Apoptosis Ultraviolet-B-Induced Human Lymphocyte Fas/Fas Ligand ...

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UV-B irradiation increased Fas and FasL expression in human lymphocytes, and that a ... UV-B irradiation was delivered using two FS-20 sun lamps, which emit.
Fas/Fas Ligand Interactions Are Involved in Ultraviolet-B-Induced Human Lymphocyte Apoptosis1 Roberto Caricchio, Elizabeth A. Reap, and Philip L. Cohen2 We wondered whether the apoptosis known to occur after UV-B irradiation might involve the Fas/Fas ligand (FasL) signaling pathway. We exposed PBLs from normal individuals, and also the Jurkat (E6-1) and U937 cell lines, to graded doses of UV-B irradiation and observed a prompt and marked increase in Fas expression at doses as low as 0.5 mJ/cm2. Increased Fas expression did not require new protein synthesis, since cycloheximide-treated cells also showed an increase in Fas after UV-B. UV-Birradiated cells cultured in the presence of zinc showed inhibition of apoptosis coincident with a marked increase in Fas1 cells, apparently indicating the accumulation of Fas-bearing cells unable to undergo apoptosis. After UV-B irradiation, PBLs showed increased expression of Fas ligand; the E6-1 lymphocytic cell line also released soluble FasL. UV-B induced apoptosis could be partially blocked by neutralizing FasL Abs, and a FasL-resistant variant of E6-1 cell line showed reduced apoptosis after UV-B irradiation, implying that the increase in Fas expression signified a role for Fas in UV-induced apoptosis. UV-induced Fas expression may serve to target stress-injured cells for removal by FasL-bearing cells or by FasL produced by the cells themselves in response to the stimuli, and may represent a general function of the Fas/FasL pathway in facilitating the apoptosis and elimination of undesirable or harmful cells. The Journal of Immunology, 1998, 161: 241–251.

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poptosis is a tightly regulated physiologic form of cell death characterized in part by nuclear condensation and cell shrinkage with preservation of an intact plasma membrane, culminating in the destruction of the nuclear chromatin and the digestion of the genomic DNA, an irreversible event (1). It occurs under a variety of physiologic, developmental conditions and in response to many cytotoxic agents. UV-B irradiation has been shown to be a particularly potent inducer of apoptosis (2), affecting various cell lines (3), keratinocytes (4), and peripheral lymphocytes (5). It also induces immune suppression and transplant tolerance by mechanisms that might involve apoptosis (6). APO-1/Fas (CD95) is a type I transmembrane glycoprotein belonging to the nerve growth factor/TNF receptor superfamily (7). Fas is expressed on activated T and B cells (7), thymocytes (8), malignant T and B cells (9), and in a variety of tissues outside the immune system such as liver, lung, and kidney (10). The interaction of Fas and its ligand has been shown to play an important role in the regulation of programmed cell death of T and B lymphocytes (11). The Fas/Fas ligand (FasL)3 pathway serves at least two functions in the immune system: first, to limit the expansion of an immune response by eliminating lymphocytes that are no longer needed, and second, to eliminate autoreactive T cells that have escaped thymic selection (12). Departments of Medicine and Microbiology/Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Received for publication November 5, 1997. Accepted for publication March 6, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grants AR33887 and AR42573 to P. L. Cohen. E. A. Reap is a fellow of the Arthritis Foundation. R. Caricchio was supported by a grant from Ministero dell’ Universita’ e della Ricerca Scientifica e Tecnologica (Italy). 2

Address correspondence and reprint requests to Dr. Philip L. Cohen, Department of Medicine, Division of Rheumatology & Immunology, CB 7280, Chapel Hill, North Carolina 27599-7280. E-mail address: [email protected]. 3 Abbreviations used in this paper: FasL, Fas ligand; sFasL, soluble FasL; CHX, cycloheximide; PI, propidium iodide; FSC, forward-angle scatter; SSC, side scatter; MFI, mean fluorescence intensity.

