common contact allergens include organic hapten

molecules found in plant products (e.g urushiol from the North American poison ivy) (Kalish

et al., 1994), drug metabolites, perfume fragrancies, rubber, plastics, additives and

preservatives (e.g methylisothiazolinones) (Gruvberger, 1997; Schnuch et al., 1997) as well as

metal ions such as nickel, chromium, cobalt, gold and palladium (Gawkrodger et al., 2000;

Thierse et al., 2005) (Table 3).

Table 3. Some important allergens included in the European standard series and mean

prevalence of positive reactions to these allergens among subjects with eczematous reactions.

Allergen name Combined prevalencea (95% CI)

Nickel 5% 18.6 (11.1-26)

Fragrance mix 5% 10.7 (5.4-16)

Myroxylon pereirae 25% 6.7 (3.5-9.9)

Cobalt chloride 1% 5.8 (3.1-8.4)

Colophonium 20% 5.2 (3.2-6.9)

Thiuram mix 1% 3.5 (2.8-4.2)

Wool alcohols 30% 3.3 (1.3-5.3)

p-Phenylenediamine 1% 3.0 (2.1-3.9)

Neomycin 20% 2.9 (2.0-3.9)

MCI & MIb 0.01% aq 2.4 (0.8-4.0)

Formaldehyde 1% aq 2.1 (1.2-3.0)

Potassium dichromate 0.5% 2.1 (1.1-3.1)

Caine mix IIIc 10% 1.5 (1.0-2.0)

Quaternium 15 1% 1.3 (0.1-2.6)

Epoxy resin 1% 1.2 (0.1-2.3)

Mercapto mix 1% 1.1 (0-2.4)

Sesquiterpene lactone mix 0.1% 1.1 (0-2.1)

Parabens mix 16% 1.1 (0-2.3)

PTBP formaldehyde resin 1% 1.0 (0-3.0)

Mercaptobenzothiazole 2% 0.8 (0.3-1.22) In petrolatum unless otherwise indicated. a Combined prevalence (95% confidence interval) of positive reactions to 20 most

important contact allergens included in the European standard series based on data from seven test centres in the United

kingdom (UK). b methylchloroisothiazolinone and methylisothiazoline; c benzocaine 5%:cinchocaine 2.5%:amethyocaine

2.5%. Modified from Britton et al., (2003).

Jacob Taku Minang

16

The risk for developing ACD is related to the exposure concentration (dose per unit area) and

the inherent allergic potency of the individual chemical (Boukhman and Maibach, 2001).

Results from a number of epidemiological studies suggest that ACD is common, with 20-25%

of the European population reported to be sensitised to one or more contact allergens (Brasch

and Geier, 1997; Schnuch et al., 1997; Meding et al., 2001; Meding and Jarvholm, 2002). It is

worth noting that allergic contact dermatitis can be prevented upon appropriate diagnosis and

avoidance of the offending agent as illustrated by the important examples of ACD to nickel

and chromium (Lachapelle et al., 1980; Avnstorp, 1989; Jensen et al., 2002).

Atopic contact dermatitis

Atopic contact dermatitis refers to an immediate-type (IgE-mediated) eczematous allergic

contact reaction in an atopic person (Hannuksela, 1980). The terms ‘atopic dermatitis’,

‘immediate contact reaction’, ‘contact urticaria’ or ‘contact urticaria syndrome’ have been

used interchangeably in the literature to describe different manifestations of atopic dermatitis

(De Waard-van et al., 1998; Aalto-Korte and Makinen-Kiljunen, 2003). The inflammatory

response in atopic contact dermatitis appears within minutes to an hour after contact with an

eliciting substance and usually disappears within a few hours. The reaction could be described

as immunologic or non-immunologic depending on whether prior sensitisation to the

offending agent is required. Sensitisation most commonly occurs via the respiratory or

gastroinstestinal tracts, but can also occur through the skin, as is the case of latex and some

foods (Guillet et al., 2005). Allergen-specific IgE antibodies, produced during primary contact

with the offending agent, bind to high-affinity Fcε receptors on the surface of mast cells and

basophils. Subsequent exposure to the same sensitising agent (allergen) and interaction

between the allergen and allergen-specific IgE antibodies on mast cells and basophils leads to

cross-linking of the high-affinity Fcε receptors and the release of a panel of

pharmacologically active mediators (Di Lorenzo et al., 1997; reviewed in Jackola et al.,

2004).

Atopic dermatitis (or atopic eczema) is characterised by skin infiltration of T lymphocytes

expressing a Th2-type profile (IL4, IL-5 or IL-13) of cytokines (Herrick et al., 2003). The

Th2-type cytokine, IL-4, plays a central role in the antibody class switch to IgE production

(See ‘IL-4’ under ‘cytokines investigated in this study’). Itching, tingling or burning

accompanied by erythema are the weakest types of reactions. A local wheal and flare suggest

contact urticarial reaction. Generalised urticaria is less common but has been reported in cases


17

of contact with agents eliciting immunologic IgE-mediated contact urticaria (Reviewed in

Wuthrich, 1998). In addition to local skin symptoms, other organs are occasionally involved

giving rise to conjunctivitis, rhinitis, an asthmatic attack or anaphylactic shock (reviewed in

Harvel et al., 1994).

Irritant contact dermatitis

Irritant contact dermatitis refers to an eczematous reaction in the skin of external origin

where, in contrast to allergic contact dermatitis, no obvious eliciting allergens can be

identified (see section on ‘allergic contact dermatitis’) (Bryld et al., 2003; Duarte et al.,

2003). Depending on the nature of the irritant, the body region that is exposed or the duration

of exposure, the irritant contact dermatitis response can be referred to as; subjective, chronic

or due to chemical burns (Tupker, 2003; Smith et al., 2004; Pedersen et al., 2005). In the

past, the pathogenesis of irritant contact dermatitis was thought to be non-immunological

(Malten et al., 1979), however, it is now generally accepted that the immune system plays a

vital role in the induction and maintenance of irritant-induced skin reactions (Wakem et al.,

2000; Levin and Maibach, 2002; Lisby and Hauser, 2002). T lymphocytes expressing the

CD4+ phenotype have been shown to be the major skin infiltrating lymphocyte population in

irritant contact dermatitis (Willis et al., 1993; Kondo et al., 1996). These skin infiltrating

CD4+ T cells secrete mainly IFN-γ and IL-2 (Hoefakker et al., 1995; Kondo et al., 1996).

Noteworthy however, in contrast to allergic skin reactions, no immunological memory seems

to be involved in eliciting irritant contact dermatitis (Moed et al., 2004a) and the development

of irritant skin reactions does not require prior sensitisation (see preceding section on ‘allergic

contact dermatitis’).

Hapten immune system interaction in allergic contact dermatitis

Haptens are small organic molecules or metal ions that are non-immunogenic by themselves

and must be able to react to or bind to proteins (larger carrier molecules) to become contact

allergens (Landsteiner and Jacobs, 1935). Thousands of potential contact sensitizers exist.

However, parameters such as whether the hapten is released from compound mixtures,

whether people are exposed, if it permeates the skin or if it has a strong sensitising capacity,

narrow down the number of interesting haptens to a few dozens of high relevance (Wahlberg,

2001). The interaction between a hapten-carrier complex and the immune system and the

ensuing inflammatory response can be divided into two phases. There is a ‘sensitization

(induction) phase’ which describes the reactions (generally asymptomatic) following primary

Jacob Taku Minang

18

application of the hapten to the skin and then an ‘elicitation (effector) phase’ during which the

clinical symptoms of ACD are manifested upon re-exposure to the hapten.

The Sensitisation phase

Following primary application to the skin, DCs, mainly epidermal LCs that reside in normal

skin in a ‘resting’ functional state, take up the hapten and process it (Fig. 1). Hapten

application also results in activation of KCs, which together with LCs and other skin-cell

types secrete inflammatory cytokines such as IL-1β, TNF-α, IL-6, IL-12 and GM-CSF (Enk

and Katz, 1992b; Kimber and Cumberbatch, 1992; Kaplan et al., 1992).

Figure 1. Sensitisation phase of contact hypersensitivity. Topical application of contact allergen induces cytokine secretion by keratinocytes (KCs), langerhans cells (LCs) and other skin cell types. The secreted cytokines activate resting LCs and promote the migration of antigen (Ag)-carrying LCs towards regional lymph nodes (step 1). In the lymph node, LCs establish contact with naïve T cells (Tnaive) (step 2) and activate them via expression of Ag-MHC complexes along with costimulatory and adhesion molecules (step 3). Primed T cells (Tsens) alter their migration pathways and begin to recirculate through peripheral tissues (step 4). Modified from Grabbe and Schwarz, (1998) with permission from Elsevier. These cytokines activate more LCs and upregulate their expression of cell-surface molecules

(e.g MHC class I and II, B7, CCR7 etc) and secretion of cytokines and chemokines (e.g. IL-8)

(Enk et al., 1993). Activated antigen-carrying LCs migrate via afferent lymphatic vessels to

the paracortical zone of regional draining lymph nodes where they establish contact with and

activate T cells (Sozzani et al., 1995). Antigen-specific activation alters the migration

pathways of primed T cells through increased expression of skin homing markers such as

cutaneous lymphocyte antigen (CLA) leading to their recirculation through peripheral tissues

Dermis

Tsens


19

(Barker et al., 1991). Other APCs, such as dermal DCs have also been implicated in priming

of naïve T cells after epicutaneous contact with hapten (Morikawa et al., 1992).

The elicitation phase

Re-exposure to a hapten leads to the same initial direct effects on the skin as primary hapten

contact during sensitization (i.e. LC activation, proinflammatory effects). Activated LCs

release cytokines that activate endothelial cells and upregulate their expression of adhesion

molecules with a resultant attraction of leukocytes to the site of hapten application (Fig. 2).

Figure 2. Elicitation phase of contact hypersensitivity (CHS). Secondary hapten application on the skin leads to the activation of Langerhans cells (LCs) and subsequent secretion of proinflammatory cytokines (step 1). The released cytokines upregulate the expression of adhesion molecules on LCs and activate endothelial cells (step 2) with a resultant attraction of leukocytes to the site of hapten application (step 3). Among these are primed T cells (Tsens), which are activated upon antigen (Ag) presentation either by resident cells or by infiltrating APCs (step 4). Ag-specific T-cell activation (step 5) induces mediator release by hapten-specific T cells (step 6), which amplifies the inflammatory process leading to further accumulation of infiltrating cells (step 7), resulting in clinically manifest allergic contact dermatitis (ACD). The time between application of allergen and elicitation of ACD is approximately 24-72 h. Modified from Grabbe and Schwarz, (1998) with permission from Elsevier.

Among these are primed T cells, which are activated upon antigen presentation either by

resident cells or by infiltrating APCs and/or LCs (Ishii et al., 1994; Ishii et al., 1995).

Activated hapten-specific T cells induce mediator release (Ptak et al., 1991a). This amplifies

the response by generating an inflammatory process that leads to further accumulation of

infiltrating cells including neutrophils, mononuclear cells and antigen-nonspecific but MHC-

restricted late acting CD3+CD4+CD8- T cells (Ptak et al., 1991b; Moed et al., 2004b). These

cells are thought to be responsible for the eczematous reaction resulting in clinically manifest

ACD. γδ TcR expressing cells have also been shown to play a role in the elicitation of contact

7

Antigen binding and internalization Epidermis

Dermis

Jacob Taku Minang

20

hypersensitivity (Askenase et al., 1995). The time between application of allergen and

elicitation of ACD is approximately 24-72 h.

RELATED BACKGROUND

Biochemistry of transition (heavy) metals

Transition metals represent a block of elements (~50 metals) in the periodic table that show

great chemical similarities within a given period as well as within a given vertical group. This

contrasts to the representative elements whose chemistry changes markedly across a given

period as the number of valence electrons changes, with chemical similarities occurring

mainly within vertical groups (Zumdahl, 1997). This difference occurs because the last

electrons added for transition metals are inner (d or f) electrons. This inner d and f electrons

cannot participate easily in bonding, as can the valence s and p electrons of the representative

elements. In forming ionic compounds with nonmetals, the transition metals exhibit two

typical characteristics; 1) more than one oxidation state is often found (e.g Cr3+ and Cr6+), 2)

the cations are often complex ions i.e. species where the transition metal ion is surrounded by

two or more ligands. For example, the compound [Co(NH3)6]Cl3 contains the [Co(NH3)6]3+

cation and Cl- anions where NH3 serves as the ligand. When dissolved in water, the solid

behaves like any ionic solid; the cations and anions are assumed to separate and move about

independently.

Transition metals turn to produce geometrically highly defined coordination complexes with

four or six electron-donators such as nitrogen or oxygen in amino acid side chains of

appropriate proteins or peptides (Fausto da Silva and Williams, 2001; Zhang and Wilcox

2002). For example, a complex of Co2+ ions has a typical tetrahedral arrangement, Ni2+ and

Pd2+ a square planar tetra-coordinated arrangement (Fig. 3) and Cr3+ a six-ligand octahedral

arrangement.

Transition metals and allergic contact dermatitis

Although several transition metal ions are known to play a vital role in living organisms as

well as in industry, a number of transition metals and their compounds also present important

occupational and health hazards (Waalkes, 1995; Garner, 2004). Some of these metals are

well known contact allergens capable of inducing a delayed type hypersensitivity response in

susceptible individuals upon prolonged direct exposure.


21

++ L ++ ++

L-----------------L L-------------------L L-----------------L

Ni Ni Pd

L------------------L L--------------------L L------------------L

L

Nickel (II) Nickel (II) Palladium (II)

Square planar Tetrahedral Square planar

Figure 3. Examples of coordination bonds to transition metal salts. L refers to a ligand i.e an electron rich hetero-atom such as nitrogen, oxygen, sulphur or phosphorous capable of donating a pair of electrons to form a coordination bond with a metal ion.

The number of ligands and the geometry of the coordination complexes, formed between the

metal ions and the electron rich atoms in amino acid side chains of some proteins or peptides,

seem to be the major factors determining the allergenicity of these metals as well as their

cross reactivities (Lepoittevin, 2001). Transition metal ions in this context serve as haptens,

with high immunogenic potential, only when in complex with cellular or matrix proteins of

the skin (Walton, 1983; Gawkrodger et al., 1986; de Fine Olivarius and Menne, 1992).

Unlike the irreversible convalent bonds formed by classical haptens and their protein carriers,

the coordination complexes formed by transition metal ions (‘non-classical’ haptens), are

reversible and allow for the exchange of the allergenic metal ions between different acceptor

sites (Thierse et al., 2005). One metal may have allergenic properties at two or more oxidation

states e.g. while Cr6+ has been assumed to be the major form of chromium involved in ACD

and is used for patch testing, Cr3+ has also recently been shown to elicit ACD (Hansen et al.,

2003). The distribution in nature, uses in industry and oxidation state relevant to sensitisation

for each of the metals investigated in the studies reported in this thesis are reviewed below.

Ni2+ is a component of many different types of alloys including white gold, German silver,

nickel plating, monel solder, gold plating and stainless steel. Its presence in such a wide

variety of products makes it an especially common contactant difficult to avoid (Liden, 1992).

Ni2+ is the single most common cause of ACD (see Table 3) (Hansen et al., 2003; reviewed in

Garner, 2004). Jewelry is the main source of Au1+ exposure (often seen as dermatitis at the

site of jewelry contact e.g. earlobes, fingers) but amalgams used in gold dental restoration are

a major cause of sensitisation (Vamnes et al., 2000). Co2+ is found in abundance in our

Jacob Taku Minang

22

environment and used with e.g. Ni2+ in various alloys but also found in pigments and paints

(Liden and Wahlberg, 1994). Cr6+ is present in cement but also found in metal alloys with e.g.

Co2+ and Ni2+ (Liden and Wahlberg, 1994). Pd2+ on the other hand, is a precious metal used in

the telecommunications industry, dental alloys, high temperature solders and jewelry. White

gold may contain up to 20% Pd2+, and dental alloys may contain up to 10% Pd2+ (Vincenzi,

1995).

Molecular and cellular mechanisms underlying metal-induced allergic contact

dermatitis

A number of models have been proposed to explain metal-protein interactions underlying the

transport and delivery of metal ions (non-peptide hapten) to APC and their interactions with

HLA and TcRs. In the first model referred to as processing-independent presentation, a metal

ion (Ni2+ in this case) either directly interacts with endogenous or exogenous peptides and the

resulting Ni2+-peptide complex then binds to MHC molecules (Romagnoli et al., 1991) or on

the other hand, Ni2+ may complex directly to an MHC-bound peptide or to the MHC molecule

itself thus circumventing processing (Fig. 4, left panel) (van den Broeke et al., 1999). The

Ni2+-MHC/peptide complexes result in conformational changes of these proteins that may

create new epitopes (neoantigens), which may be recognized by T cells (Romagnoli et al.,

1991).

In the second model referred to as processing-dependent presentation, Ni2+ is thought to form

coordination bonds with membrane-bound or soluble proteins, principally human serum

albumin (HSA), on the skin via cysteine or histidine residues (Sadler et al., 1994). The Ni2+-

carrier protein complex is then taken up by epidermal APCs (mainly LCs), processed and

presented to T cells as Ni2+-peptide complexes on MHC molecules (Fig. 4, right panel)

(Moulon et al., 1995).

A third model has recently been proposed (Thierse et al., 2004; reviewed in Thierse et al.,

2005). Here the metal, Ni2+, is suggested to bind to the protein HSA (a known shuttling

molecule for Ni2+), and the presence of the HSA- Ni2+ complex in the vicinity of transient

contacts between TcR and APC-exposed HLA molecules is thought to facilitate a specific

transfer of Ni2+ from the protein (HSA) to high-affinity coordination sites created at the

TcR/HLA-interface.


23

Figure 4. Mechanism of metal ion (e.g Ni2+) presentation. Two pathways of metal ion presentation are outlined: A) To become a complete antigen, the metal ion binds directly to a MHC-bound peptide (processing-independent presentation). B) The metal ion forms coordinative bonds to cysteine or histidine residues of soluble or membrane-bound proteins. The modified proteins are taken up by antigen-presenting cells (APC), and are processed and presented to T cells as metal-peptide complexes on MHC molecules. Modified from Büdinger and Hertl, (2000) with permission from Blackwell Publishing.

Despite evidence showing HLA restriction of Ni2+ recognition by Ni2+-specific T cells, Ni2+-

induced ACD has not been associated with the distribution of any HLA alleles (Emtestam et

al., 1993; Moulon et al., 1998). However, a preference for certain TcR-Vβ elements by both

skin- and blood derived Ni2+-reactive T cells has been demonstrated (Werfel et al., 1997;

Cederbrant et al., 2003).

