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. Author manuscript; available in PMC: 2009 Jul 1.
Published in final edited form as: J Invest Dermatol. 2008 Jan 17;128(7):1780–1787. doi: 10.1038/sj.jid.5701251

UVB Radiation-Mediated Inhibition of Contact Hypersensitivity Reactions Is Dependent on the Platelet-Activating Factor System

Qiwei Zhang 1,2,6, Yongxue Yao 1,2,6, Raymond L Konger 1,3, Anthony L Sinn 2, Shanbao Cai 2, Karen E Pollok 2,4, Jeffrey B Travers 1,2,4,5
PMCID: PMC2643301  NIHMSID: NIHMS84998  PMID: 18200048

Abstract

Through its ability to both induce immunosuppression and act as a carcinogen, UVB radiation plays a major role in cutaneous malignancies. Recent studies have indicated that UVB-mediated inhibition of delayed-type hypersensitivity reactions is mediated, in part, by the lipid mediator platelet-activating factor (PAF). The objective of this study was to further define the mechanism by which UVB inhibits contact hypersensitivity (CHS) reactions. UVB irradiation resulted in an inhibition of subsequent CHS to the chemical DNFB in wild-type, but not in PAF-R-deficient mice. UVB-mediated inhibition of CHS was also blocked by a cyclooxygenase-2 (COX-2) inhibitor or a neutralizing antibody directed against IL-10. UVB irradiation upregulated IL-10 mRNA levels in lymph nodes and spleen only to significant levels in PAF-R-expressing mice. Bone marrow transplantation studies demonstrated that UVB-mediated immunomodulatory effects were dependent on PAF-R-positive bone marrow. These studies suggest that UVB irradiation results in epidermal production of PAF agonists, which then act on PAF-R-positive bone marrow-derived cells to upregulate IL-10 through COX-2-generated prostaglandins.

INTRODUCTION

UVB radiation (290−320 nm; UVB) is the most important environmental carcinogen, and skin is the major target. Non-melanoma skin cancer is the most common type of cancer diagnosed, and UV radiation is the source (Kripke, 1977; Carroll et al., 2003). In addition to its ability to damage DNA, UVB radiation is immunosuppressive (Ullrich, 2005). UVB-induced immunosuppression is divided into two distinct types, local and systemic immunosuppression (Ullrich, 2005). Local immunosuppression is observed when UVB irradiation to an area of skin results in the inability to sensitize with an antigen in the UVB-irradiated area. Systemic immunosuppression is produced when UVB irradiation to an area results in the inability to sensitize with an antigen in skin distant from the UVB-irradiated site. Experimental studies with mice indicate that systemic immunosuppression usually necessitates higher doses of UVB than local immunosuppression. Compelling findings from a multitude of sources, including animal model systems and human patients, especially transplant patients, have confirmed that the immunosuppressive effects of UV are a major risk factor in the development of these skin cancers (Ullrich, 2002; Young and Wikonkal, 2007).

Recent studies have implicated UVB-induced soluble mediators derived from human keratinocytes in systemic immunosuppression. The lipid mediator platelet-activating factor (1-O-alkyl-2-acetyl glycerophosphocholine, PAF) is one of the mediators that have been implicated. PAF is an inflammatory phospholipid mediator that exerts its effects through a single specific G-protein-coupled receptor, the PAF receptor (PAF-R) (Ishii et al., 2002). The PAF-R is expressed by cells of the innate immune system, but also by keratinocytes of the skin (Travers et al., 1995). PAF is synthesized in response to diverse stimuli including cytokines, endotoxin, and Ca2+ ionophores (Travers et al., 1996). Notably, direct damage to keratinocytes by either heat or cold stimuli results in significant PAF production (Alappatt et al., 2000). PAF is produced enzymatically, yet PAF and sn-2 short-chained acyl glycerophosphocholines (GPCs) with PAF-R agonistic activity can also be produced through free radical-mediated damage (Marathe et al., 2000).

