Role of phagocytic macrophages in induction of contact hypersensitivity and tolerance by hapten applied to normal and ultraviolet B-irradiated skin.

Liposomes containing the drug dichloromethylene diphosphonate (Cl2MDP) can eliminate phagocytic cells, such as macrophages, when injected in vivo. In this paper we report that Cl2MDP-containing liposomes have been used experimentally to determine the extent to which cutaneous macrophages participate (1) in the induction of contact hypersensitivity (CH) when hapten is painted on normal murine skin, and (2) in the induction of CH or tolerance when hapten is painted on murine skin that has been exposed to ultraviolet B (UVB) radiation. Intradermal (i.d.) injections of Cl2MDP-containing liposomes were found to have no deleterious effects on CH induction via normal skin, whether the amount of hapten (dinitrofluorobenzene) applied to the cutaneous surface was optimal or excessive. Moreover, Cl2MDP-containing liposomes did not deplete the epidermis of Langerhans' cells. However, similar i.d. injections of Cl2MDP-containing liposomes did prevent the induction of CH when hapten was painted on UVB-irradiated skin of BALB/c mice, a strain that develops CH when hapten is applied to UVB-exposed skin. These findings indicate that the antigen-presenting cell (APC) function found in skin of UVB-resistant mice following exposure to UVB radiation can be attributed to macrophages. This explains why these mice develop and display CH after UVB radiation. By contrast, i.d. injections of Cl2MDP-containing liposomes failed to prevent the induction of the tolerance when hapten was applied to the surface of UVB-exposed skin of UVB-susceptible mice, such as C57BL/6. Since the dermis of UVB-exposed skin of these mice is known to contain a novel population of cells that can provide a tolerance-conferring signal, the current findings rule out macrophages as the responsible cell type.     

Role of F4/80+ cells during induction of hapten-specific contact hypersensitivity.

F4/80, a monoclonal antibody that binds to a surface molecule on mature macrophages and certain dendritic cells, has been used to explore the role of epidermal and dermal cells as antigen-presenting cells (APC) during the induction of contact hypersensitivity (CH) in mice. Systemic administration of the antibody appeared to have little or no physical or functional effect on intraepidermal Langerhans' cells, even though a subpopulation of these cells expressed the F4/80 ligand. None the less, systematically administered F4/80 antibodies were able to impair CH induction when dinitrofluorobenzene (DNFB) was painted on normal body wall skin of BALB/c mice [an ultraviolet B (UVB)-resistant strain]. Interestingly, systemic F4/80 antibodies did not affect CH induction in C57BL/10 mice (a UVB-susceptible strain). When a sensitizing dose of hapten was injected intracutaneously (i.c.) into F4/80-treated BALB/c and C57BL/10 mice, CH induction was impaired in both inbred strains, although the severity of impairment was greater in BALB/c mice. Following UVB radiation of body wall skin, anti-F4/80-treated BALB/c mice displayed very feeble CH, whether hapten was painted epicutaneously or injected i.c. at the irradiated site. Based on these and other recent reported results, it is concluded that (1) BALB/c mice rely partially upon dermal, F4/80+ cells as a source of APC when hapten is applied epicutaneously, whereas C57BL/10 mice rely almost exclusively upon epidermal Langerhans' cells in this circumstance; and (2) after UVB radiation of skin, BALB/c mice can use F4/80+ dermal cells as the source of APC function when hapten is painted epicutaneously. These findings are discussed with respect to the cellular basis for the differential susceptibilities of genetically defined strains of mice to the deleterious effects of UVB radiation on CH induction.     

Evidence that ultraviolet B radiation induces tolerance and impairs induction of contact hypersensitivity by different mechanisms.

Tumour necrosis factor-alpha (TNF-alpha) and cis-urocanic acid (UCA) have recently been implicated in the process by which ultraviolet B radiation (UVB) impairs the induction of contact hypersensitivity when dinitrofluorobenzene (DNFB) is painted on UVB-exposed skin. The evidence supports the hypothesis that UVB radiation converts trans- to cis-UCA in the epidermis which in turn causes the epidermis of UVB-susceptible mice to produce/contain excessive local amounts of TNF-alpha. When hapten is painted on TNF-alpha- or UVB-treated skin, contact hypersensitivity fails to develop. As UVB radiation also induces hapten-specific tolerance and suppressor T cells when hapten is applied to UVB-exposed skin of UVB-susceptible strains of mice, we examined whether TNF-alpha and/or UVB-irradiated UCA (UV-UCA) might be similarly involved in the mechanism by which UVB induces tolerance. We report that intracutaneously-injected TNF-alpha and UV-UCA altered the cutaneous environment such that when DNFB was painted on the injected site, hapten-specific tolerance was induced and suppressor cells were generated. However, the tolerance induced by UVB radiation and the tolerance that followed intracutaneous injection of UV-UCA were not reversed by neutralizing anti-TNF-alpha antibodies. Moreover, UV-UCA and TNF-alpha-induced tolerance and suppressor cells in both UVB-susceptible (UVB-S) and UVB-resistant mice, whereas UVB radiation induced tolerance only in UVB-S mice. We conclude that the mechanism by which UVB radiation induces tolerance in mice is separate and distinct from the mechanism by which UVB radiation impairs contact hypersensitivity induction. Moreover, our data support the view that the generation of suppressor cells and the development of hapten-specific tolerance may be mechanistically distinct. The possible molecular and cellular mediators of UVB-induced tolerance are discussed.     

Tumour necrosis factor-alpha mediates ultraviolet light B-enhanced expression of contact hypersensitivity.

Acute, low-dose ultraviolet B radiation (UVB) impairs the induction of contact hypersensitivity (CH) to dinitrochlorobenzene (DNCB) in certain inbred strains of mice (termed UVB-susceptible), but not in others (termed UVB-resistant). By contrast, exposure of mouse ear skin to an identical regimen of UVB has been reported to exaggerate the expression of CH. Recently, tumour necrosis factor-alpha (TNF-alpha) has been demonstrated to mediate the deleterious effects of UVB on CH induction, presumably through local release of TNF-alpha within UVB exposed skin. The present studies were conducted to determine whether TNF-alpha also mediates the exaggerated expression of CH induced by UVB radiation. It was found that TNF-alpha, injected intradermally at the ear challenge site, enhanced the expression of CH to DNFB in conventionally sensitized mice. Interestingly, TNF-alpha was able to amplify the expression of CH in the ears of both UVB-susceptible strains of mice, and UVB-resistant strains. However, anti-TNF-alpha antibodies neutralized UVB-enhanced CH in UVB-susceptible mice, but not in UVB-resistant mice. These findings support the proposition that TNF-alpha, released from UVB-exposed epidermal cells, is a critical mediator of the effects of UVB radiation on induction and expression of contact hypersensitivity. The effects of UVB radiation, intradermal (ID) TNF-alpha, and/or epicutaneously applied DNFB on epidermal Langerhans' cells were also evaluated and compared. Whereas epicutaneously applied DNFB alone profoundly depleted the epidermis of Langerhans' cells, DNFB painted on UVB-exposed or TNF-alpha-treated skin was much less effective at eliminating normal appearing Langerhans' cells. These results suggest that one direct effect of TNF-alpha on Langerhans' cells may be to immobilize these antigen-presenting cells transiently within the epidermis. It is proposed that this immobilization has the paradoxical effect (a) of interfering with sensitization, by preventing hapten-bearing Langerhans' cells from migrating to the draining lymph node, while at the same time (b) of amplifying CH expression by lengthening the interval of hapten retention and presentation with the epidermis.     

Studies on delayed systemic effects of ultraviolet B radiation on the induction of contact hypersensitivity, 3. Dendritic cells from secondary lymphoid organs are deficient in interleukin-12 production and capacity to promote activation and differentiation of T helper type 1 cells

