British Journal of Dermatology (1975) 92, 611.

Humoral and cellular immunity in atopic eczema D.I.GROVE, J.G.REID AND I.J.FORBES Department of Medicine, University of Adelaide, and Dermatology Unit, Queen Elizabeth Hospital, Woodville, South Australia 5011 Accepted for publication 13 October 1974

SUMMARY

Parameters of humoral and cellular immunity were measured in thirty-five patients with atopic eczema. The mean serum IgE level was raised but levels of the other major immunoglobulin classes were normal. Ten per cent of patients failed to respond to tetanus immunization. All patients responded to S. typhi H antigen. Fourteen per cent of patients failed to mount delayed hypersensitivity reactions to a battery of three intradermal antigens. The phytohaemagglutinin-stimulated uptake of ^H thymidine by lymphocytes was normal in the presence of autologous or of fetal calf serum, as was the spontaneous lymphocyte uptake. T and B lymphocyte numbers in the peripheral blood were normal. These results are similar to those found in asthmatic patients and support the hypothesis that, in some patients, atopic eczema is associated with an immunodeficiency state.

Immunoglobulin E antibodies are associated with the atopic disorders, asthma, hay fever and atopic eczema. There is variability in the allergens to which an atopic individual will develop hypersensitivity and variation in the organs which are affected. The fundamental reason or reasons for clinical manifestations in a proportion of the population is still not clear. Several hypotheses, not necessarily mutually exclusive, have been put forward to explain the development of the atopic state. The familial clustering of atopic disease suggests that an atopic tendency may be inherited (Sherman, 1965). Increased sensitivity to pharmacological intermediates released from mast cells or basophils as a result of the interaction of antigen with surface IgE may be an underlying fault in atopy (Townley, Dennis & Itkin, 1965). It has been suggested that in asthma there is a functional imbalance of the autonomic nervous system with partial blockade of the beta adrenergic system (Szentivanyi, 196&). There may be a defect in the mucosal barrier in atopic subjects leading to more efficient contact between antigen and immunologically competent cells (Leskowitz, Salvaggio & Schwartz, 1972). In 1937, Rostenberg & Sulzberger showed that patients with atopic dermatitis were much less likely to have delayed hypersensitivity reactions on patch testing with a wide range of antigens than patients with contact dermatitis. Kaufman & Hobbs (1970) have suggested that this may be the first record of an association between immune deficiency and atopy. Kuhns & Pappenheimer (1952) described subjects who produced reaginic but not precipitating antibodies to diphtheria toxoid. A high prevalence of atopy has been noted in immunoglobulin deficiency syndromes (Hobbs, 1969). Conversely immunoglobulin deficiencies, especially of IgA, were found in more than 7% of atopic subjects (Kaufman & Hobbs, 1970). They suggested that 'atopy develops in individuals whose immuno611

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D.I.Grove, J.G.Reid and I.J.Forbes

logical vocabulary is backward'. Taylor et al. (1973) have shown that transient IgA deficiency in infancy may be associated with the subsequent development of atopy in the children of reaginic parents. We have reported that some patients with asthma have deficiencies of humoral or cellular immunity as shown by impaired antibody response to tetanus toxoid, impaired delayed hypersensitivity reactions to antigen and altered lymphocyte response to phytohaemagglutinin when cultured in fetal calf serum (Grove et al., 1975). This study was undertaken to determine whether abnormalities of humoral and cellular immunity could be demonstrated in atopic eczema. Patients and controls

Twelve male and twenty-three female patients were studied. Ages ranged from 5 to 64 years, with i patient in the first decade and 6, 18, 6, i, 2 and i patients in each succeeding decade. All patients had been referred for dermatological opinion and had been diagnosed as suffering from atopic dermatitis on the basis of the clinical history and morphology and distribution of the lesions. Patients had either the typical fiexural eczematous lesions with lichenification, or atypical dermatitis with a definite past history of classical infantile eczema plus a past or family history of asthma or hay fever. Control values were established on normal subjects with a similar age and sex distribution to the patients studied. As an additional control, lymphocyte tritiated (^H) thymidine uptakes were measured in patients with psoriasis, some of whom were receiving topical corticosteroid therapy. METHODS Immunoglobulin levels

Serum levels of immunoglobulins (Ig) G, A and M were measured with Behringwerke immunodiffusion plates. Standard solutions were obtained from Behringwerke. Serum levels of IgD and IgE were measured by the single radial diffusion method described by Rowe (1969). IgD standard was obtained from Behringwerke; IgE standard was from a batch of pooled human sera, 69/204, supplied by WHO. Antibody responses

