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Antioxidants are potent inhibitors of spontaneous and induced bosinophil apoptosis
S162
E S D R I JSID I S I D Abstracts
0967
0970
ANTIOXIDANTS ARE POTENT INHIBITORS OF SPONTANEOUS AND INDUCED EOSINOPHIL APOPTOSIS. Bettina Wedi, Iulia Straede, Britta Wieland, Alexander Kano. Department of Dermatology, Hannover Medical School, Hannover, Oermany. Several enhancers of eosinophil survival such as IL-3, IL-5, and GM-CSF are weil known. However, the mechanisms for indaction of eosinophil apoptosis remain uncertain. Since the role of oxidatlve stress has not been investigated the present study was undertaken to determine the role of reactive oxygen species and antioxidants in eosinophil apoptosis, Eosinophils were cultured with 1-500 ,uM sodium arsenite known to induce intraceUular oxidative metabolites in cells. The proportion of apoptotic eosinophils displaying a hypodiploid DNA pank was assessed quantitatively by flow cytometric analysis and qualitatively by agarose gel alectrophoresis of nucleosomal DNA. There was a significant increase in the rate of eosioophil apoptosis with low concentrations of arsenite (optim. conc. 50 }aM), whereas high coneentrations (500 gM) showed rates of apoptosis similar to control medium. Coincubation expedments demonstratexl that arsenite induced apoptosis can be nearly completely prevented by selective antioxidants such as glutathione (GSH) and N-acetyl-cysteine (NAC) but not dimethyl sulfoxide (DMSO) or taurine (TAUR). Moreover, GSH, aad NAC signifieantly reduced eosinophil apoptosis mediated by anti-fas mAb (CD95). Next it was shown that GSH and NAC were also able to significanfly delay spontaneous apoptosis in unstimulated eosinophils. In eontrast, TAUR and DMSO had no effect. Taken together these data point to an important mle of oxygen-dependent mechanisms in the regulation of eosinophil survival and apoptosis.
PROTEIC ALLERGENS CAPTATION BY DENDRITIC CELLS GENERATF.D FROM CORD BLOOD PROGENITORS AND EFF]CIENT PREaENTATION IN PRIMARY RESPONaE. N~ß)alie Rot~er'. Clande AndN,~. Dantel Schmnt-. Claude V~ncenr.'lNSERM U346, H~~tal Edouerd Herdot, Lyon, Fmnce. "%aboratolres S t a l l e S , Fmmes, Fmnce. The safety and e f ~ of s u b l ~ a l immunob'terapyhave beeff demonstrated in moderate alleq~i¢ astllma and seaannal ~initis. In order to dofine tl~ Ixedse med~nism of a~io~nof agefgtm vdten it puaes thmagh the oral muoosa, ~~eklvesUgatedthe mle of ~ n s teils In the capture and intemallzaUon of the allergens. An/n vtb'o model uslng denddU¢ cels wtth the phenotyplc c h a ~ of epldermal Langerhans cells, m developed. Langemers Mke denddtlc ceHs (LLDC) wem generated/n v/tro fmm cord Uood C034* p~ogenitors by cultum vAth app¢ol~iate growth factom. T h e ~ L I J ~ were userl in oKler to sludy the er¢loc~.olds of two tecomlaiaam major alleegea$ (r-Bet v I and r-Phl p I) and to study the acuvagon of autologous nalve T ne# after LLDC acflvalJon. The anergens w~a taboled wlth FITC, Ludfer Yolow was used as polltNe contml of mictopkmcytosls, Oexlrln-FffC and Mannan-BaAFITe as positive ~ for receptor rnediated endocytosis. Intemalzatlon was obsmved at a hlgher rate wlth I~ffi allergensthan wlth I.udfer Yellow or FITC-Oexlran. Irllemnllzatlonwas dose dependent and wls malnly a propm~/of LLOC at Iow doses, the cegadedved from the urne progenltom and clevolclof LLDC phenotYl~Cmm'twm pctmnt«t n lotet IntemIMzagan potermy..a, clear (;ut relatlon~lp laetv~m~tl~e endo¢,/tlc potermy and the maturatlan m g a of LLIX; was dantorBIrated. The endocy~ potercy of LLDC appeamd at day 7 alter the ~ of ttm 0ultme, penked et day 10-14 mld dtsappeered plogresslvely ilner wherelm LLOC wem still pme~t. Confo(~dmlaoseop/of U.DC indl~led that allergenswere Internal~zed by the cell bOdyand by the denddtlnpfoten, furlhenn~e, the use of monanslne anggested that int~mmgzali«~ w~mdone W pln<~-'yto~asand anl ~ raneplors. LLOC I n c u l ~ ~ wlm ptolac aùergeas Induced a prol~rat'Ne response of nalve T lympl~-ym in part ~ the exp~nùmts.
