Contact Dermatitis, 1998, 38, 36–39 Copyright C Munksgaard 1998Printed in Denmark . All rights reserved

ISSN 0105-1873

Interleukin-4 and the interleukin-4 receptor inallergic contact dermatitis

A. R C. B. B

Skin Treatment and Research Trust Laboratory, Division of Medicine, Imperial College School ofMedicine, Chelsea and Westminster Hospital, London SW10 9NH, UK

Cutaneous immune responses involving T helper (TH) type 1 (TH1) and type 2 (TH2) T cells,characterised by secretion of interferon-g (Ifn-g) and interleukin-4 (IL-4), respectively, have bothbeen reported in allergic contact dermatitis (ACD). We used immunohistochemistry to localizeexpression of IL-4 in ACD lesions and unaffected skin. Atopic dermatitis (AD) and psoriasisbiopsies provided positive and negative IL-4 immunoreactivity controls. To investigate the role ofIL-4 in ACD, we investigated expression of IL-4 receptors in ACD, AD and psoriatic skin. IL-4immunoreactivity was found in cells in the dermal infiltrate in 3 out of 7 ACD lesions, but not inunaffected skin from these patients. IL-4 immunoreactivity was found in the dermal infiltrate ofall lesional and unaffected AD biopsies, but in none of the psoriatic biopsies. IL-4 receptor a chainimmunoreactivity, associated with dermal mast cells, was found in all patients. Local expression ofIL-4 in ACD indicates either TH2 or TH0 immunoregulation in some allergic contact dermatitislesions. Our findings do not support exclusive TH1 or TH2 cutaneous immune responses in ACD.Expression of IL-4 receptors by cutaneous mast cells provides a route through which local effectsof IL-4 might be mediated.

Key words: interleukin-4; interleukin-4 receptor; cytokines; atopic dermatitis; allergic contact der-matitis. C Munksgaard, 1998.

Accepted for publication 24 September 1997

The combinations of cytokines produced by T-lymphocytes have been attributed to differentfunctional subsets (T-helper type 1 (TH 1) and T-helper type 2 (TH 2) cells), characterized princi-pally by production of interferon (Ifn)-g or of in-terleukin (IL)-4, respectively (1, 2). TH1 cells andthe cytokines they produce mediate cellular im-mune reactions, whilst TH2 cells and their cyto-kines are involved in humoral immunity and 3rdstimulation of IgE production. A 3rd class of Thelper cells (TH0) has also been described. Thesecells may be progenitors of either TH1 or TH2cells or could represent distinct classes of cells pro-ducing both Ifn-g and IL-4 (3).

Studies in mouse models for allergic contact der-matitis (ACD) and with human ACD lesions havedemonstrated a variety of findings; TH1, TH2 andTH0 lymphocyte clones have been isolated fromallergic contact dermatitis lesions (4–7).

There have been conflicting observations con-cerning the expression of TH2 cytokines by ex-vivoT cell clones in allergic contact dermatitis. Wetherefore decided to investigate the expression of

IL-4 in allergic contact dermatitis lesions by im-munohistochemistry.

The immune response in acute atopic dermatitis(AD) has been characterized as TH2. High levelsof TH2 cytokines (including IL-4) are present inacute lesions and are produced by T-cell clones de-rived from lesions or from peripheral blood (8). Inchronic plaque psoriasis, several studies haveshown a TH1 response in skin lesions (9). IL-4 hasnot been detected in psoriatic lesions. We usedlesional and non-lesional AD and psoriatic skin aspositive and negative immunoreactivity controlsfor IL-4 expression.

IL-4 signalling is transduced through IL-4-speci-fic receptors which consist of 2 transmembranepeptides, a and g. While g is common to receptorsfor several cytokines, the a peptide is specific tothe IL-4 receptor (IL-4R). IL-4 signalling is alsotransduced through lower affinity IL-4/IL-13 re-ceptors. These are heterodimers consisting of theIL-4R a chain and a second IL-13 binding chain.IL-13 does not signal through the IL-4R a/gR (10).In order to examine further possible local effects

37IL-4 AND THE IL-4 RECEPTOR IN ACD

of cutaneous IL-4 expression in ACD, we haveexamined the distribution of L-4 R, with anti-bodies against the human IL-4Ra chain.