Through its ability to act as a potent pro-oxidative stressor (Peus et al., 1998), UVB has been demonstrated to trigger production of PAF and oxidized GPCs (ox-GPC) with PAF-R agonistic activity (Walterscheid et al., 2002; Marathe et al., 2005). Several lines of evidence have implicated these UVB-generated PAF agonists in UVB-mediated systemic immunosuppression. First, pretreatment of mice with a series of PAF-R antagonists inhibits UVB-mediated inhibition of delayed-type hypersensitivity (DTH) reactions to Candida albicans (Walterscheid et al., 2002). This inhibition of DTH was mimicked by intraperitoneal injection of PAF or UVB-irradiated GPC (Walterscheid et al., 2002). Second, inhibition of DTH to C. albicans induced by topical psoralen and UVA or by a solar-simulator UV source was observed in wild-type, but not in PAF-R-deficient mice (Wolf et al., 2006). Finally, in a model of contact hypersensitivity (CHS) to the chemical DNFB, intraperitoneal injection of the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl GPC (CPAF) inhibited both the sensitization and elicitation phases in wild-type but had no effect in PAF-R-negative mice (Zhang et al., 2005). Significantly, COX-2 inhibitors and IL-10-neutralizing antibody blocked PAF-mediated immunomodulatory effects (Walterscheid et al., 2002; Zhang et al., 2005). These studies indicate that UVB-generated PAF/ox-GPC results in a potentially complex series of events involving COX-2 and IL-10 resulting in an inhibition of DTH reactions.

The objective of this study was to characterize the role of the PAF system in UVB-mediated systemic immunosuppression using the model system of CHS to the chemical DNFB. This study confirms the importance of PAF agonists in this process and indicates that UVB generates PAF agonists, which then act on PAF-R-positive bone marrow-derived immune cells to inhibit DTH reactions through IL-10. Inasmuch as the immunomodulatory effects of UVB are felt to be important in its carcinogenic as well as therapeutic effects, characterization of this process could have significant clinical benefit.

RESULTS

Importance of PAF-R in UVB-mediated inhibition of contact hypersensitivity

UVB irradiation induces an inhibition of DTH reactions. Thus, our earlier studies assessed the role of PAF-R in the UVB-mediated inhibition of CHS using PAF-R-deficient mice (Ishii et al., 1998). In these studies, an approximately 2.5 × 2.5 cm area of shaved back skin was treated with 7.5 kJ m−2 UVB radiation. Identically treated mice without UVB served as controls (sham). Five days after mock or UVB irradiation, unirradiated back skin approximately 3 cm away from the irradiated site was sensitized to the chemical compound DNFB. Nine days after DNFB sensitization, the dorsal sides of ears were treated with DNFB or vehicle control. Punch biopsies were taken from ears 24 hours later and inflammation assessed by weighing and comparing DNFB with vehicle-painted biopsies. Treatment of murine ears with DNFB without previous sensitization did not result in significant inflammation (as measured by ear biopsy weight) at 24 hours (data not shown). As shown in Figure 1, there was no difference in the CHS reactions between wild-type and PAF-R−/− mice. Intraperitoneal injection of the metabolically stable PAF-R agonist CPAF followed 5 days later by DNFB sensitization resulted in an inhibition of CHS to DNFB only in wild-type mice (Figure 1a). UVB treatment also inhibited subsequent CHS reactions to DNFB in wild-type, but not in PAF-R−/− mice (Figure 1b). These studies demonstrate involvement of the PAF system in UVB-mediated inhibition of CHS.

Figure 1. Effect of UVB and CPAF on CHS in wild-type vs PAF-R−/− mice.

Figure 1

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Wild-type and PAF-R−/− mice were treated with either (a) intraperitoneal injection of 250 ng of the PAF-R agonist CPAF or vehicle control, or (b) local irradiation with 0 (sham) or 7,500 J m−2 UVB. Mice were then sensitized to DNFB and elicitation reactions performed on the dorsal ears as outlined in Materials and Methods. At 24 hours after elicitation reactions, 5 mm punch biopsies were obtained from the ears and weighed. The data listed are the mean±SEM difference in ear biopsy weights from n = 6 mice. *Statistically (P<0.05) significant changes.

