- Email: [email protected]
Cyclosporine in atopic dermatitis
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Cyclosporine in atopic dermatitis Modulation in the expression of immunologic markers in lesional skin Th. van Joost, MD, a M. M. A. Kozel, MD, b B. Tank, PhD, a R. Troost, MD, a and E. P. Prens, M D a, b Rotterdam, the Netherlands
Background: In previous studies, oral cyclosporine was highly effective in the treatment of patients with severe atopic dermatitis. In this study seven patients with severe and therapyresistant atopic dermatitis underwent therapy with cyclosporine, 5 mg/kg/day, for 6 weeks. Objective: The effect of cyclosporine on the expression of cytokines, which probably play a role in this disease, was examined. Methods: The study was performed with a panel of antibodies as markers of inflammatory cells, adhesion molecules, and cytokines (interferon-'r [IFN-'y], tumor necrosis factor-a [TNF-a] and interleukins la, I/5, and 8 [IL-la, IL-lfl, and IL-8, respectively]). They were visualized by indirect immunoperoxidase techniques. Results: After 2 weeks of cyclosporine therapy, a reduction of 60% in the disease (severity and extent) was observed. This reduction was 89% after 4 weeks and 90% after 6 weeks of therapy. Results of indirect immunoperoxidase stains performed on lesional skin sections after 2 weeks of treatment showed statistically significant reduced numbers of CD14 +, CD25 (IL-2R +) and IL-8 + inflammatory cells in the dermis and CD36(OKM5) + cells in both the epidermis and dermis. The number of cells expressing IFN-3, and TNF-a, assumed to be the products of the helper T-cell (TH) 1 subset, was unaltered despite the impressive clinical benefit observed. Keratinocytes in lesional atopic skin did not express intercellular adhesion molecule type 1 (ICAM-1). The expression of the adhesion molecules ICAM-1, lymphocyte function-associated (LFA) type 1, and LFA-3 on inflammatory cells also remained unaffected by cyclosporine treatment. Conclusion: A statistically significant reduction in the number of activated T cells and in the number of cells expressing the IL-2 receptor (CD25) paralleled a marked improvement in the disease and supports the view that atopic dermatitis is based on a T-cell-mediated immune inflammation. (J AM ACAD DERMATOL 1992;27:922-8.) Oral cyclosporine m a y be beneficial in the treatm e n t of patients with severe atopic dermatitis. 1"8 T h e beneficial effects of cyclosporine have been attributed mainly to inhibition of T-cell activation, interleuldn 2 (IL-2) production, and the IL-2 receptor expression. Cyclosporine might also inhibit the production of other cytokines such as interferon-3, (IFN-30, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, and IL-4. 9 An imbalance between two helper T ( T H ) lymphocyte sub-
From the Departments of Dermato-Venereologya and Immunology,b Erasmus University. Accepted for publication May 29, 1992, Reprint requests: Th. van Joost, Department of Dermato-Venereology, University Hospital Rotterdam--Dijkzigt, Dr, Molewaterplein 40, 3015 GD Rotterdam, the Netherlands. 16/1/39648
922
sets, namely TH1 (producing IL-2, IFN-% T N F - a ) and TH2 (producing IL-3, IL-4, IL-5, IL-6, IL-10) may play a crucial role in the regulation of IgE-mediated diseases. 1~ Apparently cyclosporine has the potential to interfere at some crucial steps in the immunopathogenesis of atopic dermatitis. The purpose of this study was to confirm the beneficial effect of cyclosporine in severe atopic dermatitis resistant to topical therapy and to evaluate the immunomodulating effect(s) of cyclosporine on the expression of certain immunologic markers that may play a role in atopic dermatitis. MATERIAL AND METHODS Patients Seven patients (2 men, 5 women), 18 to 32 years of age (mean 23 years) with severe chronic atopic dermatitis (mean duration 15 years), 11 resistant to systemic and
Volume 27 Number 6, Part 1 December 1992
topical treatment, were treated in an open study with cyclosporine (Sandimmune, Sandoz Pharmaceuticals Corp., E. Hanover, N. J.), 5 mg/kg/day, for 6 weeks. The disease in all patients was chronic and severe for at least a year before the start of cyclosporine therapy. In all patients systemic therapy had been discontinued 4 weeks before treatment and none had received topical therapy 2 weeks before treatment. No other concurrent treatment was given during cyclosporine therapy with the exception of topical petrolatum. The patients were informed that previous studies t-8 had shown that a substantial improvement in pruritus and loss of sleep were to be expected once cyclosporine treatment was started. Monitoring (blood pressure, hematologic and biochemical profiles, infection, use of drugs that influence or increase the risk of renal malfunction) used in this study has been reported previously.12"14 Disease activity was measured biweekly on the basis of six clinical features (erythema, infiltration, vesicles/papules, dryness/scaling, cracking/fissuring, and excoriations) graded at six sites (head and neck, anterior and posterior trunk, mid-upper to mid-lower arm [both sides], both hands, mid thigh to mid calf [both sides], and both feet) on a 3-point scale of 0 (none) to 3 (severe) with a maximum score of 108. The extent of eczema was assessed according to "the rule of nines" as described previously.8 Lichenification, apparently a more resistant feature to short-term cyclosporine treatment, 7 was assessed separately at the same six defined sites on a scale of 0 (none) to 3 (severe) with a maximum score of 18.
Biopsies Punch biopsy specimens were obtained from each patient from newly developed unexcoriated lesions. Subsequent, specimens were taken 2 weeks after cyclosporine treatment from lesional skin near sites from which pretreatment specimens were obtained. Biopsy specimens were embedded in Tissue-Tek (OCT compound, Miles Inc., Elkhart, Ind.), snap-frozen in liquid nitrogen, and stored at -70 ~ C.
Indirect immunoperoxidase test The biopsy specimens were cut into 5 um sections. The specificity and the sources of the 20 different antibodies that were used are shown in Table I. An indirect immunoperoxidase (IIP) technique with 3-amino-9-ethylcarbazole (AEC) as a substrate was used. The sections were fixed in acetone for 5 minutes at room temperature and were incubated for 30 minutes at 37 ~ C with an optimal dilution of the primary antibody and were subsequently incubated for 30 minutes with the appropriate peroxidase-conjugated secondary antibody. Peroxidase activity was visualized with a freshly prepared acetate buffer solution containing AEC and hydrogen peroxide. Sections
Cyclosporine in atopic dermatitis 923 were counterstained with Mayer's hematoxylin and mounted with Aquamount. Preimmune goat and sheep sera were used as controls in comparable experiments. The specificities of the antibodies that were gifts (Table I) were checked in blocking experiments with appropriate recombinant human cytokines (results not shown). Well-stained cells (• were counted independently by two observers. For almost all markers the stained cells in the epidermal and in the dermal infiltrates were graded semiquantitatively on a 4-point scale (0 to 3) with steps of 0.5, representing none/very few (grade 0), few (grade 1), moderate (grade 2), and many (grade 3). The numbers of CD25 + ce/ls were assessed per high power field (• because this marker was sporadically expressed in frozen sections of atopic dermatitis lesions. A separate grading system was used for the levels of expression of cytokines. The level of staining was scored on the following 4-point scale: no staining (grade 0), moderate focal/ faint diffuse staining (grade 1), strong focal/moderate diffuse staining (grade 2), and strong diffuse staining (grade 3). All results were expressed as the mean.
