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Differential Ultraviolet-B-Induced Immunomodulation in XPA, XPC, and CSB DNA Repair-De®cient Mice

Andre Boonstra, Adri van Oudenaren, Miranda Baert, Harry van Steeg,* Pieter J. M. Leenen, Gijsbertus T. J. van der Horst,² Jan H. J. Hoeijmakers,² Huub F. J. Savelkoul, and Johan Garssen³ Department of Immunology, Erasmus University and University Hospital Rotterdam, The Netherlands; *Laboratory for Health Effects Research, National Institute of Health and the Environment, Bilthoven, The Netherlands; ²MGC-Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, The Netherlands; ³Laboratory for Pathology and Immunobiology, National Institute of Health and the Environment, Bilthoven, The Netherlands

Ultraviolet B irradiation has serious consequences for not constitutive but could be due to external factors, cellular immunity and can suppress the rejection of such as ultraviolet B. Upon exposure to ultraviolet B, skin tumors and the resistance to infectious diseases. only XPA mice are very sensitive to ultraviolet-B- DNA damage plays a crucial role in these immuno- induced inhibition of Th1-mediated contact hyper- modulatory effects of ultraviolet B, as impaired sensitivity responses and interferon-g production in repair of ultraviolet-B-induced DNA damage has skin draining lymph nodes. Lipopolysaccharide- been shown to cause suppression of cellular immun- stimulated tumor necrosis factor a and interleukin- ity. Ultraviolet-B-induced DNA damage is repaired 10 production are signi®cantly augmented in both by the nucleotide excision repair mechanism very XPA and CSB mice after ultraviolet B exposure. ef®ciently. Nucleotide excision repair comprises two Lymph node cell numbers were increased very subpathways: transcription-coupled and global signi®cantly in XPA, mildly increased in CSB, and genome repair. In this study the immunologic conse- not in XPC mice. In general XPC mice do not exhi- quences of speci®c nucleotide excision repair defects bit any indication of enhanced ultraviolet B suscepti- in three mouse models, XPA, XPC, and CSB mutant bility with regard to the immune parameters mice, were investigated. XPA mice carry a total analyzed. These data suggest that both global nucleotide excision repair defect, whereas XPC and genome repair and transcription-coupled repair are CSB mice only lack global genome and transcrip- needed to prevent immunomodulation by ultraviolet tion-coupled nucleotide excision repair, respectively. B, whereas transcription-coupled repair is the major Our data demonstrate that cellular immune param- DNA repair subpathway of nucleotide excision repair eters in XPA, XPC, and CSB mice are normal com- that prevents the acute ultraviolet-B-induced effects pared with their wild-type (control) littermates. This such as erythema. Key words: cytokines/DNA damage/ may indicate that the reported altered cellular immunosuppression/nucleotide excision repair/ultraviolet responses in patients are light. J Invest Dermatol 117:141±146, 2001

xposure of the skin to ultraviolet B (UVB) light is well Savelkoul, 1997; Schwarz, 1999). This may explain the observed known to have deleterious effects on human health. UVB-induced suppression of Th1-driven cellular immune re- Epidemiologic studies indicate that exposure to UVB sponses, like contact hypersensitivity (CHS) and delayed type Eincreases the incidence of nonmelanoma skin hypersensitivity (DTH) responses. (Kricker et al, 1995; Urbach, 1997). In addition, UVB Numerous studies have demonstrated that the induction of DNA irradiation impairs speci®c and nonspeci®c immune responses damage plays a crucial role in the UVB-induced immunosuppres- locally (in the skin) as well as systemically. UVB irradiation can sion. The majority of DNA lesions induced by UVB are induce antigen-speci®c unresponsiveness by mechanisms involving cyclobutane pyrimidine dimers (CPD) and pyrimidine±pyrimidone impaired antigen presentation to T helper (Th) cells, resulting in a (6±4) photoproducts. The repair of these