Effects of Ultraviolet B Radiation on In Vitro Hapten T-cell Sensitization by Human Langerhans Cells

Toxicology in Vitro 12 (1998) 343±351

E€ects of Ultraviolet B Radiation on In Vitro Hapten T-cell Sensitization by Human Langerhans Cells 1

P. COURTELLEMONT1, F. RATTIS1, D. SCHMITT1 and J. PEGUET-NAVARRO2*

Laboratoire Peau Humaine et ImmuniteÂ, INSERM U346, HoÃpital E. Herriot, Lyon and 2Centre de Recherche PCD, Saint Jean de Braye, France (Accepted 7 December 1997)

AbstractÐThe deleterious e€ects of ultraviolet B radiation (UVB) on the antigen-presenting function of human epidermal Langerhans cells (LC) were studied by using the in vitro primary and secondary Tcell proliferative responses to the trinitrophenyl hapten (TNP) modi®ed autologous LC. Increasing doses of UVB radiation (100±200 J/m2) induced a dose dependent inhibition of the primary and secondary TNP-speci®c T cell response. However, this decreased T-cell proliferative response after UVB radiation, was strongly enhanced when freshly isolated LC, as compared with cultured LC, were used as antigen-presenting cells (APC), suggesting an impaired development of LC accessory function. Moreover, the exogenous addition of IL1b, TNFa, IL10 or their speci®c monoclonal antibodies neither modi®ed nor reversed the immunosuppressive e€ect of UVB radiation. Even if the low doses of UVB radiation (100 and 200 J/m2) seemed to slightly a€ect HLA-DR synthesis, the antigen-presenting function of human LC cannot be related to the decreased expression of these molecules but might be associated with an impaired development of accessory molecules such as a downregulation of B7-2 antigen. # 1998 Elsevier Science Ltd. All rights reserved Abbreviations: AMELR = autologous mixed epidermal cell lymphocyte reaction; APC = antigenpresenting cells; CHSR = contact hypersensitivity reactions; FITC = ¯uorescein isothiocyanate; HBSS = Hanks' balanced salt solutions; LC = Langerhans cells; cLC = cultured Langerhans cells; fLC = freshly isolated Langerhans cells; mAb = monoclonal antibody; MELR = mixed epidermal cell lymphocyte reaction; MFI = mean ¯uorescence intensity; PBMC = peripheral blood mononuclear cells; PBS = phosphate bu€ered saline; PE = phycoerythrin; SRBC = sheep red blood cells; TNBS = 2,4,6-trinitrobenzene sulfonic acid; TNP = trinitrophenyl; TNP-cLC = TNP-modi®ed 2-day cultured Langerhans cells; TNP-fLC = TNP-modi®ed fresh Langerhans cells; UVB = ultraviolet B.

INTRODUCTION

The immunosuppressive properties of ultraviolet B radiation (UVB) have been described for several years through in vivo studies carried out both in mice (Kripke, 1974) and in humans (Baadsgaard et al., 1990; Mommaas et al., 1993). These experiments demonstrated both local and systemic immunosuppression related to the deleterious e€ects of UVB radiation. Local tolerance was characterized by an inhibition of the contact hypersensitivity reactions (CHSR) at the irradiated site (Toews et al., 1980) whereas distant alterations of immune response occurred at unexposed radiation sites (Noonan et al., 1981). Skin-mediated immune responses are mostly dependent on the presence of Langerhans cells (LC) *Author for correspondence.

in the epidermis, and consequently this epidermal cell appeared to be the main target of UVB radiation. The involved mechanisms studied in mice included both direct e€ects of UVB radiation on LC-antigen-presenting function (Simon et al., 1992) and indirect e€ects related to the presence of soluble immunosuppressive factors secreted by keratinocytes (Rivas and Ullrich, 1992). In vitro experiments have been performed on mixed epidermal cell lymphocyte reaction (MELR) to assess the inhibitory e€ects of UVB radiation on LC allostimulatory function (Rattis et al., 1995) and, ®nally, an impaired development of LC accessory function has been proposed as a possible mechanism. Recently, we have shown that 2-day cultured LC (cLC), but not freshly isolated LC (fLC) can induce in vitro primary T-cell response to the trinitrophenyl hapten. The proliferative T-cell response was hapten speci®c, as shown in a second-

