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Elevated levels of interleukin-8 in blister fluid of bullous pemphigoidcompared with suction blisters of healthy control subjects
310
Brief communications
15. Woodward AH, Ivans JC, Soule EH. Lymphangiosarcoma arising in chronic lymphedematous extremities. Cancer 1972;30:562-72. 16. Mackenzie IJ. Angiosarcoma of the face. Arch Dermatol 1985;121:549-50. 17. Meadows AT, Baum E, Fossati-Bellani F, et al. Second malignant neoplasms in children: an update from the late effects study group. J Clin Orlcol 1985;3:532-8. 18. Mazanet R, Antman KI-I.Sarcomas of soft tissue and bone. Cancer 1991;68:463-73. 19. Popper H, Thomas LB, Telles NC, et al. Development of hepatic angiosarcoma in man induced by vinyl chloride, thorotrast, and arsenic. Am J Pathoi 1978;92:349-76.
Journal of the American Academy of Dermatology February 1996
20. Falk H, Thomas LB, Popper H, et al. Hepatic angiosarcoma associated with androgenic-anabolic steroids. Lancet 1979; 2:1120-3. 21. Hoch-Ligeti C. Angiosarcoma of the liver associated with diethylstilbestrol. JAIVIA 1978;240:1510-1. 22. Russell WO, Cohen J, Enzinger FM, et al. A clinical and pathologic staging system for soft tissue sarcomas. Cancer 1977;40:1562-70. 23. Huerter CJ, Kunkel JR, Rouse JR. Angiosarcoma of the face and scalp. Cuffs 1993;51:461-2.
Elevated levels of interleukin-8 in blister fluid of bnllous pemphigoid compared with suction blisters of healthy control subjects Enno Schmidt, BS, a Andreas Ambach, MD, b Boris Bastian, IVID,a Eva-Bettina Br'Ocker, MD, a and Detlef Zillikens, M D a Wiirzburg and Heidelberg, Germany Bullous pemphigoid (BP) is an autoimmune immunoglobulin-mediated disease characterized by subepidermal blister formation. Autoantibodies are directed against hemidesmosomal antigens, including the intracellular 230 kd, and the transmembranous 180 kd protein. 1 However, in addition to autoantibodies, complement activation and inflammatory cells are necessary for blister formation. 2 Mononuclear cells are the first cells to infiltrate B P lesions,3, 4 and subsequently, eosinophils and neutrophils also appear. Interleukin 0L)-8 is a chemotactic factor for T lym.phocytes, neutrophils, 5 and eosinophils. 6 W e assayed the IL-8 level in blister fluid and serum sampies o f patients with B P and in suction blisters o f healthy volunteers.
MATERIAL AND METHODS We Studied 10 patients (six women, four men; age range, 57 to 88 years) with typical clinical, histologic, and From the Departmentof Dermatology,Universityof WUrzburg,a and the Departmentof AppliedImmunology,GermanCancerResearch Center, Heidelberg.b Presentedat the annualmeetingof the ArbeitsgemeinschaftDermatologische Forschung,Zurich,Switzerland,Nov. 13-15, 1993. Reprint w.quests:DetlefZillikens, MD, Departmentof Dermatology, Medical Collegeof Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. J AM ACAt~D~IATOL 1996;34:310-2. Cbpydght 9 1996 by the AmericanAcademyof Dermatology,Inc. 0190-9622/96 $5.00+ 0 16/54/67562
immunofluorescent features of BP before treatment. As a control group, we generated suction blisters on the flexor side of the forearm of 10 healthy volunteers 15 to 86 years of age. Five were men and five were women. We applied a constant negative pressure of 300 mm Hg until five blisters, each 6 mm in diameter, had emerged. Blister fluid was obtained immediately after blisters had developed. In the group of patients, blister fluid was aspirated within the first 6 hours after blister formation. At the time of blister puncture, blood samples were obtained from both patients and control subjects. After centrifugation, serum samples and cell-free supematants of blister fluids were stored at -80 ~ C until used. IL-8 levels were determined by a solid-phase enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, Minn.). The minimal detectable dose was 18.1 pg/ml. For statistical analysis, we used the Mann-Whitney U test to evaluate the difference of levels in patients and control subjects and the Wilcoxon matched pairs signed rank test to demonstrate differences in concentrations of blister fluid and concurrent serum samples. For correlation analysis, we applied the product-moment correlation test. RESULTS Results are shown in Fig. 1. In blister fluid o f BP, IL-8 levels were 9032 _ 7966 pg/ml (mean _ SD) which was significantly higher than the levels in corresponding serum samples (35.1 _ 19.3 pg/ml; p < 0 . 0 0 1 ) and blister fluid o f the control group (722 - 12 pg/ml; p < 0.005). N o significant differ-
Journal of the American Academy of Dermatology Volume 34, Number 2, Part 1
Brief communications
ences were detected between the IL-8 level in serum samples of patients relative to those in the control group with suction blisters. Moreover, in suction blister fluid, the IL-8 level was significantly higher than in concurrent serum samples (p < 0.001).
