Tumor necrosis factor-α gene expression is induced by cyclobutyl pyrimidine dimers in DNA
1
ESDR I JSID I SID Abstracts s177 1057 TUMOR NECROSIS FACTOR-a GENE EXPRESSION IS INDUCED BY CYCLOBUTYL PYRIMIDINE DIMERS IN DNA. Daniel Yarosh. Jonathan Klein, Adrienne O’Connor, Jeannie Kibitel. Doualas Brash, Vidva Heimadi and Betsy Sutherland, Applied Genetics Inc., Freeport, NY, Yale University, New Haven, CT and Biology Dept., Brookhaven National Laboratory, Upton, NY. Controversy rages over whether the signals for cytokine releasa following UV originate at the cell membrane or in DNA. We have looked closely at UV induction of TNFa using the promoter-CAT reporter transgene (transcription) and quantitative immunohistochemisby (translation). Induction of the reporter gene following UV was shifted to lower fluences in DNA repair-deficient xeroderma pigmentosum cells compared to repair-proficient cells, indicating that unrepaired lesions triggered TNFa transcription. The induction kinetics after UV-C were also shifted to lower fluences compared to UV-B, but the kinetics were identical when compared by cyclobutyl pyrimidine dimers produced in DNA by each UV source. The efficiency of UV-induced lipid (membrane) peroxidation by UV-A, B and C, however, did not correlate as well with TNFa expression. Cells treated with T4N5 liposomes (containing the prokaryotic DNA repair enzyme T4 endonuclease V specific for dimers) showed increased dimer- specific repair and proportionately reduced TNFa gene expression and less anti- TNFa antibody staining. TNFa induction kinetics in UV-fos knockout mouse skin was normal. UV induction of TNFa gene expression is independent of AP- I sites and is caused at least in part by cyclobutyl pyrimidine dimers in DNA. 1058 CONTACT “WERSENSITMTY aEAcrION TO OXAZOLONE IS GwEPENDENT WHEREAS CONI‘ACT HYPER?XNSITIVITY REACTION TO DNCB IS DEPENDENT ON IL-4 Claudm Trauil’, Frank lugert’, Hans Merk’, N~colas Hunze$nann# Dept of Dennat&gy, Lhuvenlty of AadEn’, Dept. of Dermatolugy, Cologne, Germany Oxawlme and DNCB are two of the most o&m used chemicals tn induce ewerunmtal ccataa hwersmstivbv reactinns Oxazokme is known as a less ~&eat alkrw thau DNCB and seems tn u&e both a i-call and umnunoglobulm reqxmse whereas DNCB mly induces a T-&l qrnse CadWing resuh QI the role of R-4 u1 CHS readicms elicited by these c4wnucals have been obtained suppoh eaher a pormflammatury or rather fibitory mle of thlh cytokiue. To evaluate the role of hterleukin-t (II-t) m CH8 we inwsti@ed the reactiw~ to locally applicated Oxazolare or DNCB. II-l deficiat and BALB/c ccmtrol mice were sens,t~red v&h I% Oxalolone or 5% DNCB 00 the unshaved aLxlcmen. Fwe days later nuce were chalkaged with the same allergen on the right ear and ear swelling was measured the follwmg 5 days with a micrometer Cnncemmg the CHS to DNCB, the knockwt mice showed a 40-60 % reductim of ear swellmp~ 24 hours after challenge @<O,Ol) and a reduced duration of CHS @G,OOl) compared to wild&e mice lhrs attmuat~cm was accompanied by a marked reduction of cellular mtikrates m the demus In order to exclude mflumces ufthe gm&c backgrnund ca the diminished CHS response, ezwrimam were perfbnned usinr beterozvxoues II-4 (+I-) BALB/c mice Here no ditTermce in the CHS re@un.&tu DNCB wh&cumparedto wddtype &e could be observed. In camast, the magnitude and duratica of CHS to Oxazolone was similar bath iu the R-4 deficient mice and m the Balb/c control mice indicating that the CHS to oxazolone Is iudepend& of n-4 our resuits therefore m&ate that the hypersmsiti-nty response to DNCB and nxazolcme may partly w&e a different pathway being dependent and in-dent nfll4. 