Cross reactivity versus co-sensitisation in metal-induced allergic contact dermatitis

Santucci et al., reported more frequent associated patch test positive reactions among

transition metals than between these metals and other substances normally included in the

standard patch test series (see ‘in vivo diagnosis of allergic contact dermatitis’) (Santucci et

al., 1996). This may be due to concurrent exposure to different metals (co-sensitisation) or

concomitant responses of T cell clones (cross reactivity) resulting from similar chemical

properties of the metals and the consequent interactions inside the skin. Furthermore,

sensitisation to one metal ion has been suggested to increase the chances of being sensitised to

additional metals (Brasch et al., 2001).

Coordination complexes formed by Ni2+ and Pd2+ ions display marked spatial (geometric)

conformational similarity as oppose to Co2+ or Cr3+ (see ‘Biochemistry of transition (heavy)

metals’). The antigen determinants created by either metal ions with certain matrix or cellular

proteins in the skin could be very similar and hence potentially interact with the same set of

Jacob Taku Minang

24

specific TcR/MHC complex. Indeed, Moulon et al., demonstrated cross-reactivity between

some Ni2+-specific T cell clones and Pd2+ but not Co2+ or Cr3+ (Moulon et al., 1995). Later

studies also showed patch test reactivity to Pd2+ to be almost exclusively found in subjects

with reactivity to Ni2+ (Gawkrodger et al., 2000). A strong Ni2+ response may thus be

indicative of potential Pd2+ reactivity. Subjects sensitised to one metal that induces a cell-

mediated response cross-reactive to other metals, would thus be generally assumed to stand

the risk of a relapse of contact dermatitis when exposed to the cross reacting metals.

Associated patch test reactivity to other transition metals has also been suggested to be due to

co-sensitisation (concurrent exposure). Wahlberg and co-worker, using the repeated open

application tests (ROATs) in guinea pigs, showed that animals induced by Co2+ reacted in the

patch test and ROATs with Co2+ but not Ni2+. Those induced with Ni2+ also reacted in patch

testing to Ni2+ but not to Co2+ and in the ROATs to Ni2+ and less to Co2+ (Wahlberg and

Liden, 2000). A significant number of subjects with Cr3+ and almost all with Co2+ reactivity

have been shown to also react with Ni2+ in the in vivo patch test (Hegewald et al., 2005;

Gawkrodger et al., 2000). It is worth noting that, isolated Co2+ reactivity is rare and reportedly

linked, in some instances, to possible Ni2+ contamination of Co2+ patch test material (Eedy et

al., 1991; Lisi et al., 2003).

Immune cells in metal-induced allergic contact dermatitis

Antigen presenting cells

The skin is the site of immediate contact with agents capable of inducing an ACD reaction.

The epidermal LC, originally described in 1868 by Paul Langerhans, is the only cell type in

normal epidermis that exhibits all accessory cell functions. LCs form a contiguous network

within the epidermis and constitute 2%-5% of the total epidermal cell population (reviewed in

Teunissen, 1992). LCs originate from CD34+ bone marrow progenitors that enter the

epidermis via the blood stream (Dieu et al., 1998). Their numbers in the epidermis is

maintained by local proliferation (Czernielewski and Demarchez, 1987). LCs express high

levels of molecules mediating antigen presentation (e.g MHC Class I and II, CD1), as well as

cellular adhesion and costimulatory molecules (e.g CD54, CD80 and CD86) (reviewed in

Romani and Schuler, 1992). Thus, LCs represent the ‘professional’ APCs in the skin (Nestle

and Burg, 1999) and play a pivotal role in the induction of cutaneous immune responses to

infectious agents as well as contact sensitizers (Kimber et al., 1998).


25

Epidermal KCs, fibroblasts, and infiltrating mononuclear cells (e.g macrohages that mature

from blood monocytes) can upregulate their expression of MHC class I or class II molecules

in the presence of LC derived IL-1β or TNF-α (Kondo and Sauder, 1995) and can hence serve

as ‘non’professional’ APC (Enk and Katz 1992b). Haptenised KCs produce IL-1β, TNF-α and

lymphocyte-attracting chemokines, like CXCL10 (IP-10) (Flier et al., 1999) thus amplifying

the ACD reactivity in the epidermis (Sterry et al., 1991).

CD4+ T cells

T cells expressing CD4 molecules recognize hapten in complex with MHC class II molecules

(Van Seventer et al., 1986). CD4+ T cells have been shown to have a clear preponderance in

cutaneous infiltrates during skin inflammatory responses induced by allergen contact. Hence

this T cell lymphocyte subset is mostly associated with the effector phase of the ACD reaction

(Moed et al., 2004b). Recently, a lot of attention has been focused on CD4+ CD25+ T cells,

defined as Tr cells, suggested to be critical for the outcome of the ACD response in humans

(Cavani et al., 1998; Cavani et al., 2000; Sebastiani et al., 2001; Cavani et al., 2003; Moed et

al., 2005).

CD8+ T cells

CD8+ T cells recognize hapten in complex with MHC class I molecules (Van Seventer et al.,

1986). Lipophilic haptens are generally associated with MHC class I molecules due to their

ability to directly penetrate into LC, conjugate with cytoplasmic proteins and be processed

along the ‘endogenous’ processing route (Kalish et al., 1994). However, metal (e.g Ni2+)-

specific CD8+ T cell clones have been successfully generated both from skin and peripheral

blood of contact allergic subjects (Cavani et al., 1998, Moulon et al., 1998; Traidl et al.,

2000). Thus, this suggests a possible mechanism of MHC class I presentation even for

hydrophilic metal ions. CD8+ T cells have been demonstrated in epidermal mononuclear cell

infiltrates during an ongoing skin inflammatory response to contact sensitisers (Zanni et al.,

1998). CD8+ T cells are thought to mediate skin inflammation through killing of hapten-

bearing target cells (Moulon et al., 1998; Traidl et al., 2000).

γδ T cells

In humans, less than 5% of T cells bear the γδ heterodimer, and the percentage of γδ T cells in

the lymphoid organs of mice has been reported to range from 1% to 3% (Miescher et al.,

1988). Surprisingly, γδ TcR expressing cells appear to represent a major T-cell population in

Jacob Taku Minang

26

the skin, intestinal epithelium, and pulmonary epithelium (Pawankar et al., 1996). The

localization of γδ T cells at epithelial sites, suggests that they are especially suited to combat

epidermal or intestinal antigens and thus form a surveillance system that monitors the external

milieu of the epithelial cells (Reviewed in Kabelitz et al., 2005). γδ TcR expressing cells have

been shown to play a role in the elicitation of contact hypersensitivity (Askenase et al., 1995)

probably in a non-antigen-specific and non-MHC-restricted manner (Ptak et al., 1991b; Dieli

et al., 1998).

Cytokines investigated in this study

In the present study we investigated metal-induced cytokine-expression profiles representing

different polarisations of the immune response; Th1- (IL-2 and IFN-γ), Th2-type (IL-4, IL-5

and IL-13) and T-regulatory (IL-10) (Table 1).

IFN-γ

IFN-γ, the key Th1-type cytokine, is involved in the induction or upregulation of cell adhesion

molecules (Dustin et al., 1988) and exerts inflammatory effects mainly through effects on

macrophages (Arenzana-Seisdedos et al., 1985). IFN-γ has been shown to be important in

protection against mycobacterial infections (Newport et al., 1996) through the induction of

tumour necrosis factor (TNF), NO- and H2O2. The latter involved in the killing of the

intracellularly living bacteria. Several reports point to a critical role for IFN-γ in the induction

and elicitation of metal-induced ACD (Kapsenberg et al., 1992; Traidl et al., 2000).

IL-2

IL-2, produced by activated CD4+ T helper cells (Carter and Swain, 1997) has been shown to

be a growth and survival factor for antigen primed CD4+ and CD8+ T cells as well as NK cells

(Horwitz et al., 2003). IL-2 has been described as both a Th0- and a Th1-type cytokine

(Mosmann and Sad, 1996; O’Garra, 1998) due to its pleiotropic growth promoting properties

on activated Th1-, Th2-, Tc1- and Tc2-cells. Increased production of IL-2 has been reported

in ex vivo stimulated PBMC cultures from subjects allergic to Ni2+ but not control non-


27

allergics (Falsafi-Amin et al., 2000; Jakobson et al., 2002; Lindemann et al., 2003) suggesting

a role, also for this cytokine in the ACD response to metals.

IL-4

IL-4 is the signature cytokine for Th2-type immune responses. It has been implicated in a

broad spectrum of biological responses which include; regulation of the differentiation of

naïve CD4+ T cells into a Th2 phenotype (O’Garra, 1998; Chen et al., 2004) and control of

humoral immune responses by regulating switching in B cells from IgM/G to IgE and IgG4, in

humans (Del Prete et al., 1988; Punnonen et al., 1993). IL-4 is a key cytokine in the

development of IgE-mediated allergic inflammation (Steinke and Borish, 2001). IL-4 has

been shown to cause erythema and induration when released in the skin (Asherson et al.,

1996; Rowe and Bunker, 1998) suggesting a role for IL-4 in the inflammatory response to

haptens by sensitised individuals upon subsequent exposure to the offending metal hapten.

IL-5

IL-5, considered to be a Th2-type cytokine, has been shown to play a role in the

differentiation and maturation of cells involved in IgE-mediated allergic reactions, such as

mast cells and eosinophils. IL-5 also inhibits certain macrophage functions (Sanderson, 1992)

and acts as a growth factor for IgA-producing B cells (Sonoda et al., 1989). Rustemeyer et al.

recently demonstrated elevated levels of IL-5 in PBMC cultures from Ni2+ allergic but not

control subjects after stimulation with Ni2+ (Jakobson et al., 2002; Rustemeyer et al., 2004).

However, the relationship between this and the inflammatory response in vivo is not yet

known.

IL-10

IL-10, formerly described as a Th2-type cytokine that functions by down regulating Th1-cell

activity, has been shown to be produced in comparable amounts by other cell types such as

monocytes, macrophages, DCs, mast cells and keratinocytes (Enk and Katz, 1992a; Moser

and Murphy, 2000). IL-10 displays immunomodulatory effects on both Th1 and Th2 cells

(Bettelli et al., 1998) by inhibiting the production of IL-1α, IL-1β, IL-6, IL-8, IL-12 and

TNF-α, as well as its own production, and by down regulating the expression of co-

stimulatory molecules required for appropriate antigen presentation (de Waal et al., 1991;

Jacob Taku Minang

28

Akdis and Blaser, 2001). IL-10 has been shown to be produced by activated keratinocytes

during the induction (sensitisation) phase of ACD and induce clonal anergy in hapten-specific

T cells via effects on langerhans cells (LC) (Enk et al., 1993) and keratinocytes (KC) (Curiel-

Lewandrowski et al., 2003). IL-10, produced by Ni2+-specific CD4+CD25+ Tr cell clones, has

also been shown to inhibit the ability of DCs to stimulate Ni2+-specific Th1 and Tc1 responses

(Cavani et al., 2000).

IL-13

IL-13, like IL-4, has been described as a Th2-type cytokine, which promotes switching in B

cells from IgM/G to IgE (the effector molecule in IgE-mediated allergy) and IgG4 in humans

(Zurawski and de Vries, 1994; Punnonen et al., 1997). IL-13 has been shown to exert anti-

inflammatory functions on monocoytes and macrophages (McKenzie et al., 1993).

Significantly higher IL-13 production has been demonstrated in cultures of PBMC from

subjects allergic to metal (Ni2+) or organic (methylisothiazolinones) haptens but not control

subjects (Jakobson et al., 2002; Masjedi et al., 2003). However, similar to IL-5, the

relationship between the magnitude of the IL-13 response in vitro and the ACD reaction in

vivo, in terms of the patch test reactivity remains to be elucidated.

In vivo diagnosis of allergic contact dermatitis

The present diagnosis of ACD relies on the patch test first described by Jadassohn in 1895

(reviewed in Lachapelle, 2001). The patch test is a provocation test where the skin is exposed

to a panel of haptens and the ensuing reaction is graded. The patch test is performed on

normal skin on the back and involves the administration of a standard series of the most

common contact allergens, which commonly include approximately 25-30 organic

compounds as well as metal salts (Brasch and Geier, 1997) (see Table 3). There are specially

designed patch test panels ‘tailor-made’ for individuals in different occupations/professions or

with different types of dermatological problems. For example, for assessment of ACD in

subjects with lichenoid reactions in the mouth, a dental series of haptens is often used and this

panel includes, in addition to Ni2+, Co2+ and Cr6+, various acrylates and metals such as Au1+,

Pd2+ and mercury (Hg2+). Small volumes of a defined concentration of the allergen in a

suitable vehicle (usually 5% petrolatum) are applied epicutaneously and then covered with

specific strips. The strips are removed 48 h after application and the results read, scored and

recorded on days 2 and 4 or days 3 and 6-7. Patch test results are read based on a scoring

system recommended by the International Contact Dermatitis Research Group (ICDRG)


29

(Table 4) (Wahlberg, 2001). Positive reactions are defined as the appearance of erythema and

oedema and with stronger reactions also papules and vesicles. Conclusive clinical diagnosis of

ACD is based not only on patch testing but also the history of the patient, assessment of

exposure as well as clinical examination.

Table 4. The scoring system for patch test reactions recommended by the International Contact Dermatitis Research Group (ICDRG) Score Definition Clinical manifestations + Weak positive reaction Erythema, infiltration, possible papules

++ Strong positive reaction:

Erythema, infiltration, papules, possible vesicles

+++ Extreme positive reaction: Intense erythema and infiltration and coalescing vesicles

- Negative reaction†

IR Irritant reactions of different types.

Discrete patchy erythema or homogenous erythema without infiltration, patchy follicular erythema*

? Doubtful reaction Faint macular or homogenous erythema, no infiltration

†Patients who show negative reaction to the allergens may be sensitive to substances other than those tested. Testing with complementary substances may be indicated. *Petechiae and follicular pustules are usually irritant and do not normally indicate allergy. It is sometimes very difficult to distinguish between an irritant reaction and a very weak positive reaction. Retesting maybe necessary to certify contact allergy. Modified from Wahlberg, 2001.

Apart from the potential risk for the patient to be sensitised due to direct skin exposure to the

contact allergens, a number of problems have been associated with the in vivo patch test. The

use of certain drugs, especially corticosteriods, or exposure to UV radiation has been reported

to result in negative patch test reactions in subjects with a well-documented history of ACD

(Bruze, 1986; reviewed in Wahlberg, 2001). Patch test results have also been shown to

fluctuate in women with different results obtained at different times during their menstrual

cycle (Hindsen et al., 1999). Most intriguing, complete discordant reactions have been

reported when subjects are patch tested at the same time point with the same contact allergen

preparation on opposite sides of the upper back (Bourke et al., 1999). Furthermore,

interpretation of patch test results, especially with regards to discriminating between a

negative, weak (+) or doubful (?) reaction, is very difficult and tends to be subjective and

depends on the observer’s experience (reviewed in Wahlberg, 2001). Frequent reports of

Jacob Taku Minang

30

false-positive and false-negative reactions are a direct consequence of some or all of these

drawbacks. There is therefore a need for reliable in vitro diagnostic test methods.

In vitro diagnosis of allergic contact dermatitis

A number of studies has been carried out aimed at developing in vitro assays that can replace

or complement the patch test. The lymphocyte transformation test (LTT) exploits the

proliferative responses of T cells upon in vitro activation with allergens (Nordlind, 1984;

Masjedi et al., 2003). However, the LTT has been shown to have a low specificity, i.e.

proliferative responses also reported in individuals with negative patch test reactions to

specific haptens and no history of ACD (von Blomberg-van der Flier et al., 1987; Lisby et al.,

1999). Results from recent studies aimed at characterising immune responses to metal and

organic haptens by cytokine profiling, suggest that the measurement of cytokine production in

response to contact allergens could be utilized as a diagnostic tool (Jakobson et al., 2002;

Masjedi et al., 2003; Rustemeyer et al., 2004).

Cytokine Detection Assays

The enzyme-linked immunosorbent assay (ELISA) and the enzyme-linked immunospot

(ELISpot) assay were used to define the cytokine expression profiles in metal-induced ACD

in the studies reported in this thesis. The ELISA assay is very useful in determining the

amount (concentration) of cytokines produced as a result of an immune response. The ELISA

was first described by Engvall and Perlmann (1972) and employs high protein binding

microtitre plates commonly in a 96-well format. There is a first step of adsorption of capture

monoclonal antibodies (mAb) specific to an epitope of the cytokine of interest. The plates are

subsequently incubated with supernatants from cells cultured in the presence or absence of

defined antigens. Serial dilutions of recombinant human cytokines are assayed in parallel to

obtain a standard curve. Thereafter the plates are incubated with biotinylated mAb specific to

a second epitope on the captured cytokines followed by enzyme-labelled streptavidin (SA).

The plates are then incubated with a substrate that forms a coloured soluble product. The

colour intensity is read and standard plots drawn using the concentrations of the recombinant

human cytokines from which the concentrations of the samples are extrapolated.

The ELISpot assay was first described as a method to determine the number of antibody-

producing B cells (Czerkinsky et al., 1983; Sedgwick and Holt, 1983). The technique was

later modified (Czerkinsky et al., 1988) for measurement of the frequency of antigen-specific


31

cytokine-producing cells and has become a useful method when elucidating immune

responses at the single cell level. The principle is similar to that of the capture ELISA. Just as

for ELISA, the assay involves a first step of adsorption of capture mAb to 96-well ELISpot

plates utilising membranes as the solid phase for attachment of the capture mAb. However,

instead of culture supernatants being used in the subsequent step, cells are incubated for

varying periods (depending on the cell population, type of stimuli or cytokine investigated) in

the plates and the mAb captures the cytokine produced. Detection of the captured cytokine is

subsequently achieved by incubation of a biotinylated mAb followed by enzyme-labelled SA.

Spots are generated by enzymatic cleavage of a substrate that yields an insoluble precipitate.

The spots, a footprint of the locally accumulated cytokine produced by one cell, can later be

counted using a dissection microscope or an image-analysis system. Since the ELISpot assay

detects single cytokine-producing cells, and thus is dependent only on a locally high

concentration of cytokine, it is possible to identify low frequencies of e.g. antigen-specific T

cells that together produce cytokine levels below the detection level of a regular capture

ELISA (Tanguay and Killion 1994; Ewen et al., 2001; Ekerfelt et al., 2002; Masjedi et al.,

2003).

