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Growth of human cutaneous mast cells in culture
ESDR I JSID I SID Abstracts
1063 MTEBLEUKIN-17 IS RELEASED BY NICKEL-SPECIFIC T CELLS AND REGULATES ICAM- EXPRESSION AND CHEMOKINE PRODUCTION IN HUMAN KERATINOCYTES. C. Albsnesi. A Cavani. and G. Gimlomoni Lab of Immunology, Istituto Dermopatico dell’lmmacolata, IRCCS, Rome, Italy IL17 is a novel cytokine selectively produced by T lymphocytes that can regulate the functions of a variety of cell types In this study, we determined whether skin-homing T cells produce B-17, and the effects of IL-17 alone or in combination with IFN-7 or TNFa on the expression of immune-modulatory membrane molecules and chemokine synthesis by human keratinocytes. Nickel-specific CD4’ T cell clones expressing the E-selecti” l&and, cutaneous lymphocyte-associated antigen, were show” to express IL-17 mRNA and co-release lL-17, IFN-y and TNFu following activation with PMA and ionomyci”. Although IL-17 alone did not induce ICAM-I expression on keratinocytes, it synergized dose-dependently with IFN-y, but not rvith TNFa, in promoting both synthesis of membrane ICAM- and release of soluble ICAM- In addition, IL-17 directly stimulated IL-8 synthesis, and synergized with bath IF’N-yand TNFa in inducing IL-8 production These effects were confirmed at the mRNA level and could be abolished by neutralization of IL-17 with specific Ab I” contrast, IL-17 had no effects on MHC class I and class II, CD40, B7-1 or B7-2 molecule expression, and on macrophage chemotactic protein 1 synthesis by keratinocytes Finally, IL-17 inhibited specifically and in a dose-dependent manner IFI’.T- and TN@-induced production of RANTES The results suggest that IL-17 is a” important player of T cell mediated skin immune responses with synergistic or antagonist effects on IF&y- and TNFa-stimulated keratinocyte activation.
1064
1067
DELAYED EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE AFTER UV IRRADIATION IN CUTANEOUS LUPUS ERYTHEMATOSUS. Anneoret Kuhn, Percy Lehmann, Thomas Ruzicka, and Victoria Kolb-Bachofen*,
ALTF,RBD IMMUNOREGULATORY CELL AND CYTOKINE RESPONSES TO UV AND HAPTEN ALONE DIFFER FROM COMPLEX COMBINED EXPOSURE. ~vivar. C. I&~&~etnti KD Coooer. Deqatment of Dermatology, Case Western Reserve University, University I&pit& of Cleveland. Cleveland, OH Haptens and IJV each have distinct capabilities to induce cutawus immuwregulatory cells and cytokines such as lLl0 and lL-12 in skin es well as in draining lymph “odes (DLN). However, coqlex exposure to combined haptea end W may significantly alter the expected response. Ski” and DLN biopsies were collected either 18h after DNFB sensitization, 66h after IJVB (72 mJ/cm’), or 18h after DNFB Bensitization of 48h post UV exposed C3H/HeN mice. We fowd that IJV and DNFB each separately induce IL10 and IL-12 proddo” in DLN but combined exposure to UV and DNFB reduces expected IL-10 and IL-12 levels by 72+5% and 85+4% SE&l respectively. UV snd DNFB both eepewtely induced a CD1 lb’ cellular infiltration into the DLN but combined exposure resulted in a 7p+15% SEM redwtio” in CD1 I b’ cells. By cellular depletion studies, only the CD1 lb+ cell populatiin was responsible for both IL-10 and IL-12 production by DLN cells, and comb&d exposure resulted in reduced production on a cell-for*ll hasis. The relative finctionsl contributiins of IL-10 and IL12 in our system was verified wing the W &sing aad time schedule of our model. Anti-mouse IL-10 did nat block W-induced suppression of CHS end antigenio tolerance induction. However, intmperitoneal injecti”” of rewnbinaM a-12 revemmi UV inhibition of the CHS response and induction of toleratxzeto DNFB. We conclude that complex exposure reduces both the IL-IO and II_-12 response by reducing CD1 lb’ cells appkzrance in the DLN, but that the resultam reduction in IL12 is the key determinant of immunosuppression in this system.