0968
0971
DERMAL INFILTKATION BY ACTIVATED MONOCYTEs PKECEDES SKIN THICKENING AND PKOCt1(I) COLLAGEN MRNA UPKEGULATION IN EARLY MURINE SCLERODERMATOUS GKAFT VEKSUS HOST D]SEASE (GVHD), A PKOMISING MODEL FOR HUMAN SCLERODERMA. LL MCCORMICK, E TOOTELL, AND AC GILLIAM~ DEPAKTMENT OF DERMATOLOGY, CASE WESTEKN ~ESEKVE UNIVERSITY, U H C , CLEVELAND, O H Scleroderma is a ehronie debilitating autoimmune flbrosing disorder of unknown efiology with immune dysregnlation and overproduetion of dermal Type I collagen. Growth factors produced by aetivated monocytes infiltrating skin may initiate and perpetuate abnormal collagen synthesis. The disease is difficnlt to study for want of a valid aniraal modeL We are nsing borte marrow transplantation in lethally irradiated mice [B10.D2 (It-2 d) to Balb/e OI-2d)] ~ produee sclerodermatuua GV}tD, a model for scleroderma. Control animals transplanted with syngeneic borte marrow and spleen cells (Balb/c to Balb/e) do not develop skin thickening or GVItD; experimental animala develop lung fibrosis and skin thickening, measured hy ultrasonography and histologF. Meaanrahle sldn thi¢kening ocenrs by d 21-25 following early (by d 7) infiltration of skin by Mac-1+ activated monocytes idantified by immunostaining in experlmental (n=28) hut not control animals (n=7). RNase proteetion assays show prominent procd(I)collagen mRNA npregnlation in experimental but not control animals by d 49. Therefore, the s¢lerodermatoas GVI]]) mnrlne model aeeurately recapltu[ates human seleroderma and demonstrates early preelinieal involvement of activated monocyte/ma¢rophages in the disease.
S KIN-DERIVED IL-10 FOLLOWING ACUTE, LOW DOSE ULTRAVIOLET-B RADIATION CREATES A SYSTEMIC ANTIGEN PRESENTATION DEFECT Iwan Kurimoto, l--Iironori Niizeki and L Wa)'ne Streileia. Scbepens Eye Research Institute and Department of Dermatology, Harvard Medical School, Boston, MA and Departmant of Dermatology, Osaka University Schon of Medicine, Osaka, Japan. Expos•re of murine skin to neute, low dose UVR has both local and systemic immune consequences. While the local effects am mediated primaxily by TNFa and are expressed in genetically suseeptible mice. the systemic consequences occur independently of TNFa in all strains of mice. Hapten applied to an unirradiated site immediately after the last dose of UVR readily sensitizas mice, hut if hapten application is delayed for >24 hours, contact hypersensitivity (CH) falls to develop. We have conducted experimeats to determine (a) whether an antigen presenting cell deficit is responsible for this failttre, (b) where such defective cells reside, and (c) why their ftmction is aberrant. 72 hrs after UVR, cells wem prepared fmm unirradiated epidermis and lymph nodes dralning unexposed skin. When hapten-derivatized and injected s.c. into naive, syngenic mice, cells from epidermis induced vigorous CH, wbereas lymph hode adberent cells failed. In addition, splenectomy failed to reverse the UVR-imposed immune defeet. UVR-exposed mice that were treated simultaneously with anfi-IL-10 antibodies responded to delayed hapten application with intense CH, and mice that received systemic injeetions of IL-10 lost their capacity to develop CH when hapten was applied epicutaneously. Systemic failure to acquire CH after UVR was deteeted in mast cells deficient raice, but was abrogated if the irradiated skin site was excised immediately after the last UVR exposure. We propose (a) that acute, low dose UVR induces epidermal cells to secrete IL-10 in a sustained fashion, and (b) that this cytokine creates an APC defect in peripheral lymph nodes, bot not in skin or spleen. Since hapten applied to unexposed skin fails to induce CH in this situation, we suspect that APC from both skin and draining lymph node taust participate in sensitization leading to CH..