Involvement of cyclooxygenase-2 and IL-10 in UVB-mediated inhibition of contact hypersensitivity

Earlier studies have implicated IL-10 in immunosuppressive effects of UVB (Shreedhar et al., 1998; Nghiem et al., 2002). It is to be noted that PAF-R activation has been shown to stimulate IL-10 production through a COX-2-dependent process in the murine keratinocyte cell line PAM 212 (Walterscheid et al., 2002). Intradermal injection of CPAF also results in epidermal IL-10 production in PAF-R-expressing, but not in PAF-R-deficient mice (Zhang et al., 2005). Thus, IL-10 could mediate PAF-R-mediated immunomodulatory effects.

The succeeding studies tested the hypothesis that UVB-generated PAF from the skin results in COX-2-mediated prostaglandins, which then induce IL-10 production mediating UVB inhibitory effects on CHS. In those studies we tested the effect of the COX-2 antagonist SC236 and a neutralizing antibody against IL-10 on UVB-mediated inhibition of CHS. As shown in Figure 2, pretreatment with SC236 has no effect on CHS in unirradiated mice, but blocks the inhibitory effects of both UVB and CPAF. To confirm that IL-10 is involved in UVB-mediated suppression of CHS reactions, we tested the ability of a neutralizing antibody against IL-10 (Nghiem et al., 2002) to block CPAF- and UVB-induced inhibition of DNFB reactions. As shown in Figure 3, systemic treatment with neutralizing anti-IL-10 antibody blocked the CPAF- (Figure 3a) and UVB- (Figure 3b) induced inhibitory effects on CHS in wild-type mice. These studies provide support for the notion that COX-2 metabolites and IL-10 are downstream mediators of PAF in UVB-mediated inhibition of CHS.

Figure 2. Effect of a COX-2 inhibitor on CPAF- and UVB-mediated inhibition of CHS in wild-type mice.

Figure 2

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Wild-type mice were pretreated with intraperitoneal injection of 200 ng of the COX-2 antagonist SC-236 or vehicle (control) and the mice treated with intraperitoneal injection of 250 ng CPAF or local irradiation with 0 (sham) or 7,500 J m−2 UVB. Mice were then sensitized to DNFB and elicitation reactions performed on the dorsal ears as outlined in Materials and Methods. At 24 hours after elicitation reactions, 5 mm punch biopsies were obtained from the ears and weighed. The data listed are the mean±SEM difference in ear biopsy weights from n = 6 mice. *Statistically (P<0.05) significant changes.

Figure 3. Effect of a neutralizing anti-IL-10 antibody on CPAF- and UVB-mediated inhibition of CHS in wild-type mice.

Figure 3

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Wild-type mice were treated with (a) 250 ng CPAF or control vehicle or (b) local irradiation with 0 (sham) or 7,500 J m−2 UVB. Four and 24 hours after UV irradiation, the mice were injected intraperitoneally with 100 μg neutralizing rat anti-mouse IL-10 antibodies or 100 μg rat IgG1 antibody control or BSA vehicle control. Mice were then sensitized to DNFB and elicitation reactions performed on the dorsal ears as outlined in Materials and Methods. At 24 hours after elicitation reactions, 5 mm punch biopsies were obtained from the ears and weighed. The data listed are the mean±SEM difference in ear biopsy weights from n = 6 mice. *Statistically (P<0.05) significant changes.

