Journal of Clinical Immunology, Vol. 11, No. 1, 1991
Interleukin 2 Therapy in Severe Atopic Dermatitis K U E - H S I U N G HSIEH, 1'3 CHEN-CHENG CHOU, 1 and SHIU-FENG H U A N G 2
Accepted: October 25, 1990
an incidence of 1.9 to 8.3% in Caucasian (3-6). Hsieh et al. (7) found a prevalence of 1.24% among 167,373 Chinese school children. The natural course is unpredictable and approximately 15 to 20% of patients have persistent dermatitis that is unchanged or worsening in childhood (2). In those patients with severe dermatitis, the fluctuating symptoms are often unendurable and the present therapy disappointing. (8). Better understanding of the pathophysiology is essential for the rational design of a therapeutic strategy. Although the pathogenesis of atopic dermatitis remains unclear, a number of immunological abnormalities have been described. They include a significant decrease in suppressor/cytotoxic T lymphocytes and an increased CD4/CD8 ratio (9-12), decreased natural killer-cell activity (10, 13), defective capacity to generate alloreactive cytotoxic T cells (14), impaired autologous mixed lymphocyte reaction due to defective CD4 responder T cells (15), and increased cell-mediated cytotoxicity against skin fibroblasts (16). In view of its broad immunoregulatory capacity, interleukin 2 (IL-2) was tried in six children with severe atopic dermatitis which was refractory to conventional treatment, in the hope of exploring the pathogenesis of atopic dermatitis, on the one hand, and finding a new therapeutic approach, on the other hand.
Interleukin 2 (IL-2) at a dose of 10,000 to 20,000 U/kg/q 8 hr was given for 9-12 days to six patients with cases of severe atopic dermatitis (AD) which were refractory to conventional therapy. After IL-2 therapy, the clinical symptoms and signs of eczema including pruritus, scratching, papulovesicles, and lichenification were much improved, but all of them recurred 2-6 weeks after stopping treatment. Adverse reactions were similar to those reported previously, but all of them subsided after discontinuation of therapy. Laboratory findings showed decreased T-cell subsets, especially CD4+ cells, and increased IL-2R+ (CD25) cells, but there was no significant change in serum IL-2, serum IgE, or in vitro IgE production. Immunopathological studies of the skin biopsies showed decreased mononuclear-cell infiltration, depletion of CD4+ ceils, and enhanced expression of CD25 and HLA-DR antigens. As lymphokine-activated killer (LAK)-cell activity against cultured fibroblasts was similar in patients with AD and in normals and CDI+ Langerhans ceils were not decreased after IL-2 therapy, we speculate that the depletion of helper/inducer CD4+ cells and hence abrogation of the exaggerated antigen processing and cellular activation in diseased skin are the explanation for the transient efficacy of IL-2 in the treatment of atopic dermatitis. KEY WORDS: tnterleukin 2; atopic dermatitis.
INTRODUCTION Atopic dermatitis is a chronic cutaneous inflammatory disease characterized by early age of onset, severe pruritus, typical morphology and distribution of skin rash, chronic relapsing course, and personal or family history of atopy (I, 2). It is a common disease among infants and children, with
MATERIALS AND METHODS
Subjects
1Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan, Republic of China. 2Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan, Republic of China. 3To whom correspondence should be addressed at Department of Pediatrics, National Taiwan University Hospital, 1 ChangTeh Street, Taipei, 10016, Taiwan, Republic of China.
From May 1988 to March 1989, six children with severe atopic dermatitis, three males and three females, aged 2 to 11 years, were admitted for a clinical trial of IL-2. All of them had severe pruritus, skin lesions of characteristic morphology and 22 0271-9142Dlf0100-00225~6.50/0© 1991PlenumPublishingCorporation
INTERLEUKIN 2 THERAPY IN ATOPIC DERMATITIS
distribution, a chronic relapsing course, and a history of atopy which met the criteria for the diagnosis of atopic dermatitis (1). The age of onset ranged from 1 month to 1 year, and the serum IgE levels were between 578 and 4175 IU/ml. The severity of eczema was classified according to the scoring system of Clendennig et al. (17) and Sampson (18). The parameters used in the scoring system include the extent of the skin rash (0 to 6), intensity of dermatitis (0 to 6), intensity of symptoms (0 to 6), use of antihistamine, antibiotics, or steroids (0 to 9), and days missed from school or parent days missed from work because of atopic dermatitis (0 to 3). All patients had a score of more than 20 and were classified as severe. Two had bronchial asthma and one had allergic rhinitis. Moreover, all of them were refractory to conventional therapy (6, 8), including systemic steroids, elimination of food allergens, and environmental control.
Treatment Protocol Human recombinant interleukin 2 (rIL-2; Cetus Corporation, Emeryvitl, CA) was given for 9-t2 days. The initial dose of rIL-2 was 20,000 U/kg/q 8 hr via intravenous infusion over a period of 1 to 4 hr. However, the dose was adjusted (decreased) according to the tolerance of the patients and the total amount of riL-2 administered ranged from 270,000 to 720,000 U/kg/day. Systemic steroids were withheld for at least 2 weeks before inclusion in the study, but local steroids and antihistamines were used liberally. The clinical response was evaluated by one dermatologist who did not know the treatment protocol. Adverse reactions were observed carefully. Blood was drawn for immunological tests before treatment, on the seventh day of treatment (8 hr after IL-2 infusion), and 3 days after completion of therapy. Skin biopsies were also performed before and after therapy. The clinical trial was approved by the human research committee of this hospital and informed consent was obtained in each case before commencement of the study.
Immunological Tests Enumeration of lymphocyte subpopulations. The analysis of lymphocyte surface markers was performed by a direct immunofluorescence technique. Fluorescinated monoclonal antibodies (CD1, CD3,
Journal of Clinical Immunology, Vol. 1t, No. I, 1991
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CD4, CD8, CD25) were obtained from Becton Dickinson Co. (Mountain View, CA). One million peripheral blood mononuclear cells (MNC) in 0.2 ml RPMI medium were incubated with 10 ~l of each monoclonal antibody at 4°C for 30 rain and washed twice with HBSS. The cells were enumerated by a FACScan (Becton Dickinson Immunocytometry Systems). The gate window for lymphocytes was determined by the combinations of forward and right-angle light scatter. The green fluorescence signals at 488-nm excitation wavelength were measured and displayed as a histogram on the display. The results are expressed as a percentage of positive cells. Lymphoprol(ferative Responses. The mitogeninduced lymphoproliferation was measured by the method of Bradley (19). Determinations of' lgE Concentrations. In vitro IgE production was done according to the method of Saryan et a/.(20). The IgE concentrations in the sera and culture media were determined with Phadebas IgE kits (Pharmacia, Uppsala, Sweden). Quantitation of IL-2. In vitro IL-2 production was performed as previously described (21). Briefly, mononuclear cells were adjusted to a concentration of 2 × 106 cells/ml in complete culture medium containing 1% phytohemagglutinin (PHA; GIBCO) and cultured at 37°C in a humidified 5% CO2 incubator for 36 hr. The supernatants were collected by centrifugation and IL-2 was titrated by the method of Gillis et al. (22) and IntertesT-2 kit (Genzyme, Boston, MA).