photolesions by shift from Th1- to Th2-mediated responses (Boonstra and exogenously introduced T4 endonucleases prevents local immu- nosuppression and inhibits the induction of cytokine production Manuscript received December 29, 2000; revised March 8, 2001; (Kripke et al, 1996). This indicates that dimers in particular might accepted for publication March 12, 2001. be linked to UV-induced immunomodulation. Furthermore, Reprint requests to: Dr. J. Garssen, Laboratory for Pathology and immunosuppression could be induced by the introduction of Immunobiology, National Institute of Health and the Environment, PO DNA strand breaks in epidermal cells by restriction endonucleases Box 1, 3720 BA Bilthoven, The Netherlands. Email: [email protected] Abbreviations: APC, antigen-presenting cells; CPD, cyclobutane (O'Connor et al, 1996; Nishigori et al, 1998). DNA damage alone is pyrimidine dimer; CS, ; GGR, genome global repair; suf®cient to trigger epidermal cells to produce tumor necrosis factor NER, nucleotide excision repair; PCl, picrylchloride; TCR, transcription- a (TNF-a), interleukin-6 (IL-6) and IL-10, thereby modulating coupled repair; XP, xeroderma pigmentosum. the activity of Langerhans cells and subsequently the local immune

0022-202X/01/$15.00 ´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 141 142 BOONSTRA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY response (Nishigori et al, 1996; O'Connor et al, 1996; Petit-Frere et MATERIALS AND METHODS al, 1998). The UVB-induced photolesions are repaired mainly by the Mice XPA, XPC, and CSB mice refer to animals homozygous for nucleotide excision repair (NER) mechanism, a complex ``cut and the targeted allele in the respective (de Vries et al, 1995; Cheo et patch''-type reaction involving more than 25 (Bootsma et al, 1997; van der Horst et al, 1997). C57BL/6, the background strain for XPA mutants, were used as control mice for the XPA strain, whereas al, 2001). Two subpathways of NER are described: transcription- hybrid 129-C57BL/6 littermates of homozygous knockout mice were coupled repair (TCR) and global genome repair (GGR). TCR is used as control animals for XPC and CSB mice (Garssen et al, 2000). designed to clean the transcribed strand of active genes from lesions Genotyping was performed by polymerase chain reaction as described in that block transcription. GGR eliminates lesions anywhere in the the original references. All mice were bred at the animal facility of the genome, including the nontranscribed strand. UV-induced CPD National Institute of Public Health and the Environment (Bilthoven, the are poorly removed by the GGR subpathway, in contrast to 6±4 Netherlands). The mice were kept under speci®c-pathogen-free photoproducts, which are removed even faster by GGR than by conditions and were 8±10 wk old at the start of the experiments. The TCR (Cleaver and States, 1997; Citterio et al, 2000; Bootsma et al, room was illuminated with yellow ¯uorescent tubes (Philips TL40W/ 16), which did not emit any measurable UV radiation. No daylight 2001). entered the animal facilities. The experiments were approved by the The importance of the NER system in repair of DNA damage is ethical committee of the National Institute of Public Health and the illustrated by the rare, hereditary human NER-de®cient syndromes Environment. xeroderma pigmentosum (XP) and Cockayne syndrome (CS). At Monoclonal antibodies and reagents All cell cultures were least eight different defects have been described in XP (XPA performed in RPMI 1640 medium supplemented with 25 mM HEPES, to XPG and a variant), of which XPA is the most common form of 100 IU per ml penicillin, 50 mg per ml streptomycin, 1 mM pyruvate, the disease. XP patients are extremely sensitive to UV light and 50 mM 2-mercaptoethanol, and 10% heat-inactivated fetal bovine serum. have a more than 1000-fold higher risk of developing skin cancer. For polyclonal T cell stimulation, concanavalin A (ConA; Janssen In addition, they exhibit pigmentation abnormalities in sun- Chimica, Beerse, Belgium) or anti-CD3 monoclonal antibodies (MoAb; exposed areas of the skin (Kraemer et al, 1987; Bootsma et al, 2001). 145±2C11) were used. For the detection of cytokines by enzyme-linked CS is characterized by photosensitivity and severe neurologic and immunosorbent assay (ELISA), MoAb against interferon-g (IFN-g; developmental abnormalities. CS patients are not cancer-prone XMG1.2; R46A2), IL-10 (SXC-1; JES-2A5.1), and IL-4 (11B11; BVD2.4G2) were used. All recombinant mouse cytokines were obtained (Nance and Berry, 1992). from R&D Systems (Minneapolis, MN). Lipopolysaccharide (LPS) was A number of studies indicate that XP patients have an impaired obtained from Difco Laboratories (Detroit, MI; LPS E. coli O26:B6). cellular immunity with reduced DTH and CHS responses (Dupey Picrylchloride (PCl) was used as a contact sensitizer (Chemotronix, and Lafforet, 1974; Salamon et al, 1975; Wysenbeek et al, 1986). Swannanoa, NC) and was recrystallized three times from methanol/H2O There are some discrepancies in the immunologic alterations in XP before use and protected from light during storage at 4°C. patients, however. Normal numbers of peripheral T cells (Norris et UVB irradiation The animals were shaved (on the back) 1 d prior to al, 1990; Gaspari et al, 1993) as well as reduced numbers have been UV exposure using an electric clipper under light ether anesthesia. The reported (Mariani et al, 1992). In contrast to XP patients, no UV source was a Kromayer UV lamp (Hanovia, Slough, U.K.) equipped speci®c immune defects have been reported for CS patients (Norris with a Schott WG 305 ®lter. The lamp had an irradiance of 140 J per m2 per s in the UV range (280±400 nm), as measured with a Kipp E11 et al, 1990; Nance and Berry, 1992). It is dif®cult to conclude from 2 patient studies whether the possible immune defects in XP patients thermopile. Two circular areas (6.3 cm in total) were irradiated with 140, 280, 560, or 840 J per m2 UVB each day for ®ve consecutive days. are either UV-induced effects or related to active disease (some Twenty-four hours after the last exposure mice were sacri®ced and patients had skin tumors) or constitutive effects due to the defect in spleens and lymph nodes were dissected. Control mice were shaved but the NER genetic machinery. Moreover, in CS patients interpret- not exposed to UV. ation may be even more complicated due to the multisystemic CHS responses The mice were skin-sensitized 4 d after the last UVB character of the disease. irradiation by topical application of 150 ml of 5% PCl in ethanol:acetone To discriminate between the different possible causes of immune (3:1) to the non-UVB-irradiated shaved abdomen, chest, and feet. suppression in NER-de®cient individuals, we examined the effect Control mice were sham-sensitized by topical application of of UVB irradiation on various immune parameters in the transgenic ethanol:acetone (3:1). Four days after the sensitization, both ears of the mouse models for XPA, XPC, and CSB (de Vries et al, 1995; Cheo mice were challenged by topical application of one drop (27-gauge et al, 1997; van der Horst et al, 1997). Comparison of these three needle) of 0.8% PCl in olive oil. Twenty-four hours after challenge, mouse models permits a systematic analysis of the contribution of duplicate measurements of the ear thickness were made using an the TCR and the GGR subpathways to the UVB-induced engineer's micrometer (Mitutoyo digimatic 293561, Veenendaal, The Netherlands). immunomodulation. XPA mice have a complete NER defect, meaning that both TCR and GGR are de®cient. XPC mice carry a In vitro stimulation of lymph node and spleen cells Draining lymph speci®c de®ciency in the GGR, whereas CSB mice have impaired nodes (inguinal and axillary) and spleens of control and UVB-irradiated TCR with a normal, functional GGR. mice were removed under aseptic conditions and single cell suspensions were prepared. Erythrocytes were removed by incubating with Gey's We focused on the cytokine production by T cells and antigen- medium for 2 min on melting ice. The isolated