0969
0972
PERIPHERAL BLOOD T CELLS FROM ATOPIC DERMATITIS PATIENT8 DEMON$TRATE INCREASED TRANSMIGRATION ACROSS MICROVA$CULAR ENDOTHELIUM. I.J=M. de Vries. A. Sleiiffers. F.C. van Rei[ann. E.F. Knol. T. Thepen, C,~.F.M BruiinzeeI-Koomen. Dept. of Dermatology and Pathology, University Hospilal Utrecht, The Nelhedands Skin lesions of palients with atopic dermatitis (AD) are characterized by the presence of activated T cells and increased expressi•n of adhesion molecules on endothelial cells (EC). We have examined the transmigration of T cells a¢ross a mono[ayer of human dennal microvascular endothelial celMine (HMEC-1) cultured on an extracellular matnx (ECM) of lysed human skin fibroblasts. Enhaßced transmigration of T cells fmm AD patients as ¢ompared to non-atopic conlrols was observed, both across non-stimulated EC and TNFa-stimulated EC. SeMctive depletion of activated T cells, by depletion of HLA-DR÷ T cells, annulled the differences between T eells from non-atopic individuals and T cells from patients with regard to Ihe transmigration aeross non-stimulated and resting EC. Possibly, increased expression of cutaneous lymphocyte-assoeiated antigen (CLA) on the activated T cells mediated this increased T cell migration. The ligand for CLA is E-selectin on activated endothelial cells. However, T cells of AD patients did not demonstrate inereased transmigtation after activation of the endothelial cells, excluding a role of E-selectin. Therefore, we hypothesize that the stale of activation of T teils, including the state of activation of their adhesion molecutes, in peripheral blood from AD patients may be the deten'nining factor for T cell transmigration, and might explain the high T cell numbers in skin of AD patients.
RAPID REDUCTION IN THE SIZE OF MOUSE CUTANEOUSMAST CELL POPULATIONS BY APOPTOS1S AFTER CESSATION OF TREATMENTWITH SCF DOES NOT RESULT IN SKIN INFLAMMATION. Marcus Maurer and SteDhen J. Galli. Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard MedicadSchool, Boston. MA, USA. Subcutaneous administration of stem teil factor (SCF), the ligand for the c-kitreceptor, to the back skin of mice promotes the local hyperplasia of cutaneous mast cells (MC). Cessation of treatment with SCF results in the rapid leduction of MC populafions by apoptosis. We used the ml-fibrin deposifion assay, a very sensitive methcd for quantifying thcreased vascular permeability, to assess whether the clearance of large numbers of apoptotic MCs is associated with a sigaificant inflammatory skin reaction. The subcutaneous injection of rrSCF[~ (30 or 100 ~tgtkg/day) or rrSCFlU-peg (pelyethylene glycol-treatedSCF, 30 or 1130gg/kg/day) for 21 days thcreased the number of MC at the site of injeetioa from 5.1+-0.7MClmm2 to 36,4±4.1, 34.7+_ 9.7, 52.5+-5.8. and 545±97 MC/mm:, respecfively. 48 hrs after cessation ofireatment with SCF, skin MCs underwem massive apoptosis, as ~sessed by light microscopy, and MC numbers dropped to 54+_12% of day 21 levels. Most notably, lzsl-fibrindepoaition during this rapid reducfion of skth MCs was virtuany unaJiemd as compared to mice receiving continuous treatment with SCF or vehicle. In the case of treatmem with rrSCFlU-pegat 100 ktg/kg/day,which tesulted in a more than 100-foldthcrease in skin MCs at the site of injection on day 21, eessation of SCF was foUowedby a decrease to one third of this number dufing the next two days, and the sldn of these rnice contained only slighfly more total and urea thsoluble (= cross-linked) epm ~2~I-fibrinthan skin of eontrol mice receiving continuous injeetions with rrSCFl~-peg (total epm: 171.1_+60.4 rs. 134.3-+37per biopsy, urea insoluble cpm: 41.4+13.1 vs, 31.4+0.5, respectively, differences not statistically significant). We conclude that the mpid eJiminahon of even large populations of MCs by apeptosis, which would also resuh in the cleatance of the coasiderable quantities of proinflammato~ products stored by these cells, does not lead to significant local cutaneous inflarmnatory responses.