Immunopathological Studies of Skin Specimens Skin biopsies were done on the same diseased area in five of the six patients before and after IL-2 therapy. Immunochemical studies were done by avidin-biotin complex stain (23, 24). Frozen tissue sections were fixed in acetone for 10 min and overlaid with the optimal dilution of monoclonal antibody for 60 rain at room temperature, followed by incubation with biotin-labeled horse anti-mouse IgG and avidin-biotin-peroxidase complex (Vector Laboratories Inc., Burlingame, CA). A number of monoclonal antibodies including OKT3 (CD3), OKT4 (CD4), OKT8 (CD8), and OKT6 (CD1) from Orthoclone Corp. (Raritan, N J) and IL-2R (CD25) and HLA-DR from Becton Dickinson (Mountain View, CA) were used. Skin biopsies and interpretation of immunopathological findings were done by
24
HSIEH, CHOU, AND HUANG
a pathologist who was blind as to the treatment status.
Allogeneic Fibroblast Cytotoxicity Assay Chromium-51 (5~Cr) release assays were performed in four patients and four healthy adults according to the method described by Leung et al. (15). Confluent allogeneic fibroblast cultures were trypsinized and resuspended at a concentration of 5 × 104 cells/ml in complete culture medium. Then, 100 ixCi sodium chromate (sp act, 200 to 500 tzCi/txg 5~Cr; New England Nuclear, Boston, MA) was added to 10 ml of the fibroblast suspension. Twotenths milliliter of this suspension, containing 1 × 104 fibroblasts, was added to each of the wells of a 96-well microtiter plate (Linbro Scientific, Hamden, CT). The microtiter plate was incubated overnight in a 5% CO2 incubator at 37°C to allow the fibroblasts to attach to the bottom of the culture wells. The next day, the microtiter wells were aspirated and washed three times with HBSS containing 2% AB serum. Effector cells included freshly prepared MNC and lymphokine-activated killer cells (LAK) which were obtained by incubation with 1000 U/ml of rlL-2 (Cetus Corporation) for 3 days, as described previously (25). For the cytotoxicity assay, 1 million effector cells in 0.2 ml of complete medium were added to test wells, giving an effector-to-target cell ratio of 100: I. For the determination of spontaneous release of 5~Cr (S), 0.2 ml of complete medium was added to control wells. For the determination of total 5~Cr release (T), 0.2 ml of a 5% solution of Triton-X was added to a set of wells. All assays were performed in triplicate. The plates were placed on a rocking plateform (Bellco Glass, Inc., Vineland, NJ) oscillating at 5 cycles per min and were incubated at 37°C in humidified 5% CO2 incubator. After overnight incubation (12 to 14 hr), the plates were centrifuged at 200g for 10 rain. One-tenth milliliter of the supernatant was removed with a Hamilton syringe and its radioactivity was measured in a gamma counter. Cytotoxicity was calculated as follows: cytotoxicity (%) = ( E - S / T - S ) x 100, where S is spontaneous release of 51Cr from fibroblasts, E is lymphocyte-mediated release of 51Cr from fibroblasts, and T is total release of 51Cr from fibroblasts with Triton-X.
Fig. 1. The papulovesicles and lichenification of diseased skin (upper panel) were markedly improved after IL-2 therapy (lower panel).
Statistics Student's t test was used for statistical analysis throughout the study. RESULTS Clinical Response
In all six patients marked desquamation over the eczematous lesions, improved lichenification, and diminished itching and scratching occurred about 5 days after treatment commenced (Fig. 1). Local steroids could be stopped, and the dose of antihistamine required was decreased. However, eczema flared up in all cases 2 to 6 weeks after discontinuation of rlL-2. The adverse reactions of IL-2 therapy such as fever, chills, malaise, mild generalized erythematous maculopapules, and hepatomegaly were seen in all patients during the period of therapy and were similar to those reported previously. Serious side effects resulting from the vascular leak
Journal of Clinical Immunology, Vol. 1I, No. 1, I991
INTERLEUKIN 2 THERAPY tN ATOPIC DERMATITIS
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Table I. Distribution of T-Cell Subsets During IL-2 Therapy CD3 + (%) Before therapy During therapy After therapy Normal control
- 12.9 a _-2 1161)b + 10.6 +- 4154) -+ 17.7 -+ 4263) + 7.7 (2646 +- 326)
70.7 (2655 66.2 (2699 57.0 (5240 69.0
CD4+ (%) 40.0 (1513 29.5 (I 148 30.3 (1240 41.2 (1724
--- 8.7*'** - 669) -2_ 5.1" -+ 1039) - 8.3** + 1056) --+ 6.1 _+ 289)
CD8 + (%) 29.5 (1158 31.7 (1454 24.7 (2349 23.8 (968
-+ 8.9 --. 706) -+ 13.6 + 2524) -+ 9.2 - 1974) + 5.1 - 212)
CD25 + (%) 2.2 (98 6.7 (197 3.3 (223 1.2 (51
--- 2.4 --- 119) -+ 4.8 -.+ 176) - 1.8 -+ t23) -+ 0.3 +-- 15)
CD4/CD8 ratio 1.41 -+ 0.32 1.18 --- 0.70 1.35 -+ 0.46 1.80 --+ 0.40 (1.00 +- 0.26)
aMean --- SD. bAbsotute number (mean - SD). *P = 0.0t. **P = 0.05.
syndrome such as decreased urinary output, body weight gain, edema, and pleural effusion were also noted in some of the patients. All the symptoms and signs subsided within 2 to 4 days after discontinuation of therapy.
were increased after IL-2 therapy in all cases (Fig. 3, lower panel). There was, however, no remarkable change in CD3+, CD8+, or C D l + cells (data not shown).
Immunological Parameters The immunological changes in patients during the course of treatment are shown in Table I. There were no consistent changes in CD3+ cells, CD8+ cells, CD4/CD8 ratio, serum IgE, in vitro IgE production, serum IL-2, or mitogen responses. There was, however, a tendency for CD4+ cells to decrease (P < 0.0t) and IL-2R+ cells to increase during treatment when compared with the pretreatment values. It is interesting to note that although the serum IL-2 of patients was highly elevated (29.9 --+- 67.1 U/ml, compared to 1.2 - 0.8 U/ml for normals), in vitro IL-2 production could not be detected. No change in total serum IgE and in vitro IgE production was found.
lmmunohistological Changes of Skin The pathologic changes of eczematous skin are shown in Fig. 2. The specimens taken before IL-2 therapy showed hyperplastic epidermis and prominent mononuclear-cell infiltration of dermis. The skin was much less thick and the mononuclear-cell infiltration was markedly decreased after IL-2 treatment. The immunochemical study with avidinbiotin complex stain indicated that the infiltrating lymphocytes consisted of more CD4+ cells and fewer CD8+ cells, and the CDI+ Langerhans cells were increased (Fig. 3, upper panel). The CD4+ cells were markedly decreased and CD25+ cells
Journal of Clinical Immunology, Vol. 11, No. 1, 1991
Fig. 2. Histopathological findings of skin lesions before (upper panel) and after (lower panel) IL-2 therapy. The hyperplasia of epidermis and mononuclear cell infiltration of dermis were markedly decreased "after treatment (case 5). 10 x ; reduced 40% for reproduction.
