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047 Connective tissue growth factor gene expression in tissue sections from eosinofhilic fasciitis
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JSID
043 TNF-a INCREASES TYPE I AND III COLLAGEN GENE EXPRBSSIONS IN SELF-PRODUCED THREE-DIMENSIONAL PIBROBLASTS CULTURES. M. Department of Dermatology, Gunms University School of Medicine., Msebsshi. Gunma, Japan We have recently established s novel tibroblsst culture. system supplemented with L-ascorbic acid Z-phosphate. The addition of L-ascorbic acid Z-phosphate could render human dermsl tibroblasts to organize s dem-dslike structure by accumulating collagens and extrscellulsr matrices. The purpose of this study is to examine the effects of TNF-a on collagen gene expressions by human dennat fibmblssts in this culture system in comparison with those in monolayer culture. TNFa suppressed the expressions of proal(1) and proal(III) collagen mRNA in monolayer culture. In contrast, these expressions were elevated in three-dimensional culture system. TNP-a increased the mRNA expressions of collsgenase and TIMP-1 in both culture systems. These data suggest that responses of human dermsl fibroblssts to TNF-a is distinct under the different culture conditions. Since our threedimensional culture system is quite. similar to the dermis both morphologically and biochemically, TN&o. possibly regulates both collagen production and destruction in viva, if this three-dimensional culture system really reflects the human dermal fibmblssts’ nature in human dermis.
Abstracts
046 TIMP-I MAY BE AN AUTOCRINE GROWTH FACTOR, IN SCLERODERMA FIBROBLASTS. K. Kikuchi :‘, T. Kadono ‘A -‘, K.Tsmski f ’ Deof Dermatdogy. Tokyo University Brsnch Hospital, Tokyo, Japan. * Depsrtment of Dermatology. Faculty of Medicine, University of Tokyo. Tokyo, Japan. We recently reported that the semm tissue inhibitors of metalloproteinar1 aIMP-1) levels in systemic sclerosis @SC) patients were elevated compared with normsl controls. To dactine the biological significance of TIMP-1 in SSc. we compared the pmliiemtive effects of TIMP-I between normal and SSc tibroblasts. TIMP-I showed significant mitogenic activity for both nomud and SSc fibroblasts. The mitogcnic responses to TIMP-1 (33-100 nghl) in SSc fibroblasts wcn significantly greater thsn rhose in normal controls, and were completely muhalized in the presence of antiTIMP-1 IgG. Anti-TIMP-I 1%; pattially but significantly blocked tlx basal mitogenic activities of SSc fibroblrsts. Sk fib&lasts produced incrrascd amounts of TIMP-I relative to normal tibroblasts, as confirmed by Western blotting, BLISA and KT-PCR. Transforming growth factor- Bl QGF-PI) up-regulated TIMP-I production in normal tibroblssts but not in SSc fibroblssts with elevated spontaneous secretion of TIMP-1.
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047
THE EFFECTS OF INTERLEUKIN-18 AND INTERLEUKIN-10 ON THE ELASTIN GENE EXPRESSION. 5% Km*. SD. Konn. m KS. Lee I.Y. SQM,Depmtment of Dermatology, Keimyung University School of Medicine. Tssgu, Korea. The elastic fibers sre s important fibrillsr comprment of the extracellulsr matrix of skin pmvidiig elasticity rind resilience ta the skin. In photos& skin, xcumulotion of abnormal elastic fibers is a chnrscteristic histologic finding, Recent m&culsr biological studies have revealed that ultrsviolet(UV) irradiation nctivoted elastin gene expression in viva and in vitro. Following UV irrsdiotion, epidsrmal keratinocytes produce mnny cytokines such as interleukin(IL)-1, tumor necrosis factor- LI and IL-lo. So, UV inducible cytokines elaborated by kerstinocytss snd inflomn~otory cells may play s role in psthogenesis of dermal chnnge in photoaging. In this study, we hnve exsminsd the effects of IL-1 nnd IL-10 on elastin gene expression in human skin fibmblnsts in culture by Northern hybridization, trims&t transfection and indirect immunofluolescence expsriments. The results indicate up-regulation of elnstin gene sxprsssion st the transcriptionsI level, IL-l and IL-IO sloborated by msny cells such 8s kerstinacytes, inflammatory cells following UV imdiation play a role, at least in prt, in psthogenesis of photoaging.