311
100.000
10.000
DISCUSSION We found a much higher level of IL-8 in blister fluid than in concurrent serum samples, which suggests the release of IL-8 at the site of blister formation in BP, and to a lesser extent, in the control group. The detection of IL-8 in suction blister fluid is in agreement with the finding of Kuhns et al.7 In addition, these authors studied IL-8 levels in suction blister lesions for 24 hours. Although during the first 5 hours IL-8 levels changed marginally, a marked increase was noticed between 8 and 24 hours. 7 The exact cellular source of IL-8 in suction blister controis remains unclear. Possible candidates include keratinocytes, fibroblasts, or endothelial cells. 8 In a recent study, no messenger RNA for IL-8 was detected in the epidermis of suction blisters9; this finding points to nonepidermal cells as sources of IL-8 in the blister fluid of control subjects. In BP blisters, IL-8 levels were 10 times higher than in suction blisters and 200 times higher than serum levels of control subjects. Levels in BP blisters corresponded to those required to exert a chemotactic effect on T lymphocytes, neutrophils, and eosinophils.5, 6 In contrast to most other cytokines, a long duration of action has been reported for IL-8 because of its resistance to degrading proteases. 1~This property may contribute to the high levels in BP blister fluid. We also cannot state the exact cellular source of IL-8 in BP blister fluid. In addition to resident cells such as keratinocytes, fibroblasts, endothelial cells, and mast cells, infiltrating cells including monocytes/macrophages, T cells, and neutrophils are possible sources. 8'1~ H All these cells are typically found in BP lesions and,appear to be in an altered state)' 4,12-14 Stimulating agents that augment IL-8 expression, including IL- 1, IL-2, IL-6, tumor necrosis factor-a, and interferon gamma, 8' 15 have been detected in the blister fluid of BP. 16-2~Recently, we found elevated levels of 1]-,-113 in BP blisters. 16 In blisters of eight patients, assayed for both IL-1 13 and IL-8, we observed a strong correlation between levels of these cytokines, whereas in blister fluid of 10 control subjects, these did not correlate. Mediators down-regulating IL-8 activity include IL-4,
1.000
m
c
100
10
B
S
Bullous Pemphigoid
B S , Healthy volunteers
Fig. 1. IL-8 levels in blister fluid (B) and serum samples (S) of patients with buUous pemphigoid (n= 10) and healthy suction blister controls (n = 10). Bars indicate mean ___SD.