Jacob Taku Minang

32

THE PRESENT STUDY

OBJECTIVES

The present study was undertaken with the overall objective of defining the relationship

between in vivo responses in terms of patch test reactivity and in vitro responses to metals

known to cause ACD in terms of cytokine production and to evaluate the potential of the

cytokine detection assays, ELISpot and ELISA, for diagnosis of metal-induced ACD. The

study had as specific objectives:

• To relate the in vitro reactivity to the in vivo reactivity to Ni2+ in order to assess if subjects

with different degrees of allergic reactions differ in their cytokine response profile and/or

magnitude [I]

• To investigate the role of IL-10 in the regulation of cytokine responses in ACD to Ni2+ [II]

• To define the profile and magnitude of cytokine responses to other common metal contact

allergens in vitro [III]

• To optimise the ELISpot assay for diagnostic purposes [IV]


33

METHODOLOGY

More detailed descriptions of the methods used in this thesis are found in the accompanying

papers (I-IV). Below is a brief description of the subjects, the reagents and the methods used

to obtain the results discussed in this thesis.

Study subjects:

Forty subjects participated in the study described in paper I. These included 30 female

subjects with a positive patch test to NiSO4 (i.e 10 subjects each with +3, +2 or +1 patch test

reactivity) and 10 healthy volunteers (control subjects; 9 female and 1 male) with no history

of contact allergy and a negative patch test result to NiSO4. The study reported in paper III

included 31 (28 female and 3 male) subjects with a positive patch test to one or more metal

haptens and five healthy volunteers (control subjects; 4 female and 1 male) with no history of

contact allergy and a negative patch test result to metals included in the standard as well as

dental patch test series. The metal-allergic individuals and their matched controls were

recruited from the Department of Dermatology and Venereology at Karolinska University

Hospital in Stockholm. Both studies were approved by the Ethics committee at the Karolinska

Hospital, Stockholm, Sweden (Ethical permission Dnr: 00-238).

Regular blood donors (n=40) registered at the Karolinska University Hospital, Stockholm,

Sweden; either with unknown allergic status or with a clinical history of Ni2+-allergy, as

stated by the subjects, provided buffy coats used in the studies reported in papers II and IV.

Subjects included in the studies gave their informed consent to participation

Diagnosis of contact allergy by the in vivo patch test (papers I and III):

The subjects were either patch tested with a standard patch test series including the metals

nickel (NiSO4; 5% in petrolatum), cobalt (CoCl2: 1 % in petrolatum) and chromium

(K2Cr2O7: 0.5 % in petrolatum) (papers I and III) or with a dental series including the

additional metals palladium (PdCl2: 2 % in petrolatum) and gold (Na3(Au(S2O3)2); 2 % in

petrolatum) (paper III). The metal haptens in petrolatum were applied by FinnChambers

(Epitest Ltd Oy, Tuusula, Finland) epicutaneously on the backs of the subjects for 48 h. The

patches were removed and the reaction read and scored using the scoring system

recommended by the International Contact Dermatitis Research Group (ICDRG) (Brasch and

Geier, 1997) (see ‘Table 4’ p30 on the ICDRG scoring system).

Jacob Taku Minang

34

Blood sample collection and processing:

Whole blood samples were obtained from metal-allergic and healthy control subjects by

venepuncture and collected in sterile heparinised glass vials and plasma samples separated

from the whole blood [papers I and III]. Buffy coats were obtained from regular blood donors

by centrifugation to remove the erythrocyte rich fraction (papers II and IV). PBMC were

separated from the whole blood (papers I and III) or buffy coats (papers II and IV) by density-

gradient centrifugation over Ficoll-Paque and kept frozen in liquid nitrogen until use.

IgE measurement (paper I):

Total plasma IgE levels (reference range 1.6-122 kU L –1) were measured with the Pharmacia

ImmunoCAP kit (Pharmacia Diagnostics, Uppsala, Sweden).

Measurement of cytokine levels in cell supernatants by ELISA (papers I, II and III):

The capture ELISA assay was used to determine the levels of cytokines in supernatants from

cell cultures with or without antigen stimulation.

Evaluation of the detection sensitivity of one-step and two-step reagents in capture ELISA

(paper IV):

Mouse anti-human IL-4, IL-13 and IFN-γ detection mAbs directly conjugated to the enzyme

alkaline phosphatase (ALP) were developed for use in a simplified ELISpot assay for

detecting allergen-induced cytokine production. The sensitivity of these directly conjugated

mAbs was compared to that using the same mAbs conjugated to biotin requiring a second

incubation step with a SA-ALP conjugate in ELISA. Briefly, ELISA plates were coated

overnight with mouse anti-human capture mAbs [82.4 (IL-4), IL13-I (IL-13) and 1-D1K

(IFN-γ)] (Mabtech, Nacka, Sweden) and subsequently with serial dilutions (1000 pg/ml to 2.0

pg/ml) of recombinant human IL-4, IL-13 (R&D Systems, Minneapolis, Minn., USA) or IFN-

γ (Bender MedSystems GmbH, Vienna, Austria). The detection mAbs [12.1 (IL-4), IL13-II

(IL-13) or 7-B-6-1 (IFN-γ) (Mabtech)] were either directly conjugated to ALP (one-step

reagent) or biotinylated (two-step reagent). Plates incubated with the one-step reagents were

incubated directly with the substrate [para-nitro-phenyl phosphate (pNPP)] whereas those

with the two-step reagent needed an extra incubation step with SA-ALP prior to incubation

with pNPP. The concentration of recombinant cytokine required to give an absorbance value

of 2.0 for either system was compared as an indicator for the detection sensitivity.


35

Enumeration of cytokine-producing cells by ELISpot:

ELISpot assays were performed using either polyvinyl diflouride (PVDF) plates

(MAIPS4510; Millipore Corp, Bedford, MA, U.S.A) pre-coated with capture mAbs

(Mabtech, Stockholm, Sweden) [paper I and IV] or PVDF plates (ELIIP10SSP: Millipore

Corp, Bedford, MA, U.S.A) coated with the capture mAbs overnight (a day before the assay)

[papers II, III and IV]. Cell suspensions in culture medium containing antigens at defined

concentrations were applied in triplicates to the plates; unstimulated cells were included in

each plate to assess spontaneous production. The cytokines captured were detected using

matched secondary mAbs either directly conjugated to ALP [paper IV] (Fig 5) or biotinylated

with subsequent addition of SA-ALP [papers I, II, III and IV]. Plates were developed by

incubation with bromo-chloro-indolyl phosphate/nitro blue tetrazolium-Plus substrate (Moss,

Inc., Pasadena, MD, U.S.A) and spots analysed as described previously (Masjedi et al., 2003).

Determination of optimal concentration of metal salts for stimulation of PBMC (paper III).

The potential of different concentrations of a panel of metal salts to induce non-specific

cytokine production or to exert toxic effects on PBMC in vitro i.e. suppression of mitogen-

induced cytokine production, were assessed using the standard ELISpot assay. The ELISpot

assay was set up using PBMC from non-allergic subjects with or without addition of the

different metal salts and the frequency of IL-2-, IL-4- or IL-13-producing cells determined. A

metal salt was defined as mitogenic at any given concentration if it induced a three-fold

increase in the spontaneous production of all three cytokines (i.e an increment of at least 10

spots per 2.5x105 input cells over spontaneous production) at the said concentration. The

toxicity of the metal salts was evaluated by adding PHA (PHA; 2 µg/ml) to parallel cultures

and any metal salt concentration resulting in ≥ 25 % reduction in PHA-induced cytokine

production was defined as toxic. Optimal concentrations for each metal salt (i.e

concentrations that would induce optimal cytokine production with no mitogenic or toxic

effects, as defined above) were determined using standard IL-2, IL-4 and IL-13 ELISpot

assays. However, unlike above, PBMC (3.0x105 input cells/well) from metal allergic subjects

were used.

Phenotypic characterisation of Ni2+-specific cytokine producing cells using depletion

experiments (paper II):

To determine the phenotype of Ni2+-specific cytokine-producing cells, different cell

populations were depleted or enriched in PBMC from Ni2+ reactive donors and the frequency

Jacob Taku Minang

36

of Ni2+-specific IL-4 producing cells and the levels of IFN-γ production determined. Briefly,

2.0 x 107 PBMC in sterile 2% FBS/PBS were incubated with 4.0 x 107 anti-human CD3, CD4

or CD8 (Dynal Biotech, Oslo, Norway) mAb coated beads. Cytokine production in cultures of

the different cell fractions (whole PBMC, CD4+ or CD8+ enriched or CD3+, CD4+ or CD8+

depleted) with or without 50 μM NiCl2.6H2O (Merck) or PHA (2 μg/ml; Orion Diagnostics)

was determined by ELISpot or ELISA as described above. As an additional control for cell

viability, the CD3 depleted cell fractions were stimulated separately with LPS (100 pg/ml;

Sigma Aldrich, St Louis, MO, USA) or anti-human CD3 mAb (100 ng/ml; Mabtech) and IL-

6, IL-10 and IFN-γ levels measured by ELISA in supernatants of 2 day cultures.

The cell depletions were confirmed by flow cytometry using phycoerythrin (PE)-conjugated

anti-human CD3, CD4 or CD8 mAb (Becton and Dickinson) staining as described previously

(Zuber et al., 2005) with saponin permeabilisation excluded from the protocol.

Intracellular cytokine measurement by flow cytometry (paper II):

PBMC, 6 x 106 cells/ml, in culture medium (Gibco BRL) with or without either NiCl2.6H2O

(50 μM; Merck, Darmstadt, Germany) or purified protein derivative (PPD; 5 μg/ml) (Orion

Diagnostics, Trosa, Sweden), were incubated in 24 well (0.5ml/well) flat-bottom tissue

culture plates (Costar Corp., Cambridge, MA, USA) at 37oc in a humidified atmosphere of

5% CO2 for 3 days. Six hours before the end of the cultures, 20 ng/ml of phorbol myristate

acetate (PMA) plus 1 μM ionomycin (Sigma Immunochemicals, St. Louis, MO, USA) were

added to the cultures. In order to promote the accumulation of de-novo synthesised cytokines

in the Golgi apparatus of the synthesising cell, Brefeldin A (BFA) (Sigma) at a 10 μg/ml final

concentration was added to the cell cultures at the same time point as PMA-ionomycin. After

stimulation, cells were processed for intracytoplasmic cytokine analysis as described

previously (Zuber et al., 2005). Briefly, to simultaneously stain the cells with directly

conjugated anti-cytokine and surface marker antibodies, 50 μl solutions of PE-labelled

antibodies to either human IL-4, IL-10 (both from Becton and Dickinson, Franklin Lakes, NJ,

USA) or IFN-γ (Mabtech) together with FITC labelled antibodies to either human CD4

(Mabtech) or CD8 (Becton and Dickinson) in saponin buffer were added to the cell

suspensions and the plates incubated at RT for 1 h. The labelled cells were washed once with

PBS containing 0.5% FCS, resuspended in 0.3 ml of the same buffer and immediately

analyzed by flow cytometry in a FACScalibur® (Becton and Dickinson) equipped with the

CellQuest® software. List mode data for 50,000 events in a “live gate” mode were acquired.


37

Single-cell cytokine production was evaluated after forward scatter (FSC) and side scatter

(SSC) gating on lymphocytes.

Statistical methods:

Cytokine responses between groups of subjects displaying different patch test reactivity or

controls [I and III] or between blood donors whose PBMC were reactive or not with Ni2+ [II

and IV] were compared using the Mann-Whitney U-test. The Wilcoxon matched pairs test

was used to compare responses by PBMC determined by the regular and a simplified ELISpot

protocol [IV]; the same test was use to compare the change in the Ni2+-induced cytokine

responses by Ni2+-reactive PBMC in the presence or absence of human recombinant IL-10

(rhIL-10) or anti-human-IL-10 (αh-IL-10) mAb respectively [II]. To evaluate the relationship

between observed parameters, correlations were computed using the Spearman rank-order

correlation coefficient rs [I, II and IV]. A multivariate correlation analysis was performed to

test for associations between multiple parameters (patch test, cytokine and IgE levels) [I]. For

comparison of antigen-specific responses, values with background (spontaneous production)

subtracted were used. A p-value < 0.05 was considered to be statistically significant. All tests

were performed using the software STATISTICA 5.1 (StatSoft, Tulsa, OK, USA).

Jacob Taku Minang

38

RESULTS AND DISCUSSION

Ni2+ induces a mixed cytokine response profile in PBMC from Ni2+-sensitised subjects and

this correlates with the in vivo patch test score (paper I):

We investigated, in the first study, if there is an association between specific cytokine profiles

or the magnitude of the cytokines induced by Ni2+ in vitro and the severity of allergic

reactivity in vivo. The in vivo reactivity to Ni2+, defined by the subject’s patch test reactivity

to NiSO4, was graded as +1 (weak), +2 (moderate) and +3 (strong) according to

recommendations by the ICDRG (Wahlberg, 2001). The frequency of Ni2+-specific cytokine

producing cells and the levels of Ni2+-induced cytokine production in PBMC from Ni2+-

allergic and control non-allergic subjects was determined by ELISpot and ELISA,

respectively.

Our results showed significantly higher levels of Ni2+-induced production of all cytokines

[Th1- (IFN-γ), Th2-type (IL-4, IL-5 and IL-13) and T regulatory (IL-10)] in subjects patch

test positive to NiSO4 as compared to the controls. Most notably, there was a significant

correlation between the degree of patch test reactivity [+1 (weak), +2 (moderate) or +3

(strong)] and the magnitude of the Ni2+-induced production of all the cytokines.

Earlier studies investigating the cytokines implicated in ACD to Ni2+ suggested a major role

for Th1-type cytokines in the ensuing detrimental inflammatory response (Sinigalia et al.,

1985; Silvennoinen-Kassinen et al., 1991; Kapsenberg et al., 1992). Data from more recent

reports suggest a mixed cytokine profile elicited by Ni2+ in PBMC from Ni2+-allergic subjects

(Falsafi-Amin et al., 2000; Jakobson et al., 2002; Lindemann et al., 2003). Our observation of

concommittant induction of Th1-, Th2- and Tr-type cytokines by Ni2+ in PBMC from Ni2+-

sensitised subjects is thus in agreement with these recent reports. However, unique to our

study, is the fact that we further categorised the Ni2+-allergic subjects based on their different

patch test reactivities (+1, +2, +3 or controls). We observed a positive correlation between the

magnitude of the cytokine responses in vitro and the degree of reactivity in the in vivo patch

test. Thus, Ni2+ induces a similar profile of cytokine responses in subjects with different

degrees of in vivo reactivity to Ni2+ but the magnitude of the cytokine response is positively

correlated with the degree of in vivo reactivity.


39

Noteworthy, however, despite the correlations observed between patch-test reactivity and

cytokines representing both Th1- and Th2-type responses, we observed an exclusive Th2-type

cytokine response to Ni2+ in several of the Ni2+-allergic subjects. Thus, in the context of

contemporary descriptions of Ni2+-induced ACD, our results suggest that Th2-type cytokines

may have simultaneous down-regulatory effects by counterbalancing the deleterious Th1

responses during the DTH response, characteristic of ACD. On the other hand, Th2-type

cytokines may exacerbate the proinflammatory responses in ACD already set off by Th1-type

cytokine-responses or in fact may have a direct role in the pathogenesis of ACD independent

of Th1-type cytokines as is the case in atopic allergies (Parronchi et al., 1992; McHugh et al.,

1994; Asherson et al., 1996; Rowe and Bunker, 1998; Liu et al., 2003).

To investigate if there existed any relationship between atopy and the exclusive Th2-type

Ni2+-induced cytokine response profile observed in a number of the Ni2+-allergic subjects,

total plasma IgE levels were measured. Interestingly, the IgE levels of the Ni2+–allergic

subjects were found to correlate negatively with the Ni2+-induced IFN-γ responses (p=0.026,

rs=-0.46, n=23) of these subjects. The total level of plasma IgE is a crude, but nevertheless

relevant correlate for the atopic status of an individual (Kasaian et al., 1995; Di Lorenzo et

al., 1997; Di Gioacchino et al., 2000; Jackola et al., 2004). Lower proliferative as well as IL-2

responses to Ni2+ have been reported in PBMC from subjects with ACD to Ni2+ with

concomitant atopic dermatitis but not in ACD subjects with no atopic dermatitis symptoms

(Buchvald et al., 2004). It is, thus, possible that the atopic status of a subject can influence

their cytokine response to contact allergens. However, due to the small number of Ni2+-

allergic subjects in this study that displayed high IgE levels, a possible relationship between

atopy and IFN-γ responses to Ni2+ can be best assessed in studies specifically designed with

this question in mind.

Diagnostic potential of in vitro cytokine responses to Ni2+ in relation to the in vivo patch

test (paper I):

Our finding of a higher magnitude of Ni2+-induced cytokine production by PBMC from Ni2+-

allergic compared to control subjects and the correlation to the in vivo patch test score

suggests a diagnostic value for these cytokine assays. We therefore attempted a definition of

positive and negative responses to Ni2+ by setting cut off values based on the responses

displayed by control subjects. The cut off criteria included both the cytokine increment (i.e

cytokine production in cultures with Ni2+ minus the unstimulated or spontaneous production)

Jacob Taku Minang

40

and the cytokine ratio or index (i.e ratio of cytokine production in cultures with Ni2+ and the

unstimulated or spontaneous production).

Using these cut off criteria, the number of patch positive subjects responding in one or more

of the assays was 23/30 (paper I). Eighteen out of the 20 subjects in the two groups with a

more clearly defined allergy to Ni2+ [strong (+3) or moderate (+2) patch test reactivity and a

clinical history of Ni2+ allergy] in contrast to only 5/10 of the weakly (+1) reactive subjects

were positive according to the cytokine assays. All control subjects were negative. Most

notably, the highest number of positive responses was found for the IL-4 ELISpot (n=19), IL-

13 ELISA (n=19) and IL-13 ELISpot (n=17) assays, respectively. This, thus, suggests a better

diagnostic value for the Th2-type (IL-4 and IL-13) cytokine asays as in vitro correlates for an

allergic reaction to Ni2+ as opposed to IFN-γ. This would be useful especially for diagnosing

ACD in Ni2+-allergic subjects with concurrent atopic symptoms especially since diminished

Th1-type cytokine responses to Ni2+ have been shown in this category of subjects (Buchvald

et al., 2004). Our findings are in line with recent results by Rustemeyer et al. reporting better

discriminaton between Ni2+-sensitised and nonsensitised subjects using IL-5, a Th2-type

cytokine, but not the Th1-type cytokine, IFN-γ (Rustemeyer et al., 2004). Worthy of mention

is the fact that, in the aforementioned study, IL-13 was not measured and IL-4 was used

instead as a supplement in combination with IL-7 in seven days PBMC cultures to boost the

levels of Ni2+-induced IL-5 production.