Patients were immunized with tetanus toxoid (Commonwealth Serum Laboratories, C.S.L.) 0-5 ml s.c. and typhoid vaccine (C.S.L.) o-i ml s.c; blood was collected before, and 2 weeks after immunization. Haemagglutinating antibodies to tetanus antigen, and precipitating antibodies to 5. typhi H antigen, were measured as previously described (Forbes, 1971). Most South Australians have previously been exposed to tetanus toxoid, but have not usually been immunized with typhoid vaccine. Mercaptoethanol treatment of sera from normal subjects, patients with chronic infections, patients with dystrophia myotonica, and asthmatics confirmed that response to tetanus toxoid was usually of the IgG type, while 5. typhi H antigen produced an IgM response (Forbes, 1971). Delayed hypersensitivity reactions

Delayed hypersensitivity (DHS) reactions to intradermal injections of o-i ml of Candida albicans (1%) (Bencard), Mumps Skin Test Antigen (Eli Lilly), and Streptokinase-Streptodornase ('Varidase', Lederle) containing 10 units of streptokinase and 25 xmits of streptodornase were measured at 48 hours. An area of induration of 5 mm or more was considered positive. Patients were considered negative if they failed to react to at least one antigen. Tests were not performed on areas of dermatitis, nor on areas receiving topical corticosteroids.

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Lymphocyte tritiated thymidine uptake Phytohaemagglutinin (PHA)-inducedi uptake of ^H thymidine hy lymphocytes was measured in whole blood culture. Dose response curves to PHA were estabhshed. The ^H thymidine uptake at varying periods of incubation with the PHA dose producing maximum responsiveness was measured. On the basis of these results, the most appropriate PHA dosage and period of incubation were used as described below. Cultures contained 0-2 ml heparinized whole blood, 3-4 ml HEPES-buffered medium 199 (C.S.L.), 002 ml PHA (Wellcome, Reagent Grade) and 0-4 ml of fetal calf serum (C.S.L.) or autologous serum. Triplicate cultures were incubated for 4 days at 37°C. At 92 hours, 2-5 ^iCi ^H thymidine (specific activity:500 mCi/mmol) were added and incubated for another 4 h. Cultures were then kept at 4°C for 30 min; centrifuged at 250 g for 10 min, washed with 3 % acetic acid, 0 9 % saline, and 5% trichloracedc acid in 0-45% saline, in succession; centrifuged at 1,000 g for 20 min with 5% trichloracetic acid and twice with methanol. After drying, the residue was dissolved in 0-5 ml Soluene (Packard) for liquid scintillation counting. The spontaneous uptake of ^H thymidine by cultures, i.e. in the absence of PHA, was measured by incubating 0 2 ml of whole blood in 3-8 ml of medium with 25 ^Ci of ^H thymidine at 37°C for 4 h then treated as above. Results were expressed as disintegrations per culture per minute. B and T lymphocytes B cells were measured by the method of Froland & Natvig (1971) and T cells by the method of Wybran Chantler & Fudenberg (1973). Ten ml of heparinized blood was diluted with 20 ml of Dulbecco buffer pH 7-4 (C.S.L.) and layered on 10 ml of Ficoll/Hypaque mixture, 12 parts of 9% FicoU (Pharmacia) in distilled water and 5 parts of 33% Hypaque (Winthrop), in a glass centrifuge tube then spun for 40 min at 400 g. The lymphocyte layer, containing 90-95% lymphocytes, was removed, spun down and washed twice with Dulbecco buffer. Cells were resuspended in buffer and divided into five aliquots. One drop of antiserum to IgG, IgA and IgM (Hyland) and polyvalent antiserum (Roboz) was added to each tube with two drops of heat-inactivated fetal calf serum (C.S.L.). The tubes were incubated for I h at 4°C, washed three dmes with Dulbecco buffer, resuspended in fetal calf serum, then the percentage of fiuorescing lymphocytes counted under U.V. light with a Zeiss microscope. The specificity of the serum was confirmed by blocking experiments with unlabelled antiserum. Sheep red cells (C.S.L.) were washed three times in saline. A suspension of 2,000,000 lymphocytes was added to i ml of 0-5% sheep red cells and 3 drops of heat-inactivated fetal calf serum in a centrifuge tube. The cells were then spun at 150 g for 5 min and kept at 4°C for i h. The cells were resuspended gently and i drop of toluidine blue added. The percentage of rosette-forming cells was counted on a Neubauer counting chamber. RESULTS

Immunoglobulin levels

IgE levels were raised in patients with atopic eczema, while levels of IgG, IgA, IgM and IgD were normal (Table i). Thirty-seven per cent of patients had IgE levels above the normal range. Antibody responses

Antibody titres were measured before and after immunization in thirty-one patients. Haemagglutinating antibodies to tetanus toxoid were not detectable in three (10%) of the patients. Each patient was age and sex matched with two controls, all of whom responded to immunization. This difference was significant (P

Humoral and cellular immunity in atopic eczema.

Parameters of humoral and cellular immunity were measured in thirty-five patients with atopic eczema. The mean serum IgE level was raised but levels o...
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