Evidence that immunocyte IL-10 mediates UVB-mediated systemic immunosuppression

UVB-mediated PAF and PAF-like lipids (for example, ox-GPC) could potentially interact with epidermal keratinocytes (Pei et al., 1998; Walterscheid et al., 2002), or PAF-R agonists could interact with bone marrow-derived cells including dendritic cells or T/B cells (Travers et al., 1989; Mazer et al., 1992; Sozzani et al., 1997) to induce COX-2 and IL-10. The succeeding studies were designed to assess whether the IL-10 induction in response to UVB was derived from skin or lymphoid organs. To that end, wild-type and PAF-R−/− mice were irradiated with UVB as per our protocol, and irradiated skin, nearby unirradiated skin, lymph nodes, or spleen were harvested at 24, 48, or 72 hours, and IL-10 mRNA measured by quantitative real-time reverse transcriptase-PCR (qRT-PCR). As depicted in Figure 4, UVB irradiation resulted in an upregulation of IL-10 mRNA levels in lymph nodes and spleen of wild-type, but not in PAF-R−/− mice at 24 hours. IL-10 mRNA levels were only slightly increased above baseline levels in wild-type, but again none was detected in PAF-R−/− mice at 48 hours (data not shown). IL-10 mRNA was not increased in lymph nodes or spleen at 72 hours. Notably, UVB irradiation did not result in increased levels of IL-10 mRNA levels in directly irradiated nor in nearby unirradiated skin at any of the time points examined (data not shown).

Figure 4. IL-10 expression in groin lymph nodes and spleen after UVB irradiation of mouse back skin.

Figure 4

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Wild-type and PAF-R−/− mice were shaved and the lower backs were exposed to 7,500 J m−2 UVB irradiation. The control mice were shaved but sham irradiated. Twenty-four hours after exposure, the groin lymph nodes and spleen were taken and homogenized and total RNA was isolated. RNA was reverse transcribed and qRT-PCR performed with the IL-10 primer kit (reference positions 319−339) on SmartCycler PCR system with SmartCycler 1.2 software. The expression levels of IL-10 were normalized to the expression levels of 18S. The data listed are the mean±SEM in IL-10/18S from n = 4 mice. *Statistically (P<0.05) significant changes in comparison to similarly treated wild-type mice. ND, not detectable.

The succeeding studies attempted to evaluate which cells in lymph nodes expressed IL-10. To that end, an area of distal back skin of wild-type and PAF-R−/− mice were treated with 7.5 kJ m−2 UVB radiation, and 24 hours later the groin draining lymph nodes were harvested. Cytokine expression in enriched populations of dendritic cells, macrophages, B cells, and CD4 and CD8 T cells was measured by qRT-PCR. As shown in Figure 5a, baseline IL-10 expression was seen in lymph node cells from both wild-type and PAF-R−/− mice. Of the cell types that appear responsible for the IL-10 production, dendritic cells and monocytes were found to have the highest expression, with lesser amounts seen in CD4 T cells. Immunocyte production of IL-12 was also examined, as this cytokine, like IL-18, has protective effects against UVB-mediated immunosuppression through its ability to protect against DNA damage (Schwarz et al., 2006). As indicated in Figure 5b, however, IL-12 mRNA levels were not altered after UVB irradiation in both wild-type and PAF-R−/− immunocytes. In addition, levels of the pleiotropic cytokine tumor necrosis factor-α were not induced differentially between wild-type and PAF-R−/− immunocytes (Figure 5c).

Figure 5. Cytokine expression in immunocytes from lymph nodes after UVB irradiation of mouse back skin.

Figure 5

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Wild-type and PAF-R−/− mice were shaved and the lower backs exposed to 7,500 J m−2 UVB irradiation. The control mice were shaved but sham irradiated. Twenty-four hours after exposure, the groin lymph nodes were obtained and cell populations purified as described in Materials and Methods. Total RNA was isolated from enriched different cell types and cDNA were synthesized and qRT-PCR was performed by the comparative threshold cycle (ΔCT) method for (a) IL-10, (b) IL-12, and (c) TNF-α and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The data listed are the mean±SEM mRNA from two separate experiments of cell types derived from pooled lymph nodes from five mice with the PCRs conducted in triplicate. DC, dendritic cells; MQ, macrophages. P<0.05 compared to self non-UVB control; P<0.05 compared to self non-UVB control and KO counterpart.