The cytokine ELISpot assay has been shown to have potential applications as a diagnostic

tool for infectious diseases such as Mycobacterium tuberculosis infection (Lalvani et al.,

2001a; Lalvani et al., 2001b). Our data put cytokine assays into focus as potential diagnostic

tools for contact allergy to Ni2+ that could replace or complement the currently used in vivo

patch test. While the magnitude of the IL-4 and IL-13 ELISpot responses was in accordance

with those reported by Jakobson et al. (2002) it was much higher than that reported by

Lindemann et al. (2003) wherein detectable but still low levels of Ni2+-induced IL-4 measured

by ELISpot could only be obtained after pre-incubation with Ni2+ for two days. The use of

nitrocellulose membrane plates instead of PVDF plates for the ELISpot assay (Schielen et al.,

1995) may be one of several possible explanations for this discrepancy. The Lindemann study

also identified IL-2 as an additional cytokine of interest for measurement of Ni2+-induced

responses by ELISpot.


41

With regard to the correlation between the in vivo patch test reactivity and the magnitude of

the in vitro cytokine responses to Ni2+, it is useful to highlight the uncertainties involved when

describing subjects with weak (+1) as oppose to moderate (+2) or strong (+3) positive

responses in the patch test. The reliability/reproducibility of a patch test score of +1 was

evaluated by performing a second patch test on some of the subjects with a historic patch test

score of +1 (unpublished data). This group included seven of the ten subjects with a historic

patch test reactivity score of +1; the difference in time between patch test 1 and 2 was

between 1-3 years and in one case 5 years. Four of these subjects did not respond to Ni2+ in

vitro according to the cut off criteria described above. Strikingly, only one of these subjects

had a positive test score (+1) in the second test. The other six were scored as negative (Table

5).

Table 5. Discordance between patch tests, clinical history and in vitro cytokine test in +1

subjects

Subject Patch

Test

Repeated

Patch test

History of

Ni2+ allergy

IL-4

ELISpot

IL-13

ELISA

13 +1 - - + +

19 +1 - - - -

20 +1 - + - -

21 +1 NDa) - + +

22 +1 - - - -

24 +1 - + - -

30 +1 ND - + -

31 +1 +1 + + +

40 +1 - - - +

41 +1 ND - - -

a) Not done.

Most reports of false positive or irritative reactions recorded as positive or vice versal at

different times, mostly involve patch test responses described as weakly positive (+1) (Bourke

et al., 1999; Hindsen et al., 1999; Wahlberg, 2001). In line with this, we observed a poor

concordance between patch test results, history of allergy to Ni2+ and in vitro reactivity to Ni2+

in the group of subjects with +1 reactivity in the patch test. Thus, the variability of the patch

Jacob Taku Minang

42

test results makes it difficult to compare patch test and in vitro test results for patch test

positive subjects with a score of +1.

The regulatory cytokine, IL-10, downregulates both Th1- and Th2-type cytokine responses

in Ni2+-induced ACD (paper II):

A key finding in the first study was our observation of elevated levels of Ni2+-induced

production of the regulatory cytokine IL-10 in the Ni2+-allergics which, just like the Th1- and

Th2-type cytokine responses, correlated with the degree of reactivity of the subjects in the in

vivo patch test. This is in line with another report (Cedebrant et al., 2003). However,

Rustemeyer et al. recently showed elevated levels of Ni2+-induced IL-10 mainly in tolerised

subjects (Rustemeyer et al., 2003). We therefore tried to define the role of IL-10 in the ACD

response to Ni2+ in relation to its effects on other cytokines.

PBMC used in this study were from blood donors with a stated history of ACD to Ni2+ and a

confirmed in vitro responsiveness to Ni2+ (ACD PBMC) and donors with unknown allergic

status but a confirmed non-responsiveness to Ni2+ in vitro (control PBMC). In vitro

responsiveness was determined using cut off definitions for IL-4 and IL-13 ELISpot as

defined in the first study. The levels of IL-13, IL-10 and IFN-γ production and the number of

IL-4-, IL-13- and IFN-γ-producing cells after Ni2+ stimulation were assessed by ELISA and

ELISpot, respectively in the presence or absence of a concentration range of rIL-10. As

expected, we observed significantly higher and correlated Th1-, Th2-type and regulatory (IL-

10) cytokine responses to Ni2+ in ACD PBMC as compared to control PBMC. Furthermore,

addition of rIL-10 to ACD PBMC reduced the levels of Ni2+-induced IL-13 and IFN-γ; rIL-10

had the most potent down regulatory effect on Ni2+-induced IFN-γ levels with a significant

reduction seen even at the lowest concentration of rIL-10 (0.2 ng/ml), which better reflects the

endogenous levels of IL-10 induced in response to Ni2+.

To assess if also the Ni2+-induced endogenous IL-10 exerts a similar suppressive effect on

cytokine responses to Ni2+ as seen with rIL-10, we measured Ni2+-induced cytokine

production in PBMC cultures with or without α-hIL-10 mAb (10 μg/ml). If Ni2+-induced IL-

10 indeed suppresses the production of other cytokines, we reasoned, then Ni2+-induced

cytokine responses would be enhanced upon its neutralisation. Whereas we observed a

significant increase in the levels of IFN-γ in response to Ni2+ in the presence of mAb to IL-10,


43

there was no net effect on the IL-13 levels or number of IL-4-, IL-13- or IFN-γ-producing

cells upon addition of the α-hIL-10 mAb. Furthermore, a greater enhancing effect of IL-10

neutralisation on the levels of production of IFN-γ was seen in donors with high (increment

above background >30 pg/ml) IL-10 production. It is worth mentioning that, the already low

or absent production of cytokines in response to Ni2+ by control PBMC was not affected by

addition of mAb to IL-10.

Cavani and co-workers had showed previously that IL-10 produced by Ni2+-specific

regulatory T cell clones markedly diminished the capacity of monocytes to stimulate Ni2+-

specific type 1 T cell clones (Cavani et al., 2000). Our data confirms a key regulatory role for

IL-10 in the immune reactivity to Ni2+. Noteworthy, is the fact that we used ex vivo stimulated

PBMC, which more closely reflect the in vivo situation. A number of studies have reported a

protective role for IL-10 in IgE-mediated allergic immune responses (Akdis et al., 1998;

Bellinghusen et al., 2001; Royer et al., 2001). Akdis et al., showed an increase of both Th1-

(IFN-γ) and Th2-type (IL-13) allergen-specific responses in PBMC from healthy subjects

upon neutralisation of endogenous IL-10 elicited by an atopic allergen (Akdis et al., 1998).

Although we also found a pleiotropic downregulatory effect on IL-4, IL-13 and IFN-γ

responses to Ni2+ by PBMC after adding rIL-10, only the IFN-γ production levels increased

significantly upon IL-10 neutralisation. This apparent difference in the number and type of

cytokines affected by neutralisation of endogenous IL-10 in Ni2+-induced ACD compared to

atopy has a bearing on the distinct cytokine profiles elicited in atopic and non-atopic as

opposed to ACD and non-ACD subjects. Allergens causing atopic allergy would generally

elicit a mixed (Th1-, Th2- and regulatory type) cytokine response in both atopic and non-

atopic subjects (Akdis et al., 2004), with a preponderance of the Th2 responses in the atopics

and the regulatory (IL-10) type responses in non-atopics. In ACD to Ni2+, elevated Ni2+-

induced IL-10 responses tend to coincide with increased Th1- and Th2-type responses in

allergic subjects but little or no responses in healthy controls. With respect to immune

responses to M. tuberculosis, a microbe known to induce a classical Th1-type (IFN-γ)

mediated DTH response (Lalvani et al., 2001a; Lalvani et al., 2001b), endogenous IL-10

neutralisation has been shown to induce strong CD8+ cytotoxic T cell-mediated lytic activity

together with an increased expression of costimulatory molecules on target cells (De la

Barrera et al., 2004). Endogenous IL-10 neutralisation may result in similar enhanced effector

Jacob Taku Minang

44

activity on T cells involved in ACD to Ni2+ consequent to the observed increase in the levels

of Ni2+-induced IFN-γ production.

The significant reduction both in the number of cytokine producing cells and the levels of the

secreted cytokines upon addition of rIL-10 may suggest a direct suppressive effect of IL-10

on individual cytokine producing cells. IL-10 has been shown to directly suppress T cell

activation by binding to a CD28-associated IL-10 receptor on T cells thereby inhibiting

tyrosine phosphorylation of this key costimulatory molecule (Akdis et al., 2000). IL-10

neutralisation on the other hand, seems to boost cytokine production per cell without any

major effect on spontaneous (non-specific) production; hence our observation of elevated

levels of Ni2+-induced IFN-γ production in the presence of α-hIL-10 mAb without any

corresponding increase in the number of IFN-γ-producing cells.

CD4+ T cells are the lymphocyte population in PBMC solely responsible for IL-4 and IFN-γ

production after Ni2+ stimulation (paper II):

In light of our demonstration of a downregulatory effect of IL-10 on Ni2+-induced production

of both Th1- and Th2-type cytokines coupled with reports of a direct suppressive effect of IL-

10 on T cells (Akdis et al., 2000), we next investigated the phenotype of the Ni2+-reactive

cells in PBMC i.e. the cells likely affected by the immunomodulatory effects of IL-10.

To achieve this aim, PBMC were enriched for CD4+ or CD8+ or depleted of CD3+, CD4+ or

CD8+ cells and the frequency of IL-4 producing cells and the levels of IFN-γ produced upon

Ni2+ stimulation were determined in these different cell fractions. Cell depletion was

confirmed by flow cytometry and 89-99% of the CD3+, CD4+ or CD8+ cells were depleted

using our protocol with the best purity obtained following CD4+ cell depletion. Depletion of

the cells expressing the pan T cell marker, CD3 (α/β and γ/δ TcR expressing T cells)

(Miescher et al., 1988), completely abrogated both IL-4 and IFN-γ production after Ni2+

stimulation. Depletion of the Th cell fraction only (i.e. CD4+ T cell) (De Gast et al., 1985) had

a similar effect on IL-4 and IFN-γ production. On the contrary, depletion of the CD8+ cell

fraction (T cytotoxic/effector cells) (Van Seventer et al., 1986) had a positive effect on the

magnitude of the Ni2+-induced production of both cytokines. Significant levels of Ni2+-

induced production of both cytokines were found in the CD4+ enriched cell fraction after

substraction of the spontaneous background. These were however generally lower than the


45

levels seen in the whole PBMC (data not shown). There was no detectable Ni2+-induced

production of either cytokine above background in the CD8+ enriched cell fraction.

Previous studies involving long-term cultured T-cell lines or clones have attributed Th1- and

Th2-type cytokine responses to Ni2+ to both CD4+ and CD8+ T cells (Moulon et al., 1998;

Traidl et al., 2000). However, Moed et al., using cells directly isolated from PBMC, recently

showed that only CD4+ CLA+ CD45RO+ and not CD8+ T cells proliferate and produce both

Th1- (IFN-γ) and Th2-type (IL-5) cytokines in response to Ni2+ (Moed et al., 2004b). In this

study, we investigated the effect of depletion of the CD3+, CD4+ or CD8+ populations from

PBMC, a more direct way of confirming the role of these cells, on Ni2+-induced Th1- and

Th2-type cytokine production. Our results suggest that CD4+ T cells may be the cell

population mainly responsible for production of both IL-4 and IFN-γ in response to Ni2+

stimulation.

Analysis of Ni2+-induced IL-10 production following depletion of different lymphocyte

fractions, unlike IL-4 and IFN-γ measurements, yielded inconclusive results. We obtained

decreased levels of IL-10 after depletion of either CD4+ or CD8+ cells in some instances but

in most instances no significant effect was observed. Earlier studies by other workers, using

cell clones, suggested that CD4+ CD25+ T regulatory cells are responsible for the bulk of

antigen-induced IL-10 production (Cavani et al., 2000; Sebastiani et al., 2001). The mixed

results we obtained could be due to the generally low levels of Ni2+-induced IL-10 production

(around 30 pg/ml or less) seen in most donors in this study. On the other hand, IL-10

production has been demonstrated in a wide variety of cell types, besides T cells (Enk and

Katz, 1992a; Royer et al., 2001). This could account for the lack of any clear observable

differences in the levels of Ni2+-induced IL-10 after depleting the T (CD3+, CD4+ or CD8+)

cell fractions.

In a nutshell, our data confirms and extends previous results by us and others showing that

PBMC from subjects with ACD to Ni2+ produce IL-10 upon Ni2+ stimulation and that the

magnitude of the Ni2+-induced IL-10 correlated positively with Th1- and Th2-type responses.

Further, endogenous IL-10 levels resulting from Ni2+ stimulation have a major down-

regulatory effect on the IFN-γ producting capacity of Ni2+-specific CD4+ T cells.

Jacob Taku Minang

46

Transitional metals commonly used in jewelry, metal alloys in dentistry and orthopaedics,

induce significant cytokine production in PBMC from contact allergic subjects (paper III):

We as well as others have shown that Ni2+ is capable of inducing significant production of

cytokines (both Th1- and Th2-type) in ex vivo stimulated PBMC from subjects with ACD to

Ni2+ but not control non-allergics (Jakobson et al., 2002; Lindemann et al., 2003; Rustemeyer

et al., 2004). However, there is very little information regarding the cytokine profiles induced

by other transition (heavy) metals also known to cause ACD. Knowledge on the profile and

the magnitude of the cytokines induced by other metals, apart from further elucidating the

immune mechanisms involved in metal-induced ACD, could be potentially exploited in

developing in vitro methods for the diagnosis of ACD. We therefore, investigated the profile

and magnitude of cytokine responses to a panel of metals in PBMC from subjects with a

history of ACD and confirmed in vivo patch test reactivity to one or several metals. The

frequency of IL-2-, IL-4- and IL-13-producing cells and the levels of production of IL-2, IL-

13 and IFN-γ in PBMC stimulated with a panel of metal salts was measured by ELISpot and

ELISA, respectively.

In agreement with our previous results (paper I), Ni2+ induced significantly higher cytokine

responses in PBMC from Ni2+ patch test reactive subjects compared to the non-allergic

controls; a mixed Th1-/Th2-type response was seen in most of these subjects. Use of either

nickel salts (NiCl2 or NiSO4) yielded a similar magnitude and profile of cytokine responses.

Furthermore, both Ni2+ salts induced cytokine production in PBMC from non-allergic control

subjects (non-specific cytokine-inducing effects) at concentrations greater than or equal to

100 μM. Both chromium salts (CrCl3/Cr3+ or K2Cr2O7/Cr6+) induced significant cytokine

[Th1- (IL-2) and/or Th2-type (IL-4 and IL-13)] production in PBMC from Cr6+ patch test

positive subjects but not in the non-allergic controls. Cr3+ tended to induce responses of

higher magnitude. Furthermore, we also showed that Cr6+ but not Cr3+ is toxic at very low

concentrations. Low concentrations of Cr6+ have previously been reported to induce cell death

or inhibition of cytokine production in vitro and also cause skin irritation (Reinhardt et al.,

1985; Li and Wang, 2002). Moreover, Hansen et al., reported induction of eczema at low

concentrations of Cr6+ but not Cr3+ (Hansen et al., 2003). Thus, our data shows that Cr3+ just

like Cr6+ induces significant cytokine production in vitro in PBMC from chromium patch test

positive subjects and suggests that the salt with Cr3+ may be preferable for use in stimulation

of PBMC for in vitro cytokine analysis. Just under half of the CoCl2 (Co2+) patch test positive

subjects responded to Co2+ with significant cytokine production to Co2+ in vitro. PBMC


47

responses to Co2+ stimulation generally involved production of either IL-2 alone or the

complete profile of cytokines (IL-4, IL-13 and IFN-γ). Co2+ has been shown to elicit irritative

and/or doubtful reactions in the patch test (Garner et al., 2004). This inherent irritative

property may account for some of the false positive results that have been reported. Thus in

vitro cytokine assays, which measure immunological memory, may be better at confirming a

true allergic reaction as well as discriminating this from any false positive reaction seen with

the patch test.

Besides our observation of positive in vitro response to Pd2+ with production of at least one

cytokine by PBMC from all subjects patch test positive to Pd2+ in the dental series, Pd2+ also

induced significant cytokine production in PBMC from some subjects tested only with the

standard series (excluding Pd2+ and Au1+). This suggests that the prevalence of ACD to Pd2+

may be considerably higher than assumed since most population/prevalence studies are based

on patch testing with the standard series (Brasch et al., 2001; Bryld et al., 2003; Hegewald et

al., 2005). Similar to Ni2+, Pd2+ induced non-specific cytokine production at concentrations

greater than 100 μM. PBMC from gold patch test positive subjects showed significantly

higher levels of IL-13 and IFN-γ responses to either salt of the metal (Na3(Au(S2O3)2/Au1+ or

HAuCl4/Au3+) in vitro compared to the non-allergic controls. Au1+ and Au3+ had toxic effects

on mitogen (PHA)-activated PBMC at concentrations above 50 μM and 70 μM, respectively.

However, despite the observed toxicity of Au1+ at lower concentrations compared to Au3+,

PBMC stimulation with Au1+, at concentrations below 50 μM, resulted in responses of higher

magnitude with a better distinction between subjects patch test positive to Au1+ and non-

allergic controls.

Overall, the results show that other metals included in the standard and/or dental series of the

in vivo patch test, just like Ni2+, are capable of inducing specific cytokine production in vitro.

Furthermore, our data point to potential future applications of the in vitro cytokine assays, as

complemetary or confirmatory tools, in the diagnosis of ACD.

In vitro evidence of in vivo cross-reactivity and/or co-sensitisation patterns (paper III):

A significant number of subjects showed positive reactions in the in vivo patch test to more

than one metal in both the standard and dental series. We therefore investigated if PBMC

from these subjects responded in vitro to all the metals to which they were reactive in the

patch test.

Jacob Taku Minang

48

Approximately 45% (4/9) and 75% (9/12) of those patch test positive to Cr6+ and Co2+,

respectively, were also reactive to Ni2+ in the in vivo patch test. Interestingly, in vitro cytokine

responses were induced by Ni2+ stimulation of PBMC from a higher percentage of these

subjects i.e about 90% (8/9) and 85% (10/12) for the Cr6+ and Co2+ patch test positive

subjects, respectively. Some of the subjects that were positive for both Ni2+ and Co2+ in the in

vivo patch test showed positive in vitro cytokine responses only to Ni2+. Furthermore, all Co2+

reactive subjects with concurrent Ni2+ patch test reactivity showed a positive in vitro response

also to Pd2+ with in vitro responses to Ni2+ and Pd2+ seen even in one Co2+ patch test positive

subject that failed to respond to Co2+ in vitro; a few Cr6+ reactive subjects also responded to

Au1+/Au3+, Co2+ and Pd2+. There have been reports of concurrent Co2+/Cr6+, Co2+/Ni2+,

Cr6+/Ni2+ or Ni2+/Co2+/Cr6+ responses in the in vivo patch test (Gawkrodger et al., 2000; Uter

et al., 2004; Hegewald et al., 2005). Our results therefore confirm, in vitro, the reported in

vivo reactivities observed with these metals but also suggest that there may exist low levels of

subclinical Ni2+-cross reactivity in subjects, patch test positive to metals other than Ni2+.

