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The production of cytokines from peripheral blood mononuclear cells from patients with atopic dermatitis before and after treatment
E S D R / J S I D / SID Abstracts
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EFFECT OF EX-VIVO ULTRAVIOLET B [RRADIATION ON THE PHENOTYPE OF MIGRATING LANGERHANS CELLS DURING SKIN ORGAN CULTURE Satoshi Nakagawa, Jan D. Bos, and Marcel B.M. Teunissen. Departmeut of Dermatology, Academic Medical Center, University of Amsterdam, The Netherlands. Ultraviolet B (UVB) irradiation of the skin causes immunosuppression or antigenspecific tolerance in whicb Langerhans cells (LC) are considered to play a role. In this study, we tested the effect of UVB irradiation of skin explants on the phenotype of LC that migrated out of skin explants after skin organ culture. The 0.2nun-thick skin shaved by keratome was irradiated with a single variable dose (400 to 1600 J/riß) of UVB. Epidermal sheets were prepared by incubating the skin in 0.3 % dispase and separating epidermis from dermis. The sheets were cultured for 18, 42, 66 h, and the migrating cells were collected. HLA-DR ÷ eells were analyzed by flow cytometry for the co-expression of costimulatory molecules. The percentage of B7-1, B7-2, ICAM-l-positive eells from the total HLA-DR ÷ eells after overnight culture was decreased as the dose of UVB became higher, whereas that of CD la and ICAM-3 was comparable to the unirradiatod control. At low doses of irradiation, the rnean fluorescence intensity (MFI) of all these antigens showed no significant change, except for CDla whieh becarne higher by UVB. However, the highest dose of UVB (1600 J/m~ decreased the MFI of all markers as compamd to the unirradiated corttrol exeept for CDIa. There was a sub-populatioe of LC in the irradiated groups which could upregulato all costimulatory moleeules as weil as HLA-DR, equally to or eren slightly higher than controls in a time-dependent manner. These results suggest the poasibility of dual action of UVB on LC: orte enhancing the upregulation of their immunomactive molecules during their maturation upon low dose exposure, the other inhibitiag their matoration by a high dose which may lead to immunosuppression.
EXPRESSION OF PGP9.5 BY NON INNERVATED LANGERHANS CELLS: A NEURONAL PROPERTY ? Alaln Gaudillère 1. Odile Sabido2. Daniel Schmitt I , Alain Claudv I . Lanmnt Mi%~rvI , INSERM U346 and Department of Dermatology 1, Edouard Hen'im Hospital, Lyon; Flow Cytomem] Center 2, Faculty of Medicine, Saim Etienne, France. PGP9.5 (protein gene product 9.5), an ubiquitin carboxyl terminal hydrolase, is a specific marker of nerve cells. We have previously shown that Langerhans cells wem closely associated to PGP9.5 nerve fibers in the epidermis but did not express PGP9.5 (Br J Dermatol 1996, 135, 343-344). Hsieh (J Neurocytol 1996, 25, 513-524) has demonstrated that epidermal Langerhans cells expressed PGP9.5 after denervation. We hypothesized that Langerhans eells extracted from the epidermis could be able to synthesizc PGP9.5. Epidermal cell suspensions wem obtalned from normal human skin samples, through the action of trypsine. They wem enriched in Langerhans cells by suceessive density gradient centrifugations, using FicolI-Hypaque medium. We have incubated these cells with anti-PGP9.5 antibody (Ultraelone), and stainings were analyzed by flow cytometry. We have noted PGP9.5 expression in Langerhans cells enriched suspensions. A part of CDla+ cells (Langerhans cellls) wem stained by anti-PGPg.5 serum whereas no CDlacell was recognized by this antibody.The staining was cytoplasmic. Paul Langerhans (1868) thought that the cells he dcscribed were nervous cells. Langerhans cells are immune cells hut share pmperties with celts of the nervous system, like the expression of PGP9.5 or neuromediators such as POMC (pm-opio-melanocortieotropine) derived-peptides, S100 protein, and neuron-specific enolase. The function of PGP 9.5 expressed in Langerhans cells remains unknown.