Patients and Methods

PatientsLocal ethical committee permission was obtainedfor these studies and patients gave informed writ-ten consent.

Skin biopsies (4 mm punch) were taken frompositive patch test sites from patients with a diag-nosis of allergic contact dermatitis (CD) and nohistory of atopy: nΩ7 for the IL-4 studies (nickelnΩ3; neomycin nΩ2; colophony nΩ1; PTBPF resinnΩ1; primin nΩ1) nΩ6 for the IL-4R studies(nickel nΩ2; colophony nΩ1; PTBPF resin nΩ2;primin nΩ1). Subjects were patients undergoingroutine patch testing with the European standardseries of allergens and other substances as dictatedby clinical circumstances. Patches were applied for1 day and positive sites biopsied 4 days later. Unin-volved biopsies were taken at the same time fromnegative patch test sites in the same patients. Atop-ic dermatitis (AD) biopsies were taken fromlesional sites in patients (nΩ7 for the IL-4 studies;nΩ6 for the IL-4 R studies) with long-standingchronic AD whose disease had recently flared andwho were presenting to the Department to com-mence a period of day care treatment. Biopsieswere also taken from clinically uninvolved skin.

Psoriasis biopsies were taken from lesional andnon-lesional sites in patients who were presentingto the Department to commence a period of daycare treatment (nΩ7 for the IL-4 studies; nΩ6 forthe IL-4 R studies). None of the patients in anygroup were receiving any systemic or topical treat-ment apart from moisturizers. Tissue for sec-tioning was immediately snap frozen and storedunder liquid nitrogen.

Immunohistochemistry

6 mm frozen sections were prepared and fixed in icecold acetone. A rabbit polyclonal anti-human IL-4antibody (Genzyme, Cambridge, MA., USA) wasused on sections from all patients examined for IL-4 immunoreactivity. Results were confirmed on 1ACD patients and 2 AD patients with a mono-clonal anti-human IL-4 antibody (Serotec, Kid-lington Oxford UK). A rabbit polyclonal anti-hu-man IL-4 receptor a chain (IL-4Ra) antibodyraised against a peptide derived from the C ter-minal region of the IL 4Ra protein. Results wereconfirmed in a subgroup of patients with ACD(nΩ1 lesional, nΩ 2 unaffected), AD (nΩ1 lesional,2 unaffected) or psoriasis (nΩ4 lesional, nΩ6 unaf-

fected), with a 2nd rabbit polyclonal antibodyraised against a peptide derived from the N ter-minal region of the protein (both anti-IL-4R anti-bodies from Santa Cruz Biotechnology Inc., Ca.,USA). Mouse monoclonal anti-human CD3 anti-body was obtained from Sigma Aldrich (PooleDorset UK).

At least 5 non-adjacent sections from each bi-opsy were immunostained with each antibody. Sec-tions were incubated with equivalent concen-trations of purified non-immune rabbit or mouseIgG (Sigma Aldrich, Poole Dorset UK) as controlsfor antibody specificity. The specifities of the anti-IL-4Ra antibodies were tested by carrying out im-munohistochemistry with antibodies blocked bypre incubation with a 5¿ excess of specific peptide.Immunohistochemistry was carried out as de-scribed previously (11).

Mast cell studies

Distributions of mast cells and IL-4 Ra immuno-reactive cells were compared by toluidine bluestaining of adjacent sections from ACD (nΩ1affected, nΩ1 unaffected), AD (nΩ1 lesional, nΩ2unaffected) and psoriasis (nΩ2 lesional, nΩ2 unaf-fected) biopsies.

Results

IL-4 immunoreactivity was detected in the dermalinfiltrate in 3 of 7 ACD positive patch test sitebiopsies and in none of the negative patch testsites. Staining was localized mainly over areas ofdense inflammatory infiltrate. No IL-4 was de-tected in the epidermis. There appeared to be noobvious correlation between IL-4 expression andallergen. 2 of the positive lesions were reactions tonickel and 1 to neomycin. The IL-4 negative ACDbiopsies also included 1 from a nickel and 1 froma neomycin reactive site.