1059 EXPRESSION OF THE CD4* CELL-SPECIFIC CHEMOA,TRACTANT IL-16 IN CUTANEOUS T CELL LYMPHOMA. Volker Blaschke. Kdstian Reich. Susanne Hawix. Neu Michael Letachert. Thomas Juna. andQ!&ne mann. Department of Dermatology. Georg-August-Univeniiy, Gattkqen. Gernmny. IL-16 is 8 soluble llgand to the CD4 molecule with chemotactk properties fw CD4’ T cells. monocytes and eosinophils. IL-16 has also been shown to ad as a mmpetence gmw(h fador for CD4’ T cells, capable of inducing a GO to Gl cell-cycle change together with an upegula- tion of CD25 expression. The leslonal infaltrate in cutaneous T cell lymphoma (CTCL) is typi- cally CD4’, with a small mlnodty of malignant cells and a majority of inflammatory bystanders. We here tested the posslWty that IL-16 is involved in the crosstalk Lwbwen both populations. The expression of IL-16 pmteln w-as analyred by lmmunohiitcchemistry in normal skin (n=5). and in lesional and dinlcalfy uninvolved skh of patients with S.&q Syndrome w CTCL (n=lZ). The pesenca of IL-16 mRNA in the epldents and denis of clinically distinct stages of CTCL was inves8gated by RT-PCR. and mRNA expfessian was localized by in stiu hybridi- sation (ISH) In selected cases. To further identify the cellular sources of IL-16 prcdudion of IL-16 was measured by RT-PCR and ELISA in Thl- and ThP-like cell clones isolated from CTCL lesions. In normal skin and uninvolved skin of CTCL patients. a wak staining for IL-16 was detected in lymphocytes and masf cells around dermal bkmd vessels. but not In ep&r. mal cells. Correspondingly. IL-16 mRNA was found in the dermal ix4 not the epidermal layer of normal skin biopsies saperated by dispase digestion. In CTCL. IL-16 wds stmqly ex- pressed by the vast majority of ln8llratlng dermal and epidemml lymphccyles. Dermal and epi- dermal IL-16 mRNA levels increased with disease pmgresoion. ISH conRrmed mat lymph- ocytes were the predominant source of IL-16 in CTCL lesions. Furthwmore. IL-16 pmdudion wes detected in Thl- and ThZ-like clones from CTCL lesions (as dwadedzed by the sews- tion of IL-4 vs. IFN?). Our results show that IL-16 is produced in large amounts in CTCL lesions. Given the biologic activities of IL-16. this factor might be involved in the recruitment and stimulation of !&onal CM’ lymphocytes and therefore contribute to the perpetuation of cutaneous Inflammation. In addillon. IL-16 could prime lesional lymphocytes to proliferate in response to other locally synthesized cytokines, e.g. 11-7. 1060 DlFFERENTlALEXPRESSIONOF CHEMOKlNESIS CORRELATEDWlTH PHASESPEClFfC INFILTRATION OF LEUKCCYTESUBSETS, KERATlNOCYTE MlGRATlOfVPROLlFERATlON AND NEOANGIOGENESIS DURING HUMAN WOUND HEALING. Eva Enoathardt Attve oksov. Matlhtt Goabalar. Eva-Batkna Blocker. and R&hard GitJ&&Dapartman:of DarmaWogy, Unherstty of woraburg, Wirnburg, Germany. Healingof c&anaous wounds requires a complex intagratad natuork of repair mechanismswhii indudas tha action of nawiy raauited laukooytas. Using a skin repair modai in adult humans, wa investigatedtha role of &fggta& cyWf&&$ (chemddnas) for saquanttal inNbattonof laukocyta subsets during wound hasting, emdovtno in sifu hvbrWa&n and concomitant imrnunohtstokxttt stainlnas of serial &itons. At day 1 aftar injury. the C-X-C &am&nas IL-Sand GR&ara maximallyaxpmssad in the sufmrt%tal wound bed, and ara spattallyand tamporaiiy assocMtadwith m&ophtl