Statistical analysis The results of the immunohistochemical studies performed before and during treatment with cyclosporine were analyzed with the Wilcoxon sign rank test. A p value less than 0.05 was considered statistically significant (Table II). RESULTS
Clinical findings A substantial decrease in the severity of itch and loss of sleep was achieved in all patients within 1 week of therapy with cyclosporine. The degree of objective clinical improvement is summarized in Table III. In all seven patients a substantial improvement was noted after 2 weeks of treatment. The mean percentage reduction in disease activity after 2 weeks of cyclosporine treatment was 60% (5 7.8 vs 23.3, Table III), 89% after 4 weeks, and 90% after 6 weeks of treatment. An important percentage reduction in the scores indicating improvement was also observed in the extent of the disease: 65% at week 2, 86% at week 4, and about 90% at week 6. The mean values for lichenification were 10 at week 0, 8 at week 4, and 7 at week 6. The mean reduction in lichenification after 6 weeks of cyclosporine therapy was about 33%. Mean serum creatinine levels increased slightly during cyclosporine therapy but in none of the patients was an unacceptable alteration in hematologic or biochemical profile observed. 13, 14 An unacceptable rise in blood pressure (diastolic
924
Journal of the American Academy of Dermatology
van J o o s t et aL
Table I. Antibody panel Cellulartargets
Workingdilution
Cortical thymocytes, Langerhans cells, some other dendtritic cells Immature T cells (cytoplasmic expression) and mature functional T cells (membrane expression) Subpopulation of cortical thymocytes, helper/inducer T cells, subpopulation ofmonocytes and macrophages Subpopulation of cortical thymocytes, cytotoxic/suppressor T cells, subpopulation of NK cells Majority of lymphoid and myeloid cells Monocytic. cells, macrophages, follicular dendritic reticulum ceils Activated T cells, activated B cells Subpopulation of T cells (especially CD4 + T cells), megakaryocytes, platelets, subpopulafionof other leukocytes Monocytes, early erythroid cells, megakaryocytes, platelets Virgin T cells, B cells, granulocytes, monocytes Broad tissue distribution; increased expression on activation Leukocytes, broad tissue distribution, dendritic cells Hematopoletic precursor cells, B ceils, activated T cells, monocytic ceils, macrophages Active dendritic cells, subsets of B cells (class II associated)
1:10
Antibody
CDla (OKT 6) CD3 (Leu-4) CD4 (Leu-3a) CD8 (Leu-2a) CD1 la (LFA-la) CD14 (MY-4)
CD25 (IL-2RI) CD29 (4B4)
CD36 (OKM5) CD45 (2H4) CD54 (ICAM-1) CD58 (LFA-3) Anti-HLA-DR RFD 1
1:50
Becton Dickinson
1:50
Becton Dickinson
1:50
CLB, Amsterdam, the Netherlands Coulter Immunology, Hialeah, Fla. Becton Dickinson Coulter Immunology
1:100 1:50 1:100
1:100
Ortho Diagnostics, Raritan, N.J. Coulter Immunology
1:250
Boehringer, Mannheim
1:25
Behring, Marburg, Germany
1:200
Becton Dickinson
1:250
1:150
L. Poulter, Royal Free Hospital, School of Medicine, London Y. Ishii, Tokyo, Japan J. van Damme, Rega Institute, Leuven, Belgium25 W. Buurman, University of Maastricht, the Netherlands26 Glaxo, Geneva, Switzerland
1:100
Biogen, Glaxo
1:200
J. van Damme, Rega Institute25
1:200
B cells, dendritic cells Macrophages, T cells, B cells, NK cells
1:200 1:20
Anti-TNF-a
Tumor calls, fibroblasts, macrophages, neutrophils, lymphocytes, endothelium Macrophages, T cells, B cells, fibroblasts, endothelial and epithelial cells Macrophages, T cells, B cells, fibroblasts, endothelial and epithelial cells T cells, neutrophils, basophils, epithelial ceils
1:5
Anti-IL-lfl* Anfi-IL-St
Erasmus University, Rotterdam, the Netherlands Becton Dickinson, Mountain View, Calif.
1:50
L25 Anti-IFN-7
Anfi-IL-la*
Source
*Sheepantimousepolyclonalantisera. tGoat antimouse polyelonalantiserum. >95 m m H g or systolic > 150 m m Hg) was not observed in any of the patients./3, 14 Histologie and immunohistochemical studies
The histologic changes seen in the frozen sections of lesions before and after cyclosporine treatment
paralleled the clinical improvement observed in most patients. After therapy, there was a clear reduction in the dermal mononuclear infiltrate and a remarkable decrease in spongiosis and the number of intraepidermal microvesicles was reduced. After 2 weeks of treatment, a slight but statistically insig-
Volume 27 Number 6, Part 1 December 1992
nificant reduction was observed in the expression of CDla, CD4, the C D 4 / C D 8 ratio, C D l l a , CD29, CD54 (ICAM-1), HLA-DR, and RFD1 on the inflarnmatory cells in the dermis (Table II).