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Fig. 3. Immunochemical stainings of diseased skin before (upper panel) and after (lower panel) IL-2 therapy, The CD4+ cells were markedly decreased after IL-2 therapy, There was a slight increase in CD25+ cells but no remarkable change in CDI+, CD3+, and CD8+ cells (data not shown). 200 ×; reduced 40% for reproduction.
Allogeneic Fibroblast Cytotoxicity Assay The results of the cytotoxicity assay showed that the value of 21.1 - 9.2% (mean +- SD) for fresh MNC of patients was much higher than that for healthy subjects (7.6 - 4.1%). LAK cells killed allogeneic fibroblasts to a much greater degree than did the fresh MNC, but there was no difference between healthy subjects (56.1 --- 11.3%) and patients (55.6 --- 6.3%). DISCUSSION All the patients responded to IL-2 therapy. The improvement usually occurred 5 days after treatment. The skin texture improved due to desquamation and subsidence of papulovesicles and lichenification (Fig. 1). The itching and scratching were decreased markedly. However, the eczema flared up in all cases 2-6 weeks after discontinuation of
HSIEH, CHOU, AND HUANG
IL-2. As expected, the side effects seen in adults (26) also occurred in children but were much less severe in the latter. The most probable explanation for the difference may be that all of our patients had allergic disease rather than cancers and none of them had received chemotherapy. In this study, a clear-cut therapeutic effect of IL-2, although transient, had been shown on protracted severe atopic dermatitis. As the therapeutic effect was transient and the IL-2 therapy was rather toxic, the potential harm of IL-2 should be seriously considered in a nonfatal disease such as atopic eczema. The longterm administration of lower dosages of IL-2 may provide a new promising therapeutic approach for this disease, but studies with more cases and a longer period of follow-up should be done before any solid recommendation can be advocated. The dermal pathology in atopic dermatitis, which varies with the temporal nature of the clinical lesion, in both the acute and the chronic phases is characterized by a lymphocytic infiltration without significant immunoglobulin deposition, suggesting a type IV cell-mediated mechanism for skin injury (27-29). The type of infiltrating mononuclear cells was determined by using monoclonal antibodies and the results showed that most of the infiltrating lymphocytes were of the CD4+ helper/inducer phenotype (24, 25, 27-30), with a CD4/CD8 ratio of 4.8:1, which was much higher than the ratio of2:1 found in peripheral blood (30). Moreover, CDI+ Langerhans cells were also increased. The presence of increased numbers of Langerhans cells and of CD4+ cells may reflect increased antigen processing and cellular activation in the diseased skin of patients. It has been shown that antigen-activated T cells and macrophages can release soluble factors which are capable of inducing proliferation and collagen biosynthesis by fibroblasts and basophil degranulation and mediator release (31, 32). Chronic infiltration of eczematous skin with activated T cells and macrophages, as evidenced by the expression of DR antigens, may be used to account for the collagen deposition and epidermal hyperplasia with subsequent lichenification of skin, on the one hand, and severe pruritus, on the other hand. We demonstrated the predominance of CD4+ cells, rather than CD8+ cells, in diseased skin of AD patients (Figs. 2 and 3) and these findings agree with those of other investigators (24, 25, 27-30). It is important to note that CD4 + cells in both blood and diseased skin, but not CD3+ and CD8+ cells, were markedly decreased after IL-2 treatment. This Journal of Clinical Immunology, Vol. 11, No. 1, 1991
INTERLEUKIN 2 THERAPY IN ATOPIC DERMATITIS
finding strongly supports the hypothesis that IL-2 may exert its therapeutic effect in AD through the depletion of CD4+ cells. Recently, epidermal Langerhans cells (LC) were shown to be capable of inducing antigen-specific T cells of the Th-2 subtype, which were able to produce IL-4 (33). The pleuripotent activity of IL-4 is well documented: (i) induction of the expression of FcERII on B cells and monocytes and LC; (ii) induction of normal B cells to produce soluble CD23/IgE-BF; (iii) induction of differentiation of monocytes into dendritic cells; and (iv) increase in class II MHC expression of monocytes. The elimination of CD4+ cells by IL-2 treatment will abrogate the enhanced antigen processing and cellular activation which may be involved in the induction of the eczematous response. Moreover, recent studies have demonstrated that depletion of CD4+ cells correlated with the therapeutic efficacy of anti-CD4 antibody in treating experimental allergic encephalomyelitis (34), a murine model for human multiple sclerosis (35). Administration of antimurine CD4 monoclonal antibodies has also been shown to be effective in the treatment of a variety of autoimmune diseases in animal models, including systemic lupus erythematosus (36). Finally, as CD4+ cells had been shown to possess cytotoxic activity (37), the depletion of CD4+ cells may also contribute to the clinical efficacy of IL-2 in AD. Cells of NK lineage have been reported to possess enhanced cytotoxic activity against skin fibroblasts in patients with AD (16). In this study, we found that fresh MNC of AD patients possessed a much greater cytotoxicity against allogeneic fibroblasts than did those of healthy subjects, however, no difference in LAK activity against fibroblasts was found between patients and normals. These results do not support the idea of enhanced NKmediated cytotoxicity in the pathogenesis of AD because NK activity can be enhanced by IL-2 and accounts for the major portion of LAK activity (38). Elevated serum IgE concentration has been observed in approximately 80% of patients with AD (39), and a perturbation of the balance between IgE-specific T-celt help and T-cell suppression has been postulated as the most probable cause (40). In this study, no significant change in serum IgE or in in vitro IgE synthesis was found after IL-2 therapy despite an increase in CD8+ cells and a decrease in CD4+ cells (Table I). Thus, the clinical efficacy of IL-2 in AD has no relationship with IgE production.