frnikratkm. IL-3 and GROa profiles also conatata with w mtgmttcn and subside after wound dosure at day 4. Macrophagaintlux reaches htst levels at dav 2 and is mratlalad bv MCP-1 mRNA axoresston both in tha bass? layer of the pcoliierative a&annts at ttis wound margtnsas watl as by mononuckar cetts in tha wwrtd area. Other mMwcyteatbadhg chemoktnessuch as MCM, MIP-la@. RANTES, and I309 am not d&ctabN at r&avant lavals. At C-i-C chari&nes~Mtt and IP-10. Moreover, tha tfma a&se of angtogenic IL-3 XjROa and angkxtattc MigrlP-10appaaranca is In accordancewfth Initial outgmwth of vassals which subsidasaftar day 4. Thus, a dynamkcatty changing sat of chemo- kines intagratas tnflammatoryand raparattvepmmssas during sktn wound rapalr. 1061 INTRACELLULAR TNF-a , IFN-y, AND IL-2 IDENTIFY ‘ICI AND THl BPFECTOR POPULATIONS IN PSORIASIS VULGARIS PLAQUl? LYMPHOCYT!JS: SINGLE CELL ANALYSIS BY FLOW CYTOMETRY. Lisa Austin, Maki Ozawa, Toy&o Kikuchi and James Krueger, Lab for Investigative Dernmtolcgy, Rockefeller Univ. NY, NY 10021. Type I cytokines (collectively including IL-2, IFN-y, and TN&x) have been found in most psmiasis wlgaris lesions, by a variety of methods. However, the ability of individual lymphocytes of different subsets to pmduce inflammatory cytnkiues in diseased skin is not known. In this study CD8+CD3+ and CD4+CD3+ lvmobocvtes in psoriatic lesions and peripheral b&d frbm bath patients and normal voiuuiecrs~were analyzed for their overall ability to pmduce Type I and Type 2 cytokines. By a highly sensitive flow cytometric technique using four-color analysis. the ability of these tens to express intracellular cytokines was assessed, with and without, direct lymphocyte activation with ionomycin and PMA (in the presence of Brefeldin A). This analysis showed that in addition to identifying a majority of Type 1 CD3+ lymphocytes in the lesions, both subsets, CDB+CD3+ and CD4+CD3+, produced primarily TN&, IFN-g, and IL-2 with minimal IL4 and variable U-10. The CD8+ population contributed the majority of Type 1 (TCI) cytokine producing cells as compared to the CD4+ population (THI) where TCl:THl percentages were TNF-a 78:69X, IF%750:30%. and IL-2 50:35% (u=4). At the individual cell level. IFN-Y pmduction and GMPl7II1A-I, the CTL actwxtion marker, were both highly associated with TNF-a production in the activated ksional lymphocytes. Many of the TNF-a producing cells were IF?-7 negative while IL-2 wa s expressed in a subpopulation of TNF-a and IPN-7 pmducing cells. These results show that cytokine expression within the TCI and ‘IHl designations is heterogeneous at the site of inflammation. Treahnent of psoriatic plaques with narrow band (312 mu) WB emphasized this conclusioq. as the surviving lymphocytes produced a different cytukme pattern. We conclude that m chronically active psoriasis vulgaris lesions, the primary subset of epiderrnal lymphocytes are TCI and THI both producing and the primary source of TNF-a, IFt-y, and U-2. 1062 INDUCTION OF NITRIC OXIDE SYNTHASE IN ENDOTHELIAL CELLS: DIFFERENT MOLECULAR MECHANISMS FOR UVA AND UVB IRRADIATION. Damela Bruch-Ger- harz”, Chtistoph Suschek’. Verena Krischel’,‘. Thomas Ruzicka’, and Victoria K&-Bach- ofen’. Research Group Immunobiology’, Biomedical Research Center, and Depallment of Dermatology’, Heinrich-Heine-University of Duesseldorf, Duesseldorf. Germany. Nitnc oxide (NO), a molecular mediator with vasadilative and immunoregulatory proper- ties, is a candidate molecule that may play a key mle in sunburn eryihema and