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 atopiceczema. The mean serum IgE level was raised but levels of the other major immunoglobulin classeswere normal. Ten per cent of patients failed to respond to tetanus immunization. All patients re-sponded to S. typhi H antigen. Fourteen per cent of patients failed to mount delayed hypersensitivityreactions 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 wasthe spontaneous lymphocyte uptake. T and B lymphocyte numbers in the peripheral blood werenormal. These results are similar to those found in asthmatic patients and support the hypothesisthat, in some patients, atopic eczema is associated with an immunodeficiency state.

Immunoglobulin E antibodies are associated with the atopic disorders, asthma, hay fever and atopiceczema. There is variability in the allergens to which an atopic individual will develop hypersensitivityand variation in the organs which are affected. The fundamental reason or reasons for clinical mani-festations in a proportion of the population is still not clear. Several hypotheses, not necessarilymutually exclusive, have been put forward to explain the development of the atopic state. The familialclustering 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 aresult 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 theautonomic 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 bet-ween antigen and immunologically competent cells (Leskowitz, Salvaggio & Schwartz, 1972).

In 1937, Rostenberg & Sulzberger showed that patients with atopic dermatitis were much lesslikely to have delayed hypersensitivity reactions on patch testing with a wide range of antigens thanpatients with contact dermatitis. Kaufman & Hobbs (1970) have suggested that this may be the firstrecord 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 atopicsubjects (Kaufman & Hobbs, 1970). They suggested that 'atopy develops in individuals whose immuno-

611

6i2 D.I.Grove, J.G.Reid and I.J.Forbes

logical vocabulary is backward'. Taylor et al. (1973) have shown that transient IgA deficiency ininfancy may be associated with the subsequent development of atopy in the children of reaginicparents.

We have reported that some patients with asthma have deficiencies of humoral or cellular immunityas shown by impaired antibody response to tetanus toxoid, impaired delayed hypersensitivity reactionsto 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 andcellular immunity could be demonstrated in atopic eczema.

Patients and controlsTwelve male and twenty-three female patients were studied. Ages ranged from 5 to 64 years, with ipatient 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 fromatopic 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 dermatitiswith a definite past history of classical infantile eczema plus a past or family history of asthma orhay fever.

Control values were established on normal subjects with a similar age and sex distribution to thepatients studied. As an additional control, lymphocyte tritiated (^H) thymidine uptakes were measuredin patients with psoriasis, some of whom were receiving topical corticosteroid therapy.

METHODS

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

Antibody responsesPatients were immunized with tetanus toxoid (Commonwealth Serum Laboratories, C.S.L.) 0-5 mls.c. and typhoid vaccine (C.S.L.) o-i ml s.c; blood was collected before, and 2 weeks after immuniza-tion. Haemagglutinating antibodies to tetanus antigen, and precipitating antibodies to 5. typhi Hantigen, were measured as previously described (Forbes, 1971).

Most South Australians have previously been exposed to tetanus toxoid, but have not usually beenimmunized with typhoid vaccine. Mercaptoethanol treatment of sera from normal subjects, patientswith chronic infections, patients with dystrophia myotonica, and asthmatics confirmed that response totetanus toxoid was usually of the IgG type, while 5. typhi H antigen produced an IgM response(Forbes, 1971).

Delayed hypersensitivity reactionsDelayed 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 48hours. An area of induration of 5 mm or more was considered positive. Patients were considerednegative if they failed to react to at least one antigen. Tests were not performed on areas of dermatitis,nor on areas receiving topical corticosteroids.

Immunity in atopic eczema 613

Lymphocyte tritiated thymidine uptakePhytohaemagglutinin (PHA)-inducedi uptake of ^H thymidine hy lymphocytes was measured inwhole blood culture. Dose response curves to PHA were estabhshed. The ^H thymidine uptake atvarying 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 asdescribed 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 (specificactivity:500 mCi/mmol) were added and incubated for another 4 h. Cultures were then kept at 4°Cfor 30 min; centrifuged at 250 g for 10 min, washed with 3 % acetic acid, 0 9 % saline, and 5% tri-chloracedc acid in 0-45% saline, in succession; centrifuged at 1,000 g for 20 min with 5% trichlorace-tic 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 byincubating 02 ml of whole blood in 3-8 ml of medium with 25 ^Ci of ^H thymidine at 37°C for4 h then treated as above. Results were expressed as disintegrations per culture per minute.

B and T lymphocytesB cells were measured by the method of Froland & Natvig (1971) and T cells by the method of WybranChantler & Fudenberg (1973).

Ten ml of heparinized blood was diluted with 20 ml of Dulbecco buffer pH 7-4 (C.S.L.) and layeredon 10 ml of Ficoll/Hypaque mixture, 12 parts of 9% FicoU (Pharmacia) in distilled water and 5 parts of33% Hypaque (Winthrop), in a glass centrifuge tube then spun for 40 min at 400 g. The lymphocytelayer, containing 90-95% lymphocytes, was removed, spun down and washed twice with Dulbeccobuffer. 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 addedto each tube with two drops of heat-inactivated fetal calf serum (C.S.L.). The tubes were incubatedfor I h at 4°C, washed three dmes with Dulbecco buffer, resuspended in fetal calf serum, thenthe percentage of fiuorescing lymphocytes counted under U.V. light with a Zeiss microscope. Thespecificity 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 lymphocyteswas added to i ml of 0-5% sheep red cells and 3 drops of heat-inactivated fetal calf serum in a centri-fuge tube. The cells were then spun at 150 g for 5 min and kept at 4°C for i h. The cells were re-suspended gently and i drop of toluidine blue added. The percentage of rosette-forming cells wascounted 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 werenormal (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. Haemagglutinat-ing antibodies to tetanus toxoid were not detectable in three (10%) of the patients. Each patient wasage and sex matched with two controls, all of whom responded to immunization. This difference wassignificant (P<o-O4, Fisher's exact test).

6i4 D.LGrove, J.G.Reid and LJ.Forbes

TABLE I. Serum immunoglobulin levels

Number Mean s.d. Probability*

N.S.

N.S.

N.S.

N.S.

<o-ooi

* Probability by 't' test, f Geometric mean. IgG, IgA andIgM: mg/ioo ml. IgD, IgE: units/ml. N.S., not significant.

Precipitating antibodies to S. typhi H antigen were found in all patients and controls.

Delayed hypersensitivity reactionsFourteen per cent of patients failed to react to any of the three intradermal antigens, compared withno failure in the controls (Table 2). Atopic eczema patients reacted to 55% while controls reacted to73% of all the tests performed.

Patients IgGControlsPatients IgAControlsPatients IgMfControlsPatients IgDfControlsPatients IgEtControls

353535353535353535«

14601560166

195172

170

12

133481 0 0

320

32560

117366

9980

1780107

TABLE 2. Delayed hypersensitivity reactions

Reactors Non-Reactors Probability

(a) Patients who reacted to at least one antigen

Patients 30 5Controls 35 o <o-O3

(b) Total number of reactions to the three antigens

Patients 58 47Controls 77 28 <o-ooi

Probability by Fisher's exact test.

Lymphocyte trttiated thymidine uptakeThe uptake of ^H thymidine by lymphocytes is shown in Table 3. There was no significant differencein uptake, either spontaneously or stimulated with PHA when cultured in either autologous or fetalcalf serum. There were no significant differences in any of the parameters between the twenty-twopatients using betamethasone cream and the twelve not using a steroid preparation. Similarly therewere no differences in PHA-stimulated uptake between twenty-six psoriatic patients using, and thirteenpsoriatic patients not using, steroid preparations.

There were no significant differences in the PHA-stimulated uptake of DHS non-reactors comparedwith DHS reactors.

Immunity in atopic eczema

TABLE 3. Lymphocyte ^H thymidine uptake*

615

PHA ^H uptake—autol.

PHA ^H uptake—F.C.S.

Spontaneous—^H uptake

PatientsControls

PatientsControls

PatientsControls

Number

3434

3030

3535

Mean

51,00055,800

58,00051,500

42Ot376t

s.d.

26,00027,000

22,00020,000

470155

Probability

N.S.

N.S.

N.S.

* Disintegrations per culture per min. t Geometric mean. Probability by'r' test. N.S., not significant.

T and B lymphocytesThere were no significant differences in either the absolute lymphocyte count, T cell numbers, or totalIgG, IgA and IgM B cell numbers (Table 4).