Cyclosporine in atopic dermatitis 925 Table II. Mean grades of expression of various markers investigated in lesional skin of seven patients with severe atopic dermatitis before and after 2 weeks of cyclosporine treatment Epldermis/epldermal inmtratr
Statistically significant alterations seen with four different specific markers CD14 Before cyclosporine treatment, in the dermis particularly, a substantial number of clustered CD14 + cells generally with dendritic morphology were observed. After treatment the number of CD14 + cells in the dermis in all patients were significantly (p < 0.05) reduced with a scattered distribution throughout the dermis. CD25. A statistically significant (.p < 0.05) decrease in the number of CD25 + cells expressing the IL-2 receptor was observed in the dermis after treatment. The number of intraepidermal CD25 + cells was reduced insignificantly, probably because of the low number of cells. CD36 (OKM5). Before cyclosporine treatment, keratinocytes in the stratum spinosum and granulosum clearly expressed CD36 (OKM5) with the characteristic intercellular reticular staining pattern. In the dermis the majority of the CD36 + cells, some with dendritic morphology, were grouped around the upper dermal capillaries (Fig. 1). After 2 weeks of cyclosporine treatment the intercellular expression of CD36 in the epidermis was significantly (p < 0.02) reduced or even absent in all seven cases and in the dermis a reduction in the number of CD36 + cells was observed (Table II and Fig. 2). Anti-IL-8. In the lower epidermal layers of most sections, a diffuse reticular staining of low intensity and in the higher epidermal layers a more intense reticular and homogeneous staining was observed. No significant changes were observed in the epidermis after treatment, but in the dermis a statistically significant (p < 0.05) reduction in the levels of expression of IL-8 on the infiltrate and in the number of IL-8 + cells was observed after treatment with cyclosporine. After 6 weeks the studies were repeated in two patients. The results did not differ significantly from those obtained after 2 weeks of treatment. DISCUSSION This open study corroborates the observations of previous studies 18 that short-term treatment ("crisis-intervention") 1with cyclosporine can be effective and safe for severe chronic atopic dermatitis. Careful monitoring of patients receiving cyclosporine is, however, strictly indicated)' 13 The reduction in the
Before
After '
Antibody
(n= 7)
(n =7)
CDla CD3 CD4 CD8 CD1 la CD14 CD25 CD29 CD36 CD45 CD54 CD58 Anfi-HLA-DR RFD 1 L25 Anti-TNF-a:~ Anti-IFN-~/w Anti-IL-la[ Anti-IL- 1/~ Anti-IL-8 82
2.2 0.7 0.1 O.5 0.7 0.3 0.71" 2.2 1.8 2.0 0.0 0.5 1.3 0.5 5.2 1.6 1.5 1.1 1.1 1.6
2.3 0.5 0.1 0.3 0.4 0.1 0.3t 1.9 0.6* 1.8 0.0 0.8 1.3 0.8 5.5 1.8 1,3 1.0 1.3 1.3
~rmal i ~ ~re
1.8 2.6 1.4 0.6 2.1 1.9 4.3t 2.5 1.3 1.8 1.9 2.3 2.2 2.2 2.1 1.6 2.0 0.9 1.3 1.5
M~r
1.5 1.8 1.1 0.6 1.4 1.1" 1.0*'t 2.1 0.6* 1.7 1.5 2.3 1.8 1.8 2.0 1.7 1.8 0.8 1.5 0.9*
*Statistically significant (p < 0.05). ~'The figures represent the mean number of cells per high power field. SDiffuse and intercellular epidermal staining and diffuse staining in the dermal infiltrate. w and focal intercellular epidermal stainingand focal staining in the dermal infiltrate. ][Diffuseepidermal staining, diffuse and focal staining in the dermal infiltrate. 82 and intercellular epidermal staining and focal staining in the dermal infiltrate,
disease activity observed at week 2 was more pronounced than that reported recently by Sowden et al. 8 In their double-blind placebo-controlled study patients continued to use the same topical steroid treatment that they were applying before entry into the trial. An explanation for the discrepancy in the clinical improvement observed in the two studies may be that in our study some what different clinical features for the overall assessment of disease activity and extent were used. Lichenification is rather resistant to short-term treatment with cyclosporine 7 and therefore was not included as a clinical variable in the overall clinical assessment in our study. Furthermore, in contrast to the previous study, 8 we also used the occurrence of papules and/or vesicles as a variable for assessing disease activity.
926
Journal of the American Academy of Dermatology
van Joost et al.
Fig. 1. Immunohistochemical staining of lesional atopic skin with OKM5 (c~-CD36) antibody before treatment with oral cyclosporine. Note intense epidermal reticular staining. In dermis CD36 + inflammatory cells are grouped around upper dermal capillaries. (;<200.)
Table Ill. Disease activity and the extent of disease in seven patients treated with cyclosporine, 5 m g / k g / d a y , for 6 weeks Patient
1
2 3 4 5 6 7 Mean Mean % reduction in scores
~_
Before therapy ........
68 49 60 57 59 49 61 57.8 --
46 60 57 33 79 30 70 53.6 --
Week 2
I
Week 4
Week 6
Activity
Extent
I Activity ,
Extent
Activity
20 26 28 21 22 25 21 23.3 59.7
23 16 15 11 42 12 12 18.7 65.1
12 2 1l 3 4 2 11 6.4 88.9
17 0 9 9 6 4 7 7.4 86.2
12 7 6 5 0 3 7 5.7 90.1
[
Extent
11 3 6 9 0 4 5 5.4 89.9
*Six-area score (see text for details), tRule-of-nines score.
T h e reduction in the number of CD25 + T cells in the dermal infiltrate after cyclosporine treatment corroborates the findings reported by Wahlgren et al. is in atopic dermatitis and by Horrocks et al. 16 in psoriasis. A statistically significant decrease in the number of CD25 + cells expressing the IL-2 receptor in the dermal infiltrate (Table II) strongly suggests that T-cell activation is inhibited by cyclosporine. This suggests an important contribution of T lymphocytes to atopic dermatitis. In contrast to keratinocytes in healthy skin, keratinocytes in lesional atopic skin and in many other inflammatory dermatoses exhibit a strong epidermal
intercellular expression of CD36 (OKMS) (Fig. 1). The reduction in the expression of CD36 by cyclosporine treatment, therefore, may reflect a decrease in the production of certain CD36-inducing cytokine(s). The observed decrease in the number of activated T cells (CD25) suggests that the CD36-inducing cytokine(s) originate from T lymphocytes, 17 A myeloid differentiation antigen, CD14 is normally expressed on the surface of monocytes and macrophages but is expressed in low amounts on neutrophils and lymphocytes. Furthermore, CD 14 is one of the two (CD 14, CD33) monocyte differentiation antigens that are expressed on normal human
Volume 27 Number 6, Part I December 1992
Cyclosporine in atopic dermatitis 927
Fig. 2. Immunohistochemical staining of lesional atopic skin with the OKM5 (a-CD36) antibody 2 weeks after treatment with oral cyclosporine. Note reduced reticular staining in epidermis and decreased CD36 + inflammatory cells in dermis. (x200.)