Copyright © 1998 by The American Association of Immunologists

Recently, we have observed that cells from Fas-deficient lpr mice were resistant to the apoptosis induced by ionizing radiation, providing evidence that the Fas/FasL system may also be involved in surveillance of injured cells (13). Therefore, we wondered whether the apoptosis known to occur after UV-B irradiation might involve the Fas/FasL signaling pathway. We have found that UV-B irradiation increased Fas and FasL expression in human lymphocytes, and that a lymphocyte cell line released functional soluble FasL (sFasL) after UV-B irradiation. These data suggest that UV-B-induced apoptosis is at least in part mediated by Fas in human lymphocytes and monocytes. Therefore we propose a third function for the Fas/FasL pathway in the immune system: to facilitate the apoptosis and elimination of cells undergoing UV light exposure and other forms of environmental trauma.

Materials and Methods Cell isolation and culture Peripheral blood from 10 healthy donors (age 25–35) was separated by density centrifugation, the mononuclear cell interface was collected over Histopaque 1077 (Sigma, St. Louis, MO) after centrifugation at 2000 rpm for 30 min, and it was then washed two times in RPMI 1640. The mononuclear cells were resuspended at a concentration of 1 3 106/ml in RPMI 1640, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 10% FBS, and nonessential amino acids (University of North Carolina Cancer Center Tissue Culture Facility, Chapel Hill, NC). These cells were transferred to 24-well plates at 37°C for 1 h to allow monocytes to adhere. Nonadherent cells were removed, washed, resuspended under the same conditions, irradiated, and cultured at 37°C in a 5% CO2/95% air-humidified atmosphere in 24-well plates at a concentration of 1 3 106cells/ml. In some experiments ZnCl2 (1 mM) or cycloheximide (CHX) (30 mg/ml) (Sigma) was added 5 min after irradiation.

Cell lines The U937 human histiocytic lymphoma and the Jurkat human T cell leukemia clone E6-1, both constitutively expressing Fas receptor and sensitive to killing by anti-Fas, were obtained from the American Type Culture Collection (Rockville, MD). The E6-1R, an anti-Fas-resistant variant of E6-1S, was generated by continuous culture in medium containing anti-Fas (clone 7C11, 1:1000) (a generous gift from Dr. M. Robertson, Indiana University, Indianapolis, IN) for 3 mo. The E6-1R2, a FasL-resistant variant 0022-1767/98/$02.00

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of E6-1S, was selected from continuous coculture for 3 mo with a National Institutes of Health 3T3 cell line transfected with FasL (a generous gift from Dr. I. N. Crispe, Yale Medical School, New Haven, CT) (14).

Irradiation of cells UV-B irradiation was delivered using two FS-20 sun lamps, which emit most of their energy within the UV-B range (290 –320 nm) with an emission peak at 310 nm. Preliminary experiments showed equivalent UV effects on cells irradiated in complete medium compared with PBS. In subsequent studies, therefore, cells were irradiated in complete media with different doses of UV-B (0.1, 0.5, 1, 10, and 20 mJ/cm2) as determined with an IL-1700 research radiometer (International Light, Newburyport, MA). Mock UV-B-irradiated control cells were treated in an identical manner except that the UV-B lamps were turned off.

Cell staining for surface markers and apoptosis Fluoresceinated anti-Fas (clone DX2, isotype: mouse IgG1k, anti-CD4 (clone HIT3a, isotype: mouse IgG2ak, and isotype-matched FITC-labeled mouse Ab controls were obtained from PharMingen (San Diego, CA). For staining, samples were washed twice with HBSS containing 15 mM HEPES, 0.5% L-glutamine, and 3% FBS (HBSS complete) (University of North Carolina Cancer Center Tissue Culture Facility, Chapel Hill, NC), and fluoresceinated Abs were added to 1 3 106 cells for 30 min at 4°C in 96-well microtiter plates. The cells were washed once with complete HBSS followed by two washes in PBS containing 0.1% NaN3. To permeabilize cell membranes for DNA staining, 1 3 106 cells were added to 3 ml of 70% ethanol, incubated for 45 min at 4°C, and washed twice. For DNA staining, 0.1 ml of 1 mg/ml RNase A (Sigma) was added per sample, followed by 0.2 ml of 100 mg/ml propidium iodide (PI) (Sigma). Cells were incubated for 20 min in the dark at 4°C and were analyzed with a FACScan (Becton Dickinson, Mountain View, CA) with Cytomation data acquisition and software (Fort Collins, CO) for green and red fluorescence.