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No study on serum IL-2 in AD has been reported in the literature, particularly after IL-2 therapy. In this study we found that while in vitro IL-2 production could not be detected, serum IL-2 was markedly increased in patients with AD, suggesting in vivo activation of T cells. The serum IL-2 tended to decrease after IL-2 treatment and this might be due to a decrease in the number of CD4+ cells, or diminished in vivo cellular activation, or both. One major drawback of this study is the lack of control group either receiving normal saline alone or just being admitted to the hospital during the same period of time. But due to the evident side effects of IL-2, it is very difficult to blind the study. Another issue of debate is the use of IL-2 in a chronic nonfatal disease in children such as atopic eczema. The side reactions resulting from IL-2 infusion resolved completely several days after discontinuation of therapy, moreover, no clinically evident adverse effects have been observed during the past year. Thus, in view of its immune amplifying effects, long-term administration of IL-2 at a lower dose may still be worth trying in severe, refractory atopic eczema. In conclusion, transient improvement of severe atopic dermatitis has been observed after IL-2 treatment, and the clinical efficacy is possibly due to depletion of CD4+ cells and hence abrogation of the enhanced antigen processing and cellular activation and decreased production of inflammatory cytokines in diseased skin. But the mechanisms by which the CD4+ cells in atopic dermatitis are selectively depleted by IL-2 treatment need further study. REFERENCES 1. Hanifin JM, Rajka G: Diagnostic features of atopic dermatitis. Acta Derm Venerol 92(S):44-47, 1980 2. Hanifin JM: Basic and clinical aspects of atopic dermatitis. Ann Allergy 54:386-893, 1984 3. Halpern SR, Sellars WA, Johnson RB, et al.: Development of child allergy in infants fed breast, soy or cow's milk. J Allergy Clin Immunol 66:465-471, 1973 4. Kjellman N-JM: Atopic disease in seven-year-old children. Acta Pediat Scand 66:465-471, 1977 5. Walker RB, Warin RP: Incidence of eczema in early childhood. Br J Dermatol 68:182-183, 1956 6. Hanifin JM: Atopic dermatitis. In Allergy Principles and Practice, 3rd ed, E Middleton Jr, CE Reed, EF Ellis, NF Adkinson Jr, JW Yunginger (eds). St. Louis, CV Mosby, 1988, pp 1403-1428 7. Hsieh KH, Shen JC: Prevalence of childhood asthma in Taipei, Taiwan, and other Asian Pacific countries. J Asthma 25:73-82, 1988
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8. Rasmussen E: Recent development in the management of patients with atopic dermatitis. J Allergy Clin Immunol 74:771-776, 1984 9. Leung DYM, Rhodes AR, Geha RS: Enumeration of T cell subsets in atopic dermatitis using monoclonal antibodies. J Allergy Clin Immunol 67:450-455, 198t t0. Chiarelti F, Canfora G, Verrotti A, Amerio P, Morgese G: Humoral and cellular immunity in children with active and quiescent atopic dermatitis. Br J Dermatol 116:651-660, 1987 11. Braathen LR: T-cell subsets in patients with mild and severe atopic dermatitis. Acta Derm Venerol St 14:133-136, 1985 12. Delespesse G, Demauberge J, Sarfati M: Characterization of circulating T lymphocyte subpopulations in atopic eczema. J Allergy Clin Immunol 69(2):135, 1982 13. Nomiyama K, Yamashita U, Nakamura H, Suenaga Y, Nishio K: Natural killer (NK) cell activity and NK-related cell surface markers in patients with atopic dermatitis. J Dermatol 15:241-247, 1988 14. Leung DYM, Wood N, Dubey D, Rhodes AR, Geha RS: Cellular basis of defective cell-mediated lympholysis in atopic dermatitis. J Immunol 130:1678-1682, 1983 15. Leung DYM, Saryan JA, Frankel R, Lareau M, Geha RS: Impairment of the autologous mixed lymphocyte reaction in atopic dermatitis. J Clin Invest 72:1482-1486, 1983 16. Leung DYM, Parkman R, Feller J, Wood N, Geha RS: Cell-mediated cytotoxicity against skin fibroblasts in atopic dermatitis. J Immunol 128:1736-1741, 1982 17. Clendenning WE, Clack WE, Ogawa M, Ishizaka K: Serum IgE studies in atopic dermatitis. J Invest Dermatol 61:233236, 1973 18. Sampson HA: The role of food allergy and mediator release in atopic dermatitis. J Allergy Clin Immunol 8t:635-645, 1989 19. Bradley LM: Cell proliferation. In Selected Methods in Cellular Immunology, BB Mishell, SM Shiigi (eds). San Francisco, WH Freeman, 1980, pp 158-166 20. Saryan JA, Leung DYM, Geha RS: Induction of human IgE synthesis by a factor derived from T cells of patients with hyper-IgE status. J Immunol 130:242-247, 1983 21. Hsieh KH: Altered interleukin-2 production and responsiveness after hyposensitization to house dust. J Allergy Clin Immunol 76:188-194, 1985 22. Gillis S, Ferm M, Ou W, Smith KA: T cell growth factor: Parameters of production and a quantitative microassay for activity. J Immnnol 120:2027-2032, 1978 23. Shu SM, Raine L, Fanger H: Comparative study of peroxidase-antiperoxidase method and avidin-biotin-complexes method for studying polypeptide hormones with radioimmunoassays. Am J Clin Pathol 75:734-739, 1981 24. Leung DYM, Bhan AK, Schneeberger EE, Geha RS: Characterization of the mononuclearinfiltrate in atopic dermatitis using monoclonal antibodies. J Allergy Clin Immunol 71:4756, 1983
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25. Hsieh KH, Shu S, Lee CS, Chu CT, Yang CS, Chang KJ: Lysis of primary hepatic tumors by tymphokine activated killer cells. Gut 28:117-124, 1986 26. Rosenberg SA, Lotze MT, Muul LM, et al.: A progress report on the treatment of 157 patients with advanced cancer using lymphokine activated killer cells and interteukin 2 or high dose interleukin-2 alone. N Engt J Med 316:889-897, 1987 27. Braathen LR, Forte O, Natvig JB, Eeg-Larsen T: Predominance of T lymphocytes in the dermal infiltrate of atopic dermatitis. Br J Dermatol 100:511-519, t979 28. Mihm MC, Ster NA, Dvorak HF, Austin KF: The structure of normal skin and the morphology of atopic dermatitis. J Invest Dermatol 67:305-312, 1976 29. Tong AKF, Mihm MC: The pathology of atopic dermatitis. Clin Rev Allergy 4:27-42, 1986 30. Zachary CB, Allen MH, MacDonald DM: In situ quantification of T-lymphocyte subsets and Langerhans' cells in the inflammatory infiltrate of atopic eczema. Br J Dermatol 112:149-156, 1985 31. Schmidt JA, Mizel SB, Cohen D, Green D: Interteukin 1, a potential regulator of fibroblast proliferation. J Immunol 128:2177-2182, 1982 32. Thueson DO, Speck LS, Lett-Brown MA, Grant IA: Histamine releasing activity. I. Production by mitogen or antigen stimulated human mononuclear cells. J tmmunol 123:626639, 1979 33. Hauser C, Snapper J, O'Harra W, Paul W, Katz S: T helper cells grown with hapten-modified cultured Langerhans cells produce IL-4 and stimulate IgE production by B cells. Eur J Immunol 19:245-250, t989 34. Alters SE, Sakai K, Steinman L, Oi VT: Mechanisms of anti-CD4-mediated depletion and immunotherapy. A study using a set of chimeric anti-CD4 antibodies. J Immunol 144:4587-4592, 1990 35. Traugott U, Reinherz EL, Raine CS: Multiple sclerosis: Distribution of T cell subsets within active chronic lesions. Science 219:308-310, 1982 36. Wofsy D, Seaman WE: Reversal of advanced murine lupus in NZB/NZW F1 mice by treatment with monoclonal antibody to L3T4. J Immunol 138:3247-3253, 1987 37. Moretta L, Mingari MC, Sekaly PR, Moretta A, Chapuis B, Cerottini JC: Surface markers of cloned human T cells with various cytolytic activities. J Exp Med 154:569-574, 1981 38. Herberman RB, Hiserodt J, Vujanovic N, et al.: Lymphokine-activated killer cell activity. Characteristics of effector cells and their progenitors in blood and spleen. Immunol Today 8:178-181, 1987 39. Johnson E, Irons J, Patterson R, Roberts M: Serum IgE concentration in atopic dermatitis, J Allergy Clin Immunol 54:94-99, 1974 40. Leung DYM, Geha RS: Immunoregulatory abnormalities in atopic dermatitis. Clin Rev Allergy 4:6%86, 1986
Journal o f Clinical Immunology, Vol. 11, No. 1, 1991
Interleukin 2 Therapy in Severe Atopic Dermatitis K U E - H S I U N G HSIEH, 1'3 CHEN-CHENG CHOU, 1 and SHIU-FENG H U A N G 2
Accepted: October 25, 1990
an incidence of 1.9 to 8.3% in Caucasian (3-6). Hsieh et al. (7) found a prevalence of 1.24% among 167,373 Chinese school children. The natural course is unpredictable and approximately 15 to 20% of patients have persistent dermatitis that is unchanged or worsening in childhood (2). In those patients with severe dermatitis, the fluctuating symptoms are often unendurable and the present therapy disappointing. (8). Better understanding of the pathophysiology is essential for the rational design of a therapeutic strategy. Although the pathogenesis of atopic dermatitis remains unclear, a number of immunological abnormalities have been described. They include a significant decrease in suppressor/cytotoxic T lymphocytes and an increased CD4/CD8 ratio (9-12), decreased natural killer-cell activity (10, 13), defective capacity to generate alloreactive cytotoxic T cells (14), impaired autologous mixed lymphocyte reaction due to defective CD4 responder T cells (15), and increased cell-mediated cytotoxicity against skin fibroblasts (16). In view of its broad immunoregulatory capacity, interleukin 2 (IL-2) was tried in six children with severe atopic dermatitis which was refractory to conventional treatment, in the hope of exploring the pathogenesis of atopic dermatitis, on the one hand, and finding a new therapeutic approach, on the other hand.