CONNECTIVE TISSUE GROWTH FACTOR GEliE EXPRESSION IN TISSUE SECTIONS FRY: EOSINOPHILIC =,I”. -*I, N. Hay& c K. I&&‘& A Department of DermatoIigy, Fsculty dMsdi&ne, University oflbkyo, Tokyo, Japan. 2Divisicm of Dsrmatow, Kant0 %&in Hospital, lbkyo, Japan. Connective tissue growth fsctor(CTGF) wss expressed in tissue regsnemtion and f&m&z diseases. We investigated mF gsne expression in tissue sections from 6 patients with sceinopbilic fssciitis using nonrsdiosctive in situ hybridization. Digosigsnin-I&&d CmF mRNA pmbss were used. CTGF mRNA signals were observed in fascia d 2 patients who had remarkable inflammatory cell infiitrstion, and in deep dermis of 3 patients who had mild intlammstory ceII infiltration. but the rest that demonstrated no inflammation had no signals. And 4 of 5 patients which expressed CIGF mRNA revs&d high value of ssrum sldnlsse. In 2 essss d remarkable inflammatory cell infikration, C’IGF mRNA was expressed in fascia, and the value of ssrum aldolase wss relative to CTGF gene expression.
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THE EFFEm OF ELECTRICAL ACUPUNCTURE STIMULATION USING THERMOGRAPHY AND PLASMA ENDOTI-IEUN(ET-l), SEROTONIN AND NO LEVELS IN PATIENTS WITH SYSTEMIC SCLERODERMA(PSS).M. Maeda H. Kachi, N. Ichihashi. Y Kitaiima.Department of Dermatology, Gifu University, School of Medicine. Gifu, Japan. Ten patients with PSS(Bamett type I;2, 11;5, III;3 cases), 6 cases of SSSD(atypical or incomplete type of PSS accompanied with possible Sjagren syndrome) and 6 healthy controIs(HC) were entried to be evaluated the mechanism of various effects of acupuncture using thermogtaphic method and blood examination of plasma E?T-1, serotonin, nitrite and nitrate levels. Plasma ET-1 levelsof PSS, SSSD and HC were 1.9&0.69. 1.76i0.39 and l.lSiO.38 pg/ml, respectively. After the electrical acupuncture stimulation for unilateral side of hand/arm(30min.). the plasma ET-1 levels decreased in all 10 cases of PSS treated( 1.6lti.45 pg/ml), but no change of plasma serotonin levels m spite of increase of plasma uitr&s levels in 2 cases of PSS. In 4 of 6 csses of SSSD and all 6 HC, plasma ET- 1 levels incressed(2.Ch%0.39 pg/mI, 1.7%0.58 pg/ml, respectively). The decrease of plasma ET-1 levels for the stimulation was thought to induce the vasodilatatton and elevate the slan surface temperature in the patients with PSS.
AUTOANTIBODIES FROM PATIENTS WITH CICATRICIAL PEMPHIGOID RECOGNIZE LAUININ a3 CHAIN MONOMER. L T. I(itapswa. M. OhM. Morohashi. Department of Dermatology, Toyama Medical and Pharmaceutical University, Toyama, JAPAN Recent studies have identified s group of cicntricial pemphigoid patients who have IgG anli-basement mcmbrnne autoantibodies that recognize epiligrin, s hcterotrimcric protein closely related or identical to laminin 5. To further understand the pathophysiology of blister formation in these patients, we have sought to identify the specific polypcptide(s) snd epitope(s) targeted by their suto antibodies. Comparative studies show thst sera from these patients and polyclonal antibody immunoprecipitatc the same set of disulfide-linked Dolvveotides from media and cell fraction of biosynthetically ‘rsd>bl;bcled human kcrntinocytcs. Moreover, these scrs immunoprecipitstc a3 subunit monomer of laminin 5 from kcratinocytc cell fraction but show no renctivity to $3 and y2 monomers. The ssme sers precipitate ssme polypeptidcs from mucous membrane-derived kerstinocytes. The epitope mapping using bacterial fusion protein of a3 subunit is now in progress.