lymphocytes were presenting cells (APC). Recently, we and colleagues showed that seeded at a concentration of 1 3 106 cells per ml and stimulated with XPA and CSB mice were very sensitive for the local UVB effect ConA (5 mg per ml) or plate-bound anti-CD3 MoAb (10 mg per ml) in like erythema, whereas only XPA mice were susceptible to UVB- 96-well ¯at bottom plates. At 48 h, the supernatants were harvested and induced immunosuppression (Miyauchi et al, 1996; Garssen et al, the concentration of cytokines was determined by ELISA. To assess anti- 2000; Horiki et al, 2000). CD3 MoAb and LPS-induced proliferation, 3H-thymidine was added to separate cultures after 48 h and 72 h, respectively. Twenty hours later, Our ®ndings show that the immune status of nonirradiated XPA, 3 XPC, and CSB mice is not detectably affected by the . the cells were harvested and H-thymidine incorporation was measured. LPS-induced cytokine production was determined in supernatant of The UVB-induced cytokine production, however, is severely lymphocyte cultures stimulated for 24 h (10 mg per ml LPS). The cells affected in XPA mice, moderately affected in CSB mice, and were cultured in 24-well ¯at bottom tissue culture plates at a similar to wild-type littermates in XPC mice. These ®ndings concentration of 2.5 3 105 cells per ml. All cultures were performed in obtained from mouse models suggest that the immune defects in triplicate or quadruplicate. XPA, XPC, and CSB patients are UVB-induced effects and are not Flow cytometric analysis 2 3 105 cells were resuspended in intrinsic effects due to the NER mutation. Furthermore, the data phosphate-buffered saline (PBS) containing 1% bovine serum albumin indicate that TCR is the major repair pathway that prevents the (BSA) and 0.1% sodium azide. For the staining of surface antigens, spleen UVB-induced acute effects. cells were incubated with ¯uorescein isothiocyanate or phycoerythrin VOL. 117, NO. 1 JULY 2001 DIFFERENTIAL IMMUNOMODULATION IN NER-DEFICIENT MICE 143 conjugated MoAb against CD3 (145±2C11), CD4 (GK1.5), CD8 (Lyt- 2), B220 (RA3.6B2), F4/80, NK1.1, and MHC-II (M5/114), all obtained from PharMingen, San Diego, CA. After washing twice with PBS±BSA-azide, the cells were resuspended and analyzed on a FACScan (Becton Dickinson, San Jose, CA). 10 ml propidium iodide (0.2 mg per ml) was added to evaluate the viability of the cells. 104 events were collected and the expression of the markers was analyzed using CellQuest software (Becton Dickinson). Cytokine ELISA IL-4 and IL-10 were determined by ELISA as described previously (MacNeil et al, 1990; Chatelain et al, 1992). Brie¯y, 96-well microplates were coated with capture antibody (1 mg per ml SXC-1; 5 mg per ml XMG1.2) at 4°C for 18 h. Incubation of the samples and standards lasted 4 h. After addition of the biotinylated detection antibodies (0.1 mg per ml 2A5.1; 1 mg per ml R46A2), the plates were incubated overnight at 4°C. The reaction was visualized using streptavidin-peroxidase (1:1500 diluted, Jackson Immunoresearch, West Grove, PA) and ABTS (1 mg per ml, Sigma). Optical density was measured at 414 nm, using a Titertek Multiscan (Flow Laboratories, Redwood City, CA). The TNF-a concentrations were measured with a Figure 1. UVB-induced systemic suppression of CHS to PCl in commercially available ELISA kit (DuoSet, R&D Systems) according to NER-de®cient mice and their repair-competent littermates. Each the protocols provided by the manufacturer. The detection limits of the bar represents the mean value of the corrected ear swelling (i.e., swelling ELISA were: IFN-g, 20 pg per ml; IL-10, 5 pg per ml; and TNF-a, in sensitized minus swelling in nonsensitized animals of the same strain) 15 pg per ml. of at least ®ve mice 6 SEM. Units are 10±3 cm. p-values re¯ect the Statistical analysis Levels of signi®cance were calculated using the comparison with the nonirradiated group (0 s UVB). *p < 0.05. two-tailed Student's t test. p < 0.05 was taken as a signi®cant difference between groups. Each group consisted of at least ®ve mice.