These studies suggest that the source of the IL-10 in this model of UVB-induced systemic immunosuppression is from immune cells, not from keratinocytes.

PAF-R-expressing immunocytes are responsible for UVB-mediated immunosuppressive effects

The present study suggests that UVB-generated PAF from the skin stimulates IL-10 production in lymphoid cells. This would indicate two possibilities for how UVB-generated PAF agonists act: (1) PAF-R agonists act on keratinocytes to stimulate COX-2 and that prostaglandins then mediate the IL-10 production in bone marrow-derived immunocytes or (2) PAF-R agonists act directly on bone marrow-derived immunocytes to upregulate IL-10 in a COX-2-dependent manner.

To test whether the PAF-R-responsive cells are skin- or bone marrow-derived, PAF-R−/− mice were transplanted with PAF-R-positive bone marrow, and vice versa, to create chimeric mice with respect to PAF-R and bone marrow. As shown in Figure 6, bone marrow replacement of PAF-R−/− mice with PAF-R-positive cells restored the inhibitory effects of UVB on CHS. Similarly, PAF-R-positive mice transplanted with PAF-R−/− bone marrow did not respond to these immunomodulatory effects of UVB (Figure 6). These studies, along with the IL-10 studies in Figures 4 and 5a, indicate that the PAF-R-positive cells critical for UVB-mediated IL-10 production and inhibition of CHS are bone marrow-derived, not keratinocytes.

Figure 6. Effect of UVB on CHS in PAF-R chimeric mice.

Figure 6

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Wild-type (BoyJ), PAF-R−/− , or two groups of chimeric mice with PAF-R-positive bone marrow in a PAF-R-negative host and PAF-R-negative bone marrow in a PAF-R-positive host or a transplantation control (PAF-R-positive bone marrow in a PAF-R-negative host) were treated with local irradiation with 0 (sham) or 7,500 J m−2 UVB. Mice were then sensitized to DNFB and elicitation reactions performed on the dorsal ears as outlined in Materials and Methods. At 24 hours after elicitation reactions, 5 mm punch biopsies were obtained from the ears and weighed. The data listed are the mean±SEM difference in ear biopsy weights. Parentheses indicate numbers of mice used in each treatment. *Statistically (P<0.05) significant changes.

DISCUSSION

This study indicates the importance of the PAF system in UVB-mediated inhibition of murine CHS. The lack of responsiveness of PAF-R-deficient mice to the inhibitory effects of UVB on CHS to the chemical DNFB and the ability of the PAF-R agonist CPAF to mimic UVB (Figure 1) provide compelling evidence implicating PAF in this process. These studies confirm our previous findings that intraperitoneal injection of the PAF agonist CPAF inhibits CHS to DNFB in wild-type, but not in PAF-R-deficient mice (Zhang et al., 2005). These studies also fit with the findings by Ullrich and colleagues that PAF-R antagonists block UVB-mediated DTH reactions to subcutaneous killed C. albicans (Walterscheid et al., 2002). In addition, UV irradiation using a solar simulator was reported to inhibit DTH to C. albicans in wild-type, but not in PAF-R−/− mice (Wolf et al., 2006).

UVB-mediated systemic immunosuppressive effects have been linked to IL-10. The ability of PAF-R activation to stimulate production of the powerful TH2 cytokine IL-10 in epidermal and other cell types provides a mechanism by which the PAF system can inhibit TH1 and enhance TH2 responses (Walterscheid et al., 2002). The importance of IL-10 in allergic skin diseases has been identified using tools such as IL-10−/− mice, whose phenotype includes blunting of TH2 and enhanced TH1-associated reactions (Yang et al., 1999). Our findings that UVB irradiation of murine skin in PAF-R-positive mice results in detectable levels of IL-10 mRNA in lymph nodes/spleen and that UVB- and CPAF-mediated inhibition of murine CHS is inhibited by a neutralizing antibody against IL-10 both indicate involvement of IL-10 in this process. Although UVB irradiation of epithelial cells has been shown to induce IL-10 production (Moodycliffe et al., 1996; Walterscheid et al., 2002), our studies suggest that immunocyte IL-10 probably plays a more important role in UVB-mediated systemic immunosuppression.