inflamma- tion. The mechanism and cellular source of NO production in these processes, however, IS not yet known. In this study, we demonstrate for the first time the expression of inducible NO synthase (iNOS) mRNA and pmtein in endothelia of human skin organ cultures after UVA (50 J/cm? or UVB exposure (200 mJ/ cm?. To elucidate the mechanism of iNOS in- duction in response to UV irradiation. cuiiured rat aortic endothelial cells lAECI were trea- ted with UVA or UVB either alone or in the presence of IL-10, a potent inducer of iNOS ex- presson. UVA (6 J/cm? or UVB irradiation (50 mJ/ cm’) of resting AEC resulted in iNOS mRNA expression but no induced NO production. The preincubatian of AEC with IL-10 (200 U/ml) for 24 h prior to UVA irradiation did not increase iNOS mRNA exnression or NO urn- duction. However. the incubation of AEC with IL-lp Immediately afler’UVA irradiation’ led to an additive enhancement of iNOS mRNA expression and NO production when compa- red to non-irradiated cells. In contrast. preinwbation of AEC with IL-lp for 24 h prior to UVB irradiation led to an addihve enhancement of iNOS mRNA expression and NO pro- duction, but no increase could be observed when cells were mcubated with IL-1 R imme- diately after UVB irradiation. Strikingly, incubation of AEC wim IL-ID 24 h pnor and III- mediately after UV irradiation led to a synergistic inuease in iNOS mRNA ezaression and NO production only in UVB irradiated &Is,-but not in UVA-irradiated cells. \;ve conclude that me expression of iNOS IS a key component of the response of endothelial cells to uv radiation. UVA appears to enhance the iNOS-inducing activity of 11-18, whereas UVB may induce cellular mediators that synergue wth IL-ID in inducing iNOS expression.
Transcript of Tumor necrosis factor-α gene expression is induced by cyclobutyl pyrimidine dimers in DNA
ESDR I JSID I SID Abstracts s177
1057 TUMOR NECROSIS FACTOR-a GENE EXPRESSION IS INDUCED BY CYCLOBUTYL PYRIMIDINE DIMERS IN DNA. Daniel Yarosh. Jonathan Klein, Adrienne O’Connor, Jeannie Kibitel. Doualas Brash, Vidva Heimadi and Betsy Sutherland, Applied Genetics Inc., Freeport, NY, Yale University, New Haven, CT and Biology Dept., Brookhaven National Laboratory, Upton, NY.
Controversy rages over whether the signals for cytokine releasa following UV originate at the cell membrane or in DNA. We have looked closely at UV induction of TNFa using the promoter-CAT reporter transgene (transcription) and quantitative immunohistochemisby (translation). Induction of the reporter gene following UV was shifted to lower fluences in DNA repair-deficient xeroderma pigmentosum cells compared to repair-proficient cells, indicating that unrepaired lesions triggered TNFa transcription. The induction kinetics after UV-C were also shifted to lower fluences compared to UV-B, but the kinetics were identical when compared by cyclobutyl pyrimidine dimers produced in DNA by each UV source. The efficiency of UV-induced lipid (membrane) peroxidation by UV-A, B and C, however, did not correlate as well with TNFa expression. Cells treated with T4N5 liposomes (containing the prokaryotic DNA repair enzyme T4 endonuclease V specific for dimers) showed increased dimer- specific repair and proportionately reduced TNFa gene expression and less anti- TNFa antibody staining. TNFa induction kinetics in UV-fos knockout mouse skin was normal. UV induction of TNFa gene expression is independent of AP- I sites and is caused at least in part by cyclobutyl pyrimidine dimers in DNA.