RESULTS Normal T, B, and APC activity in NER-de®cient mice As only limited data are available on the immune status of nonirradiated DNA repair-de®cient mice, a broad scale of immune parameters was determined for XPA, XPC, and CSB mice in cell suspensions obtained from their spleens and lymph nodes. We tested in vitro ConA- and anti-CD3 MoAb-induced T cell proliferation and cytokine production (IFN-g, IL-4, IL-10), B cell proliferation, in vitro immunoglobulin production, serum immunoglobulin levels (IgM, IgG1, IgG2a, IgE), and LPS- induced cytokine production (TNF-a, IL-6, IL-10). Preliminary data did not indicate signi®cant differences in any of these parameters in XPA, XPC, and CSB mice compared to their own wild-type littermates (data not shown). In addition, subset ratios were determined by ¯ow cytometry (CD3, CD4, CD8, B220, NK, F4/80). Again preliminary data did not indicate signi®cant Figure 2. UVB-induced effects of IFN-g production by lymph differences comparing the mutant mice with their respective node cells stimulated with ConA in the PCl-sensitized mice. See wild-type littermates. These ®ndings suggest that the in vivo APC Fig 1. Each bar represents the mean value of at least ®ve mice 6 SEM. and T and B cell compartments of XPA, XPC, and CSB mice are p-values re¯ect the comparison with the nonirradiated group (0 s UVB). not detectably affected by the different NER gene defects. *p < 0.05. CHS responses in DNA repair-de®cient mice Besides these in vitro assays, we also examined the in vivo CHS responses to PCl. As shown in Fig 1, ear swelling in nonirradiated DNA repair- demonstrated by the reduced production of IFN-g. We determined de®cient mice (0 J per m2) is similar to the response in control the UVB-induced modulation of ConA-induced IFN-g littermates. We recently showed that besides the CHS reaction to production in the lymph nodes of XPA, XPC, and CSB mutant PCl also the DTH response to Listeria monocytogenes was similar in mice to examine their differential UVB susceptibility. Figure 2 XPA, XPC, and CSB mice compared with their wild-type shows the results of a representative experiment. The IFN-g littermates (Garssen et al, 2000). Together, these data support the production by XPA lymphocytes displays a signi®cant reduction 2 ®nding that both APC and T cell driven responses are normal in after a dose of 140 J per m UVB, whereas the levels in the wild- these mice compared with their own wild-type littermates. type littermates were not signi®cantly changed. The IFN-g With respect to the sensitivity to UVB, Fig 1 shows that XPA production of both XPC and CSB lymphocytes are not inhibited mice are more sensitive and demonstrate suppression of the CHS after 140 J per m2 UVB, but showed a reduction after a UVB dose responses at doses as low as 140 J per m2. At this dose both XPC of 840 J per m2, which is similar to the response seen in control and CSB mice display normal CHS responses. A signi®cant littermates. Taken together, the UVB-induced changes of the IFN- reduction is seen at 840 J per m2 UVB in both XPC and, to a lesser g production in these mice re¯ect the pattern seen in the CHS extent, CSB mice, as well as their respective littermates. It can be responses. concluded that XPC and CSB mice do not show an enhanced Effect of UVB on lymph node hyperplasia in DNA repair- sensitivity to UVB exposure with respect to the suppression of de®cient mice Next, we determined the effect of UVB on the CHS responses, whereas XPA mice are extremely sensitive degree of hyperplasia in the skin-draining lymph nodes of XPA, compared to their own wild-type littermates. XPC, and CSB mice. As depicted in Fig 3, already after only 140 J Effect of UVB on the IFN-g production in DNA repair- per m2 UVB a 3-fold increase in the number of lymph node cells de®cient mice A number of studies have indicated that UVB was observed in XPA mice, whereas no increase was observed in irradiation inhibits the activation of Th1 cells in the lymph nodes as wild-type littermates at any UVB dose used. In XPC mice a slight 144 BOONSTRA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Effect of UVB on TNF-a and IL-10 production in the lymph nodes of DNA