0887-2333/98/$19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(97)00009-5

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ary reaction by an accelerated response to TNP and by the absence of proliferation to an irrelevant hapten such as ¯uorescein isothiocyanate (FITC) (Moulon et al., 1993). The present study used this autologous MELR (AMELR) to investigate the mechanism by which UVB radiation can induce an inhibition of in vitro primary and secondary haptenspeci®c autologous T-cell proliferative response to trinitrophenyl (TNP).

which were incubated for 10 min at 378C and then washed extensively before use as modi®ed APC in T-cell proliferation assays. T lymphocyte isolation

The following human recombinant cytokines and their speci®c monoclonal antibodies were used: IL-1b (100 U/ml; Genzyme, Cambridge, MA, USA) and monoclonal mouse anti-human IL-1b (10 mg/ ml; Genzyme), TNFa (50 U/ml; Genzyme) and monoclonal mouse anti-human TNFa (100 ng/ml; Genzyme), IL-10 (10 ng/ml; R&D Systems, Abingdon, UK) and monoclonal mouse anti-human IL-10 (10 mg/ml; R&D Systems).

Autologous T cells were isolated from the peripheral blood by Ficoll±Paque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. After washing and counting in HBSS, peripheral blood mononuclear cells (PBMC) were layered on petri dishes for 2 hr at 378C in complete RPMI medium. As previously described (Kaplan and Clark, 1974), non-adherent cells were harvested and T cells were obtained by rosetting with 2-aminoethylisothiouronium bromide hydrobromide-treated sheep red blood cells (SRBC). SRBC were eliminated by addition of ammonium chloride solution (8.7 g/ml, pH 7.7) and then washed twice. Residual accessory cells were eliminated by a second adherence step to plastic for 2 hr at 378C. At the end, T cells were suspended at 106 cells/ml in complete RPMI medium. The viability, as assessed by trypan blue dye exclusion, always exceeded 98%.

Langerhans cells-enriched epidermal cell suspensions

T cell proliferation assays

Epidermal cell suspensions were obtained from fresh normal human skin (plastic surgery) by trypsinization: 0.05% trypsin (Difco Laboratories, Detroit, MI, USA), 1 hr at 378C. These suspensions were enriched in LC by two consecutive densitygradient centrifugations. Dispersed epidermal cells were layered on Lymphoprep (Flobio, Courbevoie, France) and centrifuged for 30 min at 400 g. The cells from the interface were washed twice and resuspended in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% heatinactivated human AB serum, 10 mg gentamicin/ml, 1 mg indomethacin/ml and 2 mM L-glutamine. These LC were used either after isolation (fLC) or after a 2-day culture in complete medium supplemented with 200 U human recombinant granulocyte-macrophage colony-stimulating factor/ml (GM-CSF, Genzyme). After incubation, viable LC (cLC) were recovered by a last gradient centrifugation on Lymphoprep (Flobio) and in some experiments, highly enriched LC suspensions were obtained (pLC > 70%). A lower percentage of LC in epidermal cell suspensions has no incidence on the outcome of the proliferative T cell response.

T cell proliferative assays were carried out in Ubottomed microtitre plates by adding 105 T cells to 5  103 UVB-irradiated or non-irradiated TNPmodi®ed autologous fresh LC (TNP-fLC) or 2-day incubated LC (TNP-cLC). In some experiments, cytokines and their speci®c monoclonal antibodies were added to the wells at the time of culture initiation. After a 5-day incubation at 378C in 5% CO2, primary T cell proliferation was determined by addition of 1 mCi [3H]thymidine (1 mCi/ml, NEN products, Boston, USA) to each well for the ®nal 18 hr of culture. T cells were then harvested and the incorporated radioactivity was determined by a liquid scintillation counter (1450 Microbeta, Wallac, Finland). After 9 days of primary cultures, secondary cultures of in vitro primed T cells were carried out in 96-well microtitre plates. Viable T cells were recovered by centrifugation and restimulated (105 T cells) for 3 days with TNP-fLC or TNP-cLC (5  103 LC). T cell proliferation was assessed as described previously. Results were expressed as the mean cpm 2SD of triplicate cultures. Percentage of inhibition of control MELR was de®ned by the following ratio:

MATERIALS AND METHODS

Cytokines and antibodies

Test chemical The allergen chosen for the study was 2,4,6-trinitrobenzene sulfonic acid (TNBS; Sigma) known as a strong contact allergen. Modi®cation of antigenpresenting cells (APC) with the trinitrophenyl hapten was performed according to the method of Shearer (1974). Brie¯y, cell pellets were resuspended in Hanks' balanced salt solution (HBSS) (pH 7.2) containing 5 mM TNBS. Previous experiments showed that these doses were not toxic for the cells