Some authors have even suggested that some cases of isolated Co2+ reactivity in the in vivo

patch test may be as a result of Ni2+ contamination of Co2+ patch test material (Rystedt, 1979;

Eedy et al., 1991; Lisi et al., 2003).

Less than half of the Au6+ reactive subjects showed a positive reaction to other metals in the

in vivo patch test (1/10 to Co2+ or Cr2+ and 3/10 to Ni2+ or Pd2+). While, a significant number

of subjects that reacted to other metals, but Au6+, in the in vivo patch test tended to also react

to Au1+/Au3+ in vitro, most subjects that were exclusively patch test positive to Au2+

responded only to Au1+/Au3+ in vitro. All except one of the Pd2+ patch test positive subjects

were also positive to Ni2+. Strikingly, all the Pd2+ patch test positive subjecst showed a strong

positive reaction to Ni2+ in vitro. In a few instances, Pd2+ reactive subjects showed a positive

reaction to Au1+/Au3+ or Co2+. A number of Pd2+ patch test negative subjects (i.e those tested

with the dental series) reacted to Pd2+ in vitro; all subjects that reacted with Pd2+ in vitro also

reacted with Ni2+ in vitro. Moulon et al., showed that Ni2+-specific T cell clones cross-react

with Pd2+ but not Co2+ or Cr2+ (Moulon et al., 1995). In addition, other studies have suggested

an overlap in responses to Ni2+ and Pd2+ in the in vivo patch test with Pd2+ reactivity found

almost entirely in subjects with an already confirmed reactivity to Ni2+ (Santucci et al., 1996;

Gawkrodger et al., 2000).


49

In conclusion therefore, apart from the potential of discriminating between metal-sensitized

and non-sensitized subjects, in vitro cytokine assays could be useful in identifying subjects

with multiple metal sensitisations. Furthermore, with respect to Pd2+ reactivity, our data

suggests that a strong Ni2+ response in vitro may be indicative of Pd2+ reactivity.

Optimisation of a simplified ELISpot assay for enumeration of allergen-specific cytokine-

producing cells (paper IV):

The above results together with data from previous studies by our group (Jakobson et al.,

2002), using the ELISpot assay point to a potential use of the cytokine ELISpot as a tool to

discrimate between Ni2+-sensitised and non-sensitised subjects. In paper IV, we aimed at

evaluating a simplified ELISpot protocol for use in the detection of Ni2+-specific cytokine

producing cells.

The simplified assay consisted of precoated plates and one-step (ALP-conjugated mAb)

detection reagents as opposed to overnight adsorption of capture mAb and two-step

(biotinylated mAb followed by SA-ALP) detection reagents in the conventional assay. To

validate the simplified assay, we compared the sensitivities of the one-step and two-step

detection reagents in parallel in a capture ELISA. The one-step detection system displayed a

higher sensitivity than the two-step detection system with approximately 1.8 and 1.4 times

lower concentrations of recombinant cytokine needed to obtain absorbance values of 2.0 for

IL-4 and IL-13 respectively. On the other hand, detection with the one-step detection system

required approximately 2.3 times higher concentration of the recombinant cytokine to yield

the same absorbance value as that obtained in the two-step detection system for IFN-γ.

Although, in order to increase the detection sensitivity, the use of enzyme-labelled detection

antibodies in sandwich-based immunoassays have to a large extent been replaced by the use

of two-step detection systems (Porstmann and Kiessig, 1992; Ahlborg and Paulie, 2002), the

detection sensitivity of the one-step and two-step detection systems investigated in this study

are quite comparable.

To further investigate the usefulness of the simplified compared with the regular ELISpot

protocol, the two protocols were used in parallel to analyse the number of antigen (Ni2+, TT or

PPD) or mitogen (PHA) –induced IL-4-, IL-13- and IFN-γ-producing PBMC from blood

donors with or without a history of ACD to Ni2+. Using both protocols, significantly higher

Ni2+-induced IL-4 and IL-13 responses in PBMC from the subjects with a history of ACD to

Jacob Taku Minang

50

Ni2+ (p=0.005 and p=0.02 for the simplified and regular IL-4 ELISpot and p=0.009 and

p=0.04 for the corresponding IL-13 ELISpot, respectively) were seen. There were no

significant differences between the two protocols in the detection of cells producing IL-4, IL-

13 or IFN-γ after activation with any of the three antigens. A highly significant correlation

was found between the two ELISpot protocols when the number of cytokine-producing cells

in response to all three antigens was compared for all the subjects (Fig. 6).

Several studies have highlighted the value of the cytokine ELISpot as a powerful tool to

monitor and measure antigen-specific T-cell responses (Czerkinsky et al., 1988; Smith et al.,

2001) in experimental research and recently also for possible diagnostic applications (Lalvani

et al., 2001a; Lalvani et al., 2001b). PBMC from Ni2+-reactive subjects have been shown,

using the cytokine ELISpot, to produce significantly higher levels of both Th1 and Th2

cytokines in response to Ni2+ compared to non-allergic subjects (Jakobson et al., 2002;

Lindemann et al. 2003). The observation in the present study of Ni2+-induced IL-4 and IL-13

responses only in PBMC from blood donors with a history of contact allergy to Ni2+ further

supports the use of the ELISpot assay as an alternative diagnostic test.

The number of cells spontaneously producing cytokine was found to be higher using the

regular ELISpot, especially for IFN-γ. This may suggest a higher detection capacity by the

regular protocol. However, the amount of spontaneously secreted cytokines is generally low

as opposed to antigen-induced cytokines (Ekerfelt et al., 2002; Mäkitalo et al., 2002). Thus, a

decreased detection of spontaneously producing cells combined with a comparable detection

of antigen-specific spots could be an advantage when distinguishing between antigen-specific

responses in responder and non-responder individuals since a better signal-to-noise (antigen-

specific/spontaneous) ratio can be obtained.

The development of a simplified ELISpot assay with an overall reduction of work and

incubation steps, apart from reducing the assay time makes it possible to use one batch of

precoated plates throughout a study, thus eliminating one source of inter-assay variation. The

simplified ELISpot assay protocol, compared to the regular assay format, would facilitate

larger clinical as well as experimental studies without any loss of sensitivity.


51

Ni2+-sensitised and non-sensitised subjects respond to recall antigens with a similar

cytokine profile (In progress and paper IV):

We have therefore established that subjects with ACD to Ni2+ but not control non-allergic

subjects show elevated Th1-, Th2- and regulatory cytokine responses to Ni2+. However,

whether this points to a tendency for subjects with ACD to Ni2+ to manifest a generalised

proinflammatory response to recall antigens has not been elucidated. We therefore

investigated if PBMC from subjects with a history of ACD and a confirmed in vitro reactivity

to Ni2+ displayed a tendency of responding with a particular cytokine pattern also to other

stimuli such as tetanus toxoid (TT) and purified protein derivative (PPD) from M.

tuberculosis or the mitogen PHA.

Whereas TT elicited production of IFN-γ, IL-13 as well as IL-4 at similar levels, PPD elicited

a higher number of cells producing IFN-γ than IL-13 and few IL-4-producing cells. However,

unlike Ni2+, TT and PPD induced significant cytokine responses in both subjects with a

history of ACD to Ni2+ and the controls with no significant difference between the ACD

subjects, as a group, and the controls. Strikingly, when the subjects with ACD to Ni2+ were

subdivided based on their patch test reactivity score (+3, +2 and +1) the TT-induced response

was generally the highest in the +1 group and lowest in +3 group (Fig. 7). Our observation of

a positive correlation between the patch test reactivity score and the magnitude of the in vitro

response to Ni2+ implies a generally higher frequency of Ni2+-specific memory T cells in ACD

subjects with a patch test score of +3 compared to those with +2, +1 or the controls. We

therefore speculated that an increase in the number of Ni2+-specific memory T cells due to a

more recent sensitisation and/or repeated exposure to Ni2+ results in a decrease in the net

proportion of memory T cells specific to recall antigens to which subjects were immunised

decades ago. In the absence of recent boosting or exposure to the pathogen a drop in the

frequency of circulating memory T cells specific to recall antigens would compensate for the

increase in the proportion of the Ni2+-specific memory T-cell pool; a sort of requirement for

immunological homeostasis (reviewed in Seder and Ahmed, 2003). This is not unreasonable

given the mean age of the subjects that participated in our studies (mostly women above 40

years of age).

Although we observed individual variations in the response to PHA, there was no general

tendency that the PHA-induced responses differed between subjects with ACD to Ni2+ and the

controls.

Jacob Taku Minang

52

These preliminary findings therefore show that subjects with ACD to Ni2+ are not prone to

elevated proinflammatory (both Th1- and Th2-type) cytokine responses to every antigen.

However, the degree of in vivo reactivity to Ni2+ may affect the balance of the memoy T cell

pool specific to recall antigens arising from old vaccination regiments. However, more studies

using a larger pool of antigens commonly used in vaccination regimens are needed to further

pursue this phenomenon.


53

GENERAL CONCLUSIONS AND PERSPECTIVES

The following general conclusions can be drawn from the results discussed in this review and

in the individual articles that follow;

• Subjects with different degrees of patch test reactivity to Ni2+ display a similar mixed

Th1/Th2-type cytokine response to Ni2+ in vitro. However, the magnitude rather than the

profile of the cytokine response is predictive of the outcome of the allergic reactivity in vivo

[paper I].

• Ni2+ induces elevated levels of IL-10 that correlate with the Th1- and Th2-type cytokine

responses in subjects with ACD to Ni2+ and endogenous IL-10 levels resulting from Ni2+

stimulation have a downregulatory effect on Th1-type (IFN-γ) responses by CD4+ T cells

[paper II].

• Other transition metals included in the standard and/or dental series of the in vivo patch test,

similar to Ni2+, also elicit a mixed Th1/Th2-type cytokine response in vitro in PBMC from

metal allergic subjects [paper III].

• From a diagnostic point of view, our data show that in vitro cytokine assays can be used to

determine the reactivity of ACD patients not only reactive with Ni2+ but also with other

metals such as Au1+/Au3+, Co2+, Cr3+/Cr6+ and Pd2+ [papers I, III and IV]. However, to further

improve the usefulness of the ELISpot technique in the diagnosing of ACD it would be

relevant to;

1) Evaluate methods of enhancing the cytokine responses in weak responders

using different mAbs that inhibit regulatory cytokines or activate costimulatory molecules.

2) Investigate similarities in cytokine responses to organic lipohilic and

hydrophilic contact allergens to ensure that an agreed-upon in vitro test would cover all or

most of the present series of compounds included in the patch test.

Jacob Taku Minang

54

Figure Legends

Figure 5

The simplified and regular ELISpot assays. There are only three major steps in the simplified

assay compared to five in the regular assay format. This saves time as well as reduces inter-

assay variation since one batch of pre-coated plates could be used for one large study (clinical

trial or experimental study).

Figure 6

Highly significant correlation between the simplified and regular ELISpot assay formats.

Cytokine responses (IL-4, IL-13 and IFN-γ) to all three antigens tested (Ni2+, TT and PPD) by

PBMC from all 14 blood donors were pooled and analysed by the Spearman rank-order

correlation coefficient rs. All analyses were done using values with background (spontaneous

responses) subtracted (i.e. 14 blood donors x 3 cytokines x 3 antigens = 126 data points).

Figure 7

Nickel (Ni2+)-allergic and non-allergic subjects display a similar cytokine profile in reponse to

a recall antigen. Peripheral blood mononuclear cells (PBMC), 2.5 x 105 cells/well, from Ni2+-

allergic subjects with different patch test scores (+3, +2 and +1; n=10 per group) and non-

allergic subjects (n=10) were cultured with or without Ni2+ or tetanus toxiod (TT). The

number of IL-4 (A), IL-5 (B) and IL-13 (C) producing cells were determined by ELISpot.

Results shown are the means + SD for each group. Statistical diffeences between the groups

following a following a Mann-Whitney U test are depicted with asterisks (∗p<0.05;∗∗ p<0.01;

∗∗∗p<0.001).

Jacob Taku Minang

58

ACKNOWLEDGEMENTS

The success of such an endearvour is impossible without the help and support of many people

and institutions. To arrive this point, I have had to stand on the ‘shoulders of giants’. First of

all, I wish to express my sincere gratitude to my supervisor, Marita Troye-Blomberg, for

giving me a window into the fascinating world of Immunological Research and for her

constant support and guidance. My co-supervisor, Niklas Ahlborg, is greatly appreciated for

introducing me to the concept of metal (hapten)-induced immune responses and for his

unlimited patience and dedication to work. Many thanks to my second co-supervisor, Lena

Lundeberg, for all the work with organising the patients that participated in the studies

reported in this thesis as well as for the excellent academic discussions.

Thanks to my co-authors, Iréne Areström, Bartek Zuber and Gun Jönsson.

Many thanks to the seniors at the department of Immunology, Klavs Berzins, Carmen

Fernandez, Manuchehr Abedi-Valugerdi, Eva Sverremark-Ekström and Eva Severinson

for sharing their wide knowledge of immunology with me. I’m particularly grateful to Peter

Perlmann (RIP) and Hedvig Perlmann for their encouragement and their constant presence

that tells me to ‘keep going, there is no end to this’.

Former students; Salah Eldin Farouk for being my first instructor in the Laboratory, Ben

Adu Gyan thanks for opening your doors to me when I just arrived and for being such an

elder brother, Ankie Söderlund, Monika Hansson, Caroline Ekberg, Elizabeth Hugosson,

Ahmed Bolad, Mounira Djerbi and Valentina Screpanti thanks for being such an

inspiration, Alice Nyakeriga and Ariane Rodríquez Muňoz my two elder sisters at the

department, Qazi Khaleda Rahmen for all the challenging questions and the many

invitations for lunch, Gawa Bidla for being there right from the start, Shiva S. Esfahani,

Izaura Ross, Karin Lindroth and Piyatida Tangteerawatana.

To all my Colleagues (current) at the department of Immunology; Khosro Masjedi for the

very incisive questions and interesting discussions, Nina-Maria Vasconcelos for being such a

kind person, Esther Julián, Anna-Karin Larsson, Manijeh Vafa, Anna Tjärnlund,

Halima Balogun, Petra Amoudruz, John Arko-Mensah, Norra Bachmayer,Yvonne

Sundström, Shanie S. Hedengren, Camilla Rydström, Elisabeth Israelsson, Amilcar


59

Reis, Nnaemeka Iriemenam, Magdi Ali, Nancy Awah, Jacqueline Calla and Kulachart

Jangpatarapongsa, I say thank you for the collaboration, exciting scientific discussions and

great times out of the department.

Many thanks to Margareta Hagstedt and Ann Sjölund for all the support on the bench.

Thanks to former, Gunilla Tillinger and Suzanne Åsbrink, and present, Gelana Yadeta,

secretaries of the department of Immunology for your readiness to help every time I knocked

on your office door. To the team at the MIM secretariate, Andreas Heddini, Olle Terenius

and Anna-Leena, I say, thanks for the nice discussions and for being part of every party!

The entire group at Mabtech (www.mabtech.com) is greatly appreciated for creating such a

stimulating and humane working environment.

Dr Achidi Eric Akum is gratefully acknowledged for introducing me into the world of

biomedical research.

My sincere thanks to Dyllis M. Sefeh (min kärlek, andra halva och hjärta), you have been

such a great source of support especially at moments that I felt the world was crashing on me!

Many thanks to my siblings and their families; Ni Joe, Ni John (RIP), Ni Tom, Ni Willie, Ni

Peter, sister Mary, sister Vero, Dr Paul, Maggie, their spouses and all my nieces and

nephews for their moral support. To my cousins; Victor N. Cheo, Fidelis C. Fru and Eric Y.

Tagha, I say thanks for your support and for the wonderful trips around Europe.

Special tribute to my dad, pa Alex Minang (RIP) and mum, mami Margaret Atih Minang

for pulling it off with such a full squad!

Finally, thanks to the almighty for giving me the strength to carry on.

The studies reported in this thesis were supported by grants from the Swedish Agency for

Innovation Systems (VINNOVA), the Swedish Council for Working Life and Social

Research, the Vårdal foundation for Health Care Sciences and Allergy Research (Stockholm),

The Asthma and Allergy Foundation and the Swedish Research Council.

http://www.mabtech.com/

Jacob Taku Minang

60

REFERENCES

Aalto-Korte, K. and Makinen-Kiljunen, S. 2003. Specific immunoglobulin E in patients with immediate persulfate hypersensitivity. Contact Dermatitis 49:22-25.

Abbas, A. K., Murphy, K. M., Sher, A. 1996. Functional diversity of helper T lymphocytes.

Nature 383:787-793. Ahlborg, N. and Paulie, S. 2002. ELISA (Enzyme-linked immunosorbent assay) Wiley

Encyclopedia of Mol Med 5:1150-1153. Akdis, C. A. and Blaser, K. 2001. Mechanisms of interleukin-10-mediated immune

suppression. Immunol 103:131-136. Akdis, C. A., Blesken, T., Akdis, M., Wüthrich, B., Blaser, K. 1998 Role of Interleukin 10 in

Specific Immunotherapy. J Clin Invest 102:98-106. Akdis, C. A., Joss, A., Akdis, M., Faith, A., Blaser, K. 2000. A molecular basis for T cell

suppression by IL-10: CD28-associated IL-10 receptor inhibits CD28 tyrosine phosphorylation and phosphotidylinositol 3-kinase binding. FASEB J 14:1666-1668.

Akdis, C., Verhagen, J., Taylor, A., Karamloo, F., Karagiannidis, C., Crameri, R., Thunberg,

S., Deniz, G., Valenta, R., Fiebig, H., Kegel, C., Disch, R., Schmidt-Weber, C. B., Blaser, K., Akdis, C. A. 2004. Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells. J Exp Med 99:1567-1575.

Akira, S., Takeda, K., Kaisho, T. 2001. Toll-like receptors: critical proteins linking innate and

acquired immunity. Nat Immunol 2:675-680. Aktas, E., Akdis, M., Bilgic, S., Disch, R., Falk, C. S., Blaser, K., Akdis, C., Deniz, G. 2005.

Different natural killer (NK) receptor expression and immunoglobulin E (IgE) regulation by NK1 and NK2 cells. Clin Exp Immunol 140:301-309.

Arenzana-Seisdedos, F., Virelizier, J. L., Fiers, W. 1985. Interferons as macrophage-

activating factors. III. Preferential effects of interferon-gamma on the interleukin 1 secretory potential of fresh or aged human monocytes. J Immunol 134:2444-2448.

Asherson, G. L., Dieli, F., Sireci, G., Salerno, A. 1996. Role of IL-4 in delayed type

hypersensitivity. Clin Exp Immunol 103:1-4. Askenase, P. W., Szczepanik, M., Ptak, M., Paliwal, V., Ptak, W. 1995. Gamma delta T cells

in normal spleen assist immunized alpha beta T cells in the adoptive cell transfer of contact sensitivity. Effect of Bordetella pertussis, cyclophosphamide, and antibodies to determinants on suppressor cells. J Immunol 154:3644-3653.