Ultraviolet-B radiation (UVR) of mouse skin promotes both local and systemic immune aberrations that are thought to be important in the pathogenesis of cutaneous malignancies. Acute, low-dose UVR regimens inhibit the induction of contact hypersensitivity (CH) in genetically susceptible mice by TNF-alpha-dependent mechanisms. In addition, these regimens also promote the development of tolerance when hapten is applied to the UVR-exposed site at the completion of the radiation treatment protocol. A third immune abnormality is also observed in mice exposed to acute, low-dose UVR. This abnormality, which develops within 48-72 hr of the completion of the UVR regimen, has been described among antigen-presenting cells within secondary lymphoid organs, including lymph nodes that do not drain the site of irradiation. Dendritic cells (DCs) from lymph nodes and spleens of mice exposed to UVR lack the capacity to induce CH if they are derivatized with hapten and injected intracutaneously into naive mice. The DC defect is related to the production of and systemic dissemination of interleukin-10 (IL-10) by keratinocytes within the epidermis of the UVR-exposed skin. We have now examined the nature of the functional aberration that exists among DCs within the secondary lymphoid organs of UVR-exposed mice by examining the capacity of DCs to express co-stimulatory molecules, and their ability to activate ovalbumin (OVA) -specific DO11.10 T-cell receptor transgenic T cells in vitro. Our results indicate that DCs from UVR-exposed mice produced insufficient amounts of IL-12. When pulsed with OVA, these cells were capable of inducing proliferation among DO11.10 T cells in vitro, but the responding cells produced neither IFN-gamma nor IL-10 and IL-4. A similar antigen-presenting cell defect was generated in mice treated with a subcutaneous injection of IL-10. We conclude that acute, low-dose UVR creates an IL-10-dependent functional deficit in DCs in secondary lymphoid organs, and that this defect robs UVR-exposed mice of the capacity to develop CH when hapten is painted epicutaneously.     

High and low doses of haptens dictate whether dermal or epidermal antigen-presenting cells promote contact hypersensitivity

In the induction of contact hypersensitivity (CH) to an epicutaneously applied hapten, we have previously proposed that low doses of hapten sensitize primarily through epidermal Langerhans' cells (LC), whereas high doses rely largely on dermal antigen-presenting cells (APC). To examine this issue further, we applied either high or low doses of dinitrofluorobenzene (DNFB) epicutaneously to mice. We observed reduced LC density at the site after 12 h (nadir), which returned to normal levels at 24 h only after a low dose of hapten. When a low dose of an unrelated hapten, oxazolone, was painted on skin that had been painted 12 h previously with high dose of DNFB, oxazolone-specific CH was impaired. When grafts of whole skin, dermis alone, and epidermis alone prepared from skin painted 2 h previously with low or high doses of DNFB were placed onto naive, syngeneic mice, CH was induced by whole skin after both types of doses, by epidermis only after a low dose, and by dermis only after high dose. When epidermal cell suspensions were derivatized in vitro with low or high doses of DNFB, only cells exposed to a low dose induced proliferation of hapten-specific T cells. Thus, only a low dose of hapten reveals the APC functions of LC without the participation of dermal APC.     

Studies of delayed systemic effects of ultraviolet B radiation (UVR) on the induction of contact hypersensitivity, 2. Evidence that interleukin-10 from UVR-treated epidermis is the critical mediator

Acute, low-dose ultraviolet B radiation (UVR) alters cutaneous immunity at the local site as well as systemically. Within 2-3 days of UVR exposure, recipient mice lose their capacity to develop contact hypersensitivity (CH) when hapten is painted on unexposed skin. This loss correlates temporally with a functional deficit among dendritic antigen-presenting cells within non-draining lymph nodes and spleen. In the experiments described, the delayed systemic immune deficiency following acute, low-dose UVR exposure was found to be eliminated with neutralizing anti-interleukin-10 (IL-10) antibody. Intracutaneous injection of IL-10 generated a deficiency of systemic immunity as well as a functional deficit among lymph node dendritic cells that was similar to that induced by UVR. The skin itself was found to be the source of the IL-10 responsible for these defects, and epidermis (presumably keratinocytes) rather than mast cells was found to be the source of IL-10 within UVR-exposed skin. The potential relationships are discussed between the delayed systemic immune deficit created by acute, low-dose UVR, and the systemic immune deficits caused by chronic, high-dose UVR and by a single, high-dose UVR exposure.     

UVB-irradiated dendritic cells are impaired in their APC function and tolerize primed Th1 cells but not naive CD4+ T cells