Department of Dermatology, and *Research Group of lmmunobiology in the Biomedical Center, Heinrich-Heine-University Duesseldorf Germany UV irradiation (UVR) plays a key role in the pathogenesis of cutaneous lupus erythematosus (LE), and since 1986 we have examined over 200 patients with different subtypes after provocative phototesting. Interestingly, the development of characteristic positive phototest reactions has been considerably slower and persisted longer as compared to other photodermatoses. Several reports have shown expression of the cytokine-inducible nitric oxide synthase (iNOS) in autoimmune diseases. We now investigated the role of iNOS expression at mRNA and protein level in the pathogenests of UV-induced LE skin lesions. In healthy controls (n=8) iNOS expression was detected in the eprdermis 24 h UVR by immunohistochemistry, in situ hybridization and RT-PCR. iNOS expression decreased 48 h after UVR before subsiding 72 h after treatment. In LE patients (n=E) exactly the reverse situation was seen: iNOS mRNA and protein were not detectable in keratinocytes for up to 48 h, but became positwe 72 h after UVR and persisted for up to 25 days. Our data suggest that delayed and prolonged local iNOS expression is involved in the pathogenesis of cutaneous LE. Further studies will evaluate this differential time course of iNOS expression as a possible diagnostic tool and target for therapeutic Intervention
1065
1068
the IL1RA in cutaoeous wound repair are not know” at present Using RN= pmtection assay. icILlRA mRNA WBSonly detectable i” “urine ski” while ““detectable in any other tissue studied. After f”lI thickoess incisio”~ woU”di”g the anmmt of iclLlRA mRNA in the wound area remained unchanged throughout the observation period of l-14 days and comparable to unwounded ski”. I” contrast, 24h after injury a stm”g signal for the sIL-1RA &form was observed which the” slowly decreased to undetectable levels after 14 days. The expression of sIL-1RA wl.¶ well the kinetics of intlamatory cell imigration (graulocytes and macmphage$ into the wound ma suggesting that these cc.IIs are the main .w”rce of sIL-1RA d”nng den”+ wound healing. I” order to dissect the relevance of the different ILlRA 1SOfOtmS I” &in biology we are cmxntly developing mice permitting specific inactiVatiOnOf ti ILIRA gene in keratinocyteaor gmnulocyte and mono+ cells.
GROWTHOF HUMANCUTANEOUSMAST CELLS IN CULTURE Ik & & ~ic&l II w Departmeritsof Dermatology,Rush F’resb$eria”-St.Luke’s MedicalCmter, Cbicagq IL; and the Ihiwa’sttyof Pittsburgh,Pittsbwgb,PA. USA. Twu pqdatia~s of human c”ta”eas mast cells are know to exist: “wst CaIls expressiq tryptasa (MC-~-Jand “mst cells eapre=i”g hub tr#ase and rhymare (MC-TO Mat masl cells is human skin 818 MC-‘TC. Previous audiss have shown mat psripheml bloodrnasrcellp~orJcancbvelopiatoMC-T~eulhupdwimreccmbinaot~~ factor @CF): hmvever, the e&us of rSCF and other @nvtb 5ct”rs haw “U bea establishadcn MC-TC. Thereiixe,the purpoasoftbia study was to &w&ate tie s&ds Of gwtb 5~~&rsco humanski” MC-TC. NecuataIski” MC-TCwem adtwed ia the p-a amceotratMos (1, lo,30 and 100 “g/ml) of OflmdiumPJ~ormedilml~g~ rSCP. Wahin tiuea weeks, MC-TC had “mm thm doubled in “umber whar co”wed to occuning at 3o-loo “@al. By nix weeks, “lKliumcaitmIswi!llamaximnlreEpmse however,the ntunberof rSCP-tre&edMC-TCshad de&cd to ccmtmllevels. More frew”t additioos of rSCF to the cultare weUs failed to incmasa MC-TC nmnter or mxvival su8ge&tgdmt rSCF alms is msufficientfor their gmatb in v&o To Ruther i”vatigPts the pwible effectsof&x 5aoz-s m MC-TC grcwtb in vitro, IL-Z, K-3, IL4 and IL-IO were addsdalmsorincombioatiolwahrSCFtothsmaEtceltcultursa. Nme”ftbesecytoki”es alme or combined&I rSCF i”cmascd MC-TC mmxbm MC-TC were also cadt”ti i” ccndiitimed“I&,“, t&m a fibmearcomaline. This mediun did si&icatly i”creasemast 0U”wnbersandhiaandneconuotafteroleweakin~whmcomparedtorSCF-mated cells. Thurrtvge~hsdemoamatethathumansldnMC-TCunbsmnintaiwdm~re for weeks,but require gmwtb hc&xs aher thm rSCF. It appears mat these 5Uc.m are prcducedfrommesencbymalwlb.