…1 - cpm of MELR with irradiated LC=cpm MELR with sham ÿ irradiated LC†  100 UVB irradiation TNP-fLC or TNP-cLC were suspended in phosphate bu€er and layered in petri dishes. The UVB source (Bio Energie, Vilbert Lourmat, Marne la valleÂe, France) emitted at 312 nm. UVB irradiation

UVB irradiation of human LC alters in vitro hapten T-cell sensitization

was administered as a single dose. Sham-irradiated cells served as control. After UVB exposure (100 and 200 J/m2), cells were washed twice with HBSS without calcium and magnesium and containing 10% foetal calf serum, and enumerated. Cell viability was assessed by trypan blue exclusion test. FACS analysis For analysis of HLA-DR expression on freshly isolated or cultured human LC, cells were incubated with ¯uorescein isothiocyanate (FITC)-coupled human anti-HLA-DR mAb (Clone B8.12.2, Immunotech) for 30 min at 48C. Negative controls were performed using the appropriate irrelevant isotype-matched mAb (Dako, Denmark). ICAM1 expression on cLC was assessed by a double staining. Cells were ®rst incubated with the mouse mAb anti-human ICAM1 (Clone F4-31C2, Biocytex, France) and subsequently with FITC goat anti-mouse IgG (Becton Dickinson). Then, LC were labelled with phycoerythrin (PE)-coupled human anti-HLA-DR mAb (Clone B8.12.2; Immunotech) to only perform the analysis on HLA-DR positive population cells. All incubation steps were carried out for 30 min at 48C and cells were washed twice between successive steps with phosphate bu€ered saline (PBS) containing 10% bovine serum albumin. Cells labelled with FITC-conjugated secondary antibody alone were used as controls. FACS analysis 1 1 was performed on a COULTER Epics XL (Coultronics France). Quanti®cation assay Quanti®cation of HLA-DR and ICAM1 expression on Langerhans cells surface was performed with the quantitative immuno¯uorescence indirect assay (QIFI) (Poncelet and Carayon, 1985). 1 QIFIKIT (Dako, Trappes, France) contains a series of beads coated with di€erent well de®ned amounts of mouse monoclonal antibody molecules (high-anity anti-human CD5). The producer calibrated the beads by comparing the amount of CD5 molecules loaded on each beach to subclones of the CEM lymphoblastoid T-cell line representing a wide range of CD5 antigen density. After washing twice, beads were stained with 100 ml F(ab')2 fragment of FITC-conjugated goat anti-mouse immunoglobulins (1:50 diluted) for 45 min at 48C. After three washes with PBS-azide and two centrifugations at 300 g for 5 min, pellets were ®xed in 500 ml 1% paraformaldehyde in PBS for 2 hr. Parallel labelling and ®xation procedures were performed in the same conditions on LC. The standard regression line between ¯uorescence intensity and antigen density was established according to the mean ¯uorescence intensity (MFI) measured with the di€erent standard beads. The MFI of each sample of LC was corrected by subtraction the MFI of the negative control measured under the same conditions and converted into antigen density which indicates

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the mean number of cell surface markers expressed by cell. Statistical evaluation of results The statistical signi®cance of di€erences in the means of each experimental group was assessed by variance analysis (ANOVA). Mean di€erences were considered to be signi®cant when P < 0.05.

RESULTS

Autologous T-cell primary and secondary proliferative response after in vitro UVB radiation of either TNP-cLC or TNP-fLC In these experiments, we ®rst con®rmed that TNP-cLC can induce in vitro autologous human Tcell primary proliferative response. As shown in Fig. 1(a), cLC exposure to low doses of UVB radiation (100 and 200 J/m2) signi®cantly a€ected, in a dose-dependent manner, the induction of T cell primary response in AMELR. At 100 J/m2, the observed UVB-induced inhibition of T-cell proliferation was about 20% and increased to 40% at 200 J/m2. In the same way, we have compared the e€ects of UVB radiation on the secondary proliferative T cell response to fLC and cLC (Fig. 1b). As reported earlier (Moulon et al., 1993), cLC are far more potent APC than fLC in secondary T cell reactions. UVB exposure of fLC and cLC (100 and 200 J/m2) signi®cantly altered the T cell response. However, in AMELR using cLC the UVB-induced inhibition was 21% and 39% at 100 and 200 J/m2, respectively, whereas it reached 55% and 88% when fLC were used as APC. Consequently, cultured LC seems to be less sensitive than freshly isolated LC to UVB radiation. Several equivalent experiments have been performed with LC from di€erent donors, and whatever the obtained percentage of pLC after isolation, similar results have been observed. The kinetic pro®le of T cell secondary response to irradiated TNP-cLC con®rmed that UVBinduced inhibitory e€ect was not due merely to a shift in the maximal T cell response (Fig. 2). Furthermore, it should be noted that low UVB exposure of cLC before or after incubation with TNP did not alter the results, suggesting that UVB did not interfere with TNP processing by LC (data not shown). E€ect of exogenous epidermal cytokines, IL-1b, TNFa and IL-10 on the APC function of UVB irradiated LC Several authors have already described the enhanced production of cytokines such as IL-1, IL10 and TNFa, by human UVB irradiated keratinocytes (Enk et al., 1995; Garhing et al., 1984; KoÈck et al., 1990; Rivas and Ullrich, 1992; Schwarz and