repair-de®cient mice As APC (macrophages and dendritic cells) play a pivotal role in governing lymphocyte responses, we determined the effect of UVB on cytokine production by APC in the lymph nodes. In order to detect possible differences between DNA repair-de®cient mice and their respective littermates, lymph node cells were stimulated in vitro with LPS and the levels of TNF-a and IL-10 were determined by ELISA. As depicted in Fig 4, LPS-stimulated lymph node cells from XPA and CSB mice showed a signi®cant increase in the production of TNF-a compared with their littermate controls. In Figure 3. The effect of UVB irradiation on the cellularity of the XPA mutants, this increase was already detectable after 140 J per draining lymph nodes in nontreated NER-de®cient mice and m2 UVB, whereas in CSB mice the response started at 280 J per m2 their littermates after UVB irradiation. Cell numbers are expressed as mean values 3 10±6 6 SEM. p-values re¯ect the comparison with the UVB. Also XPC mice showed an increase, but again this was also nonirradiated group (0 s UVB). *p < 0.05. observed in littermates. The same pattern as described for TNF-a was found for IL-10 (Fig 5): a UVB-dose-dependent increase of IL-10 production in the XPA and CSB mice, whereas the control littermates demon- strated only a mild increase in IL-10 production. In XPC mice, IL- 10 was not detectable in any of the cultures. Similar effects on TNF-a and IL-10 production were demonstrated after stimulation of splenocytes (data not shown), indicating that it is not only a local event in skin-draining lymph nodes, but has systemic consequences as well.

DISCUSSION In this study we examined the immune status of DNA repair- Figure 4. TNF-a production by draining lymph node cells from de®cient XPA, XPC, and CSB mice as well as the UVB-induced UVB-irradiated and nonirradiated NER-de®cient mice and their immune effects in these mice. We demonstrated that all three littermates. The cells were stimulated with LPS in vitro, and the transgenic mouse strains have normal APC, T, and B cell responses supernatant was harvested after 24 h. The levels of TNF-a were that are identical to their own wild-type littermates. After UVB determined by ELISA. Each bar represents the mean value of at least ®ve exposure, we found that XPA mice are extremely sensitive to mice 6 SEM. p-values re¯ect the comparison with the nonirradiated group (0 s UVB). *p < 0.05. UVB-induced immunomodulation, as demonstrated by the effect on lymph node cellularity and cytokine production by stimulated T cells and APC. Moreover, CSB mice respond intermediately and XPC mice are even insensitive to these UVB-induced effects compared to their respective wild-type littermates. A number of studies showed immunologic abnormalities in XP patients. These included weaker DTH and CHS responses, low in vitro response to phytohemagglutinin, reduced IFN-g produc- tion, decreased CD4:CD8 ratios, and reduced natural killer cell activity (Morison et al, 1985; Wysenbeek et al, 1986; Norris et al, 1990; Mariani et al, 1992; Gaspari et al, 1993). No such immune defects have been described for CS. One of the problems encountered when studying these immune responses in NER- de®cient patients is that the differences in their immune status may be either constitutive or due to external factors, such as exposure to UVB. Furthermore, the multisystemic character and low frequen- cies of the diseases, as well as the interindividual variability (caused by gender, ethnicity, etc.) further complicate the interpretation of Figure 5. In vitro IL-10 production by LPS-stimulated lymph data obtained from case studies. Therefore, examination of the node cells from UVB-irradiated and nonirradiated NER-de®cient mice and their littermates. For details see the legend to Fig 4. NER-de®cient mouse models for XPA, XPC, and CSB provides Stimulation with LPS of lymph node cells from XPC+/+ and XPC±/± an excellent tool to determine the contribution of de®ned genetic mice resulted in IL-10 values below the detection limit of the ELISA, NER defects to immune dysfunction. Our ®ndings that cytokine which are therefore not graphically depicted. *p < 0.05. production and activity