1058 CONTACT “WERSENSITMTY aEAcrION TO OXAZOLONE IS GwEPENDENT WHEREAS
CONI‘ACT HYPER?XNSITIVITY REACTION TO DNCB IS DEPENDENT ON IL-4
Claudm Trauil’, Frank lugert’, Hans Merk’, N~colas Hunze$nann# Dept of Dennat&gy, Lhuvenlty of AadEn’, Dept. of Dermatolugy, Cologne, Germany
Oxawlme and DNCB are two of the most o&m used chemicals tn induce ewerunmtal ccataa hwersmstivbv reactinns Oxazokme is known as a less ~&eat alkrw thau DNCB and seems tn u&e both a i-call and umnunoglobulm reqxmse whereas DNCB mly induces a T-&l qrnse CadWing resuh QI the role of R-4 u1 CHS readicms elicited by these c4wnucals have been obtained suppoh eaher a pormflammatury or rather fibitory mle of thlh cytokiue. To evaluate the role of hterleukin-t (II-t) m CH8 we inwsti@ed the reactiw~ to locally applicated Oxazolare or DNCB. II-l deficiat and BALB/c ccmtrol mice were sens,t~red v&h I% Oxalolone or 5% DNCB 00 the unshaved aLxlcmen. Fwe days later nuce were chalkaged with the same allergen on the right ear and ear swelling was measured the follwmg 5 days with a micrometer Cnncemmg the CHS to DNCB, the knockwt mice showed a 40-60 % reductim of ear swellmp~ 24 hours after challenge @<O,Ol) and a reduced duration of CHS @G,OOl) compared to wild&e mice lhrs attmuat~cm was accompanied by a marked reduction of cellular mtikrates m the demus In order to exclude mflumces ufthe gm&c backgrnund ca the diminished CHS response, ezwrimam were perfbnned usinr beterozvxoues II-4 (+I-) BALB/c mice Here no ditTermce in the CHS re@un.&tu DNCB wh&cumparedto wddtype &e could be observed. In camast, the magnitude and duratica of CHS to Oxazolone was similar bath iu the R-4 deficient mice and m the Balb/c control mice indicating that the CHS to oxazolone Is iudepend& of n-4 our resuits therefore m&ate that the hypersmsiti-nty response to DNCB and nxazolcme may partly w&e a different pathway being dependent and in-dent nfll4.
1059 EXPRESSION OF THE CD4* CELL-SPECIFIC CHEMOA,TRACTANT IL-16 IN CUTANEOUS T CELL LYMPHOMA. Volker Blaschke. Kdstian Reich. Susanne Hawix.
Neu Michael Letachert. Thomas Juna. andQ!&ne mann. Department of Dermatology. Georg-August-Univeniiy, Gattkqen. Gernmny.
IL-16 is 8 soluble llgand to the CD4 molecule with chemotactk properties fw CD4’ T cells. monocytes and eosinophils. IL-16 has also been shown to ad as a mmpetence gmw(h fador for CD4’ T cells, capable of inducing a GO to Gl cell-cycle change together with an upegula- tion of CD25 expression. The leslonal infaltrate in cutaneous T cell lymphoma (CTCL) is typi- cally CD4’, with a small mlnodty of malignant cells and a majority of inflammatory bystanders. We here tested the posslWty that IL-16 is involved in the crosstalk Lwbwen both populations. The expression of IL-16 pmteln w-as analyred by lmmunohiitcchemistry in normal skin (n=5). and in lesional and dinlcalfy uninvolved skh of patients with S.&q Syndrome w CTCL (n=lZ). The pesenca of IL-16 mRNA in the epldents and denis of clinically distinct stages of CTCL was inves8gated by RT-PCR. and mRNA expfessian was localized by in stiu hybridi- sation (ISH) In selected cases. To further identify the cellular sources of IL-16 prcdudion of IL-16 was measured by RT-PCR and ELISA in Thl- and ThP-like cell clones isolated from CTCL lesions. In normal skin and uninvolved skin of CTCL patients. a wak staining for IL-16 was detected in lymphocytes and masf cells around dermal bkmd vessels. but not In ep&r. mal cells. Correspondingly. IL-16 mRNA was found in the dermal ix4 not the epidermal layer of normal skin biopsies saperated by dispase digestion. In CTCL. IL-16 wds stmqly ex- pressed by the vast majority of ln8llratlng dermal and epidemml lymphccyles. Dermal and epi- dermal IL-16 mRNA levels increased with disease pmgresoion. ISH conRrmed mat lymph- ocytes were the predominant source of IL-16 in CTCL lesions. Furthwmore. IL-16 pmdudion wes detected in Thl- and ThZ-like clones from CTCL lesions (as dwadedzed by the sews- tion of IL-4 vs. IFN?). Our results show that IL-16 is produced in large amounts in CTCL lesions. Given the biologic activities of IL-16. this factor might be involved in the recruitment and stimulation of !&onal CM’ lymphocytes and therefore contribute to the perpetuation of cutaneous Inflammation. In addillon. IL-16 could prime lesional lymphocytes to proliferate in response to other locally synthesized cytokines, e.g. 11-7.