As UVB irradiation does not pass significantly through the epidermis (Garmyn and Yarosh, 2007), the initial target for UVB must reside in the most superficial aspects of the skin. Thus, UVB interacting with epidermal cells then puts into play the processes resulting in UVB-mediated systemic immunosuppression. The exact process and mediators involved have not been fully characterized, yet accumulating evidence including these studies implicates the PAF system.

The synthetic pathway for PAF consists of two enzymes; a phospholipase A2 that generates the lysolipid backbone by releasing the sn-2 fatty acyl residue from alkyl phosphatidylcholine and PAF acetyltransferase activity that transfers an acetyl residue from acetyl-CoA to this newly generated lysolipid (Prescott et al., 2000). Both activities are tightly regulated, with increased intracellular Ca2+ being the premier agent for their activation.

Ligands that activate the PAF-R are generated in a second way, and this route is not subject to cellular control. The alkyl phospholipid pool is enriched at the sn-2 position with arachidonoyl residues, a source of lyso-PAF and arachidonate for prostanoid and leukotriene synthesis, which as a polyunsaturated fatty acid is susceptible to non-enzymatic oxidation. Oxidation of esterified fatty acyl residues introduces oxy functions, rearranges bonds, and fragments carbon–carbon bonds by β-scission that generate a myriad of reaction products (Frankel, 1984). Among these are a series of phospholipids with oxidatively fragmented sn-2 acyl residues that terminate with either an Ω-oxy function or a methyl group. The latter series is one carbon atom shorter than the Ω-oxy series, as expected from the β-scission reaction. Among the phospholipid reaction products are those that are ligands for the PAF-R. The most potent of the non-enzymatically generated PAF analogs are native PAF as well as the butanoyl (C4-PAF) and butenoyl (C4:1-PAF) species, which are one tenth as potent as PAF (Heery et al., 1995).

Previous studies from our group have used mass spectrometry to identify native PAF as well as ox-GPCs including butanoyl and butenoyl PAF species produced in response to UVB in the human epithelial cell line KB (Marathe et al., 2005). Moreover, UVB irradiation of the precursor phospholipid 1-hexadecyl-2-arachidonoyl GPC in a cell-free system generates these same products, consistent with a nonenzymatic process (Marathe et al., 2005).

This study fits with the hypothesis that UVB, through its ability to act as a pro-oxidative stressor, generates PAF and ox-GPCs, which then act on bone marrow-derived immunocytes to generate IL-10 through COX-2-produced prostaglandins. Compatible with this hypothesis, recent studies by Matsumura et al. (2006) found that transferring lymph node cells from UV-irradiated, FITC-sensitized mice into normal recipients transferred immune tolerance. Elegant studies by this group demonstrated that the cell responsible was a CD19+, B220+ B cell. These studies examining the transfer of UVB-mediated tolerance indicated that inhibition of both the serotonin and PAF-R systems were necessary to block the production of these B cells (Matsumura et al., 2006). Yet, the authors also reported previously that use of PAF-R antagonists alone were adequate to block UVB-mediated immunosuppression (Walterscheid et al., 2002), indicating differences in UVB-mediated immunosuppression and tolerance. It should be pointed out that this study does not examine which cell type or mediators induces tolerance, but instead the role of the PAF system in systemic immunosuppression. Although the PAF-R status of the CD19+, B220+ B cell implicated in tolerance has not been assessed (Matsumura et al., 2006), B cells have been described as expressing functional PAF-Rs, unlike T cells (Travers et al., 1989; Mazer et al., 1992). However, other PAF-R-positive immunocytes including monocytes and dendritic cells (Sozzani et al., 1997) could also react to skin-derived PAF agonists. Although PAF-R agonists including native PAF and ox-GPCs have very short half-lives, it is indeed possible that these agonists could have systemic effects. For example, PAF-R agonists have been detected in blood of hamsters (Lehr et al., 1997) and humans (Imaizumi et al., 1991) exposed to the pro-oxidative stressor cigarette smoke.