Analysis of apoptotic cells Apoptotic cells appeared in the ,2 N DNA peak identified by PI immunofluorescence. They could also be distinguished from necrotic cells by analyzing the light scatter profile. Cell death by necrosis resulted in a large decrease in forward-angle light scatter (FSC) and side scatter (SSC), while apoptotic cells were a discrete population showing a smaller decline in FSC and an increase in SSC (13, 15–17). Samples were collected in list mode, so that when cell surface markers were used, the percentage of cells undergoing apoptosis could be determined by gating on FITC-stained cells and subsequently analyzing DNA staining. At least 30,000 events were collected per sample in all experiments.

Apoptosis detection with the JAM test To screen a large number of samples, DNA fragmentation and cell death were measured using a modified standard assay for cell proliferation, involving vacuum aspiration of labeled cells through fiberglass filters. Since high m.w. DNA is trapped in these filters, small pieces of fragmented DNA are washed through, leaving only intact DNA from living cells (18). E6-1S cells (5 3 105/well in 96-well plates) were labeled for 16 h with 2.5 mCi/ml of [3H]thymidine (9.25 3 104 Bq/ml). The supernatant was then removed, and fresh medium and anti-FasL (PharMingen) at different concentrations were added. After 5 h of incubation of 37°C in a 5% CO2 incubator, cells were harvested and counted in a liquid scintillation counter. The percentage of specific DNA fragmentation was calculated as: 100 3 (spontaneous cpm 2 experimental cpm)/spontaneous cpm. Experimental cpm is defined as cpm of retained DNA in the presence of anti-FasL, and spontaneous cpm is cpm of retained DNA in the absence of anti-FasL. Experiments were performed in triplicate.

Western blotting for FasL A total of 4 3 106 per sample of PBLs were washed in PBS and lysed for 20 min on ice in 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 10 mM NaF, 1 mM EDTA, 2 mM sodium orthovanadate, 1 mM DTT, and 1 mM PMSF. After centrifugation for 15 min at 14,000 rpm, the amount of proteins in the supernatant was determined by BCA Protein Assay Reagent (Pierce, Rockford, IL) and BSA standards. Then 30 mg of lysate in Laemmli buffer were separated per lane on 12% SDS-PAGE. Western blots were probed with mouse anti-human FasL obtained from Transduction Laboratories (Lexington, KY). Bound Abs were detected with goat anti-mouse-horseradish peroxidase conjugate. An enhanced chemiluminescence system (Renaissance, NEN Life Science Product, Boston, MA) was used for detection.

Detection of sFasL To analyze the production of sFasL by E6-1 cells in response to UV-B irradiation, cells (1 3 106/ml) were irradiated (1 mJ/cm2), resuspended in complete medium, and cultured at 37°C for 2 h. Cells were recovered after brief centrifugation and aliquots of supernatant (500 ml) assayed for their cytotoxic activity using nonirradiated E6-1S susceptible to anti-Fas killing, E6-1R2 cells resistant to FasL, or anti-Fas-mediated killing as targets (1 3 105). Supernatants from nonirradiated cells were used as additional controls. Target cells were suspended in aliquoted supernatant (500 ml) and were harvested after 5-h culture. Apoptosis was measured by PI staining as described above.

Statistical analysis Student’s t test was used to determine the statistical significance of differences between groups. Statistical significance was defined as p , 0.05.