E S D R I JSID I S I D Abstracts
0967
0970
ANTIOXIDANTS ARE POTENT INHIBITORS OF SPONTANEOUS AND INDUCED EOSINOPHIL APOPTOSIS. Bettina Wedi, Iulia Straede, Britta Wieland, Alexander Kano. Department of Dermatology, Hannover Medical School, Hannover, Oermany. Several enhancers of eosinophil survival such as IL-3, IL-5, and GM-CSF are weil known. However, the mechanisms for indaction of eosinophil apoptosis remain uncertain. Since the role of oxidatlve stress has not been investigated the present study was undertaken to determine the role of reactive oxygen species and antioxidants in eosinophil apoptosis, Eosinophils were cultured with 1-500 ,uM sodium arsenite known to induce intraceUular oxidative metabolites in cells. The proportion of apoptotic eosinophils displaying a hypodiploid DNA pank was assessed quantitatively by flow cytometric analysis and qualitatively by agarose gel alectrophoresis of nucleosomal DNA. There was a significant increase in the rate of eosioophil apoptosis with low concentrations of arsenite (optim. conc. 50 }aM), whereas high coneentrations (500 gM) showed rates of apoptosis similar to control medium. Coincubation expedments demonstratexl that arsenite induced apoptosis can be nearly completely prevented by selective antioxidants such as glutathione (GSH) and N-acetyl-cysteine (NAC) but not dimethyl sulfoxide (DMSO) or taurine (TAUR). Moreover, GSH, aad NAC signifieantly reduced eosinophil apoptosis mediated by anti-fas mAb (CD95). Next it was shown that GSH and NAC were also able to significanfly delay spontaneous apoptosis in unstimulated eosinophils. In eontrast, TAUR and DMSO had no effect. Taken together these data point to an important mle of oxygen-dependent mechanisms in the regulation of eosinophil survival and apoptosis.
PROTEIC ALLERGENS CAPTATION BY DENDRITIC CELLS GENERATF.D FROM CORD BLOOD PROGENITORS AND EFF]CIENT PREaENTATION IN PRIMARY RESPONaE. N~ß)alie Rot~er'. Clande AndN,~. Dantel Schmnt-. Claude V~ncenr.'lNSERM U346, H~~tal Edouerd Herdot, Lyon, Fmnce. "%aboratolres S t a l l e S , Fmmes, Fmnce. The safety and e f ~ of s u b l ~ a l immunob'terapyhave beeff demonstrated in moderate alleq~i¢ astllma and seaannal ~initis. In order to dofine tl~ Ixedse med~nism of a~io~nof agefgtm vdten it puaes thmagh the oral muoosa, ~~eklvesUgatedthe mle of ~ n s teils In the capture and intemallzaUon of the allergens. An/n vtb'o model uslng denddU¢ cels wtth the phenotyplc c h a ~ of epldermal Langerhans cells, m developed. Langemers Mke denddtlc ceHs (LLDC) wem generated/n v/tro fmm cord Uood C034* p~ogenitors by cultum vAth app¢ol~iate growth factom. T h e ~ L I J ~ were userl in oKler to sludy the er¢loc~.olds of two tecomlaiaam major alleegea$ (r-Bet v I and r-Phl p I) and to study the acuvagon of autologous nalve T ne# after LLDC acflvalJon. The anergens w~a taboled wlth FITC, Ludfer Yolow was used as polltNe contml of mictopkmcytosls, Oexlrln-FffC and Mannan-BaAFITe as positive ~ for receptor rnediated endocytosis. Intemalzatlon was obsmved at a hlgher rate wlth I~ffi allergensthan wlth I.udfer Yellow or FITC-Oexlran. Irllemnllzatlonwas dose dependent and wls malnly a propm~/of LLOC at Iow doses, the cegadedved from the urne progenltom and clevolclof LLDC phenotYl~Cmm'twm pctmnt«t n lotet IntemIMzagan potermy..a, clear (;ut relatlon~lp laetv~m~tl~e endo¢,/tlc potermy and the maturatlan m g a of LLIX; was dantorBIrated. The endocy~ potercy of LLDC appeamd at day 7 alter the ~ of ttm 0ultme, penked et day 10-14 mld dtsappeered plogresslvely ilner wherelm LLOC wem still pme~t. Confo(~dmlaoseop/of U.DC indl~led that allergenswere Internal~zed by the cell bOdyand by the denddtlnpfoten, furlhenn~e, the use of monanslne anggested that int~mmgzali«~ w~mdone W pln<~-'yto~asand anl ~ raneplors. LLOC I n c u l ~ ~ wlm ptolac aùergeas Induced a prol~rat'Ne response of nalve T lympl~-ym in part ~ the exp~nùmts.