In summary, these studies indicate that the target for the PAF agonists that appear to be critical for UVB-mediated inhibition of murine CHS is bone marrow-derived cells. These data also suggest that the IL-10 involved in UVB-mediated systemic immunosuppression does not come from the skin, but instead from immunocytes. Inasmuch as UVB-mediated immunosuppressive effects have been implicated in processes as diverse as carcinogenesis to recrudescence of herpes simplex virus, characterization of the mediators involved has important clinical implications.

MATERIALS AND METHODS

Reagents

All chemicals were obtained from Sigma (St Louis, MO) unless indicated otherwise.

Mice

PAF-R−/− mice on a C57BL6 background generated as described previously were a kind gift of Ishii et al. (1998). B6.SJL-PtprcaPep3b/BoyJ (BoyJ) mice were obtained from the Indiana University Cancer Center, Stem Cell Transplant Core Breeding Colony (Indianapolis, IN). Age (8−12 week)-matched PAF-R+/+ C57BL6 wild-type mice were used as controls. All mice were housed in a pathogen-free environment, and studies were approved by the Animal Care and Use Committee of Indiana University School of Medicine.

Contact hypersensitivity reactions

Contact hypersensitivity to DNFB was conducted as described previously (Zhang et al., 2005) with minor modifications. Briefly, to evaluate the effect of CPAF on sensitization reactions, both wild-type and PAF-R−/− mice were injected intraperitoneally with 50 μl CPAF (250 ng), or 50 μl BSA vehicle alone. After 5 days, the back skin of mouse was shaved and 25 μl of 0.5% DNFB in acetone:olive oil (4:1, v/v) applied to an area approximately 1 × 1 cm. Nine days later, one of the dorsal sides of ears was challenged with painting of 10 μl of 0.5% DNFB and the other ear painted with vehicle. After 24 hours, 5 mm punch biopsies were obtained from the ears and weighed. Our previous studies have demonstrated that measurement of weights of punch biopsies from ears correlated with measurement of ear thickness with calipers (Petersen et al., 2002), and our laboratory prefers this methodology due to greater reproducibility in our hands. For studies assessing the ability of UVB to affect CHS, the back skin of mice was shaved, and the anesthetized mouse was draped to allow an area of 2.5 × 2.5 cm of distal back skin exposed to UVB. The UV source was a Philips (Surrey, UK) F20T12/UV-B lamp (270−390 nm, containing 2.6% UVC, 43.6% UVB, and 53.8% UVA). The intensity of the UVB source was measured before each experiment using an IL1700 radiometer and a SED240 UVB detector (International Light, Newburyport, MA) at a distance of 8 cm from the UVB source to mice. Five days after UVB irradiation of the distal back skin, an area of 1 × 1 cm of unirradiated back skin approximately 3 cm cephalic was sensitized with DNFB as outlined above.

To evaluate the effects of COX-2 or IL-10 in the CPAF- or UVB-induced suppression of CHS, the mice were first injected intraperitoneally with 250 ng CPAF or 200 ng COX-2 antagonist SC236 or BSA vehicle control or UVB irradiated as above; 4 and 24 hours later, the mice were twice injected intraperitoneally with 100 μg of neutralizing rat anti-mouse IL-10 antibody (BD Pharmingen, San Diego, CA) (Nghiem et al., 2002) or 100 μg rat IgG1 (eBioscience, San Diego, CA) as antibody control or BSA vehicle control; after 5 days, mice were sensitized with DNFB. Nine days later, the mice were challenged by painting 10 μl 0.5% DNFB on one ear and vehicle control on the other. Five millimeter punch biopsies were performed 24 hours later and weighed.