We have shown that low-dose UVB radiation converts Langerhans cells (LC) from immunogenic to tolerogenic APC. Therefore, we questioned whether low-dose UVB irradiation of bone marrow-derived dendritic cells (DC) alters their APC function, thereby inducing tolerance in T cells. To address this issue, cocultures of DC; and naive, allogeneic T cells; na?ve, OVA-specific TCR-transgenic T cells from DO11.10 mice; or primed, antigen-specific T cells using the Th1 clone AE7 were analyzed. First, we found low-dose UVB-irradiated DC (UVB-DC) to dose-dependently (50-200 J/m2) inhibit T-cell proliferation of naive and primed T cells. In addition, supernatants harvested from cocultures of UVB-DC and naive T cells showed markedly reduced levels of IL-2 and IFN-gamma and to a lesser degree of IL-4 and IL-10, suggesting a preferential down-regulation of Th1 responses by UVB-DC. FACS analysis of UVB-DC revealed no changes in surface expression of MHC, costimulatory, and adhesion molecules. To test tolerance induction, allo- or antigen-specific T cells isolated from cocultures with unirradiated DC and UVB-DC were restimulated with unirradiated DC or IL-2. It is interesting that UVB-DC induced antigen-specific tolerance in the Th1 clone AE7. In contrast, UVB-DC induced a partial inhibition of allogeneic T-cell proliferation but no tolerance with similar unresponsiveness to restimulation with IL-2 and unirradiated DC irrespective of their haplotype. Similar observations were made when naive, TCR-transgenic T cells from DO11.10 mice were used. In conclusion, UVB-DC are impaired in their APC function and tolerize the primed antigen-specific Th1 clone AE7 but not naive allo- or OVA-specific T cells.     

Systemic low-dose UVB inhibits CD8 T cells and skin inflammation by alternative and novel mechanisms

Exposure to UVB radiation before antigen delivery at an unirradiated site inhibits functional immunological responses. Mice treated dorsally with suberythemal low-dose UVB and immunized with ova in abdominal skin generated ova-specific CD8 T cells with a significantly decreased activation, expansion, and cytotoxic activity compared with unirradiated mice. UVB also impaired the delayed-type hypersensitivity (DTH) reaction to ova. Transfer of CD4⁺CD25⁺ cells from UVB-exposed mice did not suppress the ova-specific CD8 T-cell response or DTH reaction in unexposed mice, confirming that systemic low-dose UVB does not induce long-lived functional regulatory CD4⁺CD25⁺ T cells. Repairing cyclobutane pyrimidine dimer–type DNA damage and blocking aryl hydrocarbon receptor signaling also did not reverse the immunosuppressive effect of UVB on ova-specific CD8 T cells and DTH, suggesting that cyclobutane pyrimidine dimers and the aryl hydrocarbon receptor are not required in systemic low-dose UVB-induced immunosuppression. The known UVB chromophore, cis-urocanic acid, and reactive oxygen species triggered the inhibition of DTH caused by UVB, but they were not involved in the modulation of CD8 T cells. These findings indicate that systemic low-dose UVB impedes the primary response of antigen-specific CD8 T cells by a novel mechanism that is independent of pathways known to be involved in systemic suppression of DTH.UVB radiation (290 to 320 nm) in natural sunlight is a potent immunosuppressant. UVB can inhibit the immune system from generating optimal responses to tumors, contact haptens, and various microbial antigens (Ags; viral, fungal, and parasitic) that can lead to exacerbated disease. Alternatively, immunosuppressive UVB can also be beneficial to control autoimmune diseases, such as psoriasis¹ and experimental autoimmune encephalomyelitis.² The epidermis of skin absorbs UVB through chromophores, including nuclear DNA, cytoplasmic tryptophan, and extracellular trans-urocanic acid (UCA). The molecular processes that follow trigger a cascade of events that cumulates in the phenomenon of UVB-induced immunosuppression. Some of the hallmarks of this immunosuppression include UVB-induced genetic mutations in skin cells, circulation of cis-UCA from the isomerization of trans-UCA, production of reactive oxygen species (ROS), and generation of regulatory T and B cells that can transfer suppression into UVB-naïve mice.³ Because UVB has both detrimental and beneficial effects on the immune system, it is critical to understand the mechanisms regulated by UVB so that effective prophylactic and palliative therapies can be designed for skin diseases, such as skin cancer, and immune-mediated diseases in internal organs, such as multiple sclerosis.A previous study⁴ showed that UVB can inhibit CD8 and CD4 T-cell responses to haptens. In a model of contact hypersensitivity (CHS), we demonstrated that a low dose of UVB (approximately 5 minutes of summer sunlight in Sydney, Australia, at midday) is sufficient to inhibit the activation and expansion of effector CD8 and CD4 T cells in skin-draining lymph nodes (sDLNs) after sensitization to a contact hapten at an unirradiated site (systemic, Ag, and UVB at distal sites). However, it was too low to activate functional and durable CD4⁺CD25⁺ regulatory T cells. The effector T cells generated in this environment exhibited reduced skin infiltration and interferon-γ production on CHS elicitation. Moreover, this systemic low-dose UVB regimen prevented the development of dermal memory CD8 T cells.Exposing mice to a low-dose UVB regimen, followed by transcutaneous immunization with ova protein through the same skin site (local, Ag, and UVB at the same site), inhibits the proliferation, cytotoxicity, and interferon-γ production of transgenic and endogenous ova-specific CD8 T cells.^5,6 Contrary to what we previously observed, transferable suppression of the ova-specific CD8 T-cell response was correlated to the presence of CD4⁺CD25⁺ regulatory T cells in sDLNs in this model.⁵ Other researchers^7,8 have also demonstrated that regulatory T cells derived from mice irradiated with inflammatory high doses of UVB in a systemic Ag model can impede CHS and delayed-type hypersensitivity (DTH) reactions. These UV-activated regulatory T cells may inhibit T cells and skin inflammatory reactions by altering Ag-presenting cells (APCs).⁹Several UVB chromophores have been identified that can independently trigger UVB-induced immunosuppression, as shown by various studies that have repaired DNA damage, neutralized cis-UCA, and prevented ROS production. The downstream processes initiated by UVB chromophores include production of immunosuppressive mediators [ie, IL-4, IL-10, and platelet-activating factor (PGE₂)], migration of skin APCs into sDLNs, aberrant Ag presentation, mast cell activation, and induction of regulatory T cells.¹⁰ However, it is unknown whether these pathways are involved during systemic low-dose UVB, which is representative of daily nonrecreational sunlight exposure. Given that a previous investigation showed that UVB has a long-term deleterious influence on CD8 T-cell immunity, we wanted to further examine what UVB-stimulated mechanisms contribute to the inhibition of primary CD8 T-cell responses in the absence of CD4⁺CD25⁺ regulatory T cells. In addition, we assessed the ability of systemic low-dose UVB to modulate a second separate inflammatory reaction in the skin at an unirradiated site. DTH reactions are complex skin immune reactions that are primarily driven by type 1 helper CD4 T cells and various innate cells.^11,12 For this study, we used the model protein Ag, ova, that allowed us to study the endogenous Ag-specific CD8 T-cell response and a DTH reaction after UVB irradiation and immunization. UVB significantly decreased the activation, expansion, and cytotoxic activity of splenic ova-specific CD8 T cells, but this was not attributed to known UVB chromophores considered to be critical in UVB-induced immunosuppression. However, DTH reactions were modulated by cis-UCA and ROS. These findings indicate that short-term UVB can inhibit Ag-specific CD8 T-cell responses and DTH in vivo via different mechanisms and, therefore, that a novel unknown mechanism is responsible for regulating the effects of UVB on CD8 T-cell activation in secondary lymphoid organs.     