TABLE 4. T and B lymphocytes: eosinophils*

Lymphocytes

T cells

B cells

IgG

IgA

IgM

Eosinophils

PatientsControls

PatientsControls

PatientsControls

PatientsControls

PatientsControls

PatientsControls

PatientsControls

Number

3434

3434

3434

3434

3434

3434

3434

Mean

19501840

920

910

280

2 2 0

1 7 0

1 4 0

7478

150150

i6ot42t

s.d.

750410

490350

1751 0 0

1 0 0

60

5648

1 0 0

80

1500500

Probability

N.S.

N.S.

N.S.

N.S.

N.S.

N.S.

< 0"00I

* Cells//(I. t Geometric mean. Probability by 't' test. N.S., not significant.

EosinophilsThere was a significantly higher eosinophil count in patients than controls (Table 4).

DISCUSSION

These results provide evidence of impairment of antibody production or cellular immune processesin some patients with atopic eczema. They are consistent with a well-known clinical observation whichsuggested the possibility of immunodeficiency in atopic eczema. Kaposi's varicelliform eruptionusually results from infection with herpes simplex or vaccinia viruses in patients with atopic eczema(Rook, Wilkinson & Ebling, 1972) suggesting that defence mechanisms are impaired. Moreover, manypatients with eczema vaccinatum respond to hyperimmune antivaccinial gamma globulin (Sharp &Fletcher, 1973).

6i6 D.I.Grove, J.G.Reid and I.J.Forbes

The mean IgE level was elevated as has been shown in other studies (Juhlin, Johansson & Bennich,1969J Stone, Muller & Gleich, 1973). This elevation was greater than that reported by us (Groveet al., 1975) in asthmatic patients, as has been observed by others (Johansson et al., 1970; Wood &Oliver, 1972). Serum IgG levels were normal, in contrast to the observations of Varelzidis et al. (1966)who found raised levels.

Ten per cent of patients failed to respond to immunization with tetanus toxoid. This represents notmerely failure to increase titre but failure to produce any detectable haemagglutinating antibodies 2weeks after immunization. The response to S. typhi H antigen was normal. Response to tetanus toxoidmay be a relatively sensitive indicator of humoral immunological competence or reflect the greaterthymus-dependence of tetanus antigen (Hess, 1968) compared with Salmonella flagellar antigen(Davies et al., 1970).

Fourteen per cent of patients failed to make DHS reactions to intradermal antigen. Moreover,there was a significant reduction in the total number of reactions to antigens compared with normalsubjects. These findings support those of Rajka (1970) who showed that delayed reactions to bacterialand viral extracts were impaired in atopic eczema. It may be expected that patients with a cellularimmune defect would have less contact dermatitis. Since the original observations of Rostenberg &Sulzberger, there has been considerable controversy over the prevalence of contact dermatitis inatopic eczema. A Scandinavian committee tested 1027 patients with a routine series of patch testsand found that 13% of atopies and 31% of non-atopies had positive reactions (Magnusson et al.,1969). Calnan (1956) and Caron (1964) found little evidence of atopy in nickel sensitivity. Other authorshave concluded that atopic individuals were no more likely to develop contact dermatitis than normalpersons (Wilson, 1955; Jillson, Curwen & Alexander, 1959; Fregert & Moller, 1963; Cronin et al.,1970). In contrast, some authors thought that there was a positive relationship between atopy andcontact dermatitis (Epstein & Mohajerin, 1964; Epstein, 1965; Forsbeck, Skog & Ytterborn, 1966).These interpretations, however, may reflect the greater exposure of patients with atopic eczema totopical medicaments, and any damaged skin is more likely to develop a contact dermatitis (Rooket al., 1972).

No abnormalities of lymphocyte ^H thymidine uptake were seen in patients with atopic eczema.Similarly, Fjelde & Kopecka (1967) did not demonstrate a significant reduction in PHA-stimulatedlymphocyte transformation assessed morphologically. Topical corticosteroid therapy appeared to haveno effect on lymphocyte activity either in patients with atopic eczema or in those with psoriasis. Therewere no significant abnormalities in either T or B lymphocyte numbers.