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QUANTITATIVE ANALySIS "AT THE SINGLE CELL LEVEL" OF THE NOVEL CD28CDllb- SUBPOPULATIONOF CDS+ T LYMPHOCYTES. A. Lonati rD. S. Licenziati (2). M. Marcelli (1), D. Canaris (2). G. Pa~olini(Il A. Ca~so (2), G. De Pardilis (1). Departmcnts of (1) Dermatologyand Of(2) Vitolos~,Brcscia, Italy. Accordingto reccnt evidences,the rom played in the skin by CDS+ T Iymphecytes, although not yet fully elucidate~ is likely to be impottant. We theve[ore plaaned to investigate CD8+ T cell subpopulafionsexffacted ftom nccmal and pathologicalhuraan skin. Prelindnaty to Iltis, howevcr, we intended to stmty CDS+ T cell subpopulations~ in normal human peripheral blcod (PB), hecause (i) even these ale rar from belag fully understood, and (il) they were up to now generally investigated using teclmiquesother than "at the single ceU level"; in such a way, the CDS+ T cell population had been previouslysplit into a CD28÷ T cell subpopulation(¢lassifiedas ~c/totoxic") as opposed to a CDllb+ subpopulatäon (¢tassißed as ~suppressof'). For such a lmrpose, PB from 15 healthy volunleerswas subjectedto a thlee-colorFACS analysis. It was thus possible, first, to confinn the existence in PB of the two classicalsubpolmlationsof CDS+ T cells, namely CD8+ CD28+ CDIIb- and CDS+ CD28- CDllb+. Intriguingly, moreover, a third CDS+ subset was discovered in the I?Bofal! the volunteers,shewingthe pheùotype CDS+ CD28- CD! Ib-. Such a subset, ranging from 1.5 % to 4.4 % (2.67 :h 1.32 %: mean ± SD) of CD8+ teils, had previouslyheen not ),et r¢coglüzed, although preliminarilypomulated in out laboratery by usmg "panning" techniques. Altheugh vistually all the CDS+ T cells were recetaly shown to exert cytolyticabifity, the speöialfun¢tioanltole(s), if any, of the current CDS+ CD28- CDI lb- subset of T lymphoGTtesis not known at the present. These CD25- CDIIb- cells, similar to other PB teil sul~ets showing unstable phenotype, nüght represem, indeed, a Iess acfivatcd or matote stute in comparison to the mole matore CDS+ CD28+ and CDS+ CDllb+ subsets. In conclusion, the previovslyundcscn%edsubset CD8+ CD28- CD1lb- of T lymphecyteswas detected "at the single cell I¢v¢1"in normal human PB, and its possible presence and functional rom in skin may be important for the homeostasisOfthe cutaneous immunesystemand for cutaneous pathophysiology.