Ultrastructural studies bearing on the mechanism of UVB-impaired induction of contact hypersensitivity to DNCB in man

In both murine and human experimental systems, acute, low dose exposure of skin to ultraviolet B light (UVB) impairs the induction of allergic contact dermatitis (ACD) by haptens such as dinitrochlorobenzene (DNCB) in a significant proportion of individuals. By light microscopy, epidermal Langerhans cells (LC) have been reported to be depleted by UVB exposure as well as by epicutaneous hapten application, implying that LC may be the locus of action of the effeeis of both UVB and DNCB. However, light microscopy can not readily distinguish cell density changes secondary to LC necrosis from changes resulting from down-modulation of expression of LC surface molecules. Using a highly sensitive immunogold electron microscopic approach, we have evaluated the differential effects of UVB and/or DNCB on human epidermal LC. The results reveal that DNCB alone eaused significant up-regulation of cell surface HLA elass II expression on a very small number of LC. the major fraction of LC expressing normal levels of HLA class II. Furthermore, DNCB alone caused a modest reduction in the density of LC at the treated sites without evidence of cell necrosis. Treatment with UVB alone or UVB exposure followed by DNCB resulted in a reduction in the density of LC. with widespread evidence of LC necrosis. However, the few remaining intact LC were all intensely HLA class II-positive after UVB exposure followed by DNCB. whereas treatment with UVB alone did not result in changes in LC HLA class II expression. The findings that after DNCB painting only a small proportion of the LC were strongly HLA class II-positive. but after UVB exposure followed by DNCB all intact LC displayed significant up-regulation ofcell surface HLA class II expression, imply that UVB exposure inhibits the migration of epidermal LC. This is consistent with the view that DNCB fails to induce ACD when hapten is painted on UVB-exposed skin because insufficient LC are available to initiate T cell activation in the draining lymph node.     