epidermal Langerhans cells. 18 In our study, the observed decrease in the number of CD 14+ cells in the dermal infiltrate after treatment with cyclosporine suggests decreased T-cell activation by CD14 + antigen-presenting cells. It was not possible to reconcile a statistically significant reduction in the number of CD14 + dendritic cells with other dendritic cell markers such as CDla, HLA-DR, RFD- 1, and L25 after cyclosporine treatment. No HLA-DR was expressed on the keratinocytes in any of the lesional biopsy specimens. This agrees with previous observations in atopic dermatitis. 19 Griffiths et al. 19 reported focal ICAM-1 expression on keratinocytes in atopic dermatitis. Singer et al. 2~ observed ICAM- 1 expression on keratinocytes in atopic dermatitis with an indirect immunofluoreseence technique. In contrast, in the present study CD54 (ICAM-1) was not expressed on keratinocytes, but in the same sections a strong CD54 expression was observed on endothelial cells and on cells of the dermal mononuclear infiltrate (Table II). Sections from lesional psoriatic skin, which were stained simultaneously as a control in our study, also showed a clear focal intercellular expression of ICAM-1 on keratinocytes. These results exclude the possibility of an artifact in our studies. The changes in the levels of expression of ICAM- 1, LFA- 1, and LFA-3 in the present study did not reach statistical significance during treatment with cyclosporine. Recently it was reported that, in in vitro studies in which antigens and tetanus toxoid were also used as
stimuli, cyclosporine did not affect the expression of ICAM-1, LFA-1, or LFA-3 on dendritic cells and on T cells. 21 The observation that expression of IL-1, TNF-o~, and IFN-3, remained unaltered after treatment with cyclosporine despite clinical improvement (Table II) is interesting. In mice IFN-3, produced by the TH1 subset can reduce IL-4-stimulated IgE synthesis in vitro.22, 23 Cyclosporine does not reduce (IgE-mediated) immediate-type allergic responses to inhalant allergens, r Serum IgE levels were also not affected by cyclosporine treatment in atopic dermatitis. 1 These clinical and laboratory observations suggest that IgE production-enhancing stimuli are not markedly suppressed by cyclosporine in atopic dermatitis. A diffuse and intercellular staining was observed with anti-IFN-% which suggest binding of IFN-3, to the membranes of the keratinocytes. To our knowledge, the presence of IL-8 in atopic dermatitis has not been previously reported. IL-8 is a chemotactic agent for neutrophils, macrophages, and T lymphocytes; thus it facilitates the migration of these cells into inflamed skin sites. 24 In the present study the levels of expression of IL-8 (Table II) were decreased during cyclosporine treatment. However, the role of IL-8 in atopic dermatitis requires additional studies. The excellent response of atopic dermatitis to cyclosporine and the specific inhibition of IL-2 activity suggests that atopic eczema is a T cell-mediated skin disease. The role of cytokines in atopic derma-
928 van Joost et al.
titis and the effects of cyclosporine on these mediators of inflammation m e r i t further investigation. REFERENCES 1. Van Joost Th, Stolz E, Heule F. Efficacyof low-dose eyclosporine in severe atopic skin disease. Arch Dermatol 1987; 123:166-7. 2. Logan RA, Camp RDR. Severe atopie eczema: response to oral Cyclosporin A. J R See Med 1988;81:417-8. 3. Motley R J, Whittaker JA, Holt PJA. Resolution of atopic dermatitis in a patient treated with eyclosporin. Clin Exp Dermatol 1989;14:243-4. 4. Taylor RS III, Cooper KD, Headington JT, et al. Cyclosporine therapy for severe atopic dermatitis. J AM ACid3 DERMATOL1989;21:580-3. 5. Gold MH, Picaseia DD, Roenigk HH, et al. Treatment-resistant atopic dermatitis controlled with Cyclosporin A. Int J Dermatol 1989;28:481-2. 6. Ross JS, Camp RDR. Cyclosporin A in atone dermatitis. Br J Dermatol 1990;122(suppl 36):41-5. 7. Munro CS, Higgins EM, Marks JM, et al. Cyclosporin A in atopic dermatitis: therapeutic response is dissociated from effects on allergic reactions. Br J Dermatol 1991; 124:43-8. 8. Sowden JM, Berth-Jones J, Ross JS, et al. Double-blind, controlled, crossoverstudy of Cyclosporinin adults with severe refractory atopic dermatitis. Lancet 1991;338:137-40. 9. Feutren G. Cyclosporin A: recent developments in the mechanism of action and clinical application. Curt Opin Immunol 1989;2:239-45. 10. Mosman TR, Moone KW. The role of IL-10 in crossregulation of TH1 and TH2 responses. Immunol Today 1990;12:A49-A53. 11. Hanifin TM, Rajka O. Diagnostic features of atopic dermatitis. Acta Derm Venereol (Stoekh) 1980;92:44-7. 12. Van Joost Th, Bos JD, Heule F, et al. Low-dose Cyclosporin A in severe psoriasis. Br J Dermatol 1988;118:183-90. 13. Bos JD, Meinardi MMHM, van Joest Th, et al. Use of cyclosporin in psoriasis. Lancet 1989;2:1500-2. 14. Van Joost Th, Tank B, Heule F, et al. Treatment regimens in severe psoriasis vulgaris with cyclosporin. J Dermatol Treat 1991;1:311-5. 15. Wahlgren CF, Scheynius A, H~igermark O. Antipruritic
Journal of the American Academy of Dermatology
effect of oral CyclosporinA in atopic dermatitis. Acta Derm Venereol (Stock_h) 1990;70:323-9. 16. Horrooks C, Ormerod AD, Duncan JI, et al. Influence of systemic Cyclosporin A on interleukin-2 and epidermal growth factor receptor expression in psoriatic skin lesions. Clin Exp Immunol 1989;78:166-71. 17. Hunyadi J, Simon M. Expression of OKM 5 antigen on human keratinocytes in vitro upon stimulation with "r-interferon. Acta Derm Venereol (Stockh) 1986;66:527-30. 18. Fraissinette A, Dezutter-Dambuyant C, Schmitt D, et al. Ontogeny of Langerhans cells: phenotypic differentiation from the bone marrow to the skin. Dev Comp Immunol 1990;14:335-46. 19. Griffiths CEM, Voorhees J J, Nickoloff BJ. Characterization of intercellular adhesion molecule-1 and HLA-DR expression in normal and inflamed skin: modulation by recombinant gamma interferon and tumor necrosis factor. J AM ACAD DERMATOL1989;20:617-29. 20. Singer KH, Tuck DT, Hugh A, et al. Epidermal keratinocytes express the adhesion molecule intercellular adhesion molecule 1 in inflammatory dermatosis. J Invest Dermatol 1989;92:746-50. 21. Teunissen MBM, Pistoor FHM, Rongen HAH, et al. Comparison of the inhibitory effects of immunosuppressive agents cyclosporin A, tetranactin and didemnin B on human T cell responses in vitro. In: In vitro studies on human epidermal Langerhans cells. Ph.D. thesis, 1990, University of Amsterdam. 22. Mosmann TR, Coffman RL. TH-1 and TH-2 cells: different patterns of lymphokine secretion lead to different functional properties. Ann Rev Immunol 1989;7:145-73. 23. Mosmann TR, Coffman RL, Two types of mouse helper T-cell clones. Immunol Today 1987;8:223. 24. Barker JNWN, Mitra RS, Griffiths CEM, et al. Keratinocytes as inhibitors of inflammation. Lancet 1991; 337:21 I-4. 25. Dijkmans R, Van Damme J, Cornette F, et al. Bacterial lipopolysaccharide potentiates gamma interferon-induced cytotoxicity for normal mouse and rat fibroblasts. Infect Immun 1990;58:32-36. 26. Engelberts I, Moiler A, Schoen G J, et al. Evaluation of measurement of human TNF in plasma by ELISA. Lymphokine Cytokine Res 1991;10:69-76.