Interleukin 2 (IL-2) at a dose of 10,000 to 20,000 U/kg/q 8 hr was given for 9-12 days to six patients with cases of severe atopic dermatitis (AD) which were refractory to conventional therapy. After IL-2 therapy, the clinical symptoms and signs of eczema including pruritus, scratching, papulovesicles, and lichenification were much improved, but all of them recurred 2-6 weeks after stopping treatment. Adverse reactions were similar to those reported previously, but all of them subsided after discontinuation of therapy. Laboratory findings showed decreased T-cell subsets, especially CD4+ cells, and increased IL-2R+ (CD25) cells, but there was no significant change in serum IL-2, serum IgE, or in vitro IgE production. Immunopathological studies of the skin biopsies showed decreased mononuclear-cell infiltration, depletion of CD4+ ceils, and enhanced expression of CD25 and HLA-DR antigens. As lymphokine-activated killer (LAK)-cell activity against cultured fibroblasts was similar in patients with AD and in normals and CDI+ Langerhans ceils were not decreased after IL-2 therapy, we speculate that the depletion of helper/inducer CD4+ cells and hence abrogation of the exaggerated antigen processing and cellular activation in diseased skin are the explanation for the transient efficacy of IL-2 in the treatment of atopic dermatitis. KEY WORDS: tnterleukin 2; atopic dermatitis.
INTRODUCTION Atopic dermatitis is a chronic cutaneous inflammatory disease characterized by early age of onset, severe pruritus, typical morphology and distribution of skin rash, chronic relapsing course, and personal or family history of atopy (I, 2). It is a common disease among infants and children, with
MATERIALS AND METHODS
Subjects
1Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan, Republic of China. 2Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan, Republic of China. 3To whom correspondence should be addressed at Department of Pediatrics, National Taiwan University Hospital, 1 ChangTeh Street, Taipei, 10016, Taiwan, Republic of China.
From May 1988 to March 1989, six children with severe atopic dermatitis, three males and three females, aged 2 to 11 years, were admitted for a clinical trial of IL-2. All of them had severe pruritus, skin lesions of characteristic morphology and 22 0271-9142Dlf0100-00225~6.50/0© 1991PlenumPublishingCorporation
INTERLEUKIN 2 THERAPY IN ATOPIC DERMATITIS
distribution, a chronic relapsing course, and a history of atopy which met the criteria for the diagnosis of atopic dermatitis (1). The age of onset ranged from 1 month to 1 year, and the serum IgE levels were between 578 and 4175 IU/ml. The severity of eczema was classified according to the scoring system of Clendennig et al. (17) and Sampson (18). The parameters used in the scoring system include the extent of the skin rash (0 to 6), intensity of dermatitis (0 to 6), intensity of symptoms (0 to 6), use of antihistamine, antibiotics, or steroids (0 to 9), and days missed from school or parent days missed from work because of atopic dermatitis (0 to 3). All patients had a score of more than 20 and were classified as severe. Two had bronchial asthma and one had allergic rhinitis. Moreover, all of them were refractory to conventional therapy (6, 8), including systemic steroids, elimination of food allergens, and environmental control.
Treatment Protocol Human recombinant interleukin 2 (rIL-2; Cetus Corporation, Emeryvitl, CA) was given for 9-t2 days. The initial dose of rIL-2 was 20,000 U/kg/q 8 hr via intravenous infusion over a period of 1 to 4 hr. However, the dose was adjusted (decreased) according to the tolerance of the patients and the total amount of riL-2 administered ranged from 270,000 to 720,000 U/kg/day. Systemic steroids were withheld for at least 2 weeks before inclusion in the study, but local steroids and antihistamines were used liberally. The clinical response was evaluated by one dermatologist who did not know the treatment protocol. Adverse reactions were observed carefully. Blood was drawn for immunological tests before treatment, on the seventh day of treatment (8 hr after IL-2 infusion), and 3 days after completion of therapy. Skin biopsies were also performed before and after therapy. The clinical trial was approved by the human research committee of this hospital and informed consent was obtained in each case before commencement of the study.
Immunological Tests Enumeration of lymphocyte subpopulations. The analysis of lymphocyte surface markers was performed by a direct immunofluorescence technique. Fluorescinated monoclonal antibodies (CD1, CD3,
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CD4, CD8, CD25) were obtained from Becton Dickinson Co. (Mountain View, CA). One million peripheral blood mononuclear cells (MNC) in 0.2 ml RPMI medium were incubated with 10 ~l of each monoclonal antibody at 4°C for 30 rain and washed twice with HBSS. The cells were enumerated by a FACScan (Becton Dickinson Immunocytometry Systems). The gate window for lymphocytes was determined by the combinations of forward and right-angle light scatter. The green fluorescence signals at 488-nm excitation wavelength were measured and displayed as a histogram on the display. The results are expressed as a percentage of positive cells. Lymphoprol(ferative Responses. The mitogeninduced lymphoproliferation was measured by the method of Bradley (19). Determinations of' lgE Concentrations. In vitro IgE production was done according to the method of Saryan et a/.(20). The IgE concentrations in the sera and culture media were determined with Phadebas IgE kits (Pharmacia, Uppsala, Sweden). Quantitation of IL-2. In vitro IL-2 production was performed as previously described (21). Briefly, mononuclear cells were adjusted to a concentration of 2 × 106 cells/ml in complete culture medium containing 1% phytohemagglutinin (PHA; GIBCO) and cultured at 37°C in a humidified 5% CO2 incubator for 36 hr. The supernatants were collected by centrifugation and IL-2 was titrated by the method of Gillis et al. (22) and IntertesT-2 kit (Genzyme, Boston, MA).