Systemic suppression of contact hypersensitivity by UVB radiation is unrelated to the UVB-induced alterations in the morphology and number of Langerhans cells

Exposure of mice to UVB (280-320 nm) ultraviolet radiation reduces contact hypersensitivity (CHS) reactions to chemicals that are applied subsequently to unirradiated skin. It also decreases the number and alters the morphology of Langerhans cells at the site of irradiation. We addressed the question of whether the systemic suppression of CHS was related to these modifications of Langerhans cells by UVB radiation. In mice treated on the dorsum with UVB radiation, the number and morphology of Langerhans cells in the unexposed areas of skin used for inducing and eliciting CHS appeared normal. Therefore, the depression of CHS could not be attributed to a depletion of Langerhans cells at the sites of application of the sensitizing agent. We also examined the correlation between alterations in Langerhans cells and systemic suppression of CHS after treatment with various types of nonionizing radiation. Treatment of mice with UVA (320-400 nm) radiation eliminated detectable Langerhans cells from the exposed skin, based on ATPase staining and electron microscopy, but did not reduce CHS; in fact, CHS was enhanced in these animals. Neither rose bengal nor eosin, in combination with visible (greater than 400 nm) radiation, affected the number or appearance of Langerhans cells, even though microscopic evidence of phototoxicity was present. However, rose bengal plus visible radiation depressed CHS reactions that were induced and elicited through unexposed skin. Depletion of Langerhans cells from dorsal skin by exposure to UVA radiation did not prevent suppression of CHS by subsequent exposure of the Langerhans cell-depleted skin to UVB radiation. We conclude that systemic suppression of CHS by UVB irradiation is not related to the numerical and morphological alterations in Langerhans cells that occur locally at the site of irradiation.     

Tumor necrosis factor (TNF) receptor type 1 (p55) is a main mediator for TNF-α-induced skin inflammation

Tumor necrosis factor α (TNF-α) is a pleiotropic proinflammatory cytokine that elicits a large number of biological effects, including inflammatory and immuno-regulatory responses. Biological activities of TNF-α are mediated by two distinct TNF receptors, p55 type 1 receptor (TNFR1) and p75 type 2 receptor (TNFR2). To determine the role of TNF-α in the induction of inflammatory responses in the skin, gene-targeted mutant mice lacking either TNFR1 or TNFR2 were painted with irritant chemicals. Both phenol and croton oil painting onto the ears induced less inflammation in TNFR1(?) mice than normal and TNFR2(?) mice. Intradermal injection of TNF-α (0.2–200 ng for 3 days) into the ear induced less inflammation in TNFR1(?) mice than in normal mice. TNFR2(?) mice developed a normal inflammatory reaction to high doses of TNF-α (20–200 ng for 3 days), while they showed minimal reactivity to low doses of TNF-α (0.2–2 ng for 3 days). TNF-α is known to trigger the release of a series of other cytokines and to induce the expression of cell adhesion molecules, thus contributing to the development of inflammation. The levels of protein and mRNA for interleukin (IL)-6 were elevated in keratinocytes from normal as well as TNFR2(?) mice after treatment with TNF-α, while keratinocytes from TNFR1(?) mice did not show any up-regulation of IL-6. TNF-α induced intercellular adhesion molecule (ICAM)-1 expression in the keratinocytes from normal and TNFR2(?) mice, but not in those from TNFR1(?) mice. These results indicate that TNFR1 is critical for induction of skin inflammation by TNF-α.     

Evidence that ultraviolet B radiation transiently inhibits emigration of Langerhans cells from exposed epidermis, thwarting contact hypersensitivity induction.