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Cyclosporine in atopic dermatitis Modulation in the expression of immunologic markers in lesional skin Th. van Joost, MD, a M. M. A. Kozel, MD, b B. Tank, PhD, a R. Troost, MD, a and E. P. Prens, M D a, b Rotterdam, the Netherlands
Background: In previous studies, oral cyclosporine was highly effective in the treatment of patients with severe atopic dermatitis. In this study seven patients with severe and therapyresistant atopic dermatitis underwent therapy with cyclosporine, 5 mg/kg/day, for 6 weeks. Objective: The effect of cyclosporine on the expression of cytokines, which probably play a role in this disease, was examined. Methods: The study was performed with a panel of antibodies as markers of inflammatory cells, adhesion molecules, and cytokines (interferon-'r [IFN-'y], tumor necrosis factor-a [TNF-a] and interleukins la, I/5, and 8 [IL-la, IL-lfl, and IL-8, respectively]). They were visualized by indirect immunoperoxidase techniques. Results: After 2 weeks of cyclosporine therapy, a reduction of 60% in the disease (severity and extent) was observed. This reduction was 89% after 4 weeks and 90% after 6 weeks of therapy. Results of indirect immunoperoxidase stains performed on lesional skin sections after 2 weeks of treatment showed statistically significant reduced numbers of CD14 +, CD25 (IL-2R +) and IL-8 + inflammatory cells in the dermis and CD36(OKM5) + cells in both the epidermis and dermis. The number of cells expressing IFN-3, and TNF-a, assumed to be the products of the helper T-cell (TH) 1 subset, was unaltered despite the impressive clinical benefit observed. Keratinocytes in lesional atopic skin did not express intercellular adhesion molecule type 1 (ICAM-1). The expression of the adhesion molecules ICAM-1, lymphocyte function-associated (LFA) type 1, and LFA-3 on inflammatory cells also remained unaffected by cyclosporine treatment. Conclusion: A statistically significant reduction in the number of activated T cells and in the number of cells expressing the IL-2 receptor (CD25) paralleled a marked improvement in the disease and supports the view that atopic dermatitis is based on a T-cell-mediated immune inflammation. (J AM ACAD DERMATOL 1992;27:922-8.) Oral cyclosporine m a y be beneficial in the treatm e n t of patients with severe atopic dermatitis. 1"8 T h e beneficial effects of cyclosporine have been attributed mainly to inhibition of T-cell activation, interleuldn 2 (IL-2) production, and the IL-2 receptor expression. Cyclosporine might also inhibit the production of other cytokines such as interferon-3, (IFN-30, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, and IL-4. 9 An imbalance between two helper T ( T H ) lymphocyte sub-
From the Departments of Dermato-Venereologya and Immunology,b Erasmus University. Accepted for publication May 29, 1992, Reprint requests: Th. van Joost, Department of Dermato-Venereology, University Hospital Rotterdam--Dijkzigt, Dr, Molewaterplein 40, 3015 GD Rotterdam, the Netherlands. 16/1/39648
922
sets, namely TH1 (producing IL-2, IFN-% T N F - a ) and TH2 (producing IL-3, IL-4, IL-5, IL-6, IL-10) may play a crucial role in the regulation of IgE-mediated diseases. 1~ Apparently cyclosporine has the potential to interfere at some crucial steps in the immunopathogenesis of atopic dermatitis. The purpose of this study was to confirm the beneficial effect of cyclosporine in severe atopic dermatitis resistant to topical therapy and to evaluate the immunomodulating effect(s) of cyclosporine on the expression of certain immunologic markers that may play a role in atopic dermatitis. MATERIAL AND METHODS Patients Seven patients (2 men, 5 women), 18 to 32 years of age (mean 23 years) with severe chronic atopic dermatitis (mean duration 15 years), 11 resistant to systemic and
Volume 27 Number 6, Part 1 December 1992
topical treatment, were treated in an open study with cyclosporine (Sandimmune, Sandoz Pharmaceuticals Corp., E. Hanover, N. J.), 5 mg/kg/day, for 6 weeks. The disease in all patients was chronic and severe for at least a year before the start of cyclosporine therapy. In all patients systemic therapy had been discontinued 4 weeks before treatment and none had received topical therapy 2 weeks before treatment. No other concurrent treatment was given during cyclosporine therapy with the exception of topical petrolatum. The patients were informed that previous studies t-8 had shown that a substantial improvement in pruritus and loss of sleep were to be expected once cyclosporine treatment was started. Monitoring (blood pressure, hematologic and biochemical profiles, infection, use of drugs that influence or increase the risk of renal malfunction) used in this study has been reported previously.12"14 Disease activity was measured biweekly on the basis of six clinical features (erythema, infiltration, vesicles/papules, dryness/scaling, cracking/fissuring, and excoriations) graded at six sites (head and neck, anterior and posterior trunk, mid-upper to mid-lower arm [both sides], both hands, mid thigh to mid calf [both sides], and both feet) on a 3-point scale of 0 (none) to 3 (severe) with a maximum score of 108. The extent of eczema was assessed according to "the rule of nines" as described previously.8 Lichenification, apparently a more resistant feature to short-term cyclosporine treatment, 7 was assessed separately at the same six defined sites on a scale of 0 (none) to 3 (severe) with a maximum score of 18.
Biopsies Punch biopsy specimens were obtained from each patient from newly developed unexcoriated lesions. Subsequent, specimens were taken 2 weeks after cyclosporine treatment from lesional skin near sites from which pretreatment specimens were obtained. Biopsy specimens were embedded in Tissue-Tek (OCT compound, Miles Inc., Elkhart, Ind.), snap-frozen in liquid nitrogen, and stored at -70 ~ C.
Indirect immunoperoxidase test The biopsy specimens were cut into 5 um sections. The specificity and the sources of the 20 different antibodies that were used are shown in Table I. An indirect immunoperoxidase (IIP) technique with 3-amino-9-ethylcarbazole (AEC) as a substrate was used. The sections were fixed in acetone for 5 minutes at room temperature and were incubated for 30 minutes at 37 ~ C with an optimal dilution of the primary antibody and were subsequently incubated for 30 minutes with the appropriate peroxidase-conjugated secondary antibody. Peroxidase activity was visualized with a freshly prepared acetate buffer solution containing AEC and hydrogen peroxide. Sections
Cyclosporine in atopic dermatitis 923 were counterstained with Mayer's hematoxylin and mounted with Aquamount. Preimmune goat and sheep sera were used as controls in comparable experiments. The specificities of the antibodies that were gifts (Table I) were checked in blocking experiments with appropriate recombinant human cytokines (results not shown). Well-stained cells (• were counted independently by two observers. For almost all markers the stained cells in the epidermal and in the dermal infiltrates were graded semiquantitatively on a 4-point scale (0 to 3) with steps of 0.5, representing none/very few (grade 0), few (grade 1), moderate (grade 2), and many (grade 3). The numbers of CD25 + ce/ls were assessed per high power field (• because this marker was sporadically expressed in frozen sections of atopic dermatitis lesions. A separate grading system was used for the levels of expression of cytokines. The level of staining was scored on the following 4-point scale: no staining (grade 0), moderate focal/ faint diffuse staining (grade 1), strong focal/moderate diffuse staining (grade 2), and strong diffuse staining (grade 3). All results were expressed as the mean.
