- Email: [email protected]
Expression of cytochrome P450 3A5, 2C18, 2E1 and 1A1 in normal and immortalized human keratinocytes
ESDR I JSID I SID Abstracts
0553
0556
RESPONSES OF LANGERHANS CELLS TO HIGH AND LOW DOSES OF HAPTENS IN VIVO AND IN VITRO. Stefaao Baccl. PapleBnmaaoli and J. Wavne $4&j& Department ofHumanAnatomy and Histology, University of Florence, Italy, rnrl ____~__ c,.h-n. F”P P-n-h rnpti,,,tr -on Maw%+l,,Qe,,s. _,,” _,_ _.----_ _-____, _______.._-___--_.-. We have previously shown in mice that low doses of epicutaaeously applied haptens sensitize primarily through epidermal Langerhans cells (LC), whereas sensitization to
ALTERATION OF LANGERHANS CELL FUNCTION DURING THE EARLY STAGES OF CUTANEOUS CARCINOGENESIS CONFERS IMMUNOSUPPRESSION RATHER THAN IMMUNITY __ ., d^e Y..’ * Division mad Muii I. - ’ .r ii wo0g.s. of Pathology, University of Tasmania, Hobart, Australia Exposure of the skin to chemical carcinogens such as 7,12dimethylbenz(a)anthracene (DMBA) results in a massive migration of Langerhans cells (LC) to the draining lymph node. As a consequence the epidermis is depleted of LC and any new antigen applied through this LC depleted skin fails to induce an immune response, but instead initiates antigen specific tolerance. Analysis of the residual LC revealed that these cells fail to induce T cell proliferation, they preferentially cluster with CD8’ T cells and have a reduced ability to trap and transport antigen to the draining lymph node. Although T cells were not induced to proliferate, the LC draining DMBA treated skin were able to trigger T cell activation and produce the key costimulatory cytokine, IL-12 It is concluded that a signal was generated by these modified Langerhans cells that resulted in immune-suppression rather than immunity.
high doses is mediated largely by dermal antigen presenting cells. To better understand the effects of different doses of haptens on LC, we have analyzed these cells by immunohistochemistry and electron microscopy after painting dinitrofluombenzene (DNFB) on the skin and on skin explants of BALB/c mice. Both in viva and in vitro a
significant (pcO.01) decrease was detected in LC number 2 to 4 h after treatment. This was followed by recovery within 24 h after low doses of DNFB (1.5 pg) in viva and in vltm, but no recovery was seen after high doses (185 /zg). Moreover, heavy pamnuclear labeling was recognized after treatment in vitro; in these conditions the labeling of dendrites was reduced after big4 but not after low doses of kapten. In vitro, the effects of low doses of hapten were verified also with oxawlone (1 jag). a totally unrelated molecule. By electron microscopy 2 h after in viva treatment, the number of dendrites per dendritic cells was decreased after a high dose of DNFE4, but not after a low dose. A
many,
pwiy differentiated cells high dose of DNFB was followed by the appuvaooc of d dendritic lineage in the epidermis. We conclude that DNFB at high doses damages LC and leads to influx of precursom into the epidermis, which however fail to diffenntiate
within 24 h. On the contrary, low, sensitizing doses of DNFB spare the integrity of LC. Moreover, all doses of @tens lead to redistribution ofIa molecals to the cell interior, lpssibly as a mnsqaeace of the removal ofhapteniaed molecules from the all surface.