Immunopathological Studies of Skin Specimens Skin biopsies were done on the same diseased area in five of the six patients before and after IL-2 therapy. Immunochemical studies were done by avidin-biotin complex stain (23, 24). Frozen tissue sections were fixed in acetone for 10 min and overlaid with the optimal dilution of monoclonal antibody for 60 rain at room temperature, followed by incubation with biotin-labeled horse anti-mouse IgG and avidin-biotin-peroxidase complex (Vector Laboratories Inc., Burlingame, CA). A number of monoclonal antibodies including OKT3 (CD3), OKT4 (CD4), OKT8 (CD8), and OKT6 (CD1) from Orthoclone Corp. (Raritan, N J) and IL-2R (CD25) and HLA-DR from Becton Dickinson (Mountain View, CA) were used. Skin biopsies and interpretation of immunopathological findings were done by
24
HSIEH, CHOU, AND HUANG
a pathologist who was blind as to the treatment status.
Allogeneic Fibroblast Cytotoxicity Assay Chromium-51 (5~Cr) release assays were performed in four patients and four healthy adults according to the method described by Leung et al. (15). Confluent allogeneic fibroblast cultures were trypsinized and resuspended at a concentration of 5 × 104 cells/ml in complete culture medium. Then, 100 ixCi sodium chromate (sp act, 200 to 500 tzCi/txg 5~Cr; New England Nuclear, Boston, MA) was added to 10 ml of the fibroblast suspension. Twotenths milliliter of this suspension, containing 1 × 104 fibroblasts, was added to each of the wells of a 96-well microtiter plate (Linbro Scientific, Hamden, CT). The microtiter plate was incubated overnight in a 5% CO2 incubator at 37°C to allow the fibroblasts to attach to the bottom of the culture wells. The next day, the microtiter wells were aspirated and washed three times with HBSS containing 2% AB serum. Effector cells included freshly prepared MNC and lymphokine-activated killer cells (LAK) which were obtained by incubation with 1000 U/ml of rlL-2 (Cetus Corporation) for 3 days, as described previously (25). For the cytotoxicity assay, 1 million effector cells in 0.2 ml of complete medium were added to test wells, giving an effector-to-target cell ratio of 100: I. For the determination of spontaneous release of 5~Cr (S), 0.2 ml of complete medium was added to control wells. For the determination of total 5~Cr release (T), 0.2 ml of a 5% solution of Triton-X was added to a set of wells. All assays were performed in triplicate. The plates were placed on a rocking plateform (Bellco Glass, Inc., Vineland, NJ) oscillating at 5 cycles per min and were incubated at 37°C in humidified 5% CO2 incubator. After overnight incubation (12 to 14 hr), the plates were centrifuged at 200g for 10 rain. One-tenth milliliter of the supernatant was removed with a Hamilton syringe and its radioactivity was measured in a gamma counter. Cytotoxicity was calculated as follows: cytotoxicity (%) = ( E - S / T - S ) x 100, where S is spontaneous release of 51Cr from fibroblasts, E is lymphocyte-mediated release of 51Cr from fibroblasts, and T is total release of 51Cr from fibroblasts with Triton-X.
Fig. 1. The papulovesicles and lichenification of diseased skin (upper panel) were markedly improved after IL-2 therapy (lower panel).
Statistics Student's t test was used for statistical analysis throughout the study. RESULTS Clinical Response
In all six patients marked desquamation over the eczematous lesions, improved lichenification, and diminished itching and scratching occurred about 5 days after treatment commenced (Fig. 1). Local steroids could be stopped, and the dose of antihistamine required was decreased. However, eczema flared up in all cases 2 to 6 weeks after discontinuation of rlL-2. The adverse reactions of IL-2 therapy such as fever, chills, malaise, mild generalized erythematous maculopapules, and hepatomegaly were seen in all patients during the period of therapy and were similar to those reported previously. Serious side effects resulting from the vascular leak
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Table I. Distribution of T-Cell Subsets During IL-2 Therapy CD3 + (%) Before therapy During therapy After therapy Normal control
- 12.9 a _-2 1161)b + 10.6 +- 4154) -+ 17.7 -+ 4263) + 7.7 (2646 +- 326)
70.7 (2655 66.2 (2699 57.0 (5240 69.0
CD4+ (%) 40.0 (1513 29.5 (I 148 30.3 (1240 41.2 (1724
--- 8.7*'** - 669) -2_ 5.1" -+ 1039) - 8.3** + 1056) --+ 6.1 _+ 289)
CD8 + (%) 29.5 (1158 31.7 (1454 24.7 (2349 23.8 (968
-+ 8.9 --. 706) -+ 13.6 + 2524) -+ 9.2 - 1974) + 5.1 - 212)
CD25 + (%) 2.2 (98 6.7 (197 3.3 (223 1.2 (51
--- 2.4 --- 119) -+ 4.8 -.+ 176) - 1.8 -+ t23) -+ 0.3 +-- 15)
CD4/CD8 ratio 1.41 -+ 0.32 1.18 --- 0.70 1.35 -+ 0.46 1.80 --+ 0.40 (1.00 +- 0.26)
aMean --- SD. bAbsotute number (mean - SD). *P = 0.0t. **P = 0.05.
syndrome such as decreased urinary output, body weight gain, edema, and pleural effusion were also noted in some of the patients. All the symptoms and signs subsided within 2 to 4 days after discontinuation of therapy.
were increased after IL-2 therapy in all cases (Fig. 3, lower panel). There was, however, no remarkable change in CD3+, CD8+, or C D l + cells (data not shown).
Immunological Parameters The immunological changes in patients during the course of treatment are shown in Table I. There were no consistent changes in CD3+ cells, CD8+ cells, CD4/CD8 ratio, serum IgE, in vitro IgE production, serum IL-2, or mitogen responses. There was, however, a tendency for CD4+ cells to decrease (P < 0.0t) and IL-2R+ cells to increase during treatment when compared with the pretreatment values. It is interesting to note that although the serum IL-2 of patients was highly elevated (29.9 --+- 67.1 U/ml, compared to 1.2 - 0.8 U/ml for normals), in vitro IL-2 production could not be detected. No change in total serum IgE and in vitro IgE production was found.
lmmunohistological Changes of Skin The pathologic changes of eczematous skin are shown in Fig. 2. The specimens taken before IL-2 therapy showed hyperplastic epidermis and prominent mononuclear-cell infiltration of dermis. The skin was much less thick and the mononuclear-cell infiltration was markedly decreased after IL-2 treatment. The immunochemical study with avidinbiotin complex stain indicated that the infiltrating lymphocytes consisted of more CD4+ cells and fewer CD8+ cells, and the CDI+ Langerhans cells were increased (Fig. 3, upper panel). The CD4+ cells were markedly decreased and CD25+ cells
Journal of Clinical Immunology, Vol. 11, No. 1, 1991
Fig. 2. Histopathological findings of skin lesions before (upper panel) and after (lower panel) IL-2 therapy. The hyperplasia of epidermis and mononuclear cell infiltration of dermis were markedly decreased "after treatment (case 5). 10 x ; reduced 40% for reproduction.
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Fig. 3. Immunochemical stainings of diseased skin before (upper panel) and after (lower panel) IL-2 therapy, The CD4+ cells were markedly decreased after IL-2 therapy, There was a slight increase in CD25+ cells but no remarkable change in CDI+, CD3+, and CD8+ cells (data not shown). 200 ×; reduced 40% for reproduction.
Allogeneic Fibroblast Cytotoxicity Assay The results of the cytotoxicity assay showed that the value of 21.1 - 9.2% (mean +- SD) for fresh MNC of patients was much higher than that for healthy subjects (7.6 - 4.1%). LAK cells killed allogeneic fibroblasts to a much greater degree than did the fresh MNC, but there was no difference between healthy subjects (56.1 --- 11.3%) and patients (55.6 --- 6.3%). DISCUSSION All the patients responded to IL-2 therapy. The improvement usually occurred 5 days after treatment. The skin texture improved due to desquamation and subsidence of papulovesicles and lichenification (Fig. 1). The itching and scratching were decreased markedly. However, the eczema flared up in all cases 2-6 weeks after discontinuation of
HSIEH, CHOU, AND HUANG
IL-2. As expected, the side effects seen in adults (26) also occurred in children but were much less severe in the latter. The most probable explanation for the difference may be that all of our patients had allergic disease rather than cancers and none of them had received chemotherapy. In this study, a clear-cut therapeutic effect of IL-2, although transient, had been shown on protracted severe atopic dermatitis. As the therapeutic effect was transient and the IL-2 therapy was rather toxic, the potential harm of IL-2 should be seriously considered in a nonfatal disease such as atopic eczema. The longterm administration of lower dosages of IL-2 may provide a new promising therapeutic approach for this disease, but studies with more cases and a longer period of follow-up should be done before any solid recommendation can be advocated. The dermal pathology in atopic dermatitis, which varies with the temporal nature of the clinical lesion, in both the acute and the chronic phases is characterized by a lymphocytic infiltration without significant immunoglobulin deposition, suggesting a type IV cell-mediated mechanism for skin injury (27-29). The type of infiltrating mononuclear cells was determined by using monoclonal antibodies and the results showed that most of the infiltrating lymphocytes were of the CD4+ helper/inducer phenotype (24, 25, 27-30), with a CD4/CD8 ratio of 4.8:1, which was much higher than the ratio of2:1 found in peripheral blood (30). Moreover, CDI+ Langerhans cells were also increased. The presence of increased numbers of Langerhans cells and of CD4+ cells may reflect increased antigen processing and cellular activation in the diseased skin of patients. It has been shown that antigen-activated T cells and macrophages can release soluble factors which are capable of inducing proliferation and collagen biosynthesis by fibroblasts and basophil degranulation and mediator release (31, 32). Chronic infiltration of eczematous skin with activated T cells and macrophages, as evidenced by the expression of DR antigens, may be used to account for the collagen deposition and epidermal hyperplasia with subsequent lichenification of skin, on the one hand, and severe pruritus, on the other hand. We demonstrated the predominance of CD4+ cells, rather than CD8+ cells, in diseased skin of AD patients (Figs. 2 and 3) and these findings agree with those of other investigators (24, 25, 27-30). It is important to note that CD4 + cells in both blood and diseased skin, but not CD3+ and CD8+ cells, were markedly decreased after IL-2 treatment. This Journal of Clinical Immunology, Vol. 11, No. 1, 1991
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finding strongly supports the hypothesis that IL-2 may exert its therapeutic effect in AD through the depletion of CD4+ cells. Recently, epidermal Langerhans cells (LC) were shown to be capable of inducing antigen-specific T cells of the Th-2 subtype, which were able to produce IL-4 (33). The pleuripotent activity of IL-4 is well documented: (i) induction of the expression of FcERII on B cells and monocytes and LC; (ii) induction of normal B cells to produce soluble CD23/IgE-BF; (iii) induction of differentiation of monocytes into dendritic cells; and (iv) increase in class II MHC expression of monocytes. The elimination of CD4+ cells by IL-2 treatment will abrogate the enhanced antigen processing and cellular activation which may be involved in the induction of the eczematous response. Moreover, recent studies have demonstrated that depletion of CD4+ cells correlated with the therapeutic efficacy of anti-CD4 antibody in treating experimental allergic encephalomyelitis (34), a murine model for human multiple sclerosis (35). Administration of antimurine CD4 monoclonal antibodies has also been shown to be effective in the treatment of a variety of autoimmune diseases in animal models, including systemic lupus erythematosus (36). Finally, as CD4+ cells had been shown to possess cytotoxic activity (37), the depletion of CD4+ cells may also contribute to the clinical efficacy of IL-2 in AD. Cells of NK lineage have been reported to possess enhanced cytotoxic activity against skin fibroblasts in patients with AD (16). In this study, we found that fresh MNC of AD patients possessed a much greater cytotoxicity against allogeneic fibroblasts than did those of healthy subjects, however, no difference in LAK activity against fibroblasts was found between patients and normals. These results do not support the idea of enhanced NKmediated cytotoxicity in the pathogenesis of AD because NK activity can be enhanced by IL-2 and accounts for the major portion of LAK activity (38). Elevated serum IgE concentration has been observed in approximately 80% of patients with AD (39), and a perturbation of the balance between IgE-specific T-celt help and T-cell suppression has been postulated as the most probable cause (40). In this study, no significant change in serum IgE or in in vitro IgE synthesis was found after IL-2 therapy despite an increase in CD8+ cells and a decrease in CD4+ cells (Table I). Thus, the clinical efficacy of IL-2 in AD has no relationship with IgE production.