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Fig. 1. UVB radiation inhibits the autologous T-cell primary proliferative response to human TNP-cLC (a) and the autologous T-cell secondary proliferative response to human TNP-cLC and TNP-fLC (b). Cultured LC appeared less sensitive than freshly prepared LC to the UVB deleterious e€ects. Fresh and cultured epidermal cell suspensions (30±35% LC) were exposed to a single dose of UVB radiation (312 nm) from 0 to 200 J/m2. Then, LC were treated with TNP, washed, and ®nally numerated. 5  103 viable TNP-cLC or TNP-fLC were cultured with 105 autologous naive T cells. Negative controls included T lymphocytes alone (LT) or T lymphocytes cultured with non-treated LC enriched epidermal cell suspension (EC + LT). T cell proliferation was assessed by [3H]thymidine incorporation for the ®nal 18-hr of culture. Results are expressed as the mean cpm2SD of triplicate cultures. Asterisks indicate signi®cant di€erence from control (non-irradiated) (*P < 0.05; ANOVA).

Luger, 1989) and these cytokines have been implicated in the UVB-induced immunosuppression (Rivas and Ullrich, 1994). Consequently, we investigated the ability of exogenous addition of these cytokines or their speci®c monoclonal antibodies to alter the observed UVB-induced immunosuppressive response in our in vitro model. As reported for allogeneic reactions (PeÂguet-Navarro et al., 1993), we found here that the addition of IL-1b (100 U/ml) induced a signi®cant increase of autologous T-cell proliferative response to TNP-cLC (Fig. 3a). However, this immunostimulation did not cancel the inhibition of T-cell secondary proliferative response induced by UVB-irradiated LC. The addition of monoclonal mouse anti-human IL-1b (10 mg/ml) induced a signi®cant decrease of the Tcell response in AMELR using either irradiated or non-irradiated cLC. Similar results were obtained (data not shown) in primary T cell responses using

cLC, or in secondary T cell responses using fLC as APC. These results con®rmed the immunostimulatory capacity of IL-1b and suggested the presence of this cytokine in the supernatant of AMELR either related to epidermal cells or the culture medium supplemented with human sera. As shown in Fig. 3(b), the addition of human recombinant TNFa as well as its speci®c antibody to the AMELR, produced neither a decrease of the T-cell proliferative response nor a reverse outcome of the UVB immunosuppressive e€ect. On the contrary, the same experiments carried out with IL-10, con®rmed the strong immunosuppressive capacity of this cytokine (Fig. 3c). However, the exogenous addition of anti-human IL-10 monoclonal antibody did not abrogate the immunosuppressive response related to UVB radiation, since the levels of inhibition were analogous to the control (around 24% and 46% for 100 and 200 J/m2, respectively).

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HLA-DR expression, measured in MFI on cLC, with a dose±response relationship (18% and 25% at 100 and 200 J/m2) (Fig. 4a). On the contrary, HLA-DR expression did not change on LC irradiation at the end of the incubation period (Fig. 4b). Similar results were obtained in ®ve other experiments. In contrast with previous studies performed in murine models (Tang and Udey, 1991), these experiments demonstrated that ICAM-1 expression on human 36-hr-cultured LC, was not altered by prior UVB irradiation (Fig. 5a). The estimated number of ICAM-1/cLC after a 36-hr incubation period was close to 1432 30  103 (Fig. 5b), and this con®rmed the low ICAM-1 expression on LC in comparison with the HLA-DR expression.