Avnstorp, C. 1989. Prevalence of cement eczema in Denmark before and since addition of

ferrous sulfate to Danish cement. Acta Derm Venereol 69:151-155.


61

Baecher-Allan, C., Brown, J. A., Freeman, G. J., Hafler, D. A. 2001. CD4+CD25high regulatory cells in human peripheral blood. J Immunol 167:1245-1253.

Ball, E. J. and Stastny, P. 1984. Antigen-specific HLA-restricted human T-cell lines. II. A

GAT-specific T-cell line restricted by a determinant carried by an HLA-DQ molecule. Immunogenetics. 20:547-564.

Barker, J. N., Mitra, R. S., Griffiths, C. E., Dixit, V. M., Nickoloff, B. J. 1991. Keratinocytes

as initiators of inflammation. Lancet 337:211-214. Bellinghusen, I., Brand, U., Steinbrink, K., Enk, A. H, Knop, J., Saloga, J. 2001. Inhibition of

human allergic T-cell responses by IL-10-treated dendritic cells: differences from hydrocortisone-treated dendritic cells. J Allergy Clin Immunol 108: 242-249.

Bendelac, A. and Schwartz, R. H. 1991. Th0 cells in the thymus: the question of T-helper

lineages. Immunol Rev123: 169-188. Bendiner, E. 1981. Baron von Pirquet: the aristocrat who discovered and defined allergy.

Hosp Prac 16:137–144. Bettelli, E., Das, M. P., Howard, E. D., Weiner, H. L., Sobel, R. A., Kuchroo, V. K. 1998. IL-

10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of IL-10- and IL-4-deficient and transgenic mice. J Immunol 161:3299-3306.

Boukhman, M. P. and Maibach, H. I. 2001. Thresholds in contact sensitization: immunologic

mechanisms and experimental evidence in humans--an overview. Food Chem Toxicol 39:1125-1134.

Bourke, J. F., Batta, K., Prais, L., Abdullah, A., Foulds, I. S. 1999. The reproducibility of

patch tests. Br J Dermatol 140:102-105. Braciale, T. J., Morrison, L. A., Sweetser, M. T., Sambrook, J., Gething, M. J., Braciale, V. L.

1987. Antigen presentation pathways to class I and class II MHC-restricted T lymphocytes. Immunol Rev 98:95-114.

Bradding, P., Feather, I. H., Wilson, S., Bardin, P. G., Heusser, C. H., Holgate, S. T.,

Howarth, P. H. 1993. Immunolocalization of cytokines in the nasal mucosa of normal and perennial rhinitic subjects. The mast cell as a source of IL-4, IL-5, and IL-6 in human allergic mucosal inflammation. J Immunol 151:3853-3865.

Brasch, J., Geier, J. 1997. Patch test results in schoolchildren. Results from the Information

Network of Departments of Dermatology (IVDK) and the German Contact Dermatitis Research Group (DKG). Contact Dermatitis 37:286-293.

Brasch, J., Uter, W., Geier, J., Schnuch, A., and the German Contact Dermatitis Research

Group and the Information Network of Departments of Dermatology in Germany. 2001. Associated positive patch test reactions to standard contact allergens. Am J Contact Dermatitis 12:197-202.

Jacob Taku Minang

62

Britton, J. E. R., Wilkinson, S. M., English, J. S. C., Gawkrodger, D. J., Ormerod, A. D., Sansom, J. E., Shaw, S., Statham, B. 2003. The British standard series of contact dermatitis allergens: validation in clinical practice and value for clinical governance. Br J Dermatol 148:259-264.

Bruze, M. 1986. Seasonal influence on routine patch test results. Contact Dermatitis 14:184. Bryld, L. E., Hindsberger, C., Kyvik, K. O., Agner, T., Menne, T. 2003. Risk factors

influencing the development of hand eczema in a population-based twin sample. Br J Dermatol 149:1214-1220.

Buchvald, D. and Lundeberg, L. 2004. Impaired responses of peripheral blood mononuclear

cells to nickel in patients with nickel-allergic contact dermatitis and concomitant atopic dermatitis. Br J Dermatol 150:484-492.

Büdinger, L. and Hertl, M. 2000. Immunologic mechanisms in hypersensitivity reactions to

metal ions: an overview. Allergy 55:108-115. Carter, L. L. and Swain, S. L. 1997. Single cell analyses of cytokine production. Curr Opin

Immunol 9:177-182. Cavani, A., Mei, D., Guerra, E., Corinti, S., Giani, M., Pirrotta, L., Puddu, P., Girolomoni, G.

1998. Patients with allergic contact dermatitis to nickel and nonallergic individuals display different nickel-specific T cell responses. Evidence for the presence of effector CD8+ and regulatory CD4+ T cells. J Invest Dermatol 111:621-628.

Cavani, A., Nasorri, F., Ottaviani, C., Sebastiani, S., De Pita, O., Girolomoni, G. 2003.

Human CD25+ regulatory T cells maintain immune tolerance to nickel in healthy, nonallergic individuals. J Immunol 171:5760-5768.

Cavani, A., Nasorri, F., Prezzi, C., Sebastiani, S., Albanesi, C., Girolomoni, G. 2000. Human

CD4+ T lymphocytes with remarkable regulatory functions on dendritic cells and nickel-specific Th1 immune responses. J Invest Dermatol 114:295-302.

Cederbrant, K., Anderson, C., Andersson, T., Marcusson-Stahl, M., Hultman, P. 2003.

Cytokine production, lymphocyte proliferation and T-cell receptor Vbeta expression in primary peripheral blood mononuclear cell cultures from nickel-allergic individuals. Int Arch Allergy Immunol 132:373-379.

Cederbrant, K., Hultman, P., Marcusson, J. A., Tibbling, L. 1997. In vitro lymphocyte

proliferation as compared to patch test using gold, palladium and nickel. Int Arch Allergy Immunol 112:212-217.

Chen, L., Grabowski, K.A., Xin, J. P., Coleman, J., Huang, Z., Espiritu, B., Alkan, S., Xie, H.

B., Zhu, Y., White, F. A., Clancy, J. Jr., Huang, H. 2004. IL-4 Induces Differentiation and Expansion of Th2 Cytokine-Producing Eosinophils. J Immunol 172:2059-2066.

Chen, W., Jin, W., Hardegen, N., Lei, K. J., Li, L., Marinos, N., McGrady, G., Wahl, S. M.

2003. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 198:1875-1886.


63

Chien, Y. H., Jores, R., Crowley, M. P. 1996. Recognition by gamma/delta T cells. Annu Rev

Immunol 14:511-532. Coffman, R. L., Lebman, D. A., Rothman, P. 1993. Mechanism and regulation of

immunoglobulin isotype switching. Adv Immunol 54:229-270. Coffman, R. L. and von der Weid, T. 1997. Multiple pathways for the initiation of T helper 2

(Th2) responses. J Exp Med 185:373-375. Croft, M. and Swain, S. L. 1992. Analysis of CD4+ T cells that provide contact-dependent

bystander help to B cells. J Immunol 149:3157-3165. Curiel-Lewandrowski, C., Venna, S. S., Eller, M. S., Cruikshank, W., Dougherty, I., Cruz, P.

D, Jr., Gilchrest, B. A. 2003. Inhibition of the elicitation phase of contact hypersensitivity by thymidine dinucleotides is in part mediated by increased expression of interleukin-10 in human keratinocytes. Exp Dermatol 12:145-152.

Czerkinsky, C., Andersson, G., Ekre, H. P., Nilsson, L.A., Klareskog, L., Ouchterlony, Ö.

1988. Reverse ELISPOT assay for clonal analysis of cytokine production: I. Enumeration of gamma-interferon-secreting cells. J Immunol Methods 25:29-36.

Czerkinsky, C. C., Nilsson, L. Å., Nygren, H., Ouchterlony, Ö., Tarkowski, A. 1983. A solid-

phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 65:109-121.

Czernielewski, J. M. Demarchez, M. 1987. Further evidence for the self-reproducing capacity

of Langerhans cells in human skin. J Invest Dermatol 88:17-20. De Fine Olivarius, F. and Menne, T. 1992. Skin reactivity to metallic cobalt in patients with a

positive patch test to cobalt chloride. Contact Dermatitis 27:241-243. De Gast, G. C., Verdonck, L. F., Middeldorp, J. M., The, T. H., Hekker, A., vd Linden, J. A.,

Kreeft, H. A., Bast, B. J. 1985. Recovery of T cell subsets after autologous bone marrow transplantation is mainly due to proliferation of mature T cells in the graft. Blood 66:428-431.

De la Barrera, S., Aleman, M., Musella, R., Schierloh, P., Pasquinelli, V., Garcia, V., Abbate,

E., Sasiain Mdel, C. 2004. IL-10 down-regulates costimulatory molecules on Mycobacterium tuberculosis-pulsed macrophages and impairs the lytic activity of CD4 and CD8 CTL in tuberculosis patients. Clin Exp Immunol 138:128-138.

Del Prete, G., Maggi, E., Paola, P., Ghretien, I., Tiri, A., Macchia, D., Banchereau, J., De

Vreis, J.E., Romagnani, S. 1988. IL-4 is an essential factor for the IgE synthesis induced in vitro by human T cell clones and their supernatants. J Immunol 140:4193-4198.

Dent, L. A. 2002. For better or worse: common determinants influencing health and disease in

parasitic infections, asthma and reproductive biology. J Reprod Immunol 57:255-272.

Jacob Taku Minang

64

Derocq, J. M., Segui, M., Poinot-Chazel, C., Minty, A., Caput, D., Ferrara, P., Casellas, P. 1994. Interleukin-13 stimulates interleukin-6 production by human keratinocytes. Similarity with interleukin-4. FEBS Lett 343:32-36.

De Waal Malefyt, R., Abrams, J., Bennett, B., Figdor, C. G., de Vries, J. E. 1991. Interleukin

10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med 174:1209-1220.

De Waard-van der Spek, F. B., Elst, E. F., Mulder, P. G., Munte, K., Deviller, A. C., Oranje,

A. P. 1998. Diagnostic tests in children with atopic dermatitis and food allergy. Allergy 53:1087-1091.

Dhodapkar, M. V. and Steinman, R. M. 2002. Antigen-bearing immature dendritic cells

induce peptide-specific CD8(+) regulatory T cells in vivo in humans. Blood 100:174-177. Dieli, F., Ptak, W., Sireci, G., Romano, G. C., Potestio, M, Salerno, A, Asherson, G. L. 1998.

Cross-talk between V beta 8+ and gamma delta+ T lymphocytes in contact sensitivity. Immunology 93:469-477.

Dieu, M. C., Vanbervliet, B., Vicari, A., Bridon, J. M., Oldham, E., Ait-Yahia, S., Briere, F.,

Zlotnik, A., Lebecque, S., Caux, C. 1998. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med 188:373-386.

Di Gioacchino, M., Cavallucci, E., Di Stefano, F., Verna, N., Ramondo, S., Ciuffreda, S.,

Riccioni, G., Boscolo, P. 2000. Influence of total IgE and seasonal increase of eosinophil cationic protein on bronchial hyperreactivity in asthmatic grass-sensitized farmers. Allergy 55:1030-1034.

Di Lorenzo, G., Mansueto, P., Melluso, M., Morici, G., Norrito, F., Esposito, Pellitteri, M., Di

Salvo, A., Colombo, A., Candore, G., Caruso, C. 1997. Non-specific airway hyperresponsiveness in mono-sensitive Sicilian patients with allergic rhinitis. Its relationship to total serum IgE levels and blood eosinophils during and out of the pollen season. Clin Exp Allergy 27:1052-1059.

Doherty, P. C. and Zinkernagel, R. M. 1975. A biological role for the major

histocompatibility antigens. Lancet 1:1406-1409. Duarte, I., Lazzarini, R., Kobata, C. M. 2003. Contact dermatitis in adolescents. Am J Contact

Dermat 14:200-202. Dustin, M. L., Singer, K. H., Tuck, D. T., Springer, T. A. 1988. Adhesion of T lymphoblasts

to epidermal keratinocytes is regulated by interferon gamma and is mediated by intercellular adhesion molecule 1 (ICAM-1). J Exp Med 167:1323-1340.

Eedy, D.J., Burrows, D., McMaster, D. 1991. The nickel content of certain commercially

available metallic patch test materials and its relevance in nickel-sensitive subjects. Contact Dermatitis 24:11-15.


65

Ekerfelt, C., Ermerudh, J., Jenmalm, M. C. 2002. Detection of spontaneous and antigen-induced human interleukin-4 responses in vitro: comparison of ELISPOT, a novel ELISA and real-time RT-PCR. J Immunol Methods 260:55-67.

Emtestam, L., Zetterquist, H., Olerup, O. 1993. HLA-DR, -DQ and -DP alleles in nickel,

chromium, and/or cobalt-sensitive individuals: genomic analysis based on restriction fragment length polymorphisms. J Invest Dermatol 100:271-274.

Engvall, E. and Perlmann, P. 1972. Enzyme-linked immunosorbent assay, ELISA. 3.

Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigen-coated tubes. J Immunol 109:129-135.

Enk, A. H., Angeloni, V. L., Udey, M. C., Katz, S. I. 1993. Inhibition of Langerhans cell

antigen-presenting function by IL-10. A role for IL-10 in induction of tolerance. J Immunol 151:2390-2398.

Enk, A. H. and Katz, S. I. 1992a. Identification and induction of keratinocyte-derived IL-10. J

Immunol 149:92-95. Enk, A. H. and Katz, S. I. 1992b. Early molecular events in the induction phase of contact

sensitivity. Proc Natl Acad Sci U S A 89:1398-1402. Estlander, T., Kanerva, L., Tupasela, O., Keskinen, H., Jolanki, R. 1993. Immediate and

delayed allergy to nickel with contact urticaria, rhinitis, asthma and contact dermatitis. Clin Exp Allergy 23:306-310.

Ewen, C. and Baca-Estrada, M. E. 2001. Evaluation of interleukin-4 concentration by ELISA

is influenced by the consumption of IL-4 by cultured cells. J Interferon Cytokine Res 21: 39-43.

Falsafi-Amin, H., Lundeberg, L., Bakhiet, M., Nordlind, K. 2000. Early DNA synthesis and

cytokine expression in the nickel activation of peripheral blood mononuclear cells in nickel-allergic subjects. Int Arch Allergy Immunol 123:170-176.

Fausto da Silva, J. J. R. and Williams, R. J. P. 2001. The Biological chemistry of the

elements. The inorganic chemistry of life, 2nd ed. Oxford University Press, Oxford UK. Firestein, G. S., Roeder, W. D., Laxer, J. A., Townsend, K. S., Weaver, C. T., Hom, J. T.,

Linton, J., Torbett, B. E., Glasebrook, A. L. 1989. A new murine CD4+ T cell subset with an unrestricted cytokine profile. J Immunol 143: 518-525.

Flier, J., Boorsma, D. M., Bruynzeel, D. P., Van Beek, P. J., Stoof, T. J., Scheper, R. J.,

Willemze, R, Tensen, C. P. 1999. The CXCR3 activating chemokines IP-10, Mig, and IP-9 are expressed in allergic but not in irritant patch test reactions. J Invest Dermatol 113:574-578.

Fontenot, J. D., Gavin, M. A., Rudensky, A. Y. 2003. Foxp3 programs the development and

function of CD4+CD25+ regulatory T cells. Nat Immunol 4:330-336.

Jacob Taku Minang

66

Franc, N. C., White, K., Ezekowitz, R. A. 1999. Phagocytosis and development: back to the future. Curr Opin Immunol 11:47-52.

Fujimura, T., Chamoto, K., Tsuji, T., Sato, T., Yokouchi, H., Aiba, S., Tagami, H., Tanaka, J.,

Imamura, M., Togashi, Y., Koda, T., Nishimura, T. 2004. Generation of leukemia-specific T-helper type 1 cells applicable to human leukemia cell-therapy. Immunol Lett 93:17-25.

Fukaura, H., Kent, S. C., Pietrusewicz, M. J., Khoury, S. J., Weiner, H. L., Hafler, D. A.

1996. Induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor-beta1-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients. J Clin Invest 98:70-77.

Garner, L. A. 2004. Contact dermatitis to metals. Dermatologic Ther 17:321-327. Gawkrodger, D. J., Lewis, F. M., Shah, M. 2000. Contact sensitivity to nickel and other

metals in jewelry reactors. J. Am. Acad. Dermatol 43:31-36. Gawkrodger, D. J., Vestey, J. P., Wong, W. K., Buxton, P. K. 1986. Contact clinic survey of

nickel-sensitive subjects. Contact Dermatitis 14:165-169. Gershon, R. K. and Kondo, K. 1970. Cell interactions in the induction of tolerance: the role of

thymic lymphocytes. Immunology 18:723-737. Gilliet, M. and Liu, Y. J. 2002. Generation of human CD8 T regulatory cells by CD40 ligand-

activated plasmacytoid dendritic cells. J Exp Med 195:695-704. Gorelik, L. and Flavell, R. A. 2002. Transforming growth factor-beta in T-cell biology. Nat

Rev Immunol 2:46-53. Grabbe, S. and Schwarz, T. 1998. Immunoregulatory mechanisms involved in elicitation of

allergic contact hypersensitivity. Immunol Today 19:37-44. Groux, H., O'Garra, A., Bigler, M, Rouleau, M., Antonenko, S., de Vries, J. E., Roncarolo, M.

G. 1997. A CD4+ T-cell subset inhibits antigen specific T-cell responses and prevents colitis. Nature 389:737-742.

Gruvberger, B. 1997. Methylisothiazolinones. Diagnosis and prevention of allergic contact

dermatitis. Acta Derm Venereol (suppl.) (Stockh) 200:1-42. Guillet, G., Guillet, M. H., Dagregorio, G. 2005. Allergic contact dermatitis from natural

rubber latex in atopic dermatitis and the risk of later Type I allergy. Contact Dermatitis 53:46-51.

Hannuksela, M. 1980. Atopic contact dermatitis. Contact Dermatitis. 6:30-32. Hansen, M. B., Johansen, J. D., Menne, T. 2003. Chromium allergy: significance of both

Cr(III) and Cr(VI). Contact Dermatitis 49:206-212.


67

Hart, O. M., Athie-Morales, V., O’connor, G. M., Gardiner, C. M. 2005. TLR7/8-Mediated Activation of Human NK Cells Results in Accessory Cell-Dependent IFN-gamma Production. J Immunol 175:1636-1642.