0968
0971
DERMAL INFILTKATION BY ACTIVATED MONOCYTEs PKECEDES SKIN THICKENING AND PKOCt1(I) COLLAGEN MRNA UPKEGULATION IN EARLY MURINE SCLERODERMATOUS GKAFT VEKSUS HOST D]SEASE (GVHD), A PKOMISING MODEL FOR HUMAN SCLERODERMA. LL MCCORMICK, E TOOTELL, AND AC GILLIAM~ DEPAKTMENT OF DERMATOLOGY, CASE WESTEKN ~ESEKVE UNIVERSITY, U H C , CLEVELAND, O H Scleroderma is a ehronie debilitating autoimmune flbrosing disorder of unknown efiology with immune dysregnlation and overproduetion of dermal Type I collagen. Growth factors produced by aetivated monocytes infiltrating skin may initiate and perpetuate abnormal collagen synthesis. The disease is difficnlt to study for want of a valid aniraal modeL We are nsing borte marrow transplantation in lethally irradiated mice [B10.D2 (It-2 d) to Balb/e OI-2d)] ~ produee sclerodermatuua GV}tD, a model for scleroderma. Control animals transplanted with syngeneic borte marrow and spleen cells (Balb/c to Balb/e) do not develop skin thickening or GVItD; experimental animala develop lung fibrosis and skin thickening, measured hy ultrasonography and histologF. Meaanrahle sldn thi¢kening ocenrs by d 21-25 following early (by d 7) infiltration of skin by Mac-1+ activated monocytes idantified by immunostaining in experlmental (n=28) hut not control animals (n=7). RNase proteetion assays show prominent procd(I)collagen mRNA npregnlation in experimental but not control animals by d 49. Therefore, the s¢lerodermatoas GVI]]) mnrlne model aeeurately recapltu[ates human seleroderma and demonstrates early preelinieal involvement of activated monocyte/ma¢rophages in the disease.
S KIN-DERIVED IL-10 FOLLOWING ACUTE, LOW DOSE ULTRAVIOLET-B RADIATION CREATES A SYSTEMIC ANTIGEN PRESENTATION DEFECT Iwan Kurimoto, l--Iironori Niizeki and L Wa)'ne Streileia. Scbepens Eye Research Institute and Department of Dermatology, Harvard Medical School, Boston, MA and Departmant of Dermatology, Osaka University Schon of Medicine, Osaka, Japan. Expos•re of murine skin to neute, low dose UVR has both local and systemic immune consequences. While the local effects am mediated primaxily by TNFa and are expressed in genetically suseeptible mice. the systemic consequences occur independently of TNFa in all strains of mice. Hapten applied to an unirradiated site immediately after the last dose of UVR readily sensitizas mice, hut if hapten application is delayed for >24 hours, contact hypersensitivity (CH) falls to develop. We have conducted experimeats to determine (a) whether an antigen presenting cell deficit is responsible for this failttre, (b) where such defective cells reside, and (c) why their ftmction is aberrant. 72 hrs after UVR, cells wem prepared fmm unirradiated epidermis and lymph nodes dralning unexposed skin. When hapten-derivatized and injected s.c. into naive, syngenic mice, cells from epidermis induced vigorous CH, wbereas lymph hode adberent cells failed. In addition, splenectomy failed to reverse the UVR-imposed immune defeet. UVR-exposed mice that were treated simultaneously with anfi-IL-10 antibodies responded to delayed hapten application with intense CH, and mice that received systemic injeetions of IL-10 lost their capacity to develop CH when hapten was applied epicutaneously. Systemic failure to acquire CH after UVR was deteeted in mast cells deficient raice, but was abrogated if the irradiated skin site was excised immediately after the last UVR exposure. We propose (a) that acute, low dose UVR induces epidermal cells to secrete IL-10 in a sustained fashion, and (b) that this cytokine creates an APC defect in peripheral lymph nodes, bot not in skin or spleen. Since hapten applied to unexposed skin fails to induce CH in this situation, we suspect that APC from both skin and draining lymph node taust participate in sensitization leading to CH..