Fig. 2. Kinetics of T-cell secondary response to TNP-cLC after a single UVB exposure (200 J/m2). Cultured LC were irradiated or not at 200 J/m2, modi®ed or not with TNP and added to autologous T lymphocytes. T cell proliferation was assessed each day by [3H]thymidine incorporation for the ®nal 18 hr of culture. Negative controls included T lymphocytes alone (LT) and T lymphocytes cultured with untreated LC. Results are expressed as the mean cpm2SD of triplicate cultures.

Similar results were obtained with AMELR using fLC as APC (data not shown). These results demonstrated that in the present experimental conditions, the inhibition of T-cell response to UVB irradiated LC was not due to a defective or an excessive production of these cytokines during AMELR, since the exogenous addition of IL-1b, anti-TNFa or anti-IL-10 neutralizing antibodies failed to reverse UVB-induced suppression of autologous T-cell proliferation in response to TNP. This suggests that UVB directly a€ects the antigen-presenting function of human LC. Quanti®cation of HLA-DR and ICAM1 expression on UVB-irradiated Langerhans cells after a short in vitro culture To assess the e€ects of UVB on HLA-DR and ICAM-1 expression by LC, cells were exposed to radiation, then incubated for 18±36 hr and stained. Alternatively, LC were irradiated at the end of incubation period, immediately before staining. HLADR expression was also quanti®ed on fLC (data not shown). As previously described (Meunier et al., 1996), the estimated number of HLA-DR molecules/fLC was close to 460 2 75  103 and unchanged by UVB radiation. The increased level of HLA-DR expression after in vitro incubation, was already reported (Romani et al., 1989) and con®rmed in these experiments since the number of HLA-DR molecules/cLC increased sixfold after an 18-hr culture (2.7 2 0.37  106). However, prior UVB radiation slightly inhibited the upregulation of

DISCUSSION

It has been largely demonstrated that UVB irradiation of the skin causes immunosuppression both in mice and humans. In particular, UVB suppresses CHSR to haptens, an assay which has been largely used in mice to study the mechanisms of UVB induced immunosuppression. These mechanisms have been related to migration and depletion of LC from UVB treated skin (Moodycli€e et al., 1992) and the appearance of other APC which preferentially activate suppressor T lymphocytes (Baadsgaard et al., 1988). Other mechanisms involved both direct and indirect e€ects of UVB on LC antigen-presenting function. Human studies on LC sensitivity to UVB radiation have mostly used the in vitro model of allogeneic MELR. In the present study, we have tested the e€ects of UVB in an in vitro model of human T cell sensitization to hapten because of its greater relevance to the UVBinduced cutaneous suppressive e€ect and because this assay allows the analysis of the potentially interacting e€ects of hapten and UVB on human LC function. We have documented for the ®rst time that lowdose UVB radiation can impair the in vitro T cell sensitization to a strong allergen such as TNP. UVB radiation is e€ective both in primary and secondary T cell responses to TNP-treated LC, which is relevant to the in vivo observation that UVB can suppress both the induction and the elicitation phases of contact allergy (SjoÈvall and Christensen, 1986; SjoÈvall and MoÈller, 1985). Several lines of evidence have suggested that epidermal cell-derived cytokines are mediators of UVB-induced immunosuppressive e€ects, although contradictory results have been reported. In mice, production of IL-1b by LC is regarded as a primary and essential event for epicutaneous sensitization (Enk et al., 1993). TNFa and UVB have similar e€ects on CHSR and injection of antibodies to TNFa can reverse the immune suppression in UVBsusceptible strains. However, in a recent study, suc-

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Fig. 3. Exogenous addition of human recombinant IL-1b (a) TNFa (b) IL10 (c) or their respective monoclonal antibodies did not alter the UVB-induced immunosuppressive response. Cultured epidermal cell suspensions (40% LC) were exposed to a single dose of UVB radiation (312 nm) from 0 to 200 J/m2. Then, LC were treated with TNP, washed, and ®nally numerated. 5  103 TNP-cLC were cultured with 105 autologous in vitro primed T cells. Human recombinant IL-1b (100 U/ml), TNFa (50 U/ml) or IL10 (10 ng/ml) and their respective monoclonal antibodies (IL-1b 10 mg/ml, TNFa 100 ng/ml, IL10 10 mg/ml) were added to the complete RPMI medium at the beginning of the culture using cLC as APC. T cell secondary proliferative response was assessed by [3H]thymidine incorporation for the ®nal 18-hr of culture. Results are expressed as the mean cpm2SD of triplicate cultures. Asterisks indicate signi®cant di€erence from control (non-irradiated) (*P < 0.05; ANOVA).