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No study on serum IL-2 in AD has been reported in the literature, particularly after IL-2 therapy. In this study we found that while in vitro IL-2 production could not be detected, serum IL-2 was markedly increased in patients with AD, suggesting in vivo activation of T cells. The serum IL-2 tended to decrease after IL-2 treatment and this might be due to a decrease in the number of CD4+ cells, or diminished in vivo cellular activation, or both. One major drawback of this study is the lack of control group either receiving normal saline alone or just being admitted to the hospital during the same period of time. But due to the evident side effects of IL-2, it is very difficult to blind the study. Another issue of debate is the use of IL-2 in a chronic nonfatal disease in children such as atopic eczema. The side reactions resulting from IL-2 infusion resolved completely several days after discontinuation of therapy, moreover, no clinically evident adverse effects have been observed during the past year. Thus, in view of its immune amplifying effects, long-term administration of IL-2 at a lower dose may still be worth trying in severe, refractory atopic eczema. In conclusion, transient improvement of severe atopic dermatitis has been observed after IL-2 treatment, and the clinical efficacy is possibly due to depletion of CD4+ cells and hence abrogation of the enhanced antigen processing and cellular activation and decreased production of inflammatory cytokines in diseased skin. But the mechanisms by which the CD4+ cells in atopic dermatitis are selectively depleted by IL-2 treatment need further study. REFERENCES 1. Hanifin JM, Rajka G: Diagnostic features of atopic dermatitis. Acta Derm Venerol 92(S):44-47, 1980 2. Hanifin JM: Basic and clinical aspects of atopic dermatitis. Ann Allergy 54:386-893, 1984 3. Halpern SR, Sellars WA, Johnson RB, et al.: Development of child allergy in infants fed breast, soy or cow's milk. J Allergy Clin Immunol 66:465-471, 1973 4. Kjellman N-JM: Atopic disease in seven-year-old children. Acta Pediat Scand 66:465-471, 1977 5. Walker RB, Warin RP: Incidence of eczema in early childhood. Br J Dermatol 68:182-183, 1956 6. Hanifin JM: Atopic dermatitis. In Allergy Principles and Practice, 3rd ed, E Middleton Jr, CE Reed, EF Ellis, NF Adkinson Jr, JW Yunginger (eds). St. Louis, CV Mosby, 1988, pp 1403-1428 7. Hsieh KH, Shen JC: Prevalence of childhood asthma in Taipei, Taiwan, and other Asian Pacific countries. J Asthma 25:73-82, 1988
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8. Rasmussen E: Recent development in the management of patients with atopic dermatitis. J Allergy Clin Immunol 74:771-776, 1984 9. Leung DYM, Rhodes AR, Geha RS: Enumeration of T cell subsets in atopic dermatitis using monoclonal antibodies. J Allergy Clin Immunol 67:450-455, 198t t0. Chiarelti F, Canfora G, Verrotti A, Amerio P, Morgese G: Humoral and cellular immunity in children with active and quiescent atopic dermatitis. Br J Dermatol 116:651-660, 1987 11. Braathen LR: T-cell subsets in patients with mild and severe atopic dermatitis. Acta Derm Venerol St 14:133-136, 1985 12. Delespesse G, Demauberge J, Sarfati M: Characterization of circulating T lymphocyte subpopulations in atopic eczema. J Allergy Clin Immunol 69(2):135, 1982 13. Nomiyama K, Yamashita U, Nakamura H, Suenaga Y, Nishio K: Natural killer (NK) cell activity and NK-related cell surface markers in patients with atopic dermatitis. J Dermatol 15:241-247, 1988 14. Leung DYM, Wood N, Dubey D, Rhodes AR, Geha RS: Cellular basis of defective cell-mediated lympholysis in atopic dermatitis. J Immunol 130:1678-1682, 1983 15. Leung DYM, Saryan JA, Frankel R, Lareau M, Geha RS: Impairment of the autologous mixed lymphocyte reaction in atopic dermatitis. J Clin Invest 72:1482-1486, 1983 16. Leung DYM, Parkman R, Feller J, Wood N, Geha RS: Cell-mediated cytotoxicity against skin fibroblasts in atopic dermatitis. J Immunol 128:1736-1741, 1982 17. Clendenning WE, Clack WE, Ogawa M, Ishizaka K: Serum IgE studies in atopic dermatitis. J Invest Dermatol 61:233236, 1973 18. Sampson HA: The role of food allergy and mediator release in atopic dermatitis. J Allergy Clin Immunol 8t:635-645, 1989 19. Bradley LM: Cell proliferation. In Selected Methods in Cellular Immunology, BB Mishell, SM Shiigi (eds). San Francisco, WH Freeman, 1980, pp 158-166 20. Saryan JA, Leung DYM, Geha RS: Induction of human IgE synthesis by a factor derived from T cells of patients with hyper-IgE status. J Immunol 130:242-247, 1983 21. Hsieh KH: Altered interleukin-2 production and responsiveness after hyposensitization to house dust. J Allergy Clin Immunol 76:188-194, 1985 22. Gillis S, Ferm M, Ou W, Smith KA: T cell growth factor: Parameters of production and a quantitative microassay for activity. J Immnnol 120:2027-2032, 1978 23. Shu SM, Raine L, Fanger H: Comparative study of peroxidase-antiperoxidase method and avidin-biotin-complexes method for studying polypeptide hormones with radioimmunoassays. Am J Clin Pathol 75:734-739, 1981 24. Leung DYM, Bhan AK, Schneeberger EE, Geha RS: Characterization of the mononuclearinfiltrate in atopic dermatitis using monoclonal antibodies. J Allergy Clin Immunol 71:4756, 1983
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25. Hsieh KH, Shu S, Lee CS, Chu CT, Yang CS, Chang KJ: Lysis of primary hepatic tumors by tymphokine activated killer cells. Gut 28:117-124, 1986 26. Rosenberg SA, Lotze MT, Muul LM, et al.: A progress report on the treatment of 157 patients with advanced cancer using lymphokine activated killer cells and interteukin 2 or high dose interleukin-2 alone. N Engt J Med 316:889-897, 1987 27. Braathen LR, Forte O, Natvig JB, Eeg-Larsen T: Predominance of T lymphocytes in the dermal infiltrate of atopic dermatitis. Br J Dermatol 100:511-519, t979 28. Mihm MC, Ster NA, Dvorak HF, Austin KF: The structure of normal skin and the morphology of atopic dermatitis. J Invest Dermatol 67:305-312, 1976 29. Tong AKF, Mihm MC: The pathology of atopic dermatitis. Clin Rev Allergy 4:27-42, 1986 30. Zachary CB, Allen MH, MacDonald DM: In situ quantification of T-lymphocyte subsets and Langerhans' cells in the inflammatory infiltrate of atopic eczema. Br J Dermatol 112:149-156, 1985 31. Schmidt JA, Mizel SB, Cohen D, Green D: Interteukin 1, a potential regulator of fibroblast proliferation. J Immunol 128:2177-2182, 1982 32. Thueson DO, Speck LS, Lett-Brown MA, Grant IA: Histamine releasing activity. I. Production by mitogen or antigen stimulated human mononuclear cells. J tmmunol 123:626639, 1979 33. Hauser C, Snapper J, O'Harra W, Paul W, Katz S: T helper cells grown with hapten-modified cultured Langerhans cells produce IL-4 and stimulate IgE production by B cells. Eur J Immunol 19:245-250, t989 34. Alters SE, Sakai K, Steinman L, Oi VT: Mechanisms of anti-CD4-mediated depletion and immunotherapy. A study using a set of chimeric anti-CD4 antibodies. J Immunol 144:4587-4592, 1990 35. Traugott U, Reinherz EL, Raine CS: Multiple sclerosis: Distribution of T cell subsets within active chronic lesions. Science 219:308-310, 1982 36. Wofsy D, Seaman WE: Reversal of advanced murine lupus in NZB/NZW F1 mice by treatment with monoclonal antibody to L3T4. J Immunol 138:3247-3253, 1987 37. Moretta L, Mingari MC, Sekaly PR, Moretta A, Chapuis B, Cerottini JC: Surface markers of cloned human T cells with various cytolytic activities. J Exp Med 154:569-574, 1981 38. Herberman RB, Hiserodt J, Vujanovic N, et al.: Lymphokine-activated killer cell activity. Characteristics of effector cells and their progenitors in blood and spleen. Immunol Today 8:178-181, 1987 39. Johnson E, Irons J, Patterson R, Roberts M: Serum IgE concentration in atopic dermatitis, J Allergy Clin Immunol 54:94-99, 1974 40. Leung DYM, Geha RS: Immunoregulatory abnormalities in atopic dermatitis. Clin Rev Allergy 4:6%86, 1986
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