Harvel, J., Bason, M., Maibach, H. 1994. Contact urticaria and its mechanisms. Food Chem

Toxicol 32:103-112. Hedges, J. F., Lubick, K. J., Jutila, M. A. 2005. Gamma delta T cells respond directly to

pathogen-associated molecular patterns. J Immunol 174:6045-6053. Hegewald, J., Uter, W., Pfahlberg, A., Geier, J, Schnuch, A., for the IVDK. 2005. A

multifactorial analysis of concurrent patch-test reactions to nickel, cobalt, and chromate. Allergy 60:372-378.

Herrick, C. A., Xu, L., McKenzie, A. N., Tigelaar, R. E., Bottomly, K. 2003. IL-13 is

necessary, not simply sufficient, for epicutaneously induced Th2 responses to soluble protein antigen. J Immunol 170:2488-2495.

Hessle, C., Andersson, B., Wold, A. E. 2000. Gram-positive bacteria are potent inducers of

monocytic interleukin-12 (IL-12) while gram-negative bacteria preferentially stimulate IL-10 production. Infect Immun 68: 3581-3586.

Hindsen, M., Bruze, M., Christensen, O. B. 1999. Individual variation in nickel patch test

reactivity. Am J Contact Dermat 10:62-67. Hoefakker, S., Caubo, M., van ‘t Erve, E. H., Roggeveen, M. J., Boersma, W. J., van Joost,

T., Notten, W. R., Claassen, E. 1995. In vivo cytokine profiles in allergic and irritant contact dermatitis. Contact Dermatitis 33:258-266.

Horwitz, D. A., Zheng, S. G., Gray, J. D. 2003. The role of the combination of IL-2 and TGF-

beta or IL-10 in the generation and function of CD4+ CD25+ and CD8+ regulatory T cell subsets. J Leukoc Biol 74:471-478.

Inaba, K. and Inaba, M. 2005. Antigen recognition and presentation by dendritic cells. Int J

Hematol 81:181-187. Ishii, N., Sugita, Y., Nakajima, H., Tanaka, S., Askenase, P. W. 1995. Elicitation of nickel

sulfate (NiSO4)-specific delayed-type hypersensitivity requires early-occurring and early-acting, NiSO4-specific DTH-initiating cells with an unusual mixed phenotype for an antigen-specific cell. Cell Immunol 161:244-255.

Ishii, N., Takahashi, K., Nakajima, H., Tanaka, S., Askenase, P. W. 1994. DNFB contact

sensitivity (CS) in BALB/c and C3H/He mice: requirement for early-occurring, early-acting, antigen-specific, CS-initiating cells with an unusual phenotype (Thy-1+, CD5+, CD3-, CD4-, CD8-, sIg-, B220+, MHC class II-, CD23+, IL-2R-, IL-3R+, Mel-14-, Pgp-1+, J11d+, MAC-1+, LFA-1+, and Fc gamma RII+). J Invest Dermatol 102:321-327.

Jackola, D. R., Blumenthal, M. N., Rosenberg, A. 2004. Evidence for two independent

distributions of serum immunoglobulin E in atopic families: cognate and non-cognate IgE. Hum Immunol 65:20-30.

Jacob Taku Minang

68

Jakobson, E., Masjedi, K., Ahlborg, N., Lundeberg, L., Karlberg, A. T., Scheynius, A., 2002.

Cytokine production in nickel-sensitized individuals analysed with enzyme-linked immunospot assay: possible implication for diagnosis. Br J Dermatol 147:442-449.

Jensen, C. S., Lisby, S., Baadsgaard, O., Volund, A., Menne, T. 2002. Decrease in nickel

sensitization in a Danish schoolgirl population with ears pierced after implementation of a nickel-exposure regulation. Br J Dermatol 146:636-642.

José, A. M., Borghans., Noest, A. J., De Boer, R. J. 1999. How specific should

immunological memory be? J Immunol 163:569-575. Kabelitz, D., Marischen, L., Oberg, H. H., Holtmeier, W., Wesch, D. 2005. Epithelial defence

by gamma delta T cells. Int Arch Allergy Immunol 137:73-81. Kalish, R. S., Wood, J. A., LaPorte, A. 1994. Processing of urushiol (poison ivy) hapten by

both endogenous and exogenous pathways for presentation to T cells in vitro. J Clin Invest 93:2039-2047.

Kaplan, G., Walsh, G., Guido, L. S., Meyn, P., Burkhardt, R. A., Abalos, R. M., Barker, J.,

Frindt, P. A., Fajardo, T. T., Celona, R., Cohn Z. A. 1992. Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony-stimulating factor: Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing. J Exp Med 175:1717-1728.

Kapsenberg, M. L., Wierenga, E. A., Stiekema F. E. M., Tiggelman A. B. C., Bos, J. D. 1992.

Th1 lymphokine production profiles of nickel-specific CD4+ T-lymphocyte clones from nickel contact allergic and nonallergic individuals. J Invest Dermatol 98:59-63.

Kasaian, M. T., Meyer, C. H., Nault, A. K., Bond, J. F. 1995. An increased frequency of IgE-

producing B cell precursors contributes to the elevated levels of plasma IgE in atopic subjects. Clin Exp Allergy 25:749-755.

Kemeny, D. M., Vyas, B., Vukmanovic-Stejic, M., Thomas, M. J., Noble, A., Loh, L. C.,

O'Connor, B. J. 1999. CD8(+) T cell subsets and chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 160: 33-37.

Khattri, R., Cox, T., Yasayko, S. A., Ramsdell, F. 2003. An essential role for Scurfin in

CD4+CD25+ T regulatory cells. Nat Immunol 4:337-342. Kidd, P. 2003. Th1/Th2 balance: the hypothesis, its limitations, and implications for health

and disease. Altern Med Rev 8:223-246. Kimber, I. and Cumberbatch, M. 1992. Stimulation of Langerhans cell migration by tumor

necrosis factor alpha (TNF-alpha). J Invest Dermatol 99:48-50. Kimber, I., Dearman, R. J., Cumberbatch, M., Huby, R. J. 1998. Langerhans cells and

chemical allergy. Curr Opin Immunol 10:614-619.


69

Kondo, S., Kooshesh, F., Wang, B., Fujisawa, H., Sauder, D. N. 1996. Contribution of the CD28 molecule to allergic and irritant-induced skin reactions in CD28 -/- mice. J Immunol 157:4822-4829.

Kondo, S. and Sauder, D. N. 1995. Epidermal cytokines in allergic contact dermatitis. J Am

Acad Dermatol 33:786-800. Lachapelle, J-M. 2001. Historical aspects of patch testing. In: Rycroft R. J. G., Menné, T.,

Frosch, P. J., Lepoittevin, J-P (eds) Textbook of contact dermatitis, 3rd edn. Springer, Berlin Heidelberg New York pp 1-10.

Lachapelle, J. M., Lauwerys, R., Tennstedt, D., Andanson, J., Benezra, C., Chabeau, G.,

Ducombs, G., Foussereau, J., Lacroix, M., Martin, P. 1980. Eau de Javel and prevention of chromate allergy in France. Contact Dermatitis 6:107-110.

Lafaille, J. J. 1998. The role of helper T cell subsets in autoimmune diseases. Cytokine

Growth Factor Rev 9:139-151. Lalvani, A., Pathan, A. A., Durkan, H., Wilkinson, K. A., Whelan, A., Deeks, J. J., Reece, W.

H., Latif, M., Pasvol, G., Hill, A. V., 2001a. Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Lancet 357:2017-2021.

Lalvani, A., Pathan, A. A., McShane, H., Wilkinson, R. J., Latif, M., Conlon, C. P., Pasvol,

G., Hill, A. V. 2001b. Rapid detection of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Am. J. Respir. Crit. Care Med. 163:824-828.

Landsteiner, K., Jacobs, J. L. 1935. Studies on the sensitisation of animals with simple

chemical compounds. J Exp Med 61:643. Lepoittevin, J. P. 2001. Molecular Aspects of allergic contact dermatitis. In: Rycroft R. J. G.,

Menné, T., Frosch, P. J., Lepoittevin, J-P (eds) Textbook of contact dermatitis, 3rd edn. Springer, Berlin Heidelberg New York pp 59-89.

Levin, C. Y. and Maibach, H. I. 2002. Irritant contact dermatitis: is there an immunologic

component? Int Immunopharmacol 2:183-189. Liden, C. 1992. Nickel in jewellery and associated products. Contact Dermatitis. 26:73-75. Liden, C. and Wahlberg, J. E. 1994. Cross-reactivity to metal compounds studied in guinea

pigs induced with chromate or cobalt. Acta Derm Venereol. 74:341-343. Lienhardt, C., Azzurri, A., Amedei, A., Fielding, K., Sillah, J., Sow, O. Y., Bah, B.,

Benagiano, M., Diallo, A., Manetti, R., Manneh, K., Gustafson, P., Bennett, S., D'Elios, M. M., McAdam, K., Del Prete, G. 2002. Active tuberculosis in Africa is associated with reduced Th1 and increased Th2 activity in vivo. Eur J Immunol 32:1605-1613.

Li, L. F. and Wang, J. 2002. Contact hypersensitivity in hand dermatitis. Contact Dermatitis.

47:206-209.

Jacob Taku Minang

70

Lindemann, M., Bohmer, J., Zabel, M., Grosse-Wilde, H. 2003. ELISpot: a new tool for the detection of nickel sensitization. Clin Exp Allergy 33:992-998.

Lisby, S., Hansen, L. H., Menn, T., Baadsgaard, O. 1999. Nickel-induced proliferation of both

memory and naive T cells in patch test-negative individuals. Clin Exp Immunol 117:217-222.

Lisby, S. and Hauser, C. 2002. Transcriptional regulation of tumor necrosis factor-alpha in

keratinocytes mediated by interleukin-1beta and tumor necrosis factor-alpha. Exp Dermatol 11:592-598.

Lisi, P., Brunelli, L., Stingeni, L. 2003. Co-sensitivity between cobalt and other transition

metals. Contact Dermatitis. 48:172-173. Liu, C. A., Wang, C. L., Chuang, H., Ou, C. Y., Hsu, T. Y., Yang, K. D. 2003. Prenatal

prediction of infant atopy by maternal but not paternal total IgE levels. J Allergy Clin Immunol 112:899-904.

Luster, A. D. 2002. The role of chemokines in linking innate and adaptive immunity. Curr

Opin Immunol 14: 129-135. Malten, K. E, den Arend, J. A., Wiggers, R. E. 1979. Delayed irritation: hexanediol diacrylate

and butanediol diacrylate. Contact Dermatitis 5:178-184. Marcinkiewicz, J. and Chain, B. M. 1993. Regulation of in vitro release of TH2 type

cytokines (IL-4, IL-6) in the T cell response to the trinitrophenyl (TNP) hapten. Cell Immunol 146:406-411.

Masjedi, K., Ahlborg, N., Gruvberger, B., Bruze, M., Karlberg, A. T. 2003.

Methylisothiazolinones elicit increased production of both T helper (Th)1- and Th2-like cytokines by peripheral blood mononuclear cells from contact allergic individuals. Br J Dermatol 149:1172-1182.

Mason, D. 2001. Some quantitative aspects of T-cell repertoire selection: the requirement for

regulatory T cells. Immunol Rev 182:80-88. Matzinger, R. 1994. Tolerance, danger, and the extended family. Annu Rev Immunol 12:991-

1045. McGuirk, P. and Mills, K. H. 2002. Pathogen-specific regulatory T cells provoke a shift in the

Th1/ Th2 paradigm in immunity to infectious diseases. Trends Immunol 23:450-455. McHugh, S. M., Wilson, A. B., Deighton, J., Lachmann, P. J., Ewan, P. W. 1994. The profiles

of interleukin (IL)-2, IL-6, and interferon-gamma production by peripheral blood mononuclear cells from house-dust-mite-allergic patients: a role for IL-6 in allergic disease. Allergy 49:751-759.

McKenzie, A. N., Culpepper, J. A., de Waal Malefyt, R., Briere, F., Punnonen, J., Aversa, G.,

Sato, A., Dang, W., Cocks, B. G., Menon, S., De Vries, J. E. and Zurawski G. 1993.


71

Interleukin 13, a T-cell-derived cytokine that regulates human monocyte and B-cell function. Proc Natl Acad Sci U S A. 90: 3735-3739.

Meding, B., Jarvholm, B., 2002. Hand eczema in Swedish adults - changes in prevalence

between 1983 and 1996. J Invest Dermatol 118:719-723. Meding, B., Liden, C., Berglind, N., 2001. Self-diagnosed dermatitis in adults. Results from a

population survey in Stockholm. Contact Dermatitis 45:341-345. Miescher, G. C., Howe, R. C., Lees, R. K., MacDonald, H. R. 1988. CD3-associated

alpha/beta and gamma/delta heterodimeric receptors are expressed by distinct populations of CD4- CD8- thymocytes. J Immunol 140:1779-1782.

Moed, H., Boorsma, D. M., Stoof, T. J., von Blomberg, B. M., Bruynzeel, D. P., Scheper, R.

J., Gibbs, S., Rustemeyer, T. 2004b. Nickel-responding T cells are CD4+ CLA+ CD45RO+ and express chemokine receptors CXCR3, CCR4 and CCR10. Br J Dermatol 151:32-41.

Moed, H., Boorsma, D. M., Tensen, C. P., Flier, J., Jonker, M. J., Stoof, T. J, von Blomberg,

B. M., Bruynzeel, D. P., Scheper, R. J., Rustemeyer, T., Gibbs, S. 2004a. Increased CCL27-CCR10 expression in allergic contact dermatitis: implications for local skin memory. J Pathol 204:39-46.

Moed, H., Boorsma, D. M., von Blomberg, B. M. E., Bruynzeel, D. P., Scheper, R. J., Gibbs,

S., Rustemeyer, T. 2005. Regulation of nickel-induced T-cell responsiveness by CD4+CD25+ cells in contact allergic patients and healthy individuals. Contact Dermatitis 53:71-74.

Morikawa, Y., Furotani, M., Kuribayashi, K., Matsuura, N., Kakudo, K. 1992. The role of

antigen-presenting cells in the regulation of delayed-type hypersensitivity. I. Spleen dendritic cells. Immunol 77:81-87.

Moser, M. and Murphy, K. M. 2000. Dendritic cell regulation of TH1 TH2 development. Nat

Immunol 1:199-205. Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A., and Coffman, R. L. 1986.

Two types of murine helper T cell clone. I. Definition according, to profiles of lymphokine activities and secreted proteins. J Immunol 136:2348-2357.

Mosmann, T. R. and Coffman, R. L. 1989. TH1 and TH2 cells: different patterns of

lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145-173.

Mosmann, T. R. and Sad, S. 1996. The expanding universe of T-cell subsets: Th1, Th2 and

more. Immunol Today 17:138-146. Moulon, C., Vollmer, J., Weltzien, H. U. 1995. Characterization of processing requirements

and metal cross-reactivities in T cell clones from patients with allergic contact dermatitis to nickel. Eur J Immunol 25:3308-3315.

Jacob Taku Minang

72

Moulon, C., Wild, D., Dormoy, A., Weltzien, H. U. 1998. MHC-dependent and –independent activation of human nickel-specific CD8+ cytotoxic T cells from allergic donors. J Invest Dermatol 111:360-366.

Mäkitalo, B., Andersson, M., Areström, I., Karlen, K., Villinger, F., Ansari, A., Paulie, S.,

Thorstensson, R., Ahlborg, N. 2002. ELISpot and ELISA analysis of spontaneous, mitogen-induced and antigen-specific cytokine production in cynomolgus and rhesus macagues. J Immunol Methods 270:85-97.

Möller, G. 1988. Do suppressor T cells exist? Scand J Immunol 27:247-250. Nestle, F. O. and Burg, G. 1999. Dendritic cells: role in skin diseases and therapeutic

applications. Clin Exp Dermatol 24:204-207. Newport, M. J., Huxley, C. M., Huston, S., Hawrylowicz, C. M., Oostra, B. A., Williamson,

R., Levin, M. 1996. A mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection. N Engl J Med 335:1941-1949.

Nishimura, E., Sakihama, T., Setoguchi, R., Tanaka, K., Sakaguchi, S. 2004. Induction of

antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising Foxp3+CD25+CD4+ regulatory T cells. Int Immunol 16:1189-1201.

Nordlind, K. 1984. Further studies on the lymphocyte transformation test in diagnosis of

nickel allergy. Effect of fractionation of lymphocytes into different subpopulations on the basis of density. Int Arch Allergy Appl Immunol 75:333-336.

Oberg, L., Johansson, S., Michaelsson, J., Tomasello, E., Vivier, E., Karre, K., Hoglund, P.

2004. Loss or mismatch of MHC class I is sufficient to trigger NK cell-mediated rejection of resting lymphocytes in vivo - role of KARAP/DAP12-dependent and -independent pathways. Eur J Immunol 34:1646-1653.

O'Garra, A. 1998. Cytokines induce the development of functionally heterogeneous T helper

cell subsets. Immunity 8:275-283. Pakala, S. V, Kurrer, M. O., Katz, J. D. 1997. T helper 2 (Th2) T cells induce acute

pancreatitis and diabetes in immune-compromised nonobese diabetic (NOD) mice. J Exp Med 186:299-306.

Parronchi, P., De Carli, M., Manetti, R., Simonelli, C., Piccinni, M. P., Macchia, D., Maggi,

E., Del Prete, G., Ricci, M., Romagnani, S. 1992. Aberrant interleukin (IL)-4 and IL-5 production in vitro by CD4+ helper T cells from atopic subjects. Eur J Immunol 22:1615-1620.

Pasare, C. and Medzhitov, R. 2004. Toll-like receptors and acquired immunity. Semin

Immunol 16:23-26. Pawankar, R. U., Okuda, M., Suzuki, K., Okumura, K, Ra, C. 1996. Phenotypic and

molecular characteristics of nasal mucosal gamma delta T cells in allergic and infectious rhinitis. Am J Respir Crit Care Med 153:1655-1665.


73

Pedersen, L. K., Held, E., Johansen, J. D., Agner, T. 2005. Short-term effects of alcohol-based disinfectant and detergent on skin irritation. Contact Dermatitis 52:82-87.

Porstmann, T. and Kiessig, S. T. 1992. Enzyme immunoassay techniques: An overview. J

Immunol Methods 150:5-21. Probst, P., Küntzlin, D., Fleischer, B. 1995. Th2-type infiltrating T cells in nickel-induced

contact dermatitis. Cellular Immunol 165:134-140. Ptak, W., Geba, G. P., Askenase, P. W. 1991a. Initiation of delayed-type hypersensitivity by

low doses of monoclonal IgE antibody. Mediation by serotonin and inhibition by histamine. J Immunol 146:3929-3936.

Ptak, W., Herzog, W. R., Askenase, P. W. 1991b. Delayed-type hypersensitivity initiation by

early-acting cells that are antigen mismatched or MHC incompatible with late-acting, delayed-type hypersensitivity effector T cells. J Immunol 146:469-475.