of APC, T, and B cells were similar in nonirradiated NER de®cient transgenics and their wild-type littermates suggests that the presence of mutated NER genes by itself has no consequences for the capacity of these cells to generate increase in the number of lymph node cells was seen after UVB cellular immunity, at least in the different mutant mouse models irradiation. This was also observed in the corresponding wild-type described. littermate controls, however. Finally, CSB mice showed a tendency Exposure of DNA repair-de®cient mice to UVB revealed that to an increment of the number of lymph node cells after 140 J per XPA mice are extremely susceptible to systemic suppression of m2 UVB. At higher doses no further increase was detected. No CHS responses and IFN-g production, whereas CSB and XPC change was seen in the control littermates. mice are not more sensitive to UVB than wild-type littermates. Flow cytometric analysis of the lymph node cells of all groups for This is in agreement with data obtained previously (Garssen et al, CD4, CD8, CD3, B220, and MHC II did not reveal any signi®cant 2000). These effects of UVB are well described and re¯ect an changes in the ratio of a speci®c leukocyte subset, indicating that inhibition of the initiation and effector phase of Th1-mediated the increase in cell numbers in the lymph node is not due to immune responses, important in, for example, the response to selective expansion of one or a few speci®c subsets. intracellular pathogens. VOL. 117, NO. 1 JULY 2001 DIFFERENTIAL IMMUNOMODULATION IN NER-DEFICIENT MICE 145

Exposure to UVB has been shown to increase the number of enhanced in XPA and CSB, but also in XPC mice (de Vries et al, peripheral lymphocytes that migrate into the skin-draining lymph 1995; Sands et al, 1995; van der Horst et al, 1997). Apparently, these nodes (Spangrude et al, 1983; Samlowski et al, 1988). The enhanced effects are the consequence of DNA lesions in either the transcribed migration correlated with an increase in the number and density of or the nontranscribed strand. Interestingly, in man CS(B) appears to high endothelial venules within the lymph nodes (Samlowski et al, lack the strong cancer predisposition evident in XPA and XPC 1988). Both XPA and CSB mice showed an enhanced sensitivity to patients. One interpretation for this human±mouse difference is UVB with respect to lymph node cellularity. that the effect of enhanced apoptosis after UV exposure protecting Furthermore, the LPS-induced TNF-a and IL-10 production by against onset of cancer in CSB patients outweighs the immuno- lymph node cells was also enhanced in XPA and CSB mice exposed suppressive effects that would be expected to increase oncogenesis. to low UVB doses. In XPC mice and their normal wild-type In the case of the XPC defect despite the reduced immunosuppres- littermates no UVB-induced changes were detected using these sion a dramatic increase in skin cancer is found in XPC patients. parameters. As we also observed a comparable modulation of the Thus the increased mutagenesis resulting from the GGR defect in cytokine production in the spleen, our data extend these ®ndings XPC may be solely responsible for the cancer susceptibility. A by showing that the effects are not limited to the local level, but confounding parameter in these interspecies extrapolations is the have consequences for the systemic immunosuppression as well. existence of a signi®cant difference between the human and rodent The important role of TNF-a and IL-10 in modulating immune GGR subpathway. Whereas UV-induced CPD lesions are responses has been described extensively (Rivas and Ullrich, 1994; removed from the genome overall in man ± albeit slowly ± they Boonstra and Savelkoul, 1997). In UVB-irradiated skin, augmented appear to be completely overlooked by GGR in mice. This would production of these cytokines, which was induced by DNA render humans more resistant to UV-induced neoplasia than mice, damage directly, has been reported. As Langerhans cells migrate to explaining at least in part the higher cancer susceptibility observed the draining lymph nodes after UVB exposure, and CPD+ in CSB mice compared to the human