Results Apoptosis of human lymphocytes and of the E6-1S Jurkat cell line after UV-B irradiation To test their sensitivity to UV-B irradiation, lymphocytes from healthy donors were cultured for 24 and 48 h after mock UV exposure or after 20, 10, and 1 mJ/cm2 of UV-B irradiation. Apoptosis was detected according to the FSC vs SSC profile (13, 15– 17). At the lowest UV-B dose (1 mJ/cm2), it was possible to detect a clear apoptotic population but minimal necrosis. In contrast, with the highest dose (20 mJ/cm2), after 24 and 48 h most of the cells were necrotic (Fig. 1A). The 10 mJ/cm2-dose yielded a mixed population of necrotic and apoptotic cells. The UV-B response thus differed qualitatively depending on the irradiation dose. To test if the E6-1S clone responded to UV injury in a similar fashion, these cells were also exposed to graded UV-B doses. For detecting the apoptotic population in these cells the PI staining was used, although FSC vs SSC analysis (not shown) yielded much the same results. As with normal lymphocytes, we found a remarkable difference in the outcome depending on the UV-B dose (Fig. 1B). This suggests that apoptosis can occur alone or with necrosis and that this mechanism is preserved in this cell line. Moreover, as indicated by their sensitivity to small UV-B doses (1, 0.5, 0.1, 0.05 mJ/cm2), E6-12 cells are extremely sensitive to UV-B-induced apoptosis compared with normal lymphocytes (Fig. 1B). Fas expression increases after UV-B irradiation of normal human lymphocytes To test whether the Fas/FasL system might mediate UV-B-induced apoptosis, lymphocytes from healthy donors were cultured for different intervals after UV-B irradiation at different doses and Fas expression was assessed. Figure 2A shows data from a representative experiment. We found a significant increase in Fas1 cells compared with nonirradiated cells at both 10 and 1 mJ/cm2 of UV irradiation. We found no significant increase in anti-CD41 staining, even at the highest UV-B dose, suggesting that the increase of Fas1 cells was not due to nonspecific increased staining following UV-B exposure (Fig. 2B). We found a similar increase in Fas expression even at very low doses of UV-B, which correspond to possible in vivo UV exposure. Lymphocytes from 10 healthy donors were cultured and stained for Fas-FITC at T0 (0 h), T1 (24 h) and T2 (48 h) in vitro with and without exposure to UV-B irradiation (10 mJ/cm2). Figure 3 shows the mean percentage of Fas1 cells at different time points. The difference between the UV-Birradiated and untreated groups was significant at both 24 and 48 h ( p , 0.01). Fas expression is increased on UV-B-irradiated cells cultured with Zn, and is unaffected by CHX To test if the Fas increase was affected by inhibiting UV-Binduced apoptosis or by inhibiting protein synthesis, ZnCl2 or

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FIGURE 1. Apoptosis of human lymphocytes and the E6-1 cell line after UV irradiation. A, Lymphocytes from healthy donors were cultured for 24 and 48 h without treatment, and after 20, 10, and 1 mJ/cm2 of UV-B irradiation. Panels show forward-angle vs side-scatter histograms. At the very low UV-B doses (10 and 1 mJ/cm2), it was possible to detect a clear apoptotic population. In contrast, with the highest dose (20 mJ/cm2), after 24 and 48 h most of the cells were necrotic. With no treatment, most of the cells were still viable after 24 and 48 h. B, E6-1 cells were UV-B irradiated (1, 0.5, 0.1, and 0.05 mJ/cm2) and apoptotic cells were detected by PI staining after culture at different times. DNA area histograms are shown. Apoptotic cells appeared in the ,2 N DNA peak identified by PI immunofluorescence, and percentages are indicated over brackets (upper vs lower panels).

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FIGURE 2. Fas expression increases after UV irradiation. A, Fas-FITC staining and isotype-matched FITC-labeled mouse Ab control staining are shown. Lymphocytes were stained after 24 and 48 h, without treatment and after UV-B irradiation at different doses. Mean fluorescence intensity (MFI) values are shown. B, Cells were exposed to 10 mJ/cm2 of UV-B and stained with FITC-anti-CD4. No significant difference in CD4 expression was observed. MFI values are shown.