The observations of Lobitz, Honeyman & Witikler (1972) support the concept of immunodeficiencyin atopic dermatitis. They reported two adult patients with atopic dermatitis who had depressed cell-mediated immunity as evidenced by delayed hypersensitivity reactions on skin testing, inability to besensitized with dinitrochlorobenzene, reduced lymphocyte response to PHA, and lymphadenopathy.Moreover, patients with the Wiskott-Aldrich syndrome frequently have an atopic type of eczema inassociation with impaired delayed hypersensitivity reactions (Cooper et al., 1964), and have beenshown to have raised serum IgE levels (Berglund et al., 1968).

The data presented parallel the observations in asthmatics (Grove et al., 1975), although there weresome differences. IgG levels were elevated and PHA-stimulated uptake of lymphocytes cultured infetal calf serum was depressed in asthmatics. It is of interest that this depression was less in patientswith a past history of eczema. More asthmatics failed to respond to tetanus immunization and DHSskin reactivity was less impaired than in patients with atopic eczema. These variations in the frequen-cies of different abnormalities may be chance occurrences or related to the different clinical mani-festations of atopy.

These data support the hypothesis that in some patients with atopy there are deficiencies in the

Immunity in atopic eczema 617

immunological defence mechanisms leading to increased antigenic stimulation of the reaginicsystem.

ACKNOWLEDGMENT

Dr D.I. Grove is a Postgraduate Medical Research Scholar of the National Health and MedicalResearch Council of Australia.

REFERENCES

BERGLUND, G. , FINNSTROM, O., JOHANSSON, S .G .O. & MOLLER, K . L . (1968) Wiskott-Aldrich Syndrome: a studyof 6 cases with determination of the immunoglobulins A,D,G,M. and ND. Acta Paediatrica Scandinavica,51, 89.

CALNAN, CD. (1956) Nickel dermatitis. British Journal of Dermatology, 68, 229.CARON, G.A. (1964) Nickel sensitivity and atopy. British Journal of Dermatology, 76, 384.COOPER, M.D., CHASE, P., GEME, J.S.ST.,KNIVIT, W.,& GOOD, R.A. (1964) Wiskott-Aldrich Syndrome: a model

of impaired defence mechanisms. Journal of Laboratory and Clinical Medicine, 64, 849.CRONIN, E. , BANDMANN, H.J., CALNAN, CD. , FREGERT, S., HJORTH, N . , MAGNUSSON, B. , MAIBACH, H.I.,

MALTEN, K . , MENENGHINI, V., PIRILA, V. & WILKINSON, D . S . (1970) Contact dermatitis in the atopic.Acta dermato-venereologica, 50, 183.

DAVIES, A.J.S., CARTER, R.L., LEUCHARS, E . , WALLIS,V. & DIETRICH, F . M . (1970) The morphology of immunereactions in normal, thymectomized and reconstituted mice. II. Response to bacterial antigens. Immunology,19, 945.

EPSTEIN, S. (1965) Neomycin sensitivity and atopy. Dermatologica, 130, 280.EPSTEIN, S. & MOHAJERIN, A . H . (1964) Incidence of contact sensitivity in atopic dermatitis. Archives of Dermatology,

90, 284.FjELDE, A. & KOPECKA, B. (1967) Cell transformation and mitogenic effects on blood leukocyte cultures of atopic

dermatitis patients. Dermatologica, 134, 95.FORBES, LJ . (1971) Measurement of immunological function in clinical medicine. Australian and New Zealand

Journal of Medicine, i, 160.FoRSBECK, M., SKOG, E. & YTTERBORN, K . H . (1966) The frequency of allergic disease among relatives of patients

with allergic eczematous contact dermatitis. Acta dermato-venereologica, 46, 149.FREGERT, S. & MOLLER, H . (1963) Contact allergy to balsam of Peru in children. British Journal of Dermatology,

IS, 218.FROLAND, S. & NATVIG, J .B . (1971) Surface-bound immunoglobulin as a marker of B lymphocytes in man.