IL-10, and IL-1 receptor antagonist. 8,21 These have also been described in blister fluid of BP 16' 18 and possibly limit the IL-8 effects in lesions of this disease. The most striking property of IL-8 appears to be its chemotactic effect on both T cells and neutrophils. Subcutaneous or intradermal injections of IL-8 eventually lead to infiltration with these cells. Low doses favor an infiltration of T cells and high doses, an influx of neutrophils. 8 In addition, IL-8 is a potent mediator for eosinophil chemotaxis.6 The chemoattractants C5a and LTB4 have previously been demonstrated in lesions and blister fluid of Bp,22, 23 and IL-8 may also contribute to the influx of inflammatory cells in BP lesions. IL-8 enhances expression of complement receptor type I on neutrophils as well as their activation and degranulation. 8 Peripheral blood leukocytes attach to the basement membrane zone of skin sections incubated with BP serum. 2 Therefore in BP lesions/L-8 may increase the binding of neutrophils to the basement membrane zone and their release of proteolytic en-
312
Brief communications
z y m e s such as m y e l o p e r o x i d a s e , w h i c h has b e e n detected in blister fluid. 24 REFERENCES
1. lshiko A, Shimizu H, Kikuchi A, et al. Human autoantibodies against the 230-KD builous pemphigoid antigen (BPAG1) bind only to the intracellular domain of the hemidesmosome, whereas those against the 180-KD bullous pemphigoid antigen (BPAG2) bind along the plasma membrane of the hemidesmosome in normal human and swine skin. J Clin Invest 1993;91:1608-15. 2. Gammon WR, Merrit CC, Lewis DM, et al. An in vitro model of immune complex-mediated basement membrane zone separation caused by pemphigoid antibodies, leukocytes, and complement. J Invest Dermato11982;78:285-90. 3. Wintroub BU, Mihm MC, Goetzl EJ, et al. Morphologic and functional evidence for release of mast-cell products in bullous pemphigoid. N Engl J Med 1978;298:417-21. 4. Dvorak AM, Mihm MC Jr, Osage FE, et al. Bullous pemphigoid, an ultmstmctuml study of the inflammatory response: eosinophil, basophil and mast cell granule changes in multiple biopsies from one patient. J Invest Dermatol 1982:78:91-101. 5. Larson CG, Anderson AO, Apella E, et al. The neulrophilactivating protein (NAP-I) is also cheraotactic for T lymphocytes. Science 1989;243:1464-6. 6. Eager RA, Casale TB. Interleukin-8 is a potent mediator of eosinophil chemotaxis through endothelium and epithelium. Am J Physiol 1995;268:L17-L22. 7. Kuhns DB, DeCarlo E, Hawk DM, et al. Dynamics of the cellular and humoral components of the inflammatory response elicted in skin blisters in humans. J Clin Invest 1992;89:1734-40. 8. Zachariae COC. Chemotactic cytokines and inflammation: biological properties of the lymphocyte and monocyte chemotactic factors ELCF, MCAF and IL-8. Acta Derm Venereol (Stockh) 1993;181:1-37. 9. Kristensen M, Larsen CG, Jorgensen P, et al. RNA purification from epidermal suction blisters. Acta Derm Venereol (Stockh) 1991;71:423-6. 10. Peveri P, Walz A, Dewald B, et al. A novel neutrophil-activating factor produced by human mononuclear phago.cytes. J Exp Med 1988;167:1547-59. 11. MSiler A, Lippert U, Lessmann D, et al. Human mast cells produce IL-8. J Immunol 1993;151:3261-6.