THE INTERACn~ON OF NI 2+ WITH HU,Ig[AN DENDRITIC ~ E L L S L¢na C. Heffier. Leon T. Van den Broeke'". J. Lars G. N i l s s o n , Ann-Therese Karlber~ and ~ Schevnins'. 'Dept. o f Laboratory Medicin~e Div. o f Clinical Immunology, Katolinska Institute and Hospital; Dept. o f Occupational Heallfi, Occupaüonal Dermatology, National Insütute for Wor ~[dng Life, Stoekholm, Sweden. Ni ÷ allergy is most probably inifiated by the formation o f specific coordination 2complexas ~vith ¢lectron-rieh residues o f proteins. A dir¢et anfi~enic N i +-protein interaction on antigen presenúng cells (APCs) has previously beefi suggested, bat experimental evidence are l ~ t e d . In iltis study, we investigat~d the possible ~ r e c t antuen formation o f N i + on cultured human dendritic cells OgCs): DCs from N i " conta¢t allergie subjects (n=10) ùand healm~ control subjects (n=6) were obtained by culturing the adherent cell traction oz p¢ripheral blood mononucleax celIs (PBMCs) with G M - C S F and II<.~ for 7 days. The DCs displayed a typical DC phenotype as detemüned wim immunocytochemislry and induced a vigorous allogenic lymphocyte response measured b v [ m e t h y l - a H ] - t h y m i d i n e i n c o r p o r a t i o n . DCs w e m puIsed for 20 min with i~i2+ (50 ~tM) in Hank's balanced salt solution CABSS) and added to fteshly prepared auto.[9~gous responder PBMCs. In five allergic subjects, DCs pre-incubated with Ni "~ showcd a signißcant capacity to induce cel[ proliferaUon whereas no increas¢ was o ~ e r v e d In the remaining five patients or the con~ol subjects. Addition o f Ni + to PBMCs gave rise to cell proliferafion in all allergie hut not In control subjects. In conclusion out rasults indicate that direct formation o f the anfigcmc determinant o f Ni ~* on APCs is possible. The observed differenees_in allergie subjects in the ease o f activation o f antigen specißc T-cells by Ni2+~-pulsed DCs, is might be due to the heterogeneity m the molecular basis o f Ni ~ anügcn recogniiion.
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THE PRODUCTION OF CYTOKINES FROM PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH ATOPtC DERMATITIS BEFORE AND AFTER TREATMENT Sakae Kaneko. O s a m u Koro. Kivoshi Furutani, Shoso Y a m a m o t o D e p a r t m e n t of Dermatology, H i r o s h i m a U n i v e r s i t y Schoo[ of medicine, Hiroshima, 734, J a p a n It is well known t h a t many cytokines are involved in the p a t h o g e n e s i s ofatopic d e r m a t i t i s (,4/)). To u n d e r s t a n d the re[ationship between clinical severity and the capacity of peripheral blood mononuclear cells to produce cytokines, we have determined production of cytokines from p e r i p h e r a l blood mononuclear cells from p a t i e n t s with AD in vitro before and after successfu! t r e a t m e n t . The production of IFN¥ and IL-12 w a s significantly increased after 2 week's t r e a t m e n t (!o<0.005). On the other hand, the production of IL-10 significantly decreased after 2 week's and 4 week's t r e a t m e n t (p<0.01). The production of IL-4 did not significantly change after t r e a t m e n t . These d a t a suggested t h a t T cells from p a t i e n t s w i t h active i n f l a m m a t i o n of atopic d e r m a t i t i s were predominately of the Th2 type and also s u g g e s t e d t h a t the function of these T cells was possibly affected by the condition of skin i n f l a m m a t i o n .
TI/MOUR NECROSIS FACTOR ~ MODULATES HUMAN EPIDERgL%L LANGEREANS CELL FREQU~NCY. CEM Griffiths. M cumberbatch ". S Tucker. RJ Dearman* and I Kirnber" Section of Dermatology, University of Manchester, Hope Hospital, Salford, UK and "Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, UK. We have reported previously that tumour necrosis factor (T~F-~), an inducible product of keratinocytes, provides one important signal for the initiation of Langerhans cell (LC) m i g r a t i o n in mice. In the present series of experiments we have q~/estioned whether TNF-~ provides a signal for the stimulation of LC migration in humans. Seven volunteers (2 male, 5 female) were exposed intradermally to 500U of human recombinant T N F - ~ diluted in s~erile saline (50Bl) and at a distant site to saline alone. The injection sites were removed 2 hours later hy 6mm punch b i o p s y and the frequency of CDIa + LC assessed following immunofluorescence staining of epidermal sheets. Under conditions where intradermal administration to humans of TNF-~ caused no local or systemic side effects, we observed a significant reduction (24.7%) in LC density from a mean of 1073+51 LC/mm 2 in control biopsies to 808_+45 LC/mm 2 following TNF-~ treatment (mean_+SE of counts derived from the 7 volunteers, p<0.005; Students' 2-sided t-test). These results demonstrate that TNF-~ provides a signal for LC migration in humans and that local production of this cytokine m a y represent an early event in the development of c u t a n e o u s immune responses.