JSID
043 TNF-a INCREASES TYPE I AND III COLLAGEN GENE EXPRBSSIONS IN SELF-PRODUCED THREE-DIMENSIONAL PIBROBLASTS CULTURES. M. Department of Dermatology, Gunms University School of Medicine., Msebsshi. Gunma, Japan We have recently established s novel tibroblsst culture. system supplemented with L-ascorbic acid Z-phosphate. The addition of L-ascorbic acid Z-phosphate could render human dermsl tibroblasts to organize s dem-dslike structure by accumulating collagens and extrscellulsr matrices. The purpose of this study is to examine the effects of TNF-a on collagen gene expressions by human dennat fibmblssts in this culture system in comparison with those in monolayer culture. TNFa suppressed the expressions of proal(1) and proal(III) collagen mRNA in monolayer culture. In contrast, these expressions were elevated in three-dimensional culture system. TNP-a increased the mRNA expressions of collsgenase and TIMP-1 in both culture systems. These data suggest that responses of human dermsl fibroblssts to TNF-a is distinct under the different culture conditions. Since our threedimensional culture system is quite. similar to the dermis both morphologically and biochemically, TN&o. possibly regulates both collagen production and destruction in viva, if this three-dimensional culture system really reflects the human dermal fibmblssts’ nature in human dermis.
Abstracts
046 TIMP-I MAY BE AN AUTOCRINE GROWTH FACTOR, IN SCLERODERMA FIBROBLASTS. K. Kikuchi :‘, T. Kadono ‘A -‘, K.Tsmski f ’ Deof Dermatdogy. Tokyo University Brsnch Hospital, Tokyo, Japan. * Depsrtment of Dermatology. Faculty of Medicine, University of Tokyo. Tokyo, Japan. We recently reported that the semm tissue inhibitors of metalloproteinar1 aIMP-1) levels in systemic sclerosis @SC) patients were elevated compared with normsl controls. To dactine the biological significance of TIMP-1 in SSc. we compared the pmliiemtive effects of TIMP-I between normal and SSc tibroblasts. TIMP-I showed significant mitogenic activity for both nomud and SSc fibroblasts. The mitogcnic responses to TIMP-1 (33-100 nghl) in SSc fibroblasts wcn significantly greater thsn rhose in normal controls, and were completely muhalized in the presence of antiTIMP-1 IgG. Anti-TIMP-I 1%; pattially but significantly blocked tlx basal mitogenic activities of SSc fibroblrsts. Sk fib&lasts produced incrrascd amounts of TIMP-I relative to normal tibroblasts, as confirmed by Western blotting, BLISA and KT-PCR. Transforming growth factor- Bl QGF-PI) up-regulated TIMP-I production in normal tibroblssts but not in SSc fibroblssts with elevated spontaneous secretion of TIMP-1.
044
047
THE EFFECTS OF INTERLEUKIN-18 AND INTERLEUKIN-10 ON THE ELASTIN GENE EXPRESSION. 5% Km*. SD. Konn. m KS. Lee I.Y. SQM,Depmtment of Dermatology, Keimyung University School of Medicine. Tssgu, Korea. The elastic fibers sre s important fibrillsr comprment of the extracellulsr matrix of skin pmvidiig elasticity rind resilience ta the skin. In photos& skin, xcumulotion of abnormal elastic fibers is a chnrscteristic histologic finding, Recent m&culsr biological studies have revealed that ultrsviolet(UV) irradiation nctivoted elastin gene expression in viva and in vitro. Following UV irrsdiotion, epidsrmal keratinocytes produce mnny cytokines such as interleukin(IL)-1, tumor necrosis factor- LI and IL-lo. So, UV inducible cytokines elaborated by kerstinocytss snd inflomn~otory cells may play s role in psthogenesis of dermal chnnge in photoaging. In this study, we hnve exsminsd the effects of IL-1 nnd IL-10 on elastin gene expression in human skin fibmblnsts in culture by Northern hybridization, trims&t transfection and indirect immunofluolescence expsriments. The results indicate up-regulation of elnstin gene sxprsssion st the transcriptionsI level, IL-l and IL-IO sloborated by msny cells such 8s kerstinacytes, inflammatory cells following UV imdiation play a role, at least in prt, in psthogenesis of photoaging.