Punnonen, J., Aversa, G., Cocks, B. G., McKenzie, A. N., Menon, S., Zurawski, G., de Waal

Malefyt, R., de Vries, J. E. 1993. Interleukin 13 induces interleukin 4-independent IgG4 and IgE synthesis and CD23 expression by human B cells. Proc Natl Acad Sci U S A. 90: 3730-3734.

Punnonen, J., Yssel, H., de Vries, J. E. 1997. The relative contribution of IL-4 and IL-13 to

human IgE synthesis induced by activated CD4+ or CD8+ T cells. J Allergy Clin Immunol 100:792-801.

Rajan, T. V., 2003. The Gell-Coombs classification of hypersensitivity reactions: a re-

interpretation. Trends Immunol 24:376-379. Reinhardt, C. A., Pelli, D. A., Sandvold, M. 1985. Cell detachment and growth of fibroblasts

as parameters for cytotoxicity of inorganic metal salts in vitro. Cell Biol Toxicol 1:33-43. Rich, S., Seelig, M., Lee, H. M., Lin, J. 1995. Transforming growth factor beta 1 costimulated

growth and regulatory function of staphylococcal enterotoxin B-responsive CD8+ T cells. J Immunol 155:609-618.

Rincon, M., Anguita, J., Nakamura, T., Fikrig, E., Flavell, R. A. 1997. Interleukin (IL)-6

directs the differentiation of IL-4-producing CD4+ T cells. J Exp Med 185:461-469. Rogge, L., Barberis-Maino, L., Biffi, M., Passini, N., Presky, D. H., Gubler, U., Sinigaglia, F.

1997. Selective expression of an interleukin-12 receptor component by human T helper 1 cells. J Exp Med 185:825-831.

Romagnani, G. 2000. Cytokines and the Th1/Th2 paradigm. In: The Cytokine Network.

Balkwill, F. (ed), Oxford: Oxford University Press, New York pp 71-93. Romagnoli, P., Labhardt, A. M., Sinigaglia, F. 1991. Selective interaction of Ni with an

MHC-bound peptide. EMBO J 10:1303-1306.

Jacob Taku Minang

74

Romani, N. and Schuler, G. 1992. The immunologic properties of epidermal Langerhans cells as a part of the dendritic cell system. Springer Semin Immunopathol 13:265-279.

Rosenberg, A. 1955. An anecdotal biographical history of poison ivy. AMA Arch Derm

72:438-445. Rowe, A. and Bunker, C. B. 1998. Interleukin-4 and the interleukin-4 receptor in allergic

contact dermatitis. Contact Dermatitis 38:36-39. Royer, B., Varadaradjalou, S., Saas, P., Guillosson, J. J., Kantelip, J. P, Arock, M. 2001.

Inhibition of IgE-induced activation of human mast cells by IL-10. Clin Exp Allergy 31:694-704.

Ruprecht, C. R., Gattorno, M., Ferlito, F., Gregorio, A., Martini, A., Lanzavecchia, A.,

Sallusto, F. 2005. Coexpression of CD25 and CD27 identifies FoxP3+ regulatory T cells in inflamed synovia. J Exp Med 201:1793-1803.

Rustemeyer, T., von Blomberg, B. M., van Hoogstraten, I. M, Bruynzeel, D. P., Scheper, R. J.

2004. Analysis of effector and regulatory immune reactivity to nickel. Clin Exp Allergy 34:1458-1466.

Rystedt, I. 1979. Evaluation and relevance of isolated test reactions to cobalt. Contact

Dermatitis 5:233-238. Sadler, P. J., Tucker, A., Viles, J. H. 1994. Involvement of a lysine residue in the N-terminal

Ni2+ and Cu2+ binding site of serum albumins. Comparison with Co2+, Cd2+ and Al3+. Eur J Biochem 220:193-200.

Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., Toda, M. 1995. Immunologic self-

tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155:1151-1164.

Sanderson, C.J. 1992. Interleukin-5, eosinophils, and disease. Blood 79: 3101-3109. Santucci, B., Cannistraci, C., Cristaudo, A., Picardo, M. 1996. Multiple sensitivities to

transitional metals: the nickel palladium reactions. Contact Dermatitis 35:283-286. Sato, M., Goto, S., Kaneko, R., Ito, M., Sato, S., Takeuchi, S. 1998. Impaired production of

Th1 cytokines and increased frequency of Th2 subsets in PBMC from advanced cancer patients. Anticancer Res 18:3951-3955.

Schielen, P., van Rodijnen, W., Tekstra, J., Albers, R., Seinen,W. 1995. Quantification of

natural antibody producing B cells in rats by an improved ELISPOT technique using the polyvinylidene difluoride membrane as the solid support. J Immunol Methods 188:33-41.

Schmitt, E., Van Brandwijk, R., Fischer, H. G., Rude, E. 1990. Establishment of different T

cell sublines using either interleukin 2 or interleukin 4 as growth factors. Eur J Immunol 20:1709-1715.


75

Schnuch, A., Geier, J., Uter, W., Frosch, P. J., Lehmacher, W., Aberer, W., Agathos, M., Arnold, R., Fuchs, T., Laubstein, B., Lischka, G., Pietrzyk, P. M., Rakoski, J., Richter, G., Rueff, F. 1997. National rates and regional differences in sensitization to allergens of the standard series. Population-adjusted frequencies of sensitization (PAFS) in 40,000 patients from a multicenter study (IVDK). Contact Dermatitis 37:200-209.

Schnuch, A., Uter, W., Geier, J., Frosch, P.J. 1998. Contact allergies in healthcare workers.

Results from the IVDK. Acta Derm Venereol 78:358-363. Schulze-Koops, H. and Kalden, J. R. 2001. The balance of Th1/Th2 cytokines in rheumatoid

arthritis. Best Pract Res Clin Rheumatol 15:677-691. Schwartz, R. H., David, C. S., Sachs, D. H., Paul, W. E. 1976. T lymphocyte-enriched murine

peritoneal exudate cells. III. Inhibition of antigen-induced T lymphocyte Proliferation with anti-Ia antisera. J Immunol 117:531-540.

Sebastiani, S., Allavena, P., Albanesi, C., Nasorri, F., Bianchi, G., Traidl, C., Sozzani, S.,

Girolomoni, G., Cavani, A. 2001. Chemokine receptor expression and function in CD4+ T lymphocytes with regulatory activity. J Immunol 166:996-1002.

Sebzda, E., Mariathasan, S., Ohteki, T., Jones, R., Bachmann, M. F., Ohashi, P. S. 1999.

Selection of the T cell repertoire. Annu Rev Immunol 17: 829-874. Seder, R. A. and Ahmed, R. 2003. Similarities and differences in CD4+ and CD8+ effector

and memory T cell generation. Nature Immunol 9:835-842. Sedgwick, J. D and Holt, P. G. 1983. A solid-phase immunoenzymatic technique for the

enumeration of specific antibody-secreting cells. J Immunol Methods 57:301-309. Shah, M., Lewis, F. M., Gawkrodger, D. J. 1998. Nickel as an occupational allergen. A survey

of 368 nickel-sensitive subjects. Arch Dermatol 134:1231-1236. Shevach, E. M. 2001. Certified professionals: CD4(+)CD25(+) suppressor T cells. J Exp Med

193:41-46. Shevach, E. M. 2002. CD4+ CD25+ suppressor T cells: more questions than answers. Nat

Rev Immunol 2:389-400. Shirakawa, T., Kusaka, Y., Morimoto, K. 1992. Specific IgE antibodies to nickel in workers with known reactivity to cobalt. Clin Exp Allergy 22:213-218. Silvennoinen-Kassinen, S., Poikonen, K., Ikäheimo, I. 1991. Characterisation of nickel-

specific T cell clones. Scan J Immunol 33:429-434. Singh, V. K., Mehrotra, S., Agarwal, S. S. 1999. The paradigm of Th1 and Th2 cytokines: its

relevance to autoimmunity and allergy. Immunol Res 20:147-161. Sinigalia, F., Scheidegger, D., Garotta, G., Scheper, R., Pletscher, M., Lanzavecchia, A. 1985.

Isolation and characterisation of Ni-specific T cell clones from patients with Ni-contact dermatitis. J Immunol 135:3929-3932.

Jacob Taku Minang

76

Siroux, V., Curt, F., Oryszczyn, M.P., Maccario, J., Kauffmann, F. 2004. Role of gender and

hormone-related events on IgE, atopy, and eosinophils in the Epidemiological study on the Genetics and Environment of Asthma, bronchial hyperresponsiveness and atopy. J Allergy Clin Immunol 114:491-498.

Skokowa, J., Ali, S. R., Felda, O., Kumar, V., Konrad, S., Shushakova, N., Schmidt, R. E.,

Piekorz, R. P., Nurnberg, B., Spicher, K., Birnbaumer, L., Zwirner, J., Claassens, J. W., Verbeek, J. S., van Rooijen, N., Kohl, J., Gessner, J. E. 2005. Macrophages induce the inflammatory response in the pulmonary Arthus reaction through G alpha i2 activation that controls C5aR and Fc receptor cooperation. J Immunol 174:3041-3050.

Smith, J. G., Liu, X., Kaufhold, R. M., Clair, J., Caulfield, M. J. 2001. Development and

validation of a gamma interferon ELISPOT assay for quantitation of cellular immune responses to Varicella-Zoster virus. Clin Diag Lab Immunol 8:871-879.

Smith, H. R., Rowson, M., Basketter, D. A., McFadden, J. P. 2004. Intra-individual variation

of irritant threshold and relationship to transepidermal water loss measurement of skin irritation. Contact Dermatitis 51:26-29.

Sonoda, E., Matsumoto, R., Hitoshi, Y., Ishii, T., Sugimoto, M., Araki, S., Tominaga, A.,

Yamaguchi, N., Takatsu, K. 1989. Transforming growth factor beta induces IgA production and acts additively with interleukin 5 for IgA production. J Exp Med 170: 1415-1420.

Sozzani, S., Sallusto, F., Luini, W., Zhou, D., Piemonti, L., Allavena, P., Van Damme, J.,

Valitutti, S., Lanzavecchia, A., Mantovani, A. 1995. Migration of dendritic cells in response to formyl peptides, C5a, and a distinct set of chemokines. J Immunol 155:3292-3295.

Sparwasser, T., Koch, E. S., Vabulas, R. M., Heeg, K., Lipford, G. B., Ellwart, J. W.,

Wagner, H. 1998. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur J Immunol 28: 2045-2054.

Steinke, J.W. and Borish, L. 2001. Th2 cytokines and asthma. Interleukin-4: its role in the

pathogenesis of asthma, and targeting it for asthma treatment with interleukin-4 receptor antagonists. Respir Res 2:66-70.

Sterry, W., Kunne, N., Weber-Matthiesen, K., Brasch, J., Mielke, V. 1991. Cell trafficking in

positive and negative patch-test reactions: demonstration of a stereotypic migration pathway. J Invest Dermatol 96:459-462.

Takata, M., Homma, Y., Kurosaki, T. 1995. Requirement of phospholipase C-gamma 2

activation in surface immunoglobulin M-induced B cell apoptosis. J Exp Med 182: 907-914.

Tang, L., Benjaponpitak, S., Dekruyff, R. M., Umetsu, D. T. 1998. Reduced prevalence of

allergic disease in patients with multiple sclerosis associated with enhanced IL-12 production. J Allergy Clin Immunol 102:428-435.


77

Tanguay, S. and Killion, J. J. 1994. Direct comparison of ELISPOT and ELISA-based assays for detection of individual cytokine secreting cells. Lymphokine Cytokine Res 13:259-263.

Teixeira, M. M., Almeida, I. C., Gazzinelli, R. T. 2002. Introduction: innate recognition of

bacteria and protozoan parasites. Microbes Infect 4:883-886. Teunissen, M. B. 1992. Dynamic nature and function of epidermal Langerhans cells in vivo

and in vitro: a review, with emphasis on human Langerhans cells. Histochem J 24:697-716.

Thierse, H. J., Gamerdinger, K., Junkes, C., Guerreiro, N., Weltzien, H. U. 2005. T cell

receptor (TCR) interaction with haptens: metals ions as non-classical haptens. Toxicology 209:101-107.

Thierse, H. J., Moulon, C., Allespach, Y., Zimmermann, B., Doetze, A., Kuppig, S., Wild, D.,

Herberg, F., Weltzien, H. U. 2004. Metal-protein complex-mediated transport and delivery of Ni2+ to TCR/MHC contact sites in nickel-specific human T cell activation. J Immunol 172:1926-1934.

Thomas, M.J. and Kemeny, D. M. 1998. Novel CD4 and CD8 T-cell subsets. Allergy 53:

1122-1232. Torii, I., Morikawa, S., Harada, T. 2002. MD41, a novel T helper 0 clone, mediates mast-cell

dependent delayed-type hypersensitivity in mice. Immunol 107: 426-434. Traidl, C., Sebastiani, S., Albanesi, C., Merk, H. F., Puddu, P., Girolomoni, G., Cavani, A.

2000. Disparate cytotoxic activity of nickel-specific CD8+ and CD4+ T cell subsets against keratinocytes. J Immunol 165:3058-3064.

Tupker, R. A. 2003. Prediction of irritancy in the human skin irritancy model and

occupational setting. Contact Dermatitis 49:61-69. Uter, W., Ruhl, R., Pfahlberg, A., Geier, J., Schnuch, A., Gefeller, O. 2004. Contact allergy in

contruction workers: results of a multifactorial analysis. Ann Occup Hyg 48:21-27. Von Blomberg-van der Flier, M., van der Burg, C. K., Pos, O., van de Plassche-Boers, E. M.,

Bruynzeel, D. P., Garotta, G., Scheper, R. J. 1987. In vitro studies in nickel allergy: diagnostic value of a dual parameter analysis. J Invest Dermatol 88:362-368.

Vamnes, J. S., Morken, T., Helland, S., Gjerdet, N. R. 2000. Dental gold alloys and contact

hypersensitivity. Contact Dermatitis. 42(3):128-133. Van den Broeke, L. T., Heffler, L.C., Tengvall Linder, M., Nilsson, J. L., Karlberg, A. T.,

Scheynius, A. 1999. Direct Ni2+ antigen formation on cultured human dendritic cells. Immunol 96:578-585.

Van Seventer, G.A., Van Lier, R.A., Spits, H., Ivanyi, P., Melief, C.J., 1986. Evidence for a

regulatory role of the T8 (CD8) antigen in antigen-specific and anti-T3-(CD3)-induced lytic activity of allospecific cytotoxic T lymphocyte clones. Eur J Immunol 16:1363-1371.

Jacob Taku Minang

78

Verhasselt, V., Buelens, C., Willems, F., De Groote, D., Haeffner-Cavaillon, N., Goldman,

M., 1997. Bacterial lipopolysaccharide stimulates the production of cytokines and the expression of costimulatory molecules by human peripheral blood dendritic cells: evidence for a soluble CD14-dependent pathway. J Immunol 158: 2919-2925.

Vincenzi, C., Tosti, A., Guerra, L., Kokelj, F., Nobile, C., Rivara, G., Zangrando, E. 1995.

Contact dermatitis to palladium: a study of 2300 patients. Am J Cont Dermat 6:110-112. Waalkes, M. P. 1995. Metal carcinogenesis. In:Goyer RA, Klaassen CD, Waalkes MP eds.

Metal toxicology. Academic, New York, pp 447-464. Wahlberg, J. E. 2001. Patch testing. In: Rycroft R. J. G., Menné, T., Frosch, P. J., Lepoittevin,

J-P (eds) Textbook of contact dermatitis, 3rd edn. Springer, Berlin Heidelberg New York pp435-468.

Wahlberg, J. E. and Liden, C. 2000. Cross-reactivity patterns of cobalt and nickel studied with

repeated open applications (ROATS) to the skin of guinea pigs. Am J Contact Dermat 11:42-48.

Wakem, P., Burns, R. P. Jr., Ramirez, F., Zlotnick, D., Ferbel, B., Haidaris, C. G., Gaspari, A.

A. 2000. Allergens and irritants transcriptionally upregulate CD80 gene expression in human keratinocytes. J Invest Dermatol 114:1085-1092.

Walton, S. 1983. Patch testing with copper sulphate. Contact Dermatitis 9:337. Walzer, T., Dalod, M., Robbins, S. H., Zitvogel, L., Vivier, E. 2005. Natural killer cells and

dendritic cells: "l'union fait la force". Blood 106:2252-2258. Wang, Z. E., Reiner, Zheng, S., Dalton, D. K., Locksley, R. M. 1994. CD4+ effector cells

default to the Th2 pathway in interferon gamma- deficient mice infected with Leishmania major. J Exp Med 179: 1367-1371.

Weiner, H. L. 2001. Induction and mechanism of action of transforming growth factor-beta-

secreting Th3 regulatory cells. Immunol Rev 182:207-214. Werfel, T., Hentschel, M, Kapp, A., Renz, H. 1997. Dichotomy of blood- and skin-derived

IL-4-producing allergen-specific T cells and restricted Vβ repertoir in nickel-mediated contact dermatitis. J Immunol 158:2500-2505.

West, N. Y., Fitzpatrick, J. E., Jackson, E. M. 1998. Comparison testing of the irritancy of

children's liquid bubble bath using a modified human repeat insult patch test. Am J Contact Dermat 9:212-215.

Willis, C. M., Stephens, C. J., Wilkinson, J. D. 1993. Differential patterns of epidermal

leukocyte infiltration in patch test reactions to structurally unrelated chemical irritants. J Invest Dermatol 101:364-370.

Wuthrich, B. 1998. Food-induced cutaneous adverse reactions. Allergy 53:131-135.


79

Xu, D., Komai-Koma, M., Liew, F. Y. 2005. Expression and function of Toll-like receptor on T cells. Cell Immunol 233:85-89.

Zanni, M. P., von Greyerz, S., Schnyder, B., Brander, K. A., Frutig, K., Hari, Y., Valitutti, S.,

Pichler, W. J. 1998. HLA-restricted, processing- and metabolism-independent pathway of drug recognition by human alpha beta T lymphocytes. J Clin Invest 102:1591-1598.

Zhang, Y. and Wilcox, D. E. 2002. Thermodynamic and spectroscopic study of Cu(II) and

Ni(II) binding to bovine serum albumin. J Biol Inorg Chem 7:327-337. Zumdahl, S. S. 1997. Chemistry alternate edition. R Stratton, M Stephanie & CL Brooks eds.

Houghton Mifflin company Boston 4th Edn. pp 941-993. Zurawski, G. and de Vries, J. E., 1994. Interleukin 13 elicits a subset of the activities of its

close relative interleukin 4. Stem Cells 12:169-174.

Documents

Cytokine profiles in metal-induced allergic contact