0969
0972
PERIPHERAL BLOOD T CELLS FROM ATOPIC DERMATITIS PATIENT8 DEMON$TRATE INCREASED TRANSMIGRATION ACROSS MICROVA$CULAR ENDOTHELIUM. I.J=M. de Vries. A. Sleiiffers. F.C. van Rei[ann. E.F. Knol. T. Thepen, C,~.F.M BruiinzeeI-Koomen. Dept. of Dermatology and Pathology, University Hospilal Utrecht, The Nelhedands Skin lesions of palients with atopic dermatitis (AD) are characterized by the presence of activated T cells and increased expressi•n of adhesion molecules on endothelial cells (EC). We have examined the transmigration of T cells a¢ross a mono[ayer of human dennal microvascular endothelial celMine (HMEC-1) cultured on an extracellular matnx (ECM) of lysed human skin fibroblasts. Enhaßced transmigration of T cells fmm AD patients as ¢ompared to non-atopic conlrols was observed, both across non-stimulated EC and TNFa-stimulated EC. SeMctive depletion of activated T cells, by depletion of HLA-DR÷ T cells, annulled the differences between T eells from non-atopic individuals and T cells from patients with regard to Ihe transmigration aeross non-stimulated and resting EC. Possibly, increased expression of cutaneous lymphocyte-assoeiated antigen (CLA) on the activated T cells mediated this increased T cell migration. The ligand for CLA is E-selectin on activated endothelial cells. However, T cells of AD patients did not demonstrate inereased transmigtation after activation of the endothelial cells, excluding a role of E-selectin. Therefore, we hypothesize that the stale of activation of T teils, including the state of activation of their adhesion molecutes, in peripheral blood from AD patients may be the deten'nining factor for T cell transmigration, and might explain the high T cell numbers in skin of AD patients.
RAPID REDUCTION IN THE SIZE OF MOUSE CUTANEOUSMAST CELL POPULATIONS BY APOPTOS1S AFTER CESSATION OF TREATMENTWITH SCF DOES NOT RESULT IN SKIN INFLAMMATION. Marcus Maurer and SteDhen J. Galli. Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard MedicadSchool, Boston. MA, USA. Subcutaneous administration of stem teil factor (SCF), the ligand for the c-kitreceptor, to the back skin of mice promotes the local hyperplasia of cutaneous mast cells (MC). Cessation of treatment with SCF results in the rapid leduction of MC populafions by apoptosis. We used the ml-fibrin deposifion assay, a very sensitive methcd for quantifying thcreased vascular permeability, to assess whether the clearance of large numbers of apoptotic MCs is associated with a sigaificant inflammatory skin reaction. The subcutaneous injection of rrSCF[~ (30 or 100 ~tgtkg/day) or rrSCFlU-peg (pelyethylene glycol-treatedSCF, 30 or 1130gg/kg/day) for 21 days thcreased the number of MC at the site of injeetioa from 5.1+-0.7MClmm2 to 36,4±4.1, 34.7+_ 9.7, 52.5+-5.8. and 545±97 MC/mm:, respecfively. 48 hrs after cessation ofireatment with SCF, skin MCs underwem massive apoptosis, as ~sessed by light microscopy, and MC numbers dropped to 54+_12% of day 21 levels. Most notably, lzsl-fibrindepoaition during this rapid reducfion of skth MCs was virtuany unaJiemd as compared to mice receiving continuous treatment with SCF or vehicle. In the case of treatmem with rrSCFlU-pegat 100 ktg/kg/day,which tesulted in a more than 100-foldthcrease in skin MCs at the site of injection on day 21, eessation of SCF was foUowedby a decrease to one third of this number dufing the next two days, and the sldn of these rnice contained only slighfly more total and urea thsoluble (= cross-linked) epm ~2~I-fibrinthan skin of eontrol mice receiving continuous injeetions with rrSCFl~-peg (total epm: 171.1_+60.4 rs. 134.3-+37per biopsy, urea insoluble cpm: 41.4+13.1 vs, 31.4+0.5, respectively, differences not statistically significant). We conclude that the mpid eJiminahon of even large populations of MCs by apeptosis, which would also resuh in the cleatance of the coasiderable quantities of proinflammato~ products stored by these cells, does not lead to significant local cutaneous inflarmnatory responses.