s93
0554 CONTRIBmION OF INTERLE”KINS la AND1p TOLANGERHANS CELL MIGRATION.M Cumberbatch. R J Dearman and I Kim&r, Zeneca Central Toxicology Laboratory, Macclesfield, Cheshire, UK. It has been shown previously that the migration of Langerhans cells (LC) from the epidermis and then- accumulation as dendritic cells DC1 in draining lymph nodes stimulated by exposure of mice to chemical allergens, skin irritants or ultraviolet B (WBJ radiation, is dependent upon local production of turnour necrosis factor a ITNF-a). We have reported recently, however, that LC require a second signal for the initiation of migration and that this is provided by interleukin (IL)-lp. Of interest is our observation that IL-la, a cytokine expressed constitutively by keratinocytes, is able to co-operate with TX-a to regulate LC migration. Since IL-la is normally released from cells only as a res"lt of cell damage, we have investigated the conditions under which IL-la may participate in this response. We report that prior systemic treatment of mice with a neutralizing ant=-IL-la antibody, under conditions where this antibody failed to influence allergen-induced DC accumulation ~oxazolane; 0.5%). inbzhlbited totally DC accumulation induced by exposure to the skin irritant sodium lauryl sulphate (10%). These data suggest that although IL-1D may be considered essential for the induction of skin sensitization, IL-la may be of more relevance for the development of irritant responses.
0555
0558 EXPRESSION OF CYTOCHROME P450 3A5, 2Cl8, 2El AND IA1 IN NORMAL AND IMMORTALIZED HUtvfAN KERATMOCYTES. Markus Baur, Axelle Hertz&en, Katherine Mace, Armand MaIn&, Andrea M.A. fff, Department of Life Sciences, Nestlt Research Center, Lausanne, Gwitzerland. To develop a standardized in vitro skin model for the study of xenobiotic .wn+.l.~li-“” a...,.,-Ou ‘,“YL”Y.YYL& ..1~,,‘,“1 .I.,& l”lll 0I w ,..*,.^l...mn .,,&“uL”“‘L.urnul yy-‘u IrvDd&“I^_11” CC,,D were isolated and immortalized in a new serum-f%. culture medium on coated culture plates. Human keratinocyte lines (DK-NR, FKZ-NR) were established by infection of normal cells with a recombinant retroviral construct containing the Simian virus 40 large T-antigen. In this culture system we found mRNA expression of the CYPlAl, CYP2C18, CYF’2El and CYP3A5 as detected by RT-PCR. The CYP3A5 mRNA expression in normal and immortalized human keratinocytes was shown for the first time in this study. CYP450 IA2, 2A6,286 and 2D6 were not detectable by RTPCR. The mRNA expression level of the immortalized keratinocyte lines was similar to the normal keratinocytes. The keratinocyte lines showed a normal response to the monooxygenase inducer benzo(a)pyrene, by an increase of the CYF’IAI mRNA expression and the CYPIAI activity. Thus, these keratinocyte lines retain many characteristics of their normal counterparts and present a valuable and predictive in vitro model to study the effects of xenobiotics on the CYP450 metabolism in human keratinocytes.
0553
0556
RESPONSES OF LANGERHANS CELLS TO HIGH AND LOW DOSES OF HAPTENS IN VIVO AND IN VITRO. Stefaao Baccl. PapleBnmaaoli and J. Wavne $4&j& Department ofHumanAnatomy and Histology, University of Florence, Italy, rnrl ____~__ c,.h-n. F”P P-n-h rnpti,,,tr -on Maw%+l,,Qe,,s. _,,” _,_ _.----_ _-____, _______.._-___--_.-. We have previously shown in mice that low doses of epicutaaeously applied haptens sensitize primarily through epidermal Langerhans cells (LC), whereas sensitization to
ALTERATION OF LANGERHANS CELL FUNCTION DURING THE EARLY STAGES OF CUTANEOUS CARCINOGENESIS CONFERS IMMUNOSUPPRESSION RATHER THAN IMMUNITY __ ., d^e Y..’ * Division mad Muii I. - ’ .r ii wo0g.s. of Pathology, University of Tasmania, Hobart, Australia Exposure of the skin to chemical carcinogens such as 7,12dimethylbenz(a)anthracene (DMBA) results in a massive migration of Langerhans cells (LC) to the draining lymph node. As a consequence the epidermis is depleted of LC and any new antigen applied through this LC depleted skin fails to induce an immune response, but instead initiates antigen specific tolerance. Analysis of the residual LC revealed that these cells fail to induce T cell proliferation, they preferentially cluster with CD8’ T cells and have a reduced ability to trap and transport antigen to the draining lymph node. Although T cells were not induced to proliferate, the LC draining DMBA treated skin were able to trigger T cell activation and produce the key costimulatory cytokine, IL-12 It is concluded that a signal was generated by these modified Langerhans cells that resulted in immune-suppression rather than immunity.