IL-10 measurements

RNA preparation, reverse transcription and quantitative real-time PCR (QRT2-PCR) were performed as described previously (Travers et al., 2008). Tissue was homogenized and total RNA was isolated with RNeasy Mini Kit (Qiagen Sciences, Valencia, CA), as described by the manufacturer. RNA was reverse transcribed with Reaction-Ready First Strand cDNA Synthesis Kit (SuperArray, Frederick, MD). The quantitative real-time PCR was performed with the IL-10 primer kit (reference positions 319−339) obtained from SuperArray on SmartCycler PCR system with SmartCycler 1.2 software (Cepheid, Sunnyvale, CA) at 95 °C for 15 minutes and 40 cycles of 95 °C, 30 seconds; 55 °C, 30 seconds; and 72°C, 30 seconds, as described by the manufacturer. The expression levels of IL-10 were normalized to the expression levels of housekeeping gene 18S.

Measurement of immunocyte cytokines

To identify the cell type(s) of IL-10-producing cells in the lymph nodes upon UVB radiation, the shaved lower back skin of wild-type and PAF-R−/− mice (five mice per group) was mock- or UVB-radiated (7,500 J m−2). Draining inguinal lymph nodes were harvested 24 hours later and pooled together to prepare single suspension cells. CD11c-positive dendritic cells were first isolated from total lymph node cells using the anti-CD11c magnetic beads (Miltenyi Biotech, Auburn, CA). The CD11c-negative flow was further used to isolate B cells, macrophages, CD4 and CD8 T cells with anti-CD19, anti-CD11b, anti-CD4, and anti-CD8 magnetic beads, respectively.

Total RNA was isolated from enriched different cell types and cDNA was synthesized. qRT-PCR was performed by the comparative threshold cycle (ΔCT) method and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers used for IL-10, IL-12p40, tumor necrosis factor-α, and GAPDH were as described (Yee et al., 2005; Zhao et al., 2006).

Bone marrow transplantation studies

Chimeric mice were generated as described previously (Kreklau et al., 2003). Briefly, bone marrow cells from PAF-R−/− mice (CD45.2+) were harvested and transplanted into lethally irradiated PAF-R+/+ BoyJ mice (CD45.1) at 4 × 106 cells per mouse. Irradiation was delivered as a split dose of 700 cGy followed by 400 cGy delivered 4 hours later. The peripheral blood was harvested 4 months later through the tail vein, and the contribution of the PAF-R−/− cells (CD45.2+) and the host cells (CD45.1+) were analyzed by flow cytometry. A phycoerthryin-conjugated anti-mouse CD45.1 antibody (clone A20) and a PerCp-Cy5.5-conjugated anti-mouse CD45.2 antibody (clone 104) were purchased from BD Pharmingen (Auburn, CA) for detection of CD45.1+ and CD45.2+ murine cells. In another group of transplants, PAF-R+/+ BoyJ bone marrow was transplanted into lethally irradiated PAF-R−/− or PAF-R+/+ BoyJ mice and reconstitution analyzed as described above.

Data analysis

Data from the murine studies are presented as mean±SEM. Student's t-tests were used to assess statistical significance for differences in means. Significance was set at P<0.05.

ACKNOWLEDGMENTS

This work was supported in part by grants from the Riley Memorial Association, and the National Institutes of Health Grants HL62996, U19 AI070448, and Veteran's Administration Merit Award (JBT). R.L.K. was supported by a Clarian Health Values Fund Grant. We thank Dr Mohammed Al-Hassani and Qiaofang Yi for their technical assistance and Dr Dan Spandau for his critical reading of this manuscript.

Abbreviations

CHS

contact hypersensitivity

COX-2

cyclooxygenase-2

CPAF

1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine

DTH

delayed-type hypersensitivity

GPC

glycerophosphocholine

ox-GPC

oxidized glycerophosphocholine

PAF

platelet-activating factor

PAF-R

PAF receptor

Footnotes

CONFLICT OF INTEREST

The authors state no conflict of interest.

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