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EFFECT OF EX-VIVO ULTRAVIOLET B [RRADIATION ON THE PHENOTYPE OF MIGRATING LANGERHANS CELLS DURING SKIN ORGAN CULTURE Satoshi Nakagawa, Jan D. Bos, and Marcel B.M. Teunissen. Departmeut of Dermatology, Academic Medical Center, University of Amsterdam, The Netherlands. Ultraviolet B (UVB) irradiation of the skin causes immunosuppression or antigenspecific tolerance in whicb Langerhans cells (LC) are considered to play a role. In this study, we tested the effect of UVB irradiation of skin explants on the phenotype of LC that migrated out of skin explants after skin organ culture. The 0.2nun-thick skin shaved by keratome was irradiated with a single variable dose (400 to 1600 J/riß) of UVB. Epidermal sheets were prepared by incubating the skin in 0.3 % dispase and separating epidermis from dermis. The sheets were cultured for 18, 42, 66 h, and the migrating cells were collected. HLA-DR ÷ eells were analyzed by flow cytometry for the co-expression of costimulatory molecules. The percentage of B7-1, B7-2, ICAM-l-positive eells from the total HLA-DR ÷ eells after overnight culture was decreased as the dose of UVB became higher, whereas that of CD la and ICAM-3 was comparable to the unirradiatod control. At low doses of irradiation, the rnean fluorescence intensity (MFI) of all these antigens showed no significant change, except for CDla whieh becarne higher by UVB. However, the highest dose of UVB (1600 J/m~ decreased the MFI of all markers as compamd to the unirradiated corttrol exeept for CDIa. There was a sub-populatioe of LC in the irradiated groups which could upregulato all costimulatory moleeules as weil as HLA-DR, equally to or eren slightly higher than controls in a time-dependent manner. These results suggest the poasibility of dual action of UVB on LC: orte enhancing the upregulation of their immunomactive molecules during their maturation upon low dose exposure, the other inhibitiag their matoration by a high dose which may lead to immunosuppression.
EXPRESSION OF PGP9.5 BY NON INNERVATED LANGERHANS CELLS: A NEURONAL PROPERTY ? Alaln Gaudillère 1. Odile Sabido2. Daniel Schmitt I , Alain Claudv I . Lanmnt Mi%~rvI , INSERM U346 and Department of Dermatology 1, Edouard Hen'im Hospital, Lyon; Flow Cytomem] Center 2, Faculty of Medicine, Saim Etienne, France. PGP9.5 (protein gene product 9.5), an ubiquitin carboxyl terminal hydrolase, is a specific marker of nerve cells. We have previously shown that Langerhans cells wem closely associated to PGP9.5 nerve fibers in the epidermis but did not express PGP9.5 (Br J Dermatol 1996, 135, 343-344). Hsieh (J Neurocytol 1996, 25, 513-524) has demonstrated that epidermal Langerhans cells expressed PGP9.5 after denervation. We hypothesized that Langerhans eells extracted from the epidermis could be able to synthesizc PGP9.5. Epidermal cell suspensions wem obtalned from normal human skin samples, through the action of trypsine. They wem enriched in Langerhans cells by suceessive density gradient centrifugations, using FicolI-Hypaque medium. We have incubated these cells with anti-PGP9.5 antibody (Ultraelone), and stainings were analyzed by flow cytometry. We have noted PGP9.5 expression in Langerhans cells enriched suspensions. A part of CDla+ cells (Langerhans cellls) wem stained by anti-PGPg.5 serum whereas no CDlacell was recognized by this antibody.The staining was cytoplasmic. Paul Langerhans (1868) thought that the cells he dcscribed were nervous cells. Langerhans cells are immune cells hut share pmperties with celts of the nervous system, like the expression of PGP9.5 or neuromediators such as POMC (pm-opio-melanocortieotropine) derived-peptides, S100 protein, and neuron-specific enolase. The function of PGP 9.5 expressed in Langerhans cells remains unknown.