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Fig. 4. E€ect of UVB radiation on HLA-DR upregulation by human LC (a) Viable LC were ®rst irradiated (100 and 200 J/m2), then incubated for 18-hr and ®nally stained for HLA-DR expression with ¯uorescein isothiocyanate (FITC)-coupled human anti-HLA-DR mAb for 30 min at 48C. Negative controls were performed with mouse monoclonal antibodies of the same isotype. The MFI of each HLADR+ cell population was measured by cyto¯uorimetry analysis. (b) Comparative study between HLADR molecules density on LC irradiated at the beginning (UV + Inc) or at the end (Inc + UV) of the 18-hr incubation. In each experiment, non-irradiated cLC served as controls. Results are expressed as the mean HLA-DR molecules /cell2 SD of three experiments.

cessful induction of immunosuppression by UVB exposure was observed in TNF receptor gene-targeted mice, demonstrating that TNFa was not required for UVB suppressive e€ects (Kondo et al., 1995). In the present study, neither IL-1b, TNFa nor their speci®c mAb can alter the inhibitory e€ects of irradiation, which suggests that the cytokines are not essential for these e€ects. Based on murine studies, an attractive candidate for UVB-induced immunosuppression would be the recently described IL-10. First, UVB exposure of murine keratinocytes results in production of the cytokine (Rivas and Ullrich, 1992). Secondly, intradermal injection of IL-10 before application of a contact allergen induces speci®c tolerance (Enk et al., 1994). Lastly, the inhibitory e€ect of IL-10 on APC function was well documented and the cytokine was regarded as an essential mediator of UVB-induced immunosuppression (Ullrich, 1994). However, it is not known at present whether this holds true in humans. Indeed, a recent study clearly demonstrated that normal human keratinocytes

failed to produce IL-10 even on UVB irradiation (Teunissen et al., 1997). In the present study, we showed that anti-IL-10 mAb failed to reverse the inhibitory e€ects of UVB on T cell proliferation. Taken together, these data support the concept that, in humans, IL-10 was not the principal mediator of UVB-induced immunosuppression. We found here that prior UVB exposure did not a€ect the percentage of HLA-DR positive cells after an 18-hr culture but slightly altered the density of DR antigens at the cell surface. Nevertheless this slight downregulation was unlikely to account for the observed immunosuppression e€ects. Irradiated cultured LC still express a consistant level of HLADR molecules (more than 2.5  106/cell) and, furthermore, it has been reported that APC function was not merely related to MHC class II density (Hauser et al., 1989). An interesting ®nding in this study was that cultured LC were less sensitive than fresh LC to the UVB suppressive e€ects. As cultured LC express increased levels of accessory molecules, this agrees with a deleterious e€ect of UVB

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Fig. 5. E€ect of UVB radiation on ICAM-1 upregulation by human LC (a) Experimental protocol was the same as that described in Fig. 4a, except that viable cLC were double stained for ICAM-1. Negative controls were performed with mouse monoclonal antibodies of the same isotype. (b) Comparative study between ICAM-1 molecule density on LC irradiated at the beginning (UV + Inc) or at the end (Inc + UV) of the 36-hr incubation. In each experiment, non-irradiated cLC served as controls. Results are expressed as the mean ICAM-1 mol/cell 2SD of three experiments.

radiation on human LC accessory function. Indeed, a strong downregulation of ICAM-1 expression has been reported on murine LC after UVB exposure (Tang and Udey, 1991). Studies performed on human blood dendritic cells also showed a signi®cant inhibition of ICAM-1 and B7/BB1 expression after 3000 J/m2 UVB exposure (Young et al., 1993). In contrast with results obtained in the murine system, we did not notice any impairment in the regulation of ICAM-1 expression on irradiated LC after a 36-hr culture. Recently we demonstrated that ICAM-1 expression was slightly downregulated on human LC after a 2-day culture whereas a signi®cant inhibition of B7-2 expression was observed (F.M. Rattis, M. Concha, C. Dalbiez-Gauthier, P. Courtellemont, D. Schmitt and J. PeÂguet-Navarro, unpublished data). This ®nding con®rms a recent report suggesting that suppressive e€ect of UVB radiation on LC was at least partly related to an inhibition of functional B7-2 expression (Weiss et al., 1995). We have previously described that the in vitro AMELR could be used as a screening in vitro assay to eliminate strong contact allergens (Krasteva et al., 1996).The present study demonstrates that AMELR is also an excellent model to analyse the deleterious

e€ects of UVB on the induction of hapten speci®c T cell proliferation. In conclusion, our results demonstrated that in vitro UVB exposure inhibits the capacity of human LC to induce primary and secondary in vitro T cell responses to TNP. This e€ect seems more related to an impairment of upregulation of some accessory molecules on LC than to immunosuppressive factors released by epidermal cells. AcknowledgementsÐWe thank Dr D.C. Remy for technical assistance and Mr J.P. Biesse for expert statistical assistance. REFERENCES