1063 MTEBLEUKIN-17 IS RELEASED BY NICKEL-SPECIFIC T CELLS AND REGULATES ICAM- EXPRESSION AND CHEMOKINE PRODUCTION IN HUMAN KERATINOCYTES. C. Albsnesi. A Cavani. and G. Gimlomoni Lab of Immunology, Istituto Dermopatico dell’lmmacolata, IRCCS, Rome, Italy IL17 is a novel cytokine selectively produced by T lymphocytes that can regulate the functions of a variety of cell types In this study, we determined whether skin-homing T cells produce B-17, and the effects of IL-17 alone or in combination with IFN-7 or TNFa on the expression of immune-modulatory membrane molecules and chemokine synthesis by human keratinocytes. Nickel-specific CD4’ T cell clones expressing the E-selecti” l&and, cutaneous lymphocyte-associated antigen, were show” to express IL-17 mRNA and co-release lL-17, IFN-y and TNFu following activation with PMA and ionomyci”. Although IL-17 alone did not induce ICAM-I expression on keratinocytes, it synergized dose-dependently with IFN-y, but not rvith TNFa, in promoting both synthesis of membrane ICAM- and release of soluble ICAM- In addition, IL-17 directly stimulated IL-8 synthesis, and synergized with bath IF’N-yand TNFa in inducing IL-8 production These effects were confirmed at the mRNA level and could be abolished by neutralization of IL-17 with specific Ab I” contrast, IL-17 had no effects on MHC class I and class II, CD40, B7-1 or B7-2 molecule expression, and on macrophage chemotactic protein 1 synthesis by keratinocytes Finally, IL-17 inhibited specifically and in a dose-dependent manner IFI’.T- and TN@-induced production of RANTES The results suggest that IL-17 is a” important player of T cell mediated skin immune responses with synergistic or antagonist effects on IF&y- and TNFa-stimulated keratinocyte activation.
1064
1067
DELAYED EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE AFTER UV IRRADIATION IN CUTANEOUS LUPUS ERYTHEMATOSUS. Anneoret Kuhn, Percy Lehmann, Thomas Ruzicka, and Victoria Kolb-Bachofen*,
ALTF,RBD IMMUNOREGULATORY CELL AND CYTOKINE RESPONSES TO UV AND HAPTEN ALONE DIFFER FROM COMPLEX COMBINED EXPOSURE. ~vivar. C. I&~&~etnti KD Coooer. Deqatment of Dermatology, Case Western Reserve University, University I&pit& of Cleveland. Cleveland, OH Haptens and IJV each have distinct capabilities to induce cutawus immuwregulatory cells and cytokines such as lLl0 and lL-12 in skin es well as in draining lymph “odes (DLN). However, coqlex exposure to combined haptea end W may significantly alter the expected response. Ski” and DLN biopsies were collected either 18h after DNFB sensitization, 66h after IJVB (72 mJ/cm’), or 18h after DNFB Bensitization of 48h post UV exposed C3H/HeN mice. We fowd that IJV and DNFB each separately induce IL10 and IL-12 proddo” in DLN but combined exposure to UV and DNFB reduces expected IL-10 and IL-12 levels by 72+5% and 85+4% SE&l respectively. UV snd DNFB both eepewtely induced a CD1 lb’ cellular infiltration into the DLN but combined exposure resulted in a 7p+15% SEM redwtio” in CD1 I b’ cells. By cellular depletion studies, only the CD1 lb+ cell populatiin was responsible for both IL-10 and IL-12 production by DLN cells, and comb&d exposure resulted in reduced production on a cell-for*ll hasis. The relative finctionsl contributiins of IL-10 and IL12 in our system was verified wing the W &sing aad time schedule of our model. Anti-mouse IL-10 did nat block W-induced suppression of CHS end antigenio tolerance induction. However, intmperitoneal injecti”” of rewnbinaM a-12 revemmi UV inhibition of the CHS response and induction of toleratxzeto DNFB. We conclude that complex exposure reduces both the IL-IO and II_-12 response by reducing CD1 lb’ cells appkzrance in the DLN, but that the resultam reduction in IL12 is the key determinant of immunosuppression in this system.