syndrome. Possibly, this dendritic-like cells are detected in the lymph nodes (Vink et al, repair difference may also differentially affect the immune response 1996), it is likely that they are the producers of TNF-a and IL-10 in both organisms. For instance, the loss of GGR in XPC mutants in the lymph node. The persistence of DNA damage in these cells is expected to have less severe consequences in the murine system may result in long-lasting cytokine expression. The inhibitory compared to man. Consequently, the mild effects on immunologic activity of TNF-a and IL-10 may add to the UVB-induced local as parameters in XPC mice may be more pronounced in human XPC well as systemic immunosuppression. These ®ndings are in line patients. with our previous observations that UVB-induced acute skin Taken together, (i) our data demonstrate that inactivation of the reactions (edema and erythema) are absent in XPC mice, but more XPA, XPC, and CSB genes in non-UV-irradiated mice has no vigorous in XPA and CSB mice (Garssen et al, 2000). This is most signi®cant constitutive effects on immune responses (APC, T, or B probably caused by enhanced keratinocyte-derived cytokine pro- cell responses). (ii) Putting our ®ndings in the context of the duction. corresponding human syndromes these observations suggest that The UVB-induced immune effects in XPA mice are more the immunologic alterations in patients might be ascribed to vigorous than in CSB mice, but absent in XPC mice. XPA mice external factors. A likely factor is UVB, as we observed suscep- lack both TCR and GGR, whereas XPC and CSB mice are tibility to UVB in XPA and CSB mice as determined by changes in de®cient in GGR and TCR, respectively. Functionally, GGR has T cell and APC derived cytokine production. (iii) XPC mice been associated mainly (although not exclusively) with preventing showed no increased susceptibility to UVB compared to their wild- mutagenesis and carcinogenesis, whereas TCR activity prevents type littermates. (iv) Both GGR and TCR are needed to prevent apoptosis after genotoxic injury (de Boer and Hoeijmakers, 2000). immunomodulation by UVB, whereas TCR is the major DNA The study presented here clearly demonstrates an additional repair subpathway of NER that prevents the acute UVB-induced involvement of these pathways in the UVB-induced immunomo- effects. dulation. Both TCR and GGR pathways are involved in the UVB- induced immunomodulation (as deduced from the synergistic effects seen in XPA mice) but with the strongest input from TCR We thank Prof. Dr. R. Benner for critically reading the manuscript, Diane Kegler, (as apparent from the partial effects seen in CSB and the absence of Piet van Schaaik, and Hans Strootman for animal breeding and biotechnical support, signi®cant abnormality in XPC mice). and Mariska van Dijk for her excellent technical assistance. We are grateful to Dr. Introduction of DNA lesions in the absence of TCR (as evident E.C. Friedberg for providing the XPC mouse strain. This study was supported the in XPA and CSB mice) results in a transcriptional block of speci®c IRS (Leiden, The Netherlands), the Dutch Cancer Society (project EUR 1999- genes. It is likely that the arrest of RNA synthesis of critical genes in 2004 and 1998-1774), and the Spinoza award to JHJH by the Dutch epidermal cells dysregulates the homeostasis in the skin and evokes Organization for Science. the observed immune effects. Furthermore, apoptosis is another likely mechanism explaining the severe immune effects in XPA and CSB mice. Arrest of RNA synthesis initiates p53-dependent REFERENCES apoptotic pathways (Yamaizumi and Sugano, 1994; Ljungman and de Boer J, Hoeijmakers JHJ: Nucleotide excision repair and human syndromes. Zhang, 1996; Conforti et al, 2000). Therefore, one could speculate Carcinogenesis 21:453±460, 2000 that the immune system of TCR-de®cient mice (i.e., XPA and Boonstra A, Savelkoul HFJ: The role of cytokines in ultraviolet-B induced CSB mice) is triggered by stress signals, evoked by massive death of immunosuppression. Eur Cytokine Netw 8:117±123, 1997 keratinocytes in the absence of apoptosis-preventing TCR activity. 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