Baadsgaard O., Fox D. A. and Cooper K. D. (1988) Human epidermal cells from ultraviolet light-exposed skin preferentially activate autoreactive CD4+2H4+ suppressor inducer lymphocytes and CD8+ suppressor/ cytyotoxic lymphocytes. Journal of Immunology 140, 1738±1744. Baadsgaard O., Salvo B., Mannie A., Dass B., Fox D. A. and Cooper K. D. (1990) In vivo ultravioletexposed human epidermal cells activate T suppressor cell pathways that involve CD4+CD45RA+ suppressorinducer T cells. Journal of Immunology 145, 2854±2861. Enk A. H., Angeloni V. L., Udey M. C. and Katz S. I. (1993) An essential role for Langerhans cell-derived IL-1b in the initiation of primary immune response in skin. Journal of Immunology 150, 3698±3704.

UVB irradiation of human LC alters in vitro hapten T-cell sensitization Enk A. H., Saloga J., Becker D., Mohamadzadeh M. and Knop J. (1994) Induction of hapten-speci®c tolerance by interleukin 10 in vivo. Journal of Experimental Medicine 179, 1397±1402. Enk A. H., Sredni D., Blauvet A. and Katz S. I. (1995) Induction of IL-10 gene expression in human keratinocytes by UVB exposure in vivo and in vitro. Journal of Immunology 154, 4851±4856. Garhing L., Baltz M., Pepys M. B. and Daynes R. (1984) E€ect of ultraviolet radiation on production of epidermal cell thymocyte-activating factor/interleukin 1 in vivo and in vitro. In Proceedings of the National Academy of Sciences of the U.S.A. 81, 1198±1202. Hauser C., Yokoyama W. M. and Katz S. I. (1989) Characterization of primary T helper cell activation and T helper cell lines stimulated by hapten-modi®ed cultured Langerhans cells. Journal of Investigative Dermatology 93, 649±655. Kaplan M. E. and Clark C. (1974) An improved rosetting assay for detection of human T lymphocytes. Journal of Immunological Methods 5, 131±135. Kondo S., Wang B., Fujisawa H., Shivji G. M., Echtenacher B., Mak T. W. and Sauder D. N. (1995) E€ect of gene-targeted mutation in TNF receptor (p55) on contact hypersensitivity and ultraviolet B-induced immunosuppression. Journal of Immunology 155, 3801± 3805. KoÈck A., Schwarz T., Kirnbauer R., Urbanski A., Perry P., Ansel J. C. and Luger T. A. (1990) Human keratinocytes are a source for tumor necrosis factor alpha: evidence for synthesis and release upon stimulation with endotoxin or ultraviolet light. Journal of Experimental Medicine 172, 1609±1614. Krasteva M., PeÂguet-Navarro J., Moulon C., Courtellemont P., Redziniak G. and Schmitt D. (1996) In vitro primary sensitization of hapten-speci®c T cells by cultured human epidermal Langerhans cellsÐa screening predictive assay for contact sensitizers. Clinical and Experimental Allergy 26, 563±570. Kripke M. L. (1974) Antigenicity of murine skin tumors induced by ultraviolet light. Journal of the National Cancer Institute 53, 1333±1336. Meunier L., Vian L., Lagoueyte C., Lavabre-Bertrand T., Duperray C., Meynadier J. and Cano J. P. (1996) Quanti®cation of CD1a, HLA-DR, and HLA Class I expression on viable human Langerhans cells and keratinocytes. Cytometry 26, 260±264. Mommaas A. M., Mulder A. A. and Vermeer B. J. (1993) Short-term and long-term UVB-induced immunesuppression in human skin exhibit di€erent ultrastructural features. European Journal of Immunology 31, 30±34. Moodycli€e A. M., Kimber I. and Norval M. (1992) The e€ect of ultraviolet-B irradiation and urocanic acid isomers on dendritic cell migration. Immunology 77, 394± 399. Moulon C., PeÂguet-Navarro J., Courtellemont P., Redziniak G. and Schmitt D. (1993) In vitro primary sensitization and restimulation of hapten-speci®c T cells by fresh and cultured human epidermal Langerhans' cells. Immunology 80, 373±379. Noonan F. P., De Fabo E. C. and Kripke M. L. (1981) Suppression of contact hypersensitivity by UVB radiation and its relationship to UV-induced suppression of tumor immunity. Photochemistry and Photobiology 34, 683±690. PeÂguet-Navarro J., Dalbiez-Gauthier C., DezutterDambuyant C. and Schmitt D. (1993) Dissection of human Langerhans cells' allostimulatory function: the need for an activation step for full development of accessory function. European Journal of Immunology 23, 376±382. Poncelet P. and Carayon P. (1985) Cyto¯uorometric quanti®cation of cell-surface antigens by indirect immu-