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QUANTITATIVE ANALySIS "AT THE SINGLE CELL LEVEL" OF THE NOVEL CD28CDllb- SUBPOPULATIONOF CDS+ T LYMPHOCYTES. A. Lonati rD. S. Licenziati (2). M. Marcelli (1), D. Canaris (2). G. Pa~olini(Il A. Ca~so (2), G. De Pardilis (1). Departmcnts of (1) Dermatologyand Of(2) Vitolos~,Brcscia, Italy. Accordingto reccnt evidences,the rom played in the skin by CDS+ T Iymphecytes, although not yet fully elucidate~ is likely to be impottant. We theve[ore plaaned to investigate CD8+ T cell subpopulafionsexffacted ftom nccmal and pathologicalhuraan skin. Prelindnaty to Iltis, howevcr, we intended to stmty CDS+ T cell subpopulations~ in normal human peripheral blcod (PB), hecause (i) even these ale rar from belag fully understood, and (il) they were up to now generally investigated using teclmiquesother than "at the single ceU level"; in such a way, the CDS+ T cell population had been previouslysplit into a CD28÷ T cell subpopulation(¢lassifiedas ~c/totoxic") as opposed to a CDllb+ subpopulatäon (¢tassißed as ~suppressof'). For such a lmrpose, PB from 15 healthy volunleerswas subjectedto a thlee-colorFACS analysis. It was thus possible, first, to confinn the existence in PB of the two classicalsubpolmlationsof CDS+ T cells, namely CD8+ CD28+ CDIIb- and CDS+ CD28- CDllb+. Intriguingly, moreover, a third CDS+ subset was discovered in the I?Bofal! the volunteers,shewingthe pheùotype CDS+ CD28- CD! Ib-. Such a subset, ranging from 1.5 % to 4.4 % (2.67 :h 1.32 %: mean ± SD) of CD8+ teils, had previouslyheen not ),et r¢coglüzed, although preliminarilypomulated in out laboratery by usmg "panning" techniques. Altheugh vistually all the CDS+ T cells were recetaly shown to exert cytolyticabifity, the speöialfun¢tioanltole(s), if any, of the current CDS+ CD28- CDI lb- subset of T lymphoGTtesis not known at the present. These CD25- CDIIb- cells, similar to other PB teil sul~ets showing unstable phenotype, nüght represem, indeed, a Iess acfivatcd or matote stute in comparison to the mole matore CDS+ CD28+ and CDS+ CDllb+ subsets. In conclusion, the previovslyundcscn%edsubset CD8+ CD28- CD1lb- of T lymphecyteswas detected "at the single cell I¢v¢1"in normal human PB, and its possible presence and functional rom in skin may be important for the homeostasisOfthe cutaneous immunesystemand for cutaneous pathophysiology.
THE INTERACn~ON OF NI 2+ WITH HU,Ig[AN DENDRITIC ~ E L L S L¢na C. Heffier. Leon T. Van den Broeke'". J. Lars G. N i l s s o n , Ann-Therese Karlber~ and ~ Schevnins'. 'Dept. o f Laboratory Medicin~e Div. o f Clinical Immunology, Katolinska Institute and Hospital; Dept. o f Occupational Heallfi, Occupaüonal Dermatology, National Insütute for Wor ~[dng Life, Stoekholm, Sweden. Ni ÷ allergy is most probably inifiated by the formation o f specific coordination 2complexas ~vith ¢lectron-rieh residues o f proteins. A dir¢et anfi~enic N i +-protein interaction on antigen presenúng cells (APCs) has previously beefi suggested, bat experimental evidence are l ~ t e d . In iltis study, we investigat~d the possible ~ r e c t antuen formation o f N i + on cultured human dendritic cells OgCs): DCs from N i " conta¢t allergie subjects (n=10) ùand healm~ control subjects (n=6) were obtained by culturing the adherent cell traction oz p¢ripheral blood mononucleax celIs (PBMCs) with G M - C S F and II<.~ for 7 days. The DCs displayed a typical DC phenotype as detemüned wim immunocytochemislry and induced a vigorous allogenic lymphocyte response measured b v [ m e t h y l - a H ] - t h y m i d i n e i n c o r p o r a t i o n . DCs w e m puIsed for 20 min with i~i2+ (50 ~tM) in Hank's balanced salt solution CABSS) and added to fteshly prepared auto.[9~gous responder PBMCs. In five allergic subjects, DCs pre-incubated with Ni "~ showcd a signißcant capacity to induce cel[ proliferaUon whereas no increas¢ was o ~ e r v e d In the remaining five patients or the con~ol subjects. Addition o f Ni + to PBMCs gave rise to cell proliferafion in all allergie hut not In control subjects. In conclusion out rasults indicate that direct formation o f the anfigcmc determinant o f Ni ~* on APCs is possible. The observed differenees_in allergie subjects in the ease o f activation o f antigen specißc T-cells by Ni2+~-pulsed DCs, is might be due to the heterogeneity m the molecular basis o f Ni ~ anügcn recogniiion.