CHX was added to cultured human lymphocytes immediately after UV-B irradiation (10 mJ/cm2). Zn (1 mM) inhibited apoptosis (data not shown) as expected (19), accompanied by a marked increase in the intensity of Fas expression, probably due to the accumulation of Fas1 cells unable to undergo apoptosis (Fig. 4). No substantial difference in apoptosis percentage (data not shown) or in Fas expression was noticed in cultures containing CHX (30

mg/ml), suggesting that increased Fas expression does not require protein synthesis. Histograms shown are representative of three different experiments (Fig. 4). Expression of Fas in human cell lines after UV-B irradiation The U937 and E6-1 cell lines, which express Fas constitutively and are sensitive to UV-B (Fig. 5), were irradiated and, after 5 h of

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FIGURE 3. Fas expression on normal donor lymphocytes after UV irradiation. Lymphocytes from 10 healthy donors were stained for Fas-FITC at T0 (0 h), T1 (24 h), and T2 (48 h) with and without UV-B irradiation (10 mJ/cm2). The mean percentage 6 SD of Fas1 cells at different time points is shown. The difference between UV-B-irradiated and untreated groups was significant at both 24 and 48 h ( p , 0.01).

culture, were stained with fluoresceinated monoclonal anti-Fas. After UV-B irradiation, Fas expression was greatly increased on U937 cells. The E6-1 cells showed enhanced Fas1 expression after UV-B irradiation (Fig. 5).

FIGURE 4. Fas expression is increased on UV-B-irradiated cells cultured with Zn, and is unaffected by CHX. After UV-B irradiation (10 mJ/cm2), culture in the presence of Zn (1 mM) inhibited apoptosis, while Fas expression was increased (middle panel). The marked increase in Fas1 cells could be explained by the accumulation of Fas on cells unable to undergo apoptosis. No change was noticed with CHX (30 mg/ml), both in apoptosis percentage (not shown) and in Fas expression (lower panels), suggesting that increased Fas expression does not require protein synthesis. Histograms shown are representative of three different experiments. The percentage of Fas1 cells are shown. MFI values were, respectively, 135.6, 568.2, and 192.5.

We wondered if the Fas expression noted after UV-B irradiation differed between live and apoptotic cells. Two-color staining (FasFITC and PI) was performed to assess Fas expression in both populations. The apoptotic lymphocyte population could be distinguished both with PI staining and by FSC vs SSC profile. After UV-B irradiation (10 mJ/cm2), Fas expression on live cells increased, but it was striking that the vast majority of the cells undergoing apoptosis were Fas1, as shown in Figure 6. Few apoptotic cells were detectable without UV-B irradiation. The increased Fas expression on apoptotic cells after UV-B irradiation is consistent with a functional role for the Fas molecule in bringing about their death. Histograms shown in Figure 6 are representative of eight different experiments. FasL expression increased in PBLs after UV-B irradiation To test whether FasL was also increased after UV-B irradiation, PBLs from healthy donors were irradiated with different doses (1, 10, and 20 mJ/cm2), and after 18 h of culture, FasL expression was measured by Western blot. Results are shown in Figure 7. Nonirradiated cells showed no or little FasL expression; yet, after 1 and 10 mJ/cm2 of UV-B irradiation, there was a clear FasL increase. Surprisingly, after 20 mJ/cm2, FasL did not increase, implying that the highest dose was inducing necrosis more than apoptosis, or at least that the mechanism involved was not the Fas/FasL system.

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FIGURE 5. Fas expression in human cell lines after UV-B irradiation. U937 and E6-1 cell lines, which express Fas constitutively, were irradiated, and after 5 h of culture were stained with fluoresceinated monoclonal anti-Fas and an isotype control. After UV-B irradiation, Fas expression was greatly increased on U937 cells. E6-1 cells that express Fas constitutively also showed enhanced Fas1 expression after UV-B irradiation. MFI values are shown.

UV-B-induced apoptosis in anti-Fas- and FasL-sensitive and -resistant Jurkat E6-1 clones The increased expression of Fas after UV-B exposure raised the question of whether FasL was also increased after UV-B irradiation. To approach this issue, we generated E6-1 cell lines resistant to anti-Fas or to FasL (E6-1R and E6-1R2) (Fig. 8). These cell lines served as valuable reagents to assess the FasL activity of cell supernatants, because they did not undergo apoptosis when incubated with anti-Fas or FasL, respectively, and could thus be compared with wild-type E6-1 cells. Both cell lines were stained with PI after UV-B irradiation to determine the fraction undergoing apoptosis. The E6-1R line showed a moderate decrease in the apoptotic population compared with the E6-1S (Fig. 9). In contrast, the E6-1R2 line showed a nearly complete resistance to UV-B-induced apoptosis (Fig. 9). These results are consistent with a role for the Fas/FasL system in UV-B-induced apoptosis. The difference in UV-B sensitivity between the E6-1R and E6-1R2 cell lines is probably due to the fact that, despite the resistance to anti-Fas, E6-1R was still sensitive to FasL killing (Fig. 8). Release of sFasL by UV-B irradiated cells We wondered if UV-B irradiation induced the release of sFasL. E6-1S cells were irradiated (1 mJ/cm2), washed, and cultured for 2 h. Supernatants from irradiated cells were collected and their cytotoxicity tested on nonirradiated E6-1S and E6-1R2 cells by detecting apoptosis with PI staining. There was no difference in the percentage of cells undergoing apoptosis in both clones after add-