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no¯uorescence using monoclonal antibodies. Journal of Immunological Methods 85, 65±74. Rattis F. M., PeÂguet-Navarro J., Courtellemont P., Redziniak G. and Schmitt D. (1995) In vitro e€ects of ultraviolet B radiation on human Langerhans cell antigen-presenting function. Cellular Immunology 164, 65± 72. Rivas J. M. and Ullrich S. E. (1992) Systemic suppression of delayed-type hypersensitivity from UV-irradiated keratinocytes. An essential role for keratinocyte-derived IL10. Journal of Immunology 149, 3865±3871. Rivas J. M. and Ullrich S. E. (1994) The role of IL-4, IL10, and TNFa in the immune suppression induced by ultraviolet radiation. Journal of Leukocyte Biology 56, 769±775. Romani N., Lenz A., Glassel H., Stossel H., Stanzl U., Majdic O., Fritsch P. and Schuler G. (1989) Cultured human Langerhans cells resemble lymphoid dendritic cells in phenotype and function. Journal of Investigative Dermatology 93, 600±609. Schwarz T. and Luger T. A. (1989) E€ect of UV irradiation on epidermal cell cytokine production. Journal of Photochemistry and Photobiology 4, 1±13. Shearer G. M. (1974) Cell-mediated cytotoxicity to trinitrophenyl-modi®ed syngeneic lymphocytes. European Journal of Immunology 4, 527±533. Simon J. C., Krutmann J., Elmets C. A., Bergstresser P. R. and Cruz P. D. (1992) Ultraviolet B-irradiated antigen-presenting cells display altered accessory signaling for T-cell activation: relevance to immune responses initiated in skin. Journal of Investigative Dermatology 98, 66S±69S. SjoÈvall P. and Christensen O. B. (1986) Local and systemic e€ect of ultraviolet irradiation (UVB and UVA) on human allergic contact dermatitis. Acta DermatoVenereologica 66, 290±294. SjoÈvall P. and MoÈller H. (1985) The in¯uence of locally administered UV light (UVB) on the allergic contact dermatitis in the mouse. Acta Dermato-Venereologica 65, 465±471. Tang A. and Udey M. C. (1991) Inhibition of epidermal Langerhans cell function by low dose ultraviolet B radiation. Ultraviolet B radiation selectively modulates ICAM-1 (CD54) expression by murine Langerhans cells. Journal of Immunology 146, 3347±3355. Teunissen M. B. M., Koomen C. W., Jansen J., De Wall Malefyt R., Schmitt E. and Van Den Wijngaard R. M. J. G. J. (1997) In contrast to their murine counterparts, normal human keratinocytes and human epidermoid cell lines A431 and HaCaT fail to express IL-10 mRNA and protein. Clinical and Experimental Immunology 107, 213±223. Toews G. B., Bergstresser P. R. and Streilein J. W. (1980) Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. Journal of Immunology 124, 445±453. Ullrich S. E. (1994) Mechanism involved in the systemic suppression of antigen-presenting cell function by UV irradiation. Keratinocyte-derived IL-10 modulates antigen-presenting cell function of splenic adherent cells. Journal of Immunology 152, 3410±3416. Weiss J. M., Renkl A. C., Denfeld R. W., De Roche R., Spitzlei M., SchoÈpf E. and Simon J. C. (1995) Low-dose UVB radiation perturbs the functional expression of B71 and B7±2 co-stimulatory molecules on human Langerhans cells. European Journal of Immunology 25, 2858±2862. Young J. W., Baggers J. and Soergel S. A. (1993) Highdose UV-B radiation alters human dendritic cell costimulatory activity but does not allow dendritic cells to tolerize T lymphocytes to alloantigen in vitro. Blood 81, 2987±2997.