1060 DlFFERENTlAL EXPRESSION OF CHEMOKlNES IS CORRELATED WlTH PHASESPEClFfC INFILTRATION OF LEUKCCYTE SUBSETS, KERATlNOCYTE MlGRATlOfVPROLlFERATlON AND NEOANGIOGENESIS DURING HUMAN WOUND HEALING. Eva Enoathardt Attve oksov. Matlhtt Goabalar. Eva-Batkna Blocker. and R&hard GitJ&&Dapartman:of DarmaWogy, Unherstty of woraburg, Wirnburg, Germany.
Healing of c&anaous wounds requires a complex intagratad natuork of repair mechanisms whii indudas tha action of nawiy raauited laukooytas. Using a skin repair modai in adult humans, wa investigated tha role of &fggta& cyWf&&$ (chemddnas) for saquanttal inNbatton of laukocyta subsets during wound hasting, emdovtno in sifu hvbrWa&n and concomitant imrnunohtstokxttt stainlnas of serial &itons. At day 1 aftar injury. the C-X-C &am&nas IL-Sand GR&ara maximally axpmssad in the sufmrt%tal wound bed, and ara spattally and tamporaiiy assocMtad with m&ophtl frnikratkm. IL-3 and GROa profiles also conatata with w mtgmttcn and subside after wound dosure at day 4. Macrophaga intlux reaches htst levels at dav 2 and is mratlalad bv MCP-1 mRNA axoresston both in tha bass? layer of the pcoliierative a&annts at ttis wound margtnsas watl as by mononuckar cetts in tha wwrtd area. Other mMwcyteatbadhg chemoktnes such as MCM, MIP-la@. RANTES, and I309 am not d&ctabN at r&avant lavals. At
C-i-C chari&nes~Mtt and IP-10. Moreover, tha tfma a&se of angtogenic IL-3 XjROa and angkxtattc MigrlP-10 appaaranca is In accordance wfth Initial outgmwth of vassals which subsidas aftar day 4. Thus, a dynamkcatty changing sat of chemo- kines intagratas tnflammatory and raparattve pmmssas during sktn wound rapalr.
1061 INTRACELLULAR TNF-a , IFN-y, AND IL-2 IDENTIFY ‘ICI AND THl BPFECTOR POPULATIONS IN PSORIASIS VULGARIS PLAQUl? LYMPHOCYT!JS: SINGLE CELL ANALYSIS BY FLOW CYTOMETRY. Lisa Austin, Maki Ozawa, Toy&o Kikuchi and James Krueger, Lab for Investigative Dernmtolcgy, Rockefeller Univ. NY, NY 10021.