Statistical analysis The results of the immunohistochemical studies performed before and during treatment with cyclosporine were analyzed with the Wilcoxon sign rank test. A p value less than 0.05 was considered statistically significant (Table II). RESULTS
Clinical findings A substantial decrease in the severity of itch and loss of sleep was achieved in all patients within 1 week of therapy with cyclosporine. The degree of objective clinical improvement is summarized in Table III. In all seven patients a substantial improvement was noted after 2 weeks of treatment. The mean percentage reduction in disease activity after 2 weeks of cyclosporine treatment was 60% (5 7.8 vs 23.3, Table III), 89% after 4 weeks, and 90% after 6 weeks of treatment. An important percentage reduction in the scores indicating improvement was also observed in the extent of the disease: 65% at week 2, 86% at week 4, and about 90% at week 6. The mean values for lichenification were 10 at week 0, 8 at week 4, and 7 at week 6. The mean reduction in lichenification after 6 weeks of cyclosporine therapy was about 33%. Mean serum creatinine levels increased slightly during cyclosporine therapy but in none of the patients was an unacceptable alteration in hematologic or biochemical profile observed. 13, 14 An unacceptable rise in blood pressure (diastolic
924
Journal of the American Academy of Dermatology
van J o o s t et aL
Table I. Antibody panel Cellulartargets
Workingdilution
Cortical thymocytes, Langerhans cells, some other dendtritic cells Immature T cells (cytoplasmic expression) and mature functional T cells (membrane expression) Subpopulation of cortical thymocytes, helper/inducer T cells, subpopulation ofmonocytes and macrophages Subpopulation of cortical thymocytes, cytotoxic/suppressor T cells, subpopulation of NK cells Majority of lymphoid and myeloid cells Monocytic. cells, macrophages, follicular dendritic reticulum ceils Activated T cells, activated B cells Subpopulation of T cells (especially CD4 + T cells), megakaryocytes, platelets, subpopulafionof other leukocytes Monocytes, early erythroid cells, megakaryocytes, platelets Virgin T cells, B cells, granulocytes, monocytes Broad tissue distribution; increased expression on activation Leukocytes, broad tissue distribution, dendritic cells Hematopoletic precursor cells, B ceils, activated T cells, monocytic ceils, macrophages Active dendritic cells, subsets of B cells (class II associated)
1:10
Antibody
CDla (OKT 6) CD3 (Leu-4) CD4 (Leu-3a) CD8 (Leu-2a) CD1 la (LFA-la) CD14 (MY-4)
CD25 (IL-2RI) CD29 (4B4)
CD36 (OKM5) CD45 (2H4) CD54 (ICAM-1) CD58 (LFA-3) Anti-HLA-DR RFD 1
1:50
Becton Dickinson
1:50
Becton Dickinson
1:50
CLB, Amsterdam, the Netherlands Coulter Immunology, Hialeah, Fla. Becton Dickinson Coulter Immunology
1:100 1:50 1:100
1:100
Ortho Diagnostics, Raritan, N.J. Coulter Immunology
1:250
Boehringer, Mannheim
1:25
Behring, Marburg, Germany
1:200
Becton Dickinson
1:250
1:150
L. Poulter, Royal Free Hospital, School of Medicine, London Y. Ishii, Tokyo, Japan J. van Damme, Rega Institute, Leuven, Belgium25 W. Buurman, University of Maastricht, the Netherlands26 Glaxo, Geneva, Switzerland
1:100
Biogen, Glaxo
1:200
J. van Damme, Rega Institute25
1:200
B cells, dendritic cells Macrophages, T cells, B cells, NK cells
1:200 1:20
Anti-TNF-a
Tumor calls, fibroblasts, macrophages, neutrophils, lymphocytes, endothelium Macrophages, T cells, B cells, fibroblasts, endothelial and epithelial cells Macrophages, T cells, B cells, fibroblasts, endothelial and epithelial cells T cells, neutrophils, basophils, epithelial ceils
1:5
Anti-IL-lfl* Anfi-IL-St
Erasmus University, Rotterdam, the Netherlands Becton Dickinson, Mountain View, Calif.
1:50
L25 Anti-IFN-7
Anfi-IL-la*
Source
*Sheepantimousepolyclonalantisera. tGoat antimouse polyelonalantiserum. >95 m m H g or systolic > 150 m m Hg) was not observed in any of the patients./3, 14 Histologie and immunohistochemical studies
The histologic changes seen in the frozen sections of lesions before and after cyclosporine treatment
paralleled the clinical improvement observed in most patients. After therapy, there was a clear reduction in the dermal mononuclear infiltrate and a remarkable decrease in spongiosis and the number of intraepidermal microvesicles was reduced. After 2 weeks of treatment, a slight but statistically insig-
Volume 27 Number 6, Part 1 December 1992
nificant reduction was observed in the expression of CDla, CD4, the C D 4 / C D 8 ratio, C D l l a , CD29, CD54 (ICAM-1), HLA-DR, and RFD1 on the inflarnmatory cells in the dermis (Table II).