CONNECTIVE TISSUE GROWTH FACTOR GEliE EXPRESSION IN TISSUE SECTIONS FRY: EOSINOPHILIC =,I”. -*I, N. Hay& c K. I&&‘& A Department of DermatoIigy, Fsculty dMsdi&ne, University oflbkyo, Tokyo, Japan. 2Divisicm of Dsrmatow, Kant0 %&in Hospital, lbkyo, Japan. Connective tissue growth fsctor(CTGF) wss expressed in tissue regsnemtion and f&m&z diseases. We investigated mF gsne expression in tissue sections from 6 patients with sceinopbilic fssciitis using nonrsdiosctive in situ hybridization. Digosigsnin-I&&d CmF mRNA pmbss were used. CTGF mRNA signals were observed in fascia d 2 patients who had remarkable inflammatory cell infiitrstion, and in deep dermis of 3 patients who had mild intlammstory ceII infiltration. but the rest that demonstrated no inflammation had no signals. And 4 of 5 patients which expressed CIGF mRNA revs&d high value of ssrum sldnlsse. In 2 essss d remarkable inflammatory cell infikration, C’IGF mRNA was expressed in fascia, and the value of ssrum aldolase wss relative to CTGF gene expression.
045
048
THE EFFEm OF ELECTRICAL ACUPUNCTURE STIMULATION USING THERMOGRAPHY AND PLASMA ENDOTI-IEUN(ET-l), SEROTONIN AND NO LEVELS IN PATIENTS WITH SYSTEMIC SCLERODERMA(PSS).M. Maeda H. Kachi, N. Ichihashi. Y Kitaiima.Department of Dermatology, Gifu University, School of Medicine. Gifu, Japan. Ten patients with PSS(Bamett type I;2, 11;5, III;3 cases), 6 cases of SSSD(atypical or incomplete type of PSS accompanied with possible Sjagren syndrome) and 6 healthy controIs(HC) were entried to be evaluated the mechanism of various effects of acupuncture using thermogtaphic method and blood examination of plasma E?T-1, serotonin, nitrite and nitrate levels. Plasma ET-1 levelsof PSS, SSSD and HC were 1.9&0.69. 1.76i0.39 and l.lSiO.38 pg/ml, respectively. After the electrical acupuncture stimulation for unilateral side of hand/arm(30min.). the plasma ET-1 levels decreased in all 10 cases of PSS treated( 1.6lti.45 pg/ml), but no change of plasma serotonin levels m spite of increase of plasma uitr&s levels in 2 cases of PSS. In 4 of 6 csses of SSSD and all 6 HC, plasma ET- 1 levels incressed(2.Ch%0.39 pg/mI, 1.7%0.58 pg/ml, respectively). The decrease of plasma ET-1 levels for the stimulation was thought to induce the vasodilatatton and elevate the slan surface temperature in the patients with PSS.
AUTOANTIBODIES FROM PATIENTS WITH CICATRICIAL PEMPHIGOID RECOGNIZE LAUININ a3 CHAIN MONOMER. L T. I(itapswa. M. OhM. Morohashi. Department of Dermatology, Toyama Medical and Pharmaceutical University, Toyama, JAPAN Recent studies have identified s group of cicntricial pemphigoid patients who have IgG anli-basement mcmbrnne autoantibodies that recognize epiligrin, s hcterotrimcric protein closely related or identical to laminin 5. To further understand the pathophysiology of blister formation in these patients, we have sought to identify the specific polypcptide(s) snd epitope(s) targeted by their suto antibodies. Comparative studies show thst sera from these patients and polyclonal antibody immunoprecipitatc the same set of disulfide-linked Dolvveotides from media and cell fraction of biosynthetically ‘rsd>bl;bcled human kcrntinocytcs. Moreover, these scrs immunoprecipitstc a3 subunit monomer of laminin 5 from kcratinocytc cell fraction but show no renctivity to $3 and y2 monomers. The ssme sers precipitate ssme polypeptidcs from mucous membrane-derived kerstinocytes. The epitope mapping using bacterial fusion protein of a3 subunit is now in progress.