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THE PRODUCTION OF CYTOKINES FROM PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH ATOPtC DERMATITIS BEFORE AND AFTER TREATMENT Sakae Kaneko. O s a m u Koro. Kivoshi Furutani, Shoso Y a m a m o t o D e p a r t m e n t of Dermatology, H i r o s h i m a U n i v e r s i t y Schoo[ of medicine, Hiroshima, 734, J a p a n It is well known t h a t many cytokines are involved in the p a t h o g e n e s i s ofatopic d e r m a t i t i s (,4/)). To u n d e r s t a n d the re[ationship between clinical severity and the capacity of peripheral blood mononuclear cells to produce cytokines, we have determined production of cytokines from p e r i p h e r a l blood mononuclear cells from p a t i e n t s with AD in vitro before and after successfu! t r e a t m e n t . The production of IFN¥ and IL-12 w a s significantly increased after 2 week's t r e a t m e n t (!o<0.005). On the other hand, the production of IL-10 significantly decreased after 2 week's and 4 week's t r e a t m e n t (p<0.01). The production of IL-4 did not significantly change after t r e a t m e n t . These d a t a suggested t h a t T cells from p a t i e n t s w i t h active i n f l a m m a t i o n of atopic d e r m a t i t i s were predominately of the Th2 type and also s u g g e s t e d t h a t the function of these T cells was possibly affected by the condition of skin i n f l a m m a t i o n .
TI/MOUR NECROSIS FACTOR ~ MODULATES HUMAN EPIDERgL%L LANGEREANS CELL FREQU~NCY. CEM Griffiths. M cumberbatch ". S Tucker. RJ Dearman* and I Kirnber" Section of Dermatology, University of Manchester, Hope Hospital, Salford, UK and "Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, UK. We have reported previously that tumour necrosis factor (T~F-~), an inducible product of keratinocytes, provides one important signal for the initiation of Langerhans cell (LC) m i g r a t i o n in mice. In the present series of experiments we have q~/estioned whether TNF-~ provides a signal for the stimulation of LC migration in humans. Seven volunteers (2 male, 5 female) were exposed intradermally to 500U of human recombinant T N F - ~ diluted in s~erile saline (50Bl) and at a distant site to saline alone. The injection sites were removed 2 hours later hy 6mm punch b i o p s y and the frequency of CDIa + LC assessed following immunofluorescence staining of epidermal sheets. Under conditions where intradermal administration to humans of TNF-~ caused no local or systemic side effects, we observed a significant reduction (24.7%) in LC density from a mean of 1073+51 LC/mm 2 in control biopsies to 808_+45 LC/mm 2 following TNF-~ treatment (mean_+SE of counts derived from the 7 volunteers, p<0.005; Students' 2-sided t-test). These results demonstrate that TNF-~ provides a signal for LC migration in humans and that local production of this cytokine m a y represent an early event in the development of c u t a n e o u s immune responses.