Journal of the American Academy of Dermatology February 1996
12. Kamshima T, Hachisuka H, Okubo K, et al. Epidermal keratinocytes of bullous pemphigoid express intercellular adhesion molecule-1 (ICAM-1). J Dermatol 1992;19:82-6. 13. zinikens D, Ambach A, Schuessler M, et al. The interleukin-2 receptor in lesions and semm of buUous pemphigoid. Arch Dermatol Res 1992;284:141-5. 14. Ambach A, Ziilikens D, Klingert B, et al. Immunophenotyping of the mononuclear infiltrate in bullous pemphigoid. Hautarzt 1992;43:81-5. 15. Larsen CG, Anderson AP, Oppenheim JJ, et al. Production of IL-8 by human dermal fibroblasts and keratinocytes in response to IL-1 or TNF. Immunology 1989;68:31-6. 16. Schmidt E, Mittnacht A, SchOmig H, et al. Detection of IL-lct, IL-1[3 and IL-1 receptor antagonist in blister fluid of bullous pemphigoid. J Dermatol Sci (In press.) 17. Grando SA, Glukhenky BT, Dmnnik GN, et al. Mediators of inflammation in blister fluids from patients with pemphigus vulgaris and builous pemphigoid. Arch Dermatol 1989; 125:925-30. 18. Zillikens D, Schmidt E, Depprich R, et al. Contribution of cytokines to blister formation in bullous pemphigoid [Abstract]. Arch Dermatol Res 1994;286:200. 19. Zillikens D, Schuessler M, Dummer R, et al. Tumor necrosis factor in blister fluids of bullous pemphigoid. Eur J Dermatol 1992;2:429-31. 20. Kaneko F, Minagawa T, Taldguchi Y, et al. Role of cellmediated immune reaction in blister formation of bullous pemphigoid. Dermatology 1992;184:34-9. 21. DeForge LE, Tracey DE, Kenney JS, et al. Interleukin-1 receptor antagonist protein inhibits interleukin-8 expression in lipopolysaccharide-stimulated human whole blood. Am J Pathol 1992;140:1045-54. 22. Diaz-Perez JL, Jordon RE. The complement system in bullous pemphigoid. IV. Chemotactic activity in blister fluid. Clin Immunol Immunopathol 1976;5:360-70. 23. Wana S, Ueno A, Nishiyama S. Increased levels of immunoreactive leukotriene B4 in blister fluids of bullous pemphigoid patients ahd effects of a selective 5-1ipoxygenase inhibitor on experimental skin lesions. Acta Derm Venereol (Stockh) 1990;70:281-5. 24. Czech W, Schaller J, Sch6pf E, et al. Granulocyte activation in bullous diseases: release of granular proteins in bullous pemphigoid and pemphigus vulgaris. J AM ACAD DmUVIATOL1993;29:210-5.
Brief communications
15. Woodward AH, Ivans JC, Soule EH. Lymphangiosarcoma arising in chronic lymphedematous extremities. Cancer 1972;30:562-72. 16. Mackenzie IJ. Angiosarcoma of the face. Arch Dermatol 1985;121:549-50. 17. Meadows AT, Baum E, Fossati-Bellani F, et al. Second malignant neoplasms in children: an update from the late effects study group. J Clin Orlcol 1985;3:532-8. 18. Mazanet R, Antman KI-I.Sarcomas of soft tissue and bone. Cancer 1991;68:463-73. 19. Popper H, Thomas LB, Telles NC, et al. Development of hepatic angiosarcoma in man induced by vinyl chloride, thorotrast, and arsenic. Am J Pathoi 1978;92:349-76.
Journal of the American Academy of Dermatology February 1996
20. Falk H, Thomas LB, Popper H, et al. Hepatic angiosarcoma associated with androgenic-anabolic steroids. Lancet 1979; 2:1120-3. 21. Hoch-Ligeti C. Angiosarcoma of the liver associated with diethylstilbestrol. JAIVIA 1978;240:1510-1. 22. Russell WO, Cohen J, Enzinger FM, et al. A clinical and pathologic staging system for soft tissue sarcomas. Cancer 1977;40:1562-70. 23. Huerter CJ, Kunkel JR, Rouse JR. Angiosarcoma of the face and scalp. Cuffs 1993;51:461-2.