In atopic dermatitis, IL-4 immunoreactivity as-sociated with the inflammatory infiltrate in theupper dermis was found in all cases, lesional and un-affected biopsies. Some scattered IL-4 positive cellswere also detected in the epidermis. Examination ofanti-human CD3 stained adjacent sections indi-cated that these were probably infiltrating T-lymphocytes. Far less IL-4 was detected in the unaf-fected biopsies. The amount of IL-4 immunoreac-tivity in unaffected tissue was variable, dependingon the quantity of inflammatory infiltrate. No IL-4immunoreactivity was detected in any of the lesionalor non-lesional psoriatic biopsies examined.

IL-4 receptor a chain (IL-4Ra) immunoreactiv-ity was found in the dermis in all lesional and un-affected sections examined from ACD, AD and

38 ROWE & BUNKER

psoriasis biopsies, using the anti-IL-4R C-20 anti-human IL-4Ra antibody. The distribution of im-munoreactive cells was similar in all the unaffectedsections. Cells were found in close proximity todermal blood vessels, as well as to skin appendagessuch as hair follicles and sweat glands. Isolated im-munoreactive cells were also present throughoutthe reticular dermis. The distribution of these cellssuggested that they might be mast cells, which wasconfirmed by toluidine blue staining of adjacenttissue sections.

The general distribution of IL-4Ra positive cellsin lesional and unaffected ACD sections was thesame. There was no apparent change in the quan-tities of IL-4Ra immunoreactive cells in ACD, evenin the presence of substantial numbers of infiltrat-ing inflammatory cells. There appeared to be moreIL-4Ra cells in the dermis and dermal infiltrate inlesional sections than in unaffected sections fromboth AD and psoriasis biopsies.

Discussion

Our findings of IL-4 in the dermis in AD, but notin psoriasis, confirm previous reports by otherauthors (8, 9) and validate the antibodies we haveused in our studies of ACD.

We have shown that IL-4 is present in the dermalinfiltrate in some, but not all, ACD lesions. Ourlocalization of IL-4 confirms some other studiesof cytokine expression in ACD. IL-4 mRNA andprotein have been found in some ex-vivo ACD Tcell clones (5, 6). Anti-IL-4 antibody blocking ex-periments have also demonstrated a role for IL-4in contact sensitivity in mice (7).

Our findings indicate either a TH2 or TH0mechanism in some ACD lesions. Absence of IL-4 in some ACD lesions is indicative of a TH1 im-mune response, although we have not examinedthe expression of IFN-g or any other TH1 cyto-kines in these experiments. Our results indicate nocorrelation between expression of IL-4 and theallergen involved in ACD. Both TH1 and TH2cytokine profiles have been reported previously inNi-reactive T cell clones in from ACD skin (4, 6).Further studies with larger numbers of patients,examining both IL-4 and TH1 cytokines, will berequired to establish if the cytokine profiles inACD lesions do show trends towards TH1, TH2or a non-specific. Similar studies are also requiredto establish if there is any relationship betweenallergen types and cytokine expression in ACD.

Local expression of IL-4 in the skin could haveseveral proinflammatory effects relevant to skin in-flammation. IL-4 stimulates mast cell populationexpansion and activation (12), increased express-ion of low affinity Fc receptors (CD23) on antigen

presenting cells (13), and up-regulation of VCAM(vascular cell adhesion molecule) expression, so in-creasing potential infiltration of inflammatory cellsinto tissue (14).

Constitutive expression of IL-4 receptors on cu-taneous mast cells provides a mechanism throughwhich local IL-4 signalling could be transduced,for example by stimulating the local release of in-flammatory mediators and effectors.

There was no apparent difference in numbers ofimmunoreactive cells in ACD lesions and unaf-fected skin, although our sample sizes were toosmall to conduct a meaningful statistical analysis.However, the apparent increases in immunoreac-tive cells we observed in AD and psoriasis lesionscould be related to increased numbers of mast cellsobserved in AD and psoriatic lesions (15).

Acknowledgement

This research was funded by the Skin Treatmentand Research Trust.

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Address:

A. RoweSkin Treatment and Research Trust LaboratoryDivision of MedicineImperial College School of MedicineChelsea and Westminster Hospital369 Fulham Road, London SW10 9NHUK