Cyclosporine in atopic dermatitis 925 Table II. Mean grades of expression of various markers investigated in lesional skin of seven patients with severe atopic dermatitis before and after 2 weeks of cyclosporine treatment Epldermis/epldermal inmtratr
Statistically significant alterations seen with four different specific markers CD14 Before cyclosporine treatment, in the dermis particularly, a substantial number of clustered CD14 + cells generally with dendritic morphology were observed. After treatment the number of CD14 + cells in the dermis in all patients were significantly (p < 0.05) reduced with a scattered distribution throughout the dermis. CD25. A statistically significant (.p < 0.05) decrease in the number of CD25 + cells expressing the IL-2 receptor was observed in the dermis after treatment. The number of intraepidermal CD25 + cells was reduced insignificantly, probably because of the low number of cells. CD36 (OKM5). Before cyclosporine treatment, keratinocytes in the stratum spinosum and granulosum clearly expressed CD36 (OKM5) with the characteristic intercellular reticular staining pattern. In the dermis the majority of the CD36 + cells, some with dendritic morphology, were grouped around the upper dermal capillaries (Fig. 1). After 2 weeks of cyclosporine treatment the intercellular expression of CD36 in the epidermis was significantly (p < 0.02) reduced or even absent in all seven cases and in the dermis a reduction in the number of CD36 + cells was observed (Table II and Fig. 2). Anti-IL-8. In the lower epidermal layers of most sections, a diffuse reticular staining of low intensity and in the higher epidermal layers a more intense reticular and homogeneous staining was observed. No significant changes were observed in the epidermis after treatment, but in the dermis a statistically significant (p < 0.05) reduction in the levels of expression of IL-8 on the infiltrate and in the number of IL-8 + cells was observed after treatment with cyclosporine. After 6 weeks the studies were repeated in two patients. The results did not differ significantly from those obtained after 2 weeks of treatment. DISCUSSION This open study corroborates the observations of previous studies 18 that short-term treatment ("crisis-intervention") 1with cyclosporine can be effective and safe for severe chronic atopic dermatitis. Careful monitoring of patients receiving cyclosporine is, however, strictly indicated)' 13 The reduction in the
Before
After '
Antibody
(n= 7)
(n =7)
CDla CD3 CD4 CD8 CD1 la CD14 CD25 CD29 CD36 CD45 CD54 CD58 Anfi-HLA-DR RFD 1 L25 Anti-TNF-a:~ Anti-IFN-~/w Anti-IL-la[ Anti-IL- 1/~ Anti-IL-8 82
2.2 0.7 0.1 O.5 0.7 0.3 0.71" 2.2 1.8 2.0 0.0 0.5 1.3 0.5 5.2 1.6 1.5 1.1 1.1 1.6
2.3 0.5 0.1 0.3 0.4 0.1 0.3t 1.9 0.6* 1.8 0.0 0.8 1.3 0.8 5.5 1.8 1,3 1.0 1.3 1.3
~rmal i ~ ~re
1.8 2.6 1.4 0.6 2.1 1.9 4.3t 2.5 1.3 1.8 1.9 2.3 2.2 2.2 2.1 1.6 2.0 0.9 1.3 1.5
M~r
1.5 1.8 1.1 0.6 1.4 1.1" 1.0*'t 2.1 0.6* 1.7 1.5 2.3 1.8 1.8 2.0 1.7 1.8 0.8 1.5 0.9*
*Statistically significant (p < 0.05). ~'The figures represent the mean number of cells per high power field. SDiffuse and intercellular epidermal staining and diffuse staining in the dermal infiltrate. w and focal intercellular epidermal stainingand focal staining in the dermal infiltrate. ][Diffuseepidermal staining, diffuse and focal staining in the dermal infiltrate. 82 and intercellular epidermal staining and focal staining in the dermal infiltrate,
disease activity observed at week 2 was more pronounced than that reported recently by Sowden et al. 8 In their double-blind placebo-controlled study patients continued to use the same topical steroid treatment that they were applying before entry into the trial. An explanation for the discrepancy in the clinical improvement observed in the two studies may be that in our study some what different clinical features for the overall assessment of disease activity and extent were used. Lichenification is rather resistant to short-term treatment with cyclosporine 7 and therefore was not included as a clinical variable in the overall clinical assessment in our study. Furthermore, in contrast to the previous study, 8 we also used the occurrence of papules and/or vesicles as a variable for assessing disease activity.
926
Journal of the American Academy of Dermatology
van Joost et al.
Fig. 1. Immunohistochemical staining of lesional atopic skin with OKM5 (c~-CD36) antibody before treatment with oral cyclosporine. Note intense epidermal reticular staining. In dermis CD36 + inflammatory cells are grouped around upper dermal capillaries. (;<200.)
Table Ill. Disease activity and the extent of disease in seven patients treated with cyclosporine, 5 m g / k g / d a y , for 6 weeks Patient
1
2 3 4 5 6 7 Mean Mean % reduction in scores
~_
Before therapy ........
68 49 60 57 59 49 61 57.8 --
46 60 57 33 79 30 70 53.6 --
Week 2
I
Week 4
Week 6
Activity
Extent
I Activity ,
Extent
Activity
20 26 28 21 22 25 21 23.3 59.7
23 16 15 11 42 12 12 18.7 65.1
12 2 1l 3 4 2 11 6.4 88.9
17 0 9 9 6 4 7 7.4 86.2
12 7 6 5 0 3 7 5.7 90.1
[
Extent
11 3 6 9 0 4 5 5.4 89.9
*Six-area score (see text for details), tRule-of-nines score.
T h e reduction in the number of CD25 + T cells in the dermal infiltrate after cyclosporine treatment corroborates the findings reported by Wahlgren et al. is in atopic dermatitis and by Horrocks et al. 16 in psoriasis. A statistically significant decrease in the number of CD25 + cells expressing the IL-2 receptor in the dermal infiltrate (Table II) strongly suggests that T-cell activation is inhibited by cyclosporine. This suggests an important contribution of T lymphocytes to atopic dermatitis. In contrast to keratinocytes in healthy skin, keratinocytes in lesional atopic skin and in many other inflammatory dermatoses exhibit a strong epidermal
intercellular expression of CD36 (OKMS) (Fig. 1). The reduction in the expression of CD36 by cyclosporine treatment, therefore, may reflect a decrease in the production of certain CD36-inducing cytokine(s). The observed decrease in the number of activated T cells (CD25) suggests that the CD36-inducing cytokine(s) originate from T lymphocytes, 17 A myeloid differentiation antigen, CD14 is normally expressed on the surface of monocytes and macrophages but is expressed in low amounts on neutrophils and lymphocytes. Furthermore, CD 14 is one of the two (CD 14, CD33) monocyte differentiation antigens that are expressed on normal human
Volume 27 Number 6, Part I December 1992
Cyclosporine in atopic dermatitis 927
Fig. 2. Immunohistochemical staining of lesional atopic skin with the OKM5 (a-CD36) antibody 2 weeks after treatment with oral cyclosporine. Note reduced reticular staining in epidermis and decreased CD36 + inflammatory cells in dermis. (x200.)