high doses is mediated largely by dermal antigen presenting cells. To better understand the effects of different doses of haptens on LC, we have analyzed these cells by immunohistochemistry and electron microscopy after painting dinitrofluombenzene (DNFB) on the skin and on skin explants of BALB/c mice. Both in viva and in vitro a
significant (pcO.01) decrease was detected in LC number 2 to 4 h after treatment. This was followed by recovery within 24 h after low doses of DNFB (1.5 pg) in viva and in vltm, but no recovery was seen after high doses (185 /zg). Moreover, heavy pamnuclear labeling was recognized after treatment in vitro; in these conditions the labeling of dendrites was reduced after big4 but not after low doses of kapten. In vitro, the effects of low doses of hapten were verified also with oxawlone (1 jag). a totally unrelated molecule. By electron microscopy 2 h after in viva treatment, the number of dendrites per dendritic cells was decreased after a high dose of DNFE4, but not after a low dose. A
many,
pwiy differentiated cells high dose of DNFB was followed by the appuvaooc of d dendritic lineage in the epidermis. We conclude that DNFB at high doses damages LC and leads to influx of precursom into the epidermis, which however fail to diffenntiate
within 24 h. On the contrary, low, sensitizing doses of DNFB spare the integrity of LC. Moreover, all doses of @tens lead to redistribution ofIa molecals to the cell interior, lpssibly as a mnsqaeace of the removal ofhapteniaed molecules from the all surface.
s93
0554 CONTRIBmION OF INTERLE”KINS la AND1p TOLANGERHANS CELL MIGRATION.M Cumberbatch. R J Dearman and I Kim&r, Zeneca Central Toxicology Laboratory, Macclesfield, Cheshire, UK. It has been shown previously that the migration of Langerhans cells (LC) from the epidermis and then- accumulation as dendritic cells DC1 in draining lymph nodes stimulated by exposure of mice to chemical allergens, skin irritants or ultraviolet B (WBJ radiation, is dependent upon local production of turnour necrosis factor a ITNF-a). We have reported recently, however, that LC require a second signal for the initiation of migration and that this is provided by interleukin (IL)-lp. Of interest is our observation that IL-la, a cytokine expressed constitutively by keratinocytes, is able to co-operate with TX-a to regulate LC migration. Since IL-la is normally released from cells only as a res"lt of cell damage, we have investigated the conditions under which IL-la may participate in this response. We report that prior systemic treatment of mice with a neutralizing ant=-IL-la antibody, under conditions where this antibody failed to influence allergen-induced DC accumulation ~oxazolane; 0.5%). inbzhlbited totally DC accumulation induced by exposure to the skin irritant sodium lauryl sulphate (10%). These data suggest that although IL-1D may be considered essential for the induction of skin sensitization, IL-la may be of more relevance for the development of irritant responses.
0555
0558 EXPRESSION OF CYTOCHROME P450 3A5, 2Cl8, 2El AND IA1 IN NORMAL AND IMMORTALIZED HUtvfAN KERATMOCYTES. Markus Baur, Axelle Hertz&en, Katherine Mace, Armand MaIn&, Andrea M.A. fff, Department of Life Sciences, Nestlt Research Center, Lausanne, Gwitzerland. To develop a standardized in vitro skin model for the study of xenobiotic .wn+.l.~li-“” a...,.,-Ou ‘,“YL”Y.YYL& ..1~,,‘,“1 .I.,& l”lll 0I w ,..*,.^l...mn .,,&“uL”“‘L.urnul yy-‘u IrvDd