ing supernatants from nonirradiated E6-1S (1.9% vs 2.5%). In contrast, the supernatant from the irradiated cells induced apoptosis in the E6-1S and not in the E6-1R2 clone (17.6% vs 3.5%) (Fig. 10), suggesting that sFasL was released upon irradiation. Inhibition of UV-B-mediated apoptosis with anti-FasL To test the functional role of the Fas/FasL system after UV-B irradiation, we tested the effect of blocking Fas/FasL interactions with an anti-FasL Ab. E6-1S cells were labeled with [3H]thymidine. After 16 h, cells were UV-B irradiated (1 mJ/cm2) and cultured with anti-FasL (clone NOK-1, PharMingen) or the isotype control at different concentrations for 5 h. Apoptosis was detected using the JAM test. We found significant inhibition of apoptosis dependent on the concentration of anti-FasL, confirming that UVB-induced apoptosis was, in part, Fas mediated (Fig. 11).

Discussion Because the Fas/FasL system plays a major role in the homeostasis of the immune system (11) and because lymphocytes are exquisitely sensitive to UV-B (20, 21), we tested whether the lymphocytic response to UV-B irradiation involved the Fas/FasL system. We found increased Fas expression in human peripheral lymphocytes, and in a T leukemia cell line and a monocytic cell line after exposure to a wide range of UV-B irradiation doses. Fas expression was especially high on apoptotic cells after UV-B irradiation, indicating that Fas expression and cell death were correlated. We found also an increase of FasL expression after UV-B irradiation

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FIGURE 6. Fas expression on live and apoptotic lymphocytes. The apoptotic cell population from human PBLs was detected with PI staining (left panels). After UV-B irradiation (10 mJ/cm2), most of the apoptotic cells were Fas1 (right panels). Few apoptotic cells were detectable without UV-B irradiation.

in PBLs. Finally, a FasL-resistant mutant Jurkat cell line was also resistant to UV-induced apoptosis, and UV-B-induced apoptosis could be blocked by anti-FasL. These results taken together are consistent with a functional role for the Fas molecule and its ligand in UV-B-induced apoptosis.

FIGURE 7. UV-B irradiation induces FasL expression in human lymphocytes. PBLs from healthy donors were UV-B irradiated (1, 10, and 20 mJ/cm2), and mock irradiated cells were used as a negative control. After 18 h, cells were harvested, lysed, and FasL was detected by Western blot. Lysate from the Jurkat cell line E6-1 was used as a positive control. An experiment representative of three is shown. The anti-FasL mAb used recognized in cell extracts the full-length FasL protein of 37 kDa. A basal level of FasL expression was detectable in nonirradiated lymphocytes, and after exposure to 1 and 10 mJ/cm2 of UV-B dose, FasL expression increased considerably. In contrast, after 20 mJ/cm2 of UV-B dose, no difference was noticed in comparison with the negative control.