Elevated levels of interleukin-8 in blister fluid of bnllous pemphigoid compared with suction blisters of healthy control subjects Enno Schmidt, BS, a Andreas Ambach, MD, b Boris Bastian, IVID,a Eva-Bettina Br'Ocker, MD, a and Detlef Zillikens, M D a Wiirzburg and Heidelberg, Germany Bullous pemphigoid (BP) is an autoimmune immunoglobulin-mediated disease characterized by subepidermal blister formation. Autoantibodies are directed against hemidesmosomal antigens, including the intracellular 230 kd, and the transmembranous 180 kd protein. 1 However, in addition to autoantibodies, complement activation and inflammatory cells are necessary for blister formation. 2 Mononuclear cells are the first cells to infiltrate B P lesions,3, 4 and subsequently, eosinophils and neutrophils also appear. Interleukin 0L)-8 is a chemotactic factor for T lym.phocytes, neutrophils, 5 and eosinophils. 6 W e assayed the IL-8 level in blister fluid and serum sampies o f patients with B P and in suction blisters o f healthy volunteers.
MATERIAL AND METHODS We Studied 10 patients (six women, four men; age range, 57 to 88 years) with typical clinical, histologic, and From the Departmentof Dermatology,Universityof WUrzburg,a and the Departmentof AppliedImmunology,GermanCancerResearch Center, Heidelberg.b Presentedat the annualmeetingof the ArbeitsgemeinschaftDermatologische Forschung,Zurich,Switzerland,Nov. 13-15, 1993. Reprint w.quests:DetlefZillikens, MD, Departmentof Dermatology, Medical Collegeof Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. J AM ACAt~D~IATOL 1996;34:310-2. Cbpydght 9 1996 by the AmericanAcademyof Dermatology,Inc. 0190-9622/96 $5.00+ 0 16/54/67562
immunofluorescent features of BP before treatment. As a control group, we generated suction blisters on the flexor side of the forearm of 10 healthy volunteers 15 to 86 years of age. Five were men and five were women. We applied a constant negative pressure of 300 mm Hg until five blisters, each 6 mm in diameter, had emerged. Blister fluid was obtained immediately after blisters had developed. In the group of patients, blister fluid was aspirated within the first 6 hours after blister formation. At the time of blister puncture, blood samples were obtained from both patients and control subjects. After centrifugation, serum samples and cell-free supematants of blister fluids were stored at -80 ~ C until used. IL-8 levels were determined by a solid-phase enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, Minn.). The minimal detectable dose was 18.1 pg/ml. For statistical analysis, we used the Mann-Whitney U test to evaluate the difference of levels in patients and control subjects and the Wilcoxon matched pairs signed rank test to demonstrate differences in concentrations of blister fluid and concurrent serum samples. For correlation analysis, we applied the product-moment correlation test. RESULTS Results are shown in Fig. 1. In blister fluid o f BP, IL-8 levels were 9032 _ 7966 pg/ml (mean _ SD) which was significantly higher than the levels in corresponding serum samples (35.1 _ 19.3 pg/ml; p < 0 . 0 0 1 ) and blister fluid o f the control group (722 - 12 pg/ml; p < 0.005). N o significant differ-
Journal of the American Academy of Dermatology Volume 34, Number 2, Part 1
Brief communications
ences were detected between the IL-8 level in serum samples of patients relative to those in the control group with suction blisters. Moreover, in suction blister fluid, the IL-8 level was significantly higher than in concurrent serum samples (p < 0.001).