Langerhans cells (LC) play a critical role in the induction of contact hypersensitivity (CH), and ultraviolet B radiation (UVR) impairs CH induction in UVB-susceptible (UVB-S) mice via a TNF-alpha-dependent mechanism. A possible explanation of this effect is that UVR impairs CH in UVB-S mice by immobilizing LC transiently in the epidermis and upper dermis, thereby preventing their timely migration to draining lymph nodes. To test this hypothesis we examined in vitro and in vivo the effects of in vivo UVR on migration of Ia(+) cells from skin of UVB-S and UVB-resistant (UVB-R) mice. Dorsal surfaces of ears of mice were irradiated with 400 J/cm(2) UVB and either explanted in vitro or transplanted orthotopically to the thoracic wall of syngeneic recipients. After 24, 48, and 72 h the epidermis was recovered from these explants/grafts and the number of Ia(+) cells determined by immunohistochemistry. Culture medium obtained after explants were removed was also evaluated for content of Ia(+) cells. The density of Ia(+)-bearing cells in the epidermis of cultured untreated skin explants and of grafted skin fell progressively for both UVB-S and UVB-R skin during the observation period. The rate of decline in Ia(+) cells density during this interval was greatly impaired if the skin was exposed to UVR prior to excision; this effect was seen equally in UVB-S and UVB-R skin. Recovery of Ia(+) cells in the medium after removal of cultured untreated skin explants was maximum after 24 h and comparable in UVB-S and UVB-R skin. However, the number of Ia(+) cells recovered in the medium from UVB-exposed skin was significantly reduced only if the skin donor was UVB-S. We conclude that the ability of UVR to impede LC migration from epidermis is significantly greater for UVB-S mice, accounting in part for the failure of these mice after UVR to acquire CH.     

Ultraviolet B suppresses immunity by inhibiting effector and memory T cells

Contact hypersensitivity is a T-cell-mediated response to a hapten. Exposing C57BL/6 mice to UV B radiation systemically suppresses both primary and secondary contact hypersensitivity responses. The effects of UVB on in vivo T-cell responses during UVB-induced immunosuppression are unknown. We show here that UVB exposure, before contact sensitization, inhibits the expansion of effector CD4+ and CD8+ T cells in skin-draining lymph nodes and reduces the number of CD4+ and IFN-gamma+ CD8+ T cells infiltrating challenged ear skin. In the absence of UVB, at 10 weeks after initial hapten exposure, the ear skin of sensitized mice was infiltrated by dermal effector memory CD8+ T cells at the site of challenge. However, if mice were previously exposed to UVB, this cell population was absent, suggesting an impaired development of peripheral memory T cells. This finding occurred in the absence of UVB-induced regulatory CD4+ T cells and did not involve prostaglandin E2, suggesting that the importance of these two factors in mediating or initiating UVB-induced immunosuppression is dependent on UVB dose. Together these data indicate that in vivo T-cell responses are prone to immunoregulation by UVB, including a novel effect on both the activated T-cell pool size and the development of memory T cells in peripheral compartments.     

Hapten-specific tolerance induced by acute, low-dose ultraviolet B radiation of skin requires mast cell degranulation

The deleterious effects of ultraviolet B radiation (UVR) on cutaneous immunity are mediated in part by cytokines released from cutaneous cells following radiation exposure. On the one hand, TNF-alpha has been advocated as the primary mediator of failed contact hypersensitivity induction, and, on the other hand, IL-10 has been held responsible for tolerance. While keratinocytes exposed to UVR have been found to produce both TNF-alpha and IL-10, there is reason to question whether these major cellular constituents of the epidermis are the relevant source of immunomodulatory cytokines after UVR. Dermal mast cells also produce TNF-alpha and IL-10, and we have recently reported that mast cell-derived TNF-alpha is required for UVR-induced impairment of CH induction. In this study, we have examined whether mast cells are also a relevant source of IL-10 in UVR-dependent tolerance. We found that (a) UVR fails to induce tolerance in mast cell-deficient mice, and (b) that tolerance occurs if mast cells are triggered to degranulate after ligation of the IgE receptor. Both types of tolerance were neutralized with anti-IL-10 antibodies, are hapten specific, and are associated with regulatory lymphoid cells. We conclude that mast cells are required in UVR-induced tolerance and may be one of the major sources of IL-10 that mediates the tolerance induced by acute, low-dose UVR.     

Role of tumor necrosis factor-α in the regulation of keratinocyte cell cycle and DNA repair after ultraviolet-B radiation