The cellular response to UV irradiation is dependent upon the induced expression of many genes, the so called “UV response” (22), which includes the immediate early genes c-fos (23, 24) and c-jun (23, 25) and transcription factors SAP-1 and nuclear factor kB (26). UV irradiation also suppresses lymphocyte proliferative, cytotoxic, and accessory functions (27). The mechanism is not entirely clear, but may involve alteration of cytokine production and secretion (27), calcium homeostasis (28), MHC class II Ag expression (28) and, depending upon wavelength and duration of exposure, direct effects on DNA integrity (29). Circulating human T lymphocytes are exquisitively sensitive to the DNA-damaging and lethal effects of UV-B irradiation, raising the possibility that UV-B may contribute to immune suppression via a direct effect on extracapillary T lymphocytes (20, 21). Finally UV-B irradiation is a strong inducer of apoptosis (21, 28), but this occurs via incompletely understood pathways. Our results confirmed the susceptibility of human lymphocytes, human T (E6-1), and histiocytic (U937) cell lines to UV-B irradiation-induced apoptosis. Unexpectedly, we noticed a remarkable difference in the nature of the UV-B response depending on the dose. In normal lymphocytes, a dose of 20 mJ/cm2 induced mainly necrosis, yet exposure to 10 and 1 mJ/cm2 yielded a clear apoptotic population. The results were even more marked with the E6-1 cell line using 1 and 0.1 mJ/cm2. These results are in agreement with previous observations in which a very low UV-B dose was

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FIGURE 8. Response of E6-1 mutants to anti-Fas and to FasL. The E6-1R and E6-1R2 cell lines were generated by continuous culturing with anti-Fas or with the 3T3-FasL transfected cell lines, respectively (middle and right panels). Cells were cultured for 5 h with anti-Fas or with 3T3-FasL cell line and were then stained with PI. DNA area histograms are shown. E6-1R2 cells failed to undergo apoptosis when cultured with anti-Fas or FasL-transfected 3T3 cells. The E6-1R variant, in contrast, was still sensitive to the FasL killing despite resistance to anti-Fas-induced apoptosis (middle panels). E6-1 variant cell lines were stained with anti-Fas-FITC and the isotype control (low panels).

able to induce apoptosis in PBMC (21). One explanation for the response to different UV-B doses is that lymphocytes are physiologically protected against UV irradiation by the skin tissue layers. It is possible that in our experiments, with the lowest UV-B dose, which corresponds to a possible in vivo UV exposure, the apoptotic machinery was exhibiting the normal cellular UV-B response. In contrast, the direct lymphocyte irradiation at the highest UV dose may have induced diffuse cellular damage, resulting mainly in necrosis instead of apoptosis. UV-B stress may thus involve the apoptotic machinery only below certain doses. An important finding in the present work was the markedly increased Fas expression following UV-B. Recently it has been shown that exposure to UV-B light induced clustering and internalization of cell surface receptors for epidermal growth factor, TNF and IL-1, and UV-B-induced activation of these receptors (30). An analogous mechanism could only in part explain the increases of Fas expression in our experiments since CHX-treated cells, despite protein synthesis inhibition, showed increased Fas expression after UV-B irradiation. Another explanation could be the expression of an intracellular pool of

preformed Fas receptor, although this remains to be established. The increased Fas expression very likely renders the UV exposed cells sensitive to FasL, which is also induced by UV-B. Whether Fas-mediated killing occurs via an autocrine or paracrine pathway remains to be established. Since completion of this work, a report has appeared showing that UV exposure of MCF7 and BJAB cells induced apoptosis apparently via a Fas-dependent mechanism (31). Our data, using freshly isolated PBL as well as cell lines, are consistent with these findings, but the increase in fluorescence intensity by flow cytometry in our studies is in contrast to the receptor aggregation reported in this study. The differences in UV exposure and in the cell lines used in this report may account for these differences. Another important finding in the present work was the increased expression of FasL in PBLs after a low dose of UV-B, but not with the highest dose (20 mJ/cm2). The results are in contrast with a recent report (32) in which UV light induced apoptosis by activating directly the Fas receptor, independently of its ligand. Once again differences in UV exposure may account for discrepancies, especially because in our experiments only low doses of UV-B

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FIGURE 9. FasL-resistant E6-1 mutant cells are also resistant to UV-B induced apoptosis. The anti-Fas-sensitive E6-1S cell line, and its anti-Fas-resistant (E6-1R) and FasL-resistant (E6-1R2) variants were cultured for 5 h after UV-B irradiation (0.5 mJ/cm2) and stained with PI. Left panels show forward-angle vs side-scatter histograms; right panels show DNA area histograms. Although the E6-1R cells were resistant to anti-Fas killing (see Fig. 6), they showed only moderate resistance to UV-B irradiation (lower panels). In contrast, the E6-1R2 variant, selected for resistance to the FasL killing (see Fig. 6), showed little or no apoptosis following UV-B irradiation (lower panels).

involved the Fas/FasL pathway. Our results imply, depending on the UV-B dose, a more complex scenario with multiple features for the so-called UV response, and not just a simple gradient of dose/response. It has been recently reported that Jurkat T cells constitutively express intracellular FasL, which is rapidly secreted as functional sFasL after stimulation with anti-CD3 or phytohemagglutinin; moreover, Jurkat supernatants induce apoptosis in Fasexpressing cells (33). In our experiments, the supernatant from UV-B-irradiated Jurkat cells was able to induce apoptosis only in the E6-1 clone sensitive to Fas killing, indicating that UV-B also triggers FasL release by Jurkat cells. The role of the Fas/ FasL system in UV-B induced apoptosis was also confirmed by the blocking with anti-FasL Ab of the induction of apoptosis. To investigate CD95-signaling requirements, several groups have created mutant cell lines incapable of transmitting apoptotic signals through CD95 by continuous culture in medium containing anti-Fas (34 –36). In most cases the phenotypic differences between the wild-type and the mutant cell line were minor (9, 35,

36). Because we have shown that the anti-Fas-resistant cell line was still sensitive to FasL, these findings could explain why previously reported differences between wild-type and mutant cell lines were difficult to detect. Recently, we have observed that cells from Fas-deficient lpr mice were resistant to the apoptosis induced by ionizing radiation. Because the lpr mice differed from controls only in their lack of Fas expression, these observations raised the possibility that Fas itself participated in the apoptosis induced by ionizing radiation (13). Furthermore, two different groups (37, 38) have shown that the Fas system contributes to keratinocyte apoptosis in both type A and B UV-irradiated human skin. These findings are consistent with the important role for Fas/FasL interactions in the present work. The Fas/FasL system can be added to the list of the mechanisms involved in the UV response. Since UV-B damaged lymphocytes are no longer needed and since they could become dangerous, activation of the Fas/FasL system may facilitate the apoptosis and elimination of cells undergoing this and other

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FIGURE 10. Jurkat cells release sFasL after UV-B irradiation. E6-1S cells were UV-B irradiated (1 mJ/cm2), washed, and cultured for 2 h. Supernatants from irradiated cells were collected and added to E6-1S or E6-1R2 cells. Left panels show forward-angle vs side-scatter histograms; right panels show DNA area histograms. There was no difference in percentage of apoptosis in both clones after adding supernatants from nonirradiated E6-1R2 (1.9 vs 2.5%). In contrast, the supernatants from irradiated cells induced apoptosis in the E6-1S and not in the E6-1R2 clone (17.6 vs 3.5%).

forms of environmental trauma. Its malfunction could be involved in the pathogenesis of autoimmune or neoplastic disorders. Exposure to sunlight has long been associated with exac-

erbation of systemic lupus erythematosus, the prototype of the autoimmune diseases (39, 40). It is possible that inadequate UV response due to lesions in the Fas/FasL system might contribute to systemic lupus erythematosus photosensitivity.

Acknowledgments We thank Dr. D. P. McCauliffe for assistance with UV-B irradiation. We are grateful to E. McMahon for her valuable help in cell culture. We thank J. Weintraub, Dr. K. Fecho, and Dr. S. Gallucci for critically reviewing the manuscript.

References

FIGURE 11. Inhibition of UV-B mediated apoptosis with anti-FasL. The percentage of apoptosis detected with the JAM test is shown (mean 6 SD). E6-1S cells were labeled with [H3]thymidine. After 16 h, cells were UV-B irradiated (1 mJ/cm2) and cultured with anti-FasL Ab or the isotype control at different concentrations for 5 h. Cells were harvested and counted in a liquid scintillation counter. A significant inhibition of apoptosis was found with the anti-FasL Ab (left panels). In contrast, no difference was noted with the isotype control (right panels). These results are representative of three similar experiments.

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