epidermal Langerhans cells. 18 In our study, the observed decrease in the number of CD 14+ cells in the dermal infiltrate after treatment with cyclosporine suggests decreased T-cell activation by CD14 + antigen-presenting cells. It was not possible to reconcile a statistically significant reduction in the number of CD14 + dendritic cells with other dendritic cell markers such as CDla, HLA-DR, RFD- 1, and L25 after cyclosporine treatment. No HLA-DR was expressed on the keratinocytes in any of the lesional biopsy specimens. This agrees with previous observations in atopic dermatitis. 19 Griffiths et al. 19 reported focal ICAM-1 expression on keratinocytes in atopic dermatitis. Singer et al. 2~ observed ICAM- 1 expression on keratinocytes in atopic dermatitis with an indirect immunofluoreseence technique. In contrast, in the present study CD54 (ICAM-1) was not expressed on keratinocytes, but in the same sections a strong CD54 expression was observed on endothelial cells and on cells of the dermal mononuclear infiltrate (Table II). Sections from lesional psoriatic skin, which were stained simultaneously as a control in our study, also showed a clear focal intercellular expression of ICAM-1 on keratinocytes. These results exclude the possibility of an artifact in our studies. The changes in the levels of expression of ICAM- 1, LFA- 1, and LFA-3 in the present study did not reach statistical significance during treatment with cyclosporine. Recently it was reported that, in in vitro studies in which antigens and tetanus toxoid were also used as
stimuli, cyclosporine did not affect the expression of ICAM-1, LFA-1, or LFA-3 on dendritic cells and on T cells. 21 The observation that expression of IL-1, TNF-o~, and IFN-3, remained unaltered after treatment with cyclosporine despite clinical improvement (Table II) is interesting. In mice IFN-3, produced by the TH1 subset can reduce IL-4-stimulated IgE synthesis in vitro.22, 23 Cyclosporine does not reduce (IgE-mediated) immediate-type allergic responses to inhalant allergens, r Serum IgE levels were also not affected by cyclosporine treatment in atopic dermatitis. 1 These clinical and laboratory observations suggest that IgE production-enhancing stimuli are not markedly suppressed by cyclosporine in atopic dermatitis. A diffuse and intercellular staining was observed with anti-IFN-% which suggest binding of IFN-3, to the membranes of the keratinocytes. To our knowledge, the presence of IL-8 in atopic dermatitis has not been previously reported. IL-8 is a chemotactic agent for neutrophils, macrophages, and T lymphocytes; thus it facilitates the migration of these cells into inflamed skin sites. 24 In the present study the levels of expression of IL-8 (Table II) were decreased during cyclosporine treatment. However, the role of IL-8 in atopic dermatitis requires additional studies. The excellent response of atopic dermatitis to cyclosporine and the specific inhibition of IL-2 activity suggests that atopic eczema is a T cell-mediated skin disease. The role of cytokines in atopic derma-
928 van Joost et al.
titis and the effects of cyclosporine on these mediators of inflammation m e r i t further investigation. REFERENCES 1. Van Joost Th, Stolz E, Heule F. Efficacyof low-dose eyclosporine in severe atopic skin disease. Arch Dermatol 1987; 123:166-7. 2. Logan RA, Camp RDR. Severe atopie eczema: response to oral Cyclosporin A. J R See Med 1988;81:417-8. 3. Motley R J, Whittaker JA, Holt PJA. Resolution of atopic dermatitis in a patient treated with eyclosporin. Clin Exp Dermatol 1989;14:243-4. 4. Taylor RS III, Cooper KD, Headington JT, et al. Cyclosporine therapy for severe atopic dermatitis. J AM ACid3 DERMATOL1989;21:580-3. 5. Gold MH, Picaseia DD, Roenigk HH, et al. Treatment-resistant atopic dermatitis controlled with Cyclosporin A. Int J Dermatol 1989;28:481-2. 6. Ross JS, Camp RDR. Cyclosporin A in atone dermatitis. Br J Dermatol 1990;122(suppl 36):41-5. 7. Munro CS, Higgins EM, Marks JM, et al. Cyclosporin A in atopic dermatitis: therapeutic response is dissociated from effects on allergic reactions. Br J Dermatol 1991; 124:43-8. 8. Sowden JM, Berth-Jones J, Ross JS, et al. Double-blind, controlled, crossoverstudy of Cyclosporinin adults with severe refractory atopic dermatitis. Lancet 1991;338:137-40. 9. Feutren G. Cyclosporin A: recent developments in the mechanism of action and clinical application. Curt Opin Immunol 1989;2:239-45. 10. Mosman TR, Moone KW. The role of IL-10 in crossregulation of TH1 and TH2 responses. Immunol Today 1990;12:A49-A53. 11. Hanifin TM, Rajka O. Diagnostic features of atopic dermatitis. Acta Derm Venereol (Stoekh) 1980;92:44-7. 12. Van Joost Th, Bos JD, Heule F, et al. Low-dose Cyclosporin A in severe psoriasis. Br J Dermatol 1988;118:183-90. 13. Bos JD, Meinardi MMHM, van Joest Th, et al. Use of cyclosporin in psoriasis. Lancet 1989;2:1500-2. 14. Van Joost Th, Tank B, Heule F, et al. Treatment regimens in severe psoriasis vulgaris with cyclosporin. J Dermatol Treat 1991;1:311-5. 15. Wahlgren CF, Scheynius A, H~igermark O. Antipruritic
Journal of the American Academy of Dermatology
effect of oral CyclosporinA in atopic dermatitis. Acta Derm Venereol (Stock_h) 1990;70:323-9. 16. Horrooks C, Ormerod AD, Duncan JI, et al. Influence of systemic Cyclosporin A on interleukin-2 and epidermal growth factor receptor expression in psoriatic skin lesions. Clin Exp Immunol 1989;78:166-71. 17. Hunyadi J, Simon M. Expression of OKM 5 antigen on human keratinocytes in vitro upon stimulation with "r-interferon. Acta Derm Venereol (Stockh) 1986;66:527-30. 18. Fraissinette A, Dezutter-Dambuyant C, Schmitt D, et al. Ontogeny of Langerhans cells: phenotypic differentiation from the bone marrow to the skin. Dev Comp Immunol 1990;14:335-46. 19. Griffiths CEM, Voorhees J J, Nickoloff BJ. Characterization of intercellular adhesion molecule-1 and HLA-DR expression in normal and inflamed skin: modulation by recombinant gamma interferon and tumor necrosis factor. J AM ACAD DERMATOL1989;20:617-29. 20. Singer KH, Tuck DT, Hugh A, et al. Epidermal keratinocytes express the adhesion molecule intercellular adhesion molecule 1 in inflammatory dermatosis. J Invest Dermatol 1989;92:746-50. 21. Teunissen MBM, Pistoor FHM, Rongen HAH, et al. Comparison of the inhibitory effects of immunosuppressive agents cyclosporin A, tetranactin and didemnin B on human T cell responses in vitro. In: In vitro studies on human epidermal Langerhans cells. Ph.D. thesis, 1990, University of Amsterdam. 22. Mosmann TR, Coffman RL. TH-1 and TH-2 cells: different patterns of lymphokine secretion lead to different functional properties. Ann Rev Immunol 1989;7:145-73. 23. Mosmann TR, Coffman RL, Two types of mouse helper T-cell clones. Immunol Today 1987;8:223. 24. Barker JNWN, Mitra RS, Griffiths CEM, et al. Keratinocytes as inhibitors of inflammation. Lancet 1991; 337:21 I-4. 25. Dijkmans R, Van Damme J, Cornette F, et al. Bacterial lipopolysaccharide potentiates gamma interferon-induced cytotoxicity for normal mouse and rat fibroblasts. Infect Immun 1990;58:32-36. 26. Engelberts I, Moiler A, Schoen G J, et al. Evaluation of measurement of human TNF in plasma by ELISA. Lymphokine Cytokine Res 1991;10:69-76.