1 Department of Immunology, Erasmus University and University Hospital Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands
2 Department of Biology, Shandong Normal University, Jinan, Shandong, PRC
Correspondence to: A. Boonstra
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Abstract |
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Keywords: antigen-presenting cell, cytokine, immunomodulation, IL, UVB
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Introduction |
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The mechanisms responsible for local immunosuppression in the UVB-irradiated skin are well documented (7,8). Keratinocyte-derived cytokines are generally considered to be the initiators of the local effects of UVB resulting in the production of a plethora of cytokines within the epidermis (7) and crucial for the observed local immunological unresponsiveness. Keratinocyte-derived IL-10 has been demonstrated to affect B7 expression on Langerhans cells directly, thereby modifying the presentation of antigen to Th cells in the draining lymph nodes (8). Based on their cytokine secretion patterns, the CD4+ Th cells can be divided into at least two effector populations: Th1 and Th2 cells. The Th1 population produces IL-2, lymphotoxin and IFN-, whereas the Th2 cells produce IL-4, IL-5, IL-6 and IL-10 (9). It is generally assumed that especially Th1-mediated responses are sensitive to UVB exposure (1012), but the mechanisms by which UVB affects Th1 responses and the consequences for Th2-mediated responses are not yet clear.
The effects of UVB irradiation on Th cells are thought to be partly caused by altered antigen presentation. When spleen cells from UVB-irradiated mice were used as antigen-presenting cells (APC), the cytokine production by Th1 clones was suppressed, whereas the production by Th2 clones was increased. This effect could be reversed by injection of anti-IL-10 mAb in UVB-irradiated mice (13). Furthermore, anti-IL-10 mAb treatment inhibited UVB-induced suppression of DTH responses (13,14). Also, treatment of UVB-irradiated mice with anti-CD86 mAb, but not with anti-CD80, was shown to overcome the UVB-induced impairment of antigen presentation to Th1 cells (15). However, the expression of CD86 or CD80 was not modulated on splenic APC after UVB irradiation. In addition, both the injection of IL-12 as well as the neutralization of prostaglandin E2 (PGE2) were shown to restore the UVB-induced suppression of DTH responses (16,17). These in vivo studies therefore clearly showed that the APC activity of UVB-irradiated animals is modulated. However, at present it is unknown by which mechanisms the splenic antigen-presenting capacity is altered by UVB irradiation resulting in the suppressed activation of Th1 cells.
Apart from the UVB-induced effects of the APC on Th cells, it was shown that specific macrophage functions, like phagocytosis and killing of intracellular organisms, were impaired (18,19). The suppressed activity of macrophages could be restored by in vivo treatment with anti-IL-10 and anti-transforming growth factor-ß mAb (20). Interestingly, it was reported recently that after whole-body irradiation human neutrophils showed an impaired phagocytosis and a reduced adherence in vitro (21).
Although the involvement of cytokines in the modulation of APC activity by UVB irradiation is recognized, the source and targets of these factors are not well understood. In this study we address the UVB-induced modulation of cytokine production by the APC, both in vivo and in vitro, as well as the possible consequences of the altered APC activity on the micro-environment of the spleen.
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Methods |
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mAb and reagents
All cultures were performed in RPMI 1640 supplemented with 25 mM HEPES, 100 IU/ml penicillin, 50 µg/ml streptomycin, 1 mM pyruvate, 50 mM 2-mercaptoethanol and 10% heat-inactivated FCS. mAb used in the various assays were directed against CD3 (145.2C11), CD28 (37.51), heat stable antigen (J11D.B1), CD16/32 (2.4G2), MHC class II (M5/114), GR-1 (RB6.8C5), CD11b (M1/70), B220 (RA3.6B2), CD8 (YTS-169) and CD40 (FGK4.5). For the detection and neutralization of cytokines, mAb against IL-4 (11B11; BVD6.24G2), IFN- (XMG1.2; R46A2), IL-6 (32C11; 20F3), IL-12p40 (C15.6; C17.15, kindly provided by G. Trinchieri) and IL-10 (SXC-1; 2A5.1) were used. Indomethacin (Sigma, St Louis, MO) was used at a concentration of 106 M. Lipopolysaccharide (LPS) was obtained from Difco (Detroit, MI; Escherichia coli O26:B6).
UVB irradiation
UVB irradiation was essentially performed as described previously (23). Briefly, 1 day prior to UVB irradiation, the dorsal hair of mice was shaved using electric clippers. Mice were exposed on 4 consecutive days to 1500 J/m2/day UVB using two Philips TL-12 tubes (40 W), which represents a suberythemal dose for the mouse strains tested. The tubes had a broad emission spectrum (280350 nm) with a maximum emission at a wavelength of 306 nm. Around 60% of the energy emitted was within the UVB range. The tubetarget distance was 25 cm. Irradiation measurements were calibrated using a Waldmann UV meter (Schwenningen, Germany). Twenty-four hours after the last exposure mice were sacrificed and spleens were dissected. Control mice were shaven but not exposed.