Nature New Biology, 234, 251.GROVE, D.I., BURSTON, T.O., WELLBY, M.L., FORD, R . M . & FORBES, I .J . (1975). Humoral and cellular immunity

in asthma. Journal of Allergy and Clinical Immunology, (in press).HESS, M . W . (1968) Experimental Thymectomy. Springer, Berlin.HOBBS, J.R. (1969) In: Immunology and Development (Ed. by M.Adinolfi), London.JiLLSON, O.F., CuRWEN, W.L. & ALEXANDER, B.R. (i959) Problems of contact dermatitis in the atopic individual

with reference to neomycin and ragweed oil sensitivity. Annals of Allergy, 17, 215.JOHANSSON, S.G.O., BENNICH, H . , BERG, T . & HOGMAN, C (1970) Some factors influencing the serum IgE levels

in atopic diseases. Clinical and Experimental Immunology, 6, 43.JuHLiN, L., JOHANSSON, S.G.O. & BENNICH, H . (1969) IgE in dermatoses: levels in atopic dermatitis and urticaria.

Archives of Dermatology, 100, 12.KAUFMAN, H . S . & HOBBS, J.R. (1970) Immunoglobulin deficiencies in an atopic population. Lancet, ii, 1061.KUHNS, S.J. & PAPPENHEIMER, A.M. (1952) Immunochemical studies of antitoxin produced in normal and allergic

individuals hyperimmunized with diphtheria toxoid. _7oMrna/ of Experimental Medicine, 4, 363.LESKOWITZ, S., SALVAGGIO, J .E . & SCHARTZ, H . J . (1972) An hypothesis for the development of atopic allergy in

man. Clinical Allergy, 2, 237.LOBITZ, W.C, HONEYMAN, J.F. & WiNKLER, N.W. (1972) Suppressed cell-mediated immunity in two adults with

atopic dermatitis. British Journal of Dermatology, 86, 317.MAGNUSSON, B., FREGERT, S., HJORTH, N . , HOUDING, G. , PIRILA, V. & SKOG, E . (1969) Routine patch testing.

V. Fifth report by the Scandinavian committee for standardization of routine patch testing. Acta dermato-venereologica, 49, 556.

6i8 D.I.Grove, J.G.Reid and I.J.Forbes

RAJKA, G. (1970) Delayed reactivity to bacterial and viral extracts in different dermatoses. Acta dermato-venereolo-gica, 50, 28.

ROOK, A., WILKINSON, D.S. & EBLING, F.J.G. (1972) Textbook of Dermatology, 2nd edn,p. 569. Blackwell, Oxford.ROSTENBERG, A. & SuLZBERGER, M.B. (1937) Some results of patch tests. Archives of Dermatology and Syphilology,

35. 433-RowE, D.S. (1969) Radioactive single radial diffusion. A method for increasing the sensitivity of immunochemical

quantification of proteins on agar gel. Bulletin of the World Health Organization, 40,613.SHARP, J.C.M. & FLETCHER, W.B. (1973). Experience of antivaccinia immunoglobulin in the United Kingdom.

Lancet, i, 656.SHERMAN, W.B. (1965). Heredity and antigen exposure in the development of atopic disease. Immunological

Diseases. (Ed. by M.Souter, and H.L.Alexander). Little Brown, Boston.STONE, S.P., MULLER, S.A. & GLEICH, G.J. (1973) IgE levels in atopic dermatitis. Archives of Dermatology, 108,

806.SzENTiVANYi, A. (1968) The beta adrenergic theory of the atopic abnormality in bronchial asthma. Journal of

Allergy, 42, 203.TAYLOR, B., NORMAN, A.P., OGEL, H.A., STOKES, C.R., TURNER, M.W. & SOOTHILL, J.F. (1973) Transient IgA

deficiency and pathogenesis of infantile atopy. Lancet, ii, i i i .TowNLEY, R.G., DENNIS, M . M . & ITKIN, I.H. (1965) Comparative action of acetyl-beta-methylcholine, histamine

and pollen antigens in subjects with hay fever and bronchial asthma. Jowrna/ of Allergy, 36, 121.VARELZIDIS, A., WILSON, A.B., MEARA, R.H. & TURK, J.L. (1966) Immunoglobulin levels in atopic eczema

British Medical Journal, 2, 925.WILSON, H.T.H. (1955) Standard patch tests in eczema and dermatitis. British Journal of Dermatology, 67, 291.WOOD, C.B.S. & OLIVER, J. (1972) Serum IgE in asthmatic children in relation to age, sex, eczema and skin

sensitivity tests. Archives of Diseases of Childhood, 47, 890.WYBRAN, J., CHANTLER, S. & FUDENBERG, H.H. (1973). Human blood T cells: Response to phytohemagglutinin.

Journal of Immunology, n o , 1157.