Department of Dermatology, and *Research Group of lmmunobiology in the Biomedical Center, Heinrich-Heine-University Duesseldorf Germany UV irradiation (UVR) plays a key role in the pathogenesis of cutaneous lupus erythematosus (LE), and since 1986 we have examined over 200 patients with different subtypes after provocative phototesting. Interestingly, the development of characteristic positive phototest reactions has been considerably slower and persisted longer as compared to other photodermatoses. Several reports have shown expression of the cytokine-inducible nitric oxide synthase (iNOS) in autoimmune diseases. We now investigated the role of iNOS expression at mRNA and protein level in the pathogenests of UV-induced LE skin lesions. In healthy controls (n=8) iNOS expression was detected in the eprdermis 24 h UVR by immunohistochemistry, in situ hybridization and RT-PCR. iNOS expression decreased 48 h after UVR before subsiding 72 h after treatment. In LE patients (n=E) exactly the reverse situation was seen: iNOS mRNA and protein were not detectable in keratinocytes for up to 48 h, but became positwe 72 h after UVR and persisted for up to 25 days. Our data suggest that delayed and prolonged local iNOS expression is involved in the pathogenesis of cutaneous LE. Further studies will evaluate this differential time course of iNOS expression as a possible diagnostic tool and target for therapeutic Intervention
1065
1068
the IL1RA in cutaoeous wound repair are not know” at present Using RN= pmtection assay. icILlRA mRNA WBSonly detectable i” “urine ski” while ““detectable in any other tissue studied. After f”lI thickoess incisio”~ woU”di”g the anmmt of iclLlRA mRNA in the wound area remained unchanged throughout the observation period of l-14 days and comparable to unwounded ski”. I” contrast, 24h after injury a stm”g signal for the sIL-1RA &form was observed which the” slowly decreased to undetectable levels after 14 days. The expression of sIL-1RA wl.¶ well the kinetics of intlamatory cell imigration (graulocytes and macmphage$ into the wound ma suggesting that these cc.IIs are the main .w”rce of sIL-1RA d”nng den”+ wound healing. I” order to dissect the relevance of the different ILlRA 1SOfOtmS I” &in biology we are cmxntly developing mice permitting specific inactiVatiOnOf ti ILIRA gene in keratinocyteaor gmnulocyte and mono+ cells.
GROWTHOF HUMANCUTANEOUSMAST CELLS IN CULTURE Ik & & ~ic&l II w Departmeritsof Dermatology,Rush F’resb$eria”-St.Luke’s MedicalCmter, Cbicagq IL; and the Ihiwa’sttyof Pittsburgh,Pittsbwgb,PA. USA. Twu pqdatia~s of human c”ta”eas mast cells are know to exist: “wst CaIls expressiq tryptasa (MC-~-Jand “mst cells eapre=i”g hub tr#ase and rhymare (MC-TO Mat masl cells is human skin 818 MC-‘TC. Previous audiss have shown mat psripheml bloodrnasrcellp~orJcancbvelopiatoMC-T~eulhupdwimreccmbinaot~~ factor @CF): hmvever, the e&us of rSCF and other @nvtb 5ct”rs haw “U bea establishadcn MC-TC. Thereiixe,the purpoasoftbia study was to &w&ate tie s&ds Of gwtb 5~~&rsco humanski” MC-TC. NecuataIski” MC-TCwem adtwed ia the p-a amceotratMos (1, lo,30 and 100 “g/ml) of OflmdiumPJ~ormedilml~g~ rSCP. Wahin tiuea weeks, MC-TC had “mm thm doubled in “umber whar co”wed to occuning at 3o-loo “@al. By nix weeks, “lKliumcaitmIswi!llamaximnlreEpmse however,the ntunberof rSCP-tre&edMC-TCshad de&cd to ccmtmllevels. More frew”t additioos of rSCF to the cultare weUs failed to incmasa MC-TC nmnter or mxvival su8ge&tgdmt rSCF alms is msufficientfor their gmatb in v&o To Ruther i”vatigPts the pwible effectsof&x 5aoz-s m MC-TC grcwtb in vitro, IL-Z, K-3, IL4 and IL-IO were addsdalmsorincombioatiolwahrSCFtothsmaEtceltcultursa. Nme”ftbesecytoki”es alme or combined&I rSCF i”cmascd MC-TC mmxbm MC-TC were also cadt”ti i” ccndiitimed“I&,“, t&m a fibmearcomaline. This mediun did si&icatly i”creasemast 0U”wnbersandhiaandneconuotafteroleweakin~whmcomparedtorSCF-mated cells. Thurrtvge~hsdemoamatethathumansldnMC-TCunbsmnintaiwdm~re for weeks,but require gmwtb hc&xs aher thm rSCF. It appears mat these 5Uc.m are prcducedfrommesencbymalwlb.