LPS-induced cytokine production in vivo
Mice were injected i.p. with 5 µg LPS in a volume of 200 µl. After 4 h mice were sacrificed and blood was collected. The dose and time point of collection were found to be optimal for the detection of IL-12 levels.
Preparation of spleen cells and purified CD4+ cells
Spleens of control and UVB-irradiated mice were removed under aseptic conditions and single-cell suspensions were prepared. Erythrocytes were removed by incubating with Gey's medium for 5 min on melting ice. Total spleen cells were either used as a source to purify CD4+ cells or stimulated with LPS to determine cytokine levels or Ig production by splenic cells. Purified CD4+ T cells from the spleen were obtained by complement depletion with mAb to HSA, CD16/32, MHC class II and GR-1. Cells were further purified using MACS with a cocktail of biotinylated mAb against CD11b, B220, CD8 and CD40, followed by incubation with streptavidin-conjugated microbeads (Milteny Biotech, Bergisch Gladbach, Germany). CD4+ cells used for experiments were always 9095% pure as determined by flow cytometry.
In vitro stimulation of splenocytes
Splenic cell suspensions were stimulated with LPS (10 µg/ml) in the presence of different reagents and anti-cytokine mAb as indicated in the text. Cells were cultured in 96-well flat-bottom tissue culture plates at a concentration of 2.5x105 cells/ml. After incubation for 48 h, supernatant was harvested and assayed for cytokine content by ELISA. Cultures were performed in triplicate or quadruplicate.
Flow-cytometric analysis
Cells (2 x105) were resuspended in PBS containing 1% BSA and 0.1% sodium azide. For the staining of surface antigens, spleen cells were incubated with FITC- or phycoerythrin-conjugated mAb against CD80, CD86, CD40, F4/80 and MHC-II (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). Then 10 µl propidium iodide (0.2 mg/ml) was added to evaluate the viability of the cells. In total, 104 events were collected and the expression of the markers analyzed using CellQuest software (Becton Dickinson).
Cytokine ELISA
Flat-bottom microplates (96-well, Falcon 3912, Microtest II flexible assay plate; Becton Dickinson, Oxnard, CA) were coated with capture antibody diluted in PBS (1 µg/ml 20F3 or SXC-1; 5 µg/ml 11B11, XMG1.2 or C15.6) at 4°C for 18 h. After coating, plates were washed (PBS/0.1% BSA/0.05% Tween-20) and blocked with PBS supplemented with 1% BSA at room temperature for 1 h. After washing, samples and standards were added and incubation was continued for at least 4 h at room temperature. Thereafter, plates were washed and biotinylated detection antibodies were added (1 µg/ml 32C11; 0.1 µg/ml 2A5.1 or BVD6.24G2; 1 µg/ml R46A2; 2 µg/ml C17.15) and incubated overnight at 4°C. After washing, streptavidinperoxidase (1/1500 diluted; Jackson ImmunoResearch, West Grove, PA) was added. After 1 h, plates were washed and the reaction was visualized using 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (1 mg/ml; Sigma). Optical density was measured at 414 nm, using a Titertek Multiscan (Flow, Redwood City, CA). The amounts of IL-12p70 and tumor necrosis factor (TNF)- were measured with commercially available ELISA kits (Genzyme, Cambridge, MA) according to the protocols provided by the manufacturer. PGE2 levels were determined as previously described (24). The detection limits of the ELISA were: IL-4 (80 pg/ml), IFN-
(0.3 ng/ml), IL-10 (80 pg/ml), IL-12p70 (12.5 pg/ml), IL-12p40 (75 pg/ml), IL-6 (3.9 U/ml) and TNF-
(15.6 pg/ml).
IL-1 bioassay
IL-1 activity was measured by bioassay using a sub-line of the murine T cell line D10.G4.1, designated D10(N4)M (D10) (kindly provided by Dr S. J. Hopkins, Manchester, UK) (25). Proliferation of the D10 cells was measured via [3H]thymidine incorporation. Recombinant IL-1ß (UBI, Lake Placid, NY) served as a positive control. IL-1 activity was corrected for background activity of the culture medium and expressed as c.p.m.