311
100.000
10.000
DISCUSSION We found a much higher level of IL-8 in blister fluid than in concurrent serum samples, which suggests the release of IL-8 at the site of blister formation in BP, and to a lesser extent, in the control group. The detection of IL-8 in suction blister fluid is in agreement with the finding of Kuhns et al.7 In addition, these authors studied IL-8 levels in suction blister lesions for 24 hours. Although during the first 5 hours IL-8 levels changed marginally, a marked increase was noticed between 8 and 24 hours. 7 The exact cellular source of IL-8 in suction blister controis remains unclear. Possible candidates include keratinocytes, fibroblasts, or endothelial cells. 8 In a recent study, no messenger RNA for IL-8 was detected in the epidermis of suction blisters9; this finding points to nonepidermal cells as sources of IL-8 in the blister fluid of control subjects. In BP blisters, IL-8 levels were 10 times higher than in suction blisters and 200 times higher than serum levels of control subjects. Levels in BP blisters corresponded to those required to exert a chemotactic effect on T lymphocytes, neutrophils, and eosinophils.5, 6 In contrast to most other cytokines, a long duration of action has been reported for IL-8 because of its resistance to degrading proteases. 1~This property may contribute to the high levels in BP blister fluid. We also cannot state the exact cellular source of IL-8 in BP blister fluid. In addition to resident cells such as keratinocytes, fibroblasts, endothelial cells, and mast cells, infiltrating cells including monocytes/macrophages, T cells, and neutrophils are possible sources. 8'1~ H All these cells are typically found in BP lesions and,appear to be in an altered state)' 4,12-14 Stimulating agents that augment IL-8 expression, including IL- 1, IL-2, IL-6, tumor necrosis factor-a, and interferon gamma, 8' 15 have been detected in the blister fluid of BP. 16-2~Recently, we found elevated levels of 1]-,-113 in BP blisters. 16 In blisters of eight patients, assayed for both IL-1 13 and IL-8, we observed a strong correlation between levels of these cytokines, whereas in blister fluid of 10 control subjects, these did not correlate. Mediators down-regulating IL-8 activity include IL-4,
1.000
m
c
100
10
B
S
Bullous Pemphigoid
B S , Healthy volunteers
Fig. 1. IL-8 levels in blister fluid (B) and serum samples (S) of patients with buUous pemphigoid (n= 10) and healthy suction blister controls (n = 10). Bars indicate mean ___SD.
IL-10, and IL-1 receptor antagonist. 8,21 These have also been described in blister fluid of BP 16' 18 and possibly limit the IL-8 effects in lesions of this disease. The most striking property of IL-8 appears to be its chemotactic effect on both T cells and neutrophils. Subcutaneous or intradermal injections of IL-8 eventually lead to infiltration with these cells. Low doses favor an infiltration of T cells and high doses, an influx of neutrophils. 8 In addition, IL-8 is a potent mediator for eosinophil chemotaxis.6 The chemoattractants C5a and LTB4 have previously been demonstrated in lesions and blister fluid of Bp,22, 23 and IL-8 may also contribute to the influx of inflammatory cells in BP lesions. IL-8 enhances expression of complement receptor type I on neutrophils as well as their activation and degranulation. 8 Peripheral blood leukocytes attach to the basement membrane zone of skin sections incubated with BP serum. 2 Therefore in BP lesions/L-8 may increase the binding of neutrophils to the basement membrane zone and their release of proteolytic en-
312
Brief communications
z y m e s such as m y e l o p e r o x i d a s e , w h i c h has b e e n detected in blister fluid. 24 REFERENCES
1. lshiko A, Shimizu H, Kikuchi A, et al. Human autoantibodies against the 230-KD builous pemphigoid antigen (BPAG1) bind only to the intracellular domain of the hemidesmosome, whereas those against the 180-KD bullous pemphigoid antigen (BPAG2) bind along the plasma membrane of the hemidesmosome in normal human and swine skin. J Clin Invest 1993;91:1608-15. 2. Gammon WR, Merrit CC, Lewis DM, et al. An in vitro model of immune complex-mediated basement membrane zone separation caused by pemphigoid antibodies, leukocytes, and complement. J Invest Dermato11982;78:285-90. 3. Wintroub BU, Mihm MC, Goetzl EJ, et al. Morphologic and functional evidence for release of mast-cell products in bullous pemphigoid. N Engl J Med 1978;298:417-21. 4. Dvorak AM, Mihm MC Jr, Osage FE, et al. Bullous pemphigoid, an ultmstmctuml study of the inflammatory response: eosinophil, basophil and mast cell granule changes in multiple biopsies from one patient. J Invest Dermatol 1982:78:91-101. 5. Larson CG, Anderson AO, Apella E, et al. The neulrophilactivating protein (NAP-I) is also cheraotactic for T lymphocytes. Science 1989;243:1464-6. 6. Eager RA, Casale TB. Interleukin-8 is a potent mediator of eosinophil chemotaxis through endothelium and epithelium. Am J Physiol 1995;268:L17-L22. 7. Kuhns DB, DeCarlo E, Hawk DM, et al. Dynamics of the cellular and humoral components of the inflammatory response elicted in skin blisters in humans. J Clin Invest 1992;89:1734-40. 8. Zachariae COC. Chemotactic cytokines and inflammation: biological properties of the lymphocyte and monocyte chemotactic factors ELCF, MCAF and IL-8. Acta Derm Venereol (Stockh) 1993;181:1-37. 9. Kristensen M, Larsen CG, Jorgensen P, et al. RNA purification from epidermal suction blisters. Acta Derm Venereol (Stockh) 1991;71:423-6. 10. Peveri P, Walz A, Dewald B, et al. A novel neutrophil-activating factor produced by human mononuclear phago.cytes. J Exp Med 1988;167:1547-59. 11. MSiler A, Lippert U, Lessmann D, et al. Human mast cells produce IL-8. J Immunol 1993;151:3261-6.