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine produced in the skin in response to ultraviolet B radiation (UVB). TNF-α facilitates UVB-induced apoptosis and probably contributes to removal of damaged cells. Surprisingly, murine TNF-α-knockout models have demonstrated that TNF-α is necessary for the early stages of skin carcinogenesis and development of squamous cell carcinoma. In the present PhD thesis, we examined the effects of TNF-α on DNA repair and cell cycle regulation in UVB-irradiated keratinocytes. In the model of premalignant keratinocytes (HaCaT), TNF-α abolished the UVB-induced G2/M checkpoint and diminished the DNA repair despite induction of apoptosis. TNF-α activated the protein kinase B/Akt and regulation of its downstream targets, mTOR, Bad and FoxO3a. This effect was dependent on atypical protein kinase C species (aPKC) since a specific peptide blocking the activity of the PKCξ and ι/λ abrogated the activation of Akt by TNF-α. The aPKC-Akt axis was likely to be responsible for the TNF-α-induced decrease in DNA repair since blocking of Akt activity restored DNA repair. Since anti-TNF-α approaches are increasingly used in the therapy of autoimmune diseases and one of the safety concerns is the potential enhancement of skin carcinogenesis, we investigated the effect of the chimeric monoclonal anti-TNF-α antibody infliximab on UVB-irradiated HaCaT cells. Cells treated with infliximab had significantly increased levels of DNA damage despite enhanced G2/M checkpoint arrest, increased apoptosis and inhibition of Akt. In conclusion, we identified a possible novel mechanism by which TNF-α promotes UVB-induced skin carcinogenesis. This depends on aPKC-Akt activation and inhibition of DNA repair. TNF-α-treated cells are prone to escape checkpoint control and are possibly more likely to accumulate mutations, which may constitute a relevant mechanism enhancing tumor development. The effect of anti-TNF-α therapy on skin carcinogenesis warrants further investigation as our study indicates that, in contrast to what had been expected, infliximab may impair DNA repair.     

Sensitizing capacity of Langerhans' cells obtained from ultraviolet-B-exposed murine skin.

Acute low-dose ultraviolet B (UVB) radiation impairs contact hypersensitivity induction in some strains of mice (called UVB-susceptible, UVB-S), but not in others (called UVB-resistant, UVB-R). In order to determine whether these UVB-dependent phenotypes are inherent properties of epidermal Langerhans' cells, Ia-enriched epidermal cell suspensions were prepared from normal and UVB-exposed skin of C57BL/6 (UVB-S) and BALB/c (UVB-R) mice. After derivatization with dinitrofluorobenzene (DNFB), the cells were injected into footpads of naive syngeneic mice, and the recipients were evaluated for contact hypersensitivity and for in vitro evidence of hapten-specific T-cell priming. The results indicate that DNFB-conjugated Ia-enriched epidermal cells from normal mice, and from UVB-exposed skin of UVB-R mice induced contact hypersensitivity and primed hapten-specific T cells in the draining lymph node. By contrast, epidermal cells from UVB-exposed skin of UVB-S mice failed to induce contact hypersensitivity, even though hapten-specific T cells were still detectable in the draining lymph node. In addition, UVB radiation impaired the ability of hapten-bearing Langerhans' cells from UVB-S mice to activate hapten-specific, primed T cells in vitro. We conclude the traits of UVB-S and UVB-R can be expressed directly by Langerhans' cells, and that these effects are at least in part responsible for the deleterious consequences of UVB radiation on cutaneous immunity in UVB-S mice.     

Lack of metallothionein-I and -II exacerbates the immunosuppressive effect of ultraviolet B radiation and cis-urocanic acid in mice

The effect of a null mutation for the metallothionein (MT)-I and -II isoforms in mice on the immunosuppressive action of ultraviolet B (UVB; 280-320 nm) radiation has been examined. Mice were exposed to a series of increasing daily UVB doses, each dose administered to the dorsum on 3 consecutive days. Erythema was assessed, and measured as its oedema component by the post-irradiation dorsal skinfold thickness, but there was no effect of the null mutation (MT-/-) observed after 3 x 3.4 kJ/m2 of UVB radiation. Immune function was assessed by the contact hypersensitivity (CHS) response, which was initiated by sensitization on unirradiated abdominal skin, and thus demonstrated the systemic effects of dorsal treatments. In comparison with the wild-type MT+/+ mouse, the MT-/- mouse was significantly more immunosuppressed by moderate daily UVB doses (1. 75-5.9 kJ/m2). When topically applied cis-urocanic acid (cis-UCA) replaced UVB radiation as the immunosuppressive agent, contact hypersensitivity in MT-/- mice was again markedly more suppressed than in MT+/+ mice, in a dose-responsive manner. The results infer that MT, which was shown immunohistochemically to be strongly induced in the epidermis of MT+/+ mice, but to be absent in MT-/- epidermis, has the potential to protect from photoimmunosuppression, and that the mechanism of action may be via the inactivation of the epidermal UVB-photoproduct, cis-UCA.