Isotype-specific ELISA
Total supernatant IgM was measured by isotype-specific ELISA as described previously (26). Goat anti-mouse IgM (Southern Biotechnology, Birmingham, AL) was used at 1.0 µg/ml and biotinylated goat anti-mouse IgM antibody at 0.5 µg/ml as a second step. The detection limit for the IgM ELISA was 0.2 ng/ml.
In vitro APC exchange cultures
Cell suspensions were prepared from the spleens of UVB-irradiated and control BALB/c nude mice. Cells (2x105) from both experimental groups were added to 96-well flat-bottom plates and incubated for 2 h at 37°C allowing the APC to adhere to the plastic surface of the culture plates. The adherent cells were washed gently to remove remaining non-adherent cells. To assess the effect on Th cells, purified CD4+ cells were added to the cultures in the presence of 100 ng/ml soluble anti-CD3 mAb. After 2 days, supernatant was collected and IFN- levels were determined by ELISA. To assess the effect on Ig production, cultures were prepared containing adherent cells from UVB or control mice and incubated with non-adherent B cells from UVB or control mice in all possible combinations. These cultures were stimulated with 25 µg/ml LPS. After 7 days, supernatant was collected and Ig production was determined by ELISA.
Statistical analysis
Data were analyzed by Student's t-test and differences were considered significant at P < 0.05.
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Results |
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To determine the involvement of other cytokines in the observed up-regulation of IL-6 production, we added neutralizing anti-cytokine mAb to the LPS-stimulated spleen cell cultures. As shown in Fig. 4, addition of anti-IL-10 mAb strongly enhanced the production of IL-6. Anti-IL-4 mAb had no effect on the IL-6 production by spleen cells from control mice, but did increase the IL-6 production when added to cells from in vivo UVB-irradiated mice. Incubation with the prostaglandin inhibitor indomethacin significantly inhibited the IL-6 production in both experimental groups.
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Modulation of the Th cell activity by APC from UVBirradiated mice
Having demonstrated that the splenic APC compartment is altered by UVB irradiation with regard to their cytokine profile, we tested whether this affected the activation of Th cells. To examine this we incubated adherent APC with purified CD4+ cells in the presence of a suboptimal concentration of soluble anti-CD3 mAb (100 ng/ml). After 48 h, supernatant was collected and IFN- levels were determined. Figure 5
shows that the IFN-
production after co-culture with APC from UVB-exposed mice was significantly reduced as compared to culture with APC from control mice. The IFN-
levels of cultures containing UVB APC as well as cultures containing only CD4+ cells and soluble anti-CD3 were below the detection limit of the ELISA.
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Discussion |
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IL-12 is a key regulatory cytokine in directing the development of Th1 cells producing high levels of IFN- (30). In the present study, we showed that UVB irradiation reduced the LPS-induced IL-12p70 production. This reduction of the bioactive form of IL-12 is a likely explanation for the reduction of systemic Th1 responses, such as the responses to bacterial and viral infection after UVB irradiation. Since it was shown that injection of IL-12 in vivo overcame UVB-induced immunosuppression (31), our data fit in the current dogma and show for the first time the critical role of IL-12 in UVB-induced suppression of systemic Th1 responses. In vitro UVB irradiation of human monocytes also showed a reduction of IL-12p70 production (32). However, this system using in vitro irradiation of cells has limited value for the evaluation of in vivo systemic immune responses. Murine IL-12p40 was found to inhibit the bioactivity of IL-12 (33). Since we did not observe any changes in the production of LPS-induced IL-12p40 after UVB exposure, it is likely that the inhibition of Th1 responses after UVB exposure is primarily due to a reduced IL-12p70 heterodimer formation.
Several studies reported on the induction of IL-10 after UVB irradiation, both locally in the skin as well as systemically (29,34,35). In the skin, cross-regulation of IL-12 and IL-10 is important in establishing a Th2-dominated environment after UVB irradiation (35). Furthermore, neutralization of IL-10 in vivo was found to overcome systemic UVB-induced immunosuppression (14). IL-10 has been demonstrated to inhibit the production of a.o. IL-12 (36). One could speculate that the reported increased levels of serum IL-10 may negatively affect the production of IL-12, which might explain our findings. However, using our protocol of UVB irradiation (suberythemal dose) we were unable to detect modulation of the IL-10 levels in serum of UVB-irradiated animals, which is not in agreement with other studies (34,37). In these studies, however, a supra-erythemal, single dose of UVB was given.
In vitro stimulation of splenocytes from UVB-irradiated mice with LPS showed no effect on the production of IL-10 and IL-12 as compared to the control group. Interestingly, we found that following UVB treatment the LPS-induced production of IL-1, IL-6, TNF- and PGE2 was consistently increased. This was rather surprising since previous reports described a reduction of the number of Ia+ F4/80+ cells in the spleen and a reduced APC function (20,38). We therefore expected a reduction in the production of these immunoregulatory and pro-inflammatory cytokines.
Enhanced production of PGE2, IL-1, IL-6 and TNF- may have important implications for the priming conditions of CD4+ T cells in vivo resulting in enhanced Th2 cell polarization, since all of these cytokines have been described to be involved in the process of Th cell differentiation. Since we observed this increased LPS induced cytokine production in BALB/c nu/nu mice as well as in IL-4/ mice, we exclude the possibility that the modulated cytokine profile is due to an effect on T cells rather than a direct UVB effect on the APC. We anticipate that UVB irradiation will affect (sub)-populations of APC differentially, depending on the phase of the immune response, type and dose of antigen etc. Both IL-1 and IL-6 are known as growth factors for Th2, but not for Th1 cells (39). IL-6 has been reported, by means of its transcription factors, to activate the transcription of the IL-4 gene, thereby promoting the development of Th2 cells (40). PGE2 is a potent inhibitor of IL-12 production, but also facilitates Th2 development by directly inhibiting IFN-
production (41,42). In addition, PGE2 stimulates the differentiation of dendritic cells towards effective APC with Th2-skewing capabilities (43).
The enhanced systemic production of PGE2, IL-1, IL-6 and TNF- after UVB exposure, as shown in our in vitro studies, may therefore have important implications for the Th cell differentiation and the maintenance of a micro-milieu promoting Th2 activity as observed after UVB exposure.
Interestingly, PGE2 has been detected in serum of UVB-irradiated mice and inhibition of PGE2 production abrogates the induction of immunosuppression (17) by a mechanism involving the induction of IL-4 and subsequent IL-10 production (29). We found that inhibition of the production of PGE2 reduced the production of IL-6 as well as TNF- and IL-1 by splenocytes (data not shown). Since we observed increased production of PGE2 by splenocytes of UVB-irradiated mice, our findings suggest that PGE2 plays a role in the enhanced APC-derived cytokine production following UVB irradiation, thereby contributing to the enhanced Th2 polarization. Besides up-regulation of the IL-4 production, we showed that PGE2 may well be responsible for the enhanced production of pro-inflammatory, immunoregulatory cytokines in the spleens of UVB-irradiated mice. These activities suggest multiple mechanisms whereby PGE2 favors the development of Th2 cells.
We and others showed that antigen presentation by splenic APC to Th cells is modulated by UVB exposure as demonstrated by the reduced IFN- production (13). Since the expression of the activation markers tested on APC was not altered, it is likely that the modulated cytokine profile of the APC is the most important mechanism that affects Th cell activity. Although obtained from in vitro culture systems, these data are likely representative for the APCTh cell interaction in the micro-environment of the spleen, as in vitro and in vivo data are consistent.
Furthermore, we showed that the effect of UVB on the splenic APC compartment resulted in a reduction of the IgM production by B cells in vitro. The production of the other isotypes was reduced as well (data not shown). We could not detect a selective reduction of Th1-associated isotypes (IgG2a) as compared to Th2-associated isotypes (IgG1 and IgE). Interestingly, our studies show that not only APC, but also B cells are intrinsically modulated by UVB exposure. This effect on B cells has not been described before. Since B cells are rarely found in the skin, they are likely affected by a yet undefined indirect mechanism, involving soluble factors.
There are two possible mechanisms that may account for the UVB-induced alteration of the cytokine profile of splenic APC. Firstly, it is possible that the cytokine production of the individual APC is altered, influenced by immunoregulatory mediators like PGE2. Secondly, it is possible that the APC population as such in the spleen has changed. It is known that UVB irradiation causes a redistribution of APC due to migration to peripheral lymphoid tissues and a subsequent replenishment by newly immigrating cells in the skin (38). Due to the extensive functional and phenotypic heterogeneity of the APC it is tempting to speculate that after UVB irradiation the spleen is repopulated with functionally distinct subpopulations of APC (44). We are currently testing these possibilities.
In conclusion, we showed that UVB irradiation affects the APC-derived cytokines IL-1, IL-6, TNF-, IL-12p70 and PGE2. The coordinated action of IL-1, IL-6, TNF-
and PGE2 together with the reduced IL-12p70 production may well be responsible for the UVB-induced systemic selective suppression of Th1-driven immune responses as well as the effects on B cell activity.
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Acknowledgments |
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Abbreviations |
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APC antigen-presenting cells |
DTH delayed-type hypersensitivity |
PGE2 prostaglandin E2 |
TNF tumor necrosis factor |
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Notes |
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Received 11 January 2000, accepted 13 July 2000.
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References |
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