Journal of the American Academy of Dermatology February 1996
12. Kamshima T, Hachisuka H, Okubo K, et al. Epidermal keratinocytes of bullous pemphigoid express intercellular adhesion molecule-1 (ICAM-1). J Dermatol 1992;19:82-6. 13. zinikens D, Ambach A, Schuessler M, et al. The interleukin-2 receptor in lesions and semm of buUous pemphigoid. Arch Dermatol Res 1992;284:141-5. 14. Ambach A, Ziilikens D, Klingert B, et al. Immunophenotyping of the mononuclear infiltrate in bullous pemphigoid. Hautarzt 1992;43:81-5. 15. Larsen CG, Anderson AP, Oppenheim JJ, et al. Production of IL-8 by human dermal fibroblasts and keratinocytes in response to IL-1 or TNF. Immunology 1989;68:31-6. 16. Schmidt E, Mittnacht A, SchOmig H, et al. Detection of IL-lct, IL-1[3 and IL-1 receptor antagonist in blister fluid of bullous pemphigoid. J Dermatol Sci (In press.) 17. Grando SA, Glukhenky BT, Dmnnik GN, et al. Mediators of inflammation in blister fluids from patients with pemphigus vulgaris and builous pemphigoid. Arch Dermatol 1989; 125:925-30. 18. Zillikens D, Schmidt E, Depprich R, et al. Contribution of cytokines to blister formation in bullous pemphigoid [Abstract]. Arch Dermatol Res 1994;286:200. 19. Zillikens D, Schuessler M, Dummer R, et al. Tumor necrosis factor in blister fluids of bullous pemphigoid. Eur J Dermatol 1992;2:429-31. 20. Kaneko F, Minagawa T, Taldguchi Y, et al. Role of cellmediated immune reaction in blister formation of bullous pemphigoid. Dermatology 1992;184:34-9. 21. DeForge LE, Tracey DE, Kenney JS, et al. Interleukin-1 receptor antagonist protein inhibits interleukin-8 expression in lipopolysaccharide-stimulated human whole blood. Am J Pathol 1992;140:1045-54. 22. Diaz-Perez JL, Jordon RE. The complement system in bullous pemphigoid. IV. Chemotactic activity in blister fluid. Clin Immunol Immunopathol 1976;5:360-70. 23. Wana S, Ueno A, Nishiyama S. Increased levels of immunoreactive leukotriene B4 in blister fluids of bullous pemphigoid patients ahd effects of a selective 5-1ipoxygenase inhibitor on experimental skin lesions. Acta Derm Venereol (Stockh) 1990;70:281-5. 24. Czech W, Schaller J, Sch6pf E, et al. Granulocyte activation in bullous diseases: release of granular proteins in bullous pemphigoid and pemphigus vulgaris. J AM ACAD DmUVIATOL1993;29:210-5.