Type I cytokines (collectively including IL-2, IFN-y, and TN&x) have been found in most psmiasis wlgaris lesions, by a variety of methods. However, the ability of individual lymphocytes of different subsets to pmduce inflammatory cytnkiues in diseased skin is not known. In this study CD8+CD3+ and CD4+CD3+ lvmobocvtes in psoriatic lesions and peripheral b&d frbm bath patients and normal voiuuiecrs~were analyzed for their overall ability to pmduce Type I and Type 2 cytokines. By a highly sensitive flow cytometric technique using four-color analysis. the ability of these tens to express intracellular cytokines was assessed, with and without, direct lymphocyte activation with ionomycin and PMA (in the presence of Brefeldin A). This analysis showed that in addition to identifying a majority of Type 1 CD3+ lymphocytes in the lesions, both subsets, CDB+CD3+ and CD4+CD3+, produced primarily TN&, IFN-g, and IL-2 with minimal IL4 and variable U-10. The CD8+ population contributed the majority of Type 1 (TCI) cytokine producing cells as compared to the CD4+ population (THI) where TCl:THl percentages were TNF-a 78:69X, IF%750:30%. and IL-2 50:35% (u=4). At the individual cell level. IFN-Y pmduction and GMPl7II1A-I, the CTL actwxtion marker, were both highly associated with TNF-a production in the activated ksional lymphocytes. Many of the TNF-a producing cells were IF?-7 negative while IL-2 wa s expressed in a subpopulation of TNF-a and IPN-7 pmducing cells. These results show that cytokine expression within the TCI and ‘IHl designations is heterogeneous at the site of inflammation. Treahnent of psoriatic plaques with narrow band (312 mu) WB emphasized this conclusioq. as the surviving lymphocytes produced a different cytukme pattern. We conclude that m chronically active psoriasis vulgaris lesions, the primary subset of epiderrnal lymphocytes are TCI and THI both producing and the primary source of TNF-a, IFt-y, and U-2.
1062 INDUCTION OF NITRIC OXIDE SYNTHASE IN ENDOTHELIAL CELLS: DIFFERENT MOLECULAR MECHANISMS FOR UVA AND UVB IRRADIATION. Damela Bruch-Ger- harz”, Chtistoph Suschek’. Verena Krischel’,‘. Thomas Ruzicka’, and Victoria K&-Bach- ofen’. Research Group Immunobiology’, Biomedical Research Center, and Depallment of Dermatology’, Heinrich-Heine-University of Duesseldorf, Duesseldorf. Germany.
Nitnc oxide (NO), a molecular mediator with vasadilative and immunoregulatory proper- ties, is a candidate molecule that may play a key mle in sunburn eryihema and inflamma- tion. The mechanism and cellular source of NO production in these processes, however, IS not yet known. In this study, we demonstrate for the first time the expression of inducible NO synthase (iNOS) mRNA and pmtein in endothelia of human skin organ cultures after UVA (50 J/cm? or UVB exposure (200 mJ/ cm?. To elucidate the mechanism of iNOS in- duction in response to UV irradiation. cuiiured rat aortic endothelial cells lAECI were trea- ted with UVA or UVB either alone or in the presence of IL-10, a potent inducer of iNOS ex- presson. UVA (6 J/cm? or UVB irradiation (50 mJ/ cm’) of resting AEC resulted in iNOS mRNA expression but no induced NO production. The preincubatian of AEC with IL-10 (200 U/ml) for 24 h prior to UVA irradiation did not increase iNOS mRNA exnression or NO urn- duction. However. the incubation of AEC with IL-lp Immediately afler’UVA irradiation’ led to an additive enhancement of iNOS mRNA expression and NO production when compa- red to non-irradiated cells. In contrast. preinwbation of AEC with IL-lp for 24 h prior to UVB irradiation led to an addihve enhancement of iNOS mRNA expression and NO pro- duction, but no increase could be observed when cells were mcubated with IL-1 R imme- diately after UVB irradiation. Strikingly, incubation of AEC wim IL-ID 24 h pnor and III- mediately after UV irradiation led to a synergistic inuease in iNOS mRNA ezaression and NO production only in UVB irradiated &Is,-but not in UVA-irradiated cells. \;ve conclude that me expression of iNOS IS a key component of the response of endothelial cells to uv radiation. UVA appears to enhance the iNOS-inducing activity of 11-18, whereas UVB may induce cellular mediators that synergue wth IL-ID in inducing iNOS expression.
