Irradiation with ultraviolet (UV) B radiation results in the formation of apoptotic keratinocytes called sunburn cells. Recently, it was demonstrated that keratinocytes can release tumor necrosis factor-alpha (TNF-alpha), which is known to cause apoptosis in particular cells. In addition, it has been shown that UVB light induces the release of TNF-alpha by keratinocytes and that keratinocytes express the 55-kD receptor for TNF-alpha. Therefore, we investigated whether TNF-alpha is involved in UV-induced apoptosis of keratinocytes. Normal human keratinocytes and HaCaT cells were exposed to UVB light, and apoptosis was examined by nick translation evaluated by fluorescence-activated cell sorter analysis. UVB induced apoptosis in a dose-dependent manner, which was confirmed by electron microscopy. Addition of a polyclonal antibody directed against human TNF-alpha immediately after UVB exposure was able to reduce DNA fragmentation. However, it was not possible to rescue all cells from apoptosis. To prove whether TNF-alpha is also involved in vivo in UVB-induced apoptosis of keratinocytes, Balb/c mice were exposed to UVB on their abdomens, skin biopsies were performed 24 h later, and sunburn cells were counted. A single dose of 2000 J/m2 caused a significant induction of sunburn cells. Subcutaneous injection of a polyclonal antibody directed against murine TNF-alpha immediately after UVB treatment resulted in a significant but incomplete reduction of sunburn cells, whereas injection of a rabbit IgG as a control had no effect. In both the in vitro and in vivo systems, application of recombinant TNF-alpha alone either to untreated keratinocytes or into normal murine skin did not induce sunburn cells. Thus, these data demonstrate that TNF-alpha is involved in UVB-induced apoptosis, but by itself is not able to induce sunburn cells. This further supports the notion that UVB-induced apoptosis of keratinocytes is a multifactorial event.

Endotoxin tolerance, the transient, secondary down-regulation of a subset of endotoxin-driven responses after exposure to bacterial products, is thought to be an adaptive response providing protection from pathological hyperactivation of the innate immune system during bacterial infection. However, although protecting from the development of sepsis, endotoxin tolerance also can lead to fatal blunting of immunological responses to subsequent infections in survivors of septic shock. Despite considerable experimental effort aimed at characterizing the molecular mechanisms responsible for a variety of endotoxin tolerance-related phenomena, no consensus has been achieved yet. IL-12 is a macrophage- and dendritic cell (DC)-derived cytokine that plays a key role in pathological responses to endotoxin as well as in the induction of protective responses to pathogens. It recently has been shown that IL-12 production is suppressed in endotoxin tolerance, providing a likely partial mechanism for the increased risk of secondary infections in sepsis survivors. We examined the development of IL-12 suppression during endotoxin tolerance in mice. Decreased IL-12 production in vivo is clearly multifactorial, involving both loss of CD11chigh DCs as well as alterations in the responsiveness of macrophages and remaining splenic DCs. We find no demonstrable mechanistic role for B or T lymphocytes, the soluble mediators IL-10, TNF-α, IFN-αβ, or nitric oxide, or the NF-κB family members p50, p52, or RelB.

Ultraviolet (UV) light abrogates contact hypersensitivity (CHS) responses and induces hapten-specific tolerance. Because Th-1 cells are critically involved in CHS and are induced to develop by the cytokine interleukin (IL)-12, we asked whether IL-12 might overcome UV-induced local immunosuppression. C3H/HeN mice exposed to low doses of UV light over 4 d and hapten sensitized through the irradiated skin area with dinitrofluorobenzene showed profound inhibition of the CHS response, which was completely prevented upon intraperitoneal injection of murine recombinant IL-12 (rIL-12) after the last UV exposure. UV-treated mice resensitized 14 d after the first challenge displayed hapten-specific tolerance, whereas UV-exposed mice injected with rIL-12 before the first sensitization exhibited a vigorous CHS response. Furthermore, mice that were initially sensitized through UV-exposed skin also produced a significant CHS reaction when they received rIL-12 before resensitization. Adoptive transfer of spleen and lymph node cells from UV-irradiated mice treated with rIL-12 had no effect on the CHS response in recipient mice, whereas transfer of cells from UV-treated mice inhibited the immune response. These findings demonstrate that rIL-12 can prevent UV-induced local immunosuppression and overcome UV-induced hapten-specific tolerance.

Toll-like receptor (TLR) ligands lead to the induction of proinflammatory cytokines and are potent enhancers of specific immune responses. We show here that a single systemic dose of R-848, a ligand for TLR7, potently enhanced hapten sensitization during the induction of contact hypersensitivity (CHS). However, R-848 administration also resulted in a rapid and almost complete depletion of leukocytes from the blood. This effect was transient and was associated with general induction of endothelial adhesiveness. In response to R-848, endothelial cells up-regulated adhesion molecules in vitro and in vivo and leukocytes exhibited increased rolling on endothelia in R-848-treated animals. Adhesion molecule induction appeared to be a direct effect, because endothelial cells expressed TLR7 in vitro and in vivo. After R-848 treatment, the tissue residence time of leukocytes was markedly prolonged in all major peripheral organs. The resulting transiently reduced availability of peripheral-blood leukocytes (PBLs) ( IntroductionToll-like receptors (TLRs) are a family of mammalian proteins expressed on a variety of cell types of the immune system. 1 TLRs are able to recognize specific patterns conserved in microorganisms. TLR triggering leads to the induction of inflammatory responses and induces the development of specific immunity. 1 Consequently, specific TLR ligands, such as polyinosinicpolycytidylic acid (poly I:C), lipopolysaccharide (LPS), imidazoquinolines, or CpG oligodeoxynucleotides, have been employed as powerful immune adjuvants and may enhance specific antitumor and antiviral immunity. 2,3 TLR7 is predominantly expressed by plasmacytoid dendritic cells (DCs), myeloid DCs, 4 and B cells 5 and recognizes ssRNA. 6,7 R-848 and the structurally related compound imiquimod (the active substance of the drug Aldara; 3M Pharmaceuticals, St Paul, MN) are artificial ligands for TLR7. 8,9 These imidazoquinolines are immune response modifiers that possess potent antiviral 10 and antitumoral activity when applied topically to the skin. 11 This activity is in part mediated by the induction of type I interferons and inflammatory cytokines such as tumor necrosis factor-␣ (TNF-␣) and interleukin-1␣ (IL-1␣). 12 Plasmacytoid DCs have been found to be major targets of R-848, producing large amounts of interferon-␣ and IL-12p40 as well as up-regulating activation markers in response to R-848 both in vitro 13,14 and in vivo. 14 In addition, topical imiquimod has been shown to induce emigration of resident LCs from murine skin, thereby amplifying allergic contact hypersensitivity (CHS) reactions 15 as well as inducing the migration of immature human DCs into draining lymph nodes of cancer patients. 16 Imidazoquinolines also strongly enhance T helper-1 (Th1)-type immune responses: They induce IL-12 in human Langerhans cells (LCs) 17 and lead to production of high amounts of IFN-␥ and to suppression of IL-4 and IL-5 production by T cells in cultures of human peripheral-blood leukocytes (PBLs) and murine spleen cells. 18 Interestingly, pe...

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IL-12 is a pivotal cytokine that links the innate and adaptive immune responses. TNF-α also plays a key role in orchestrating inflammation and immunity. The reciprocal influence of these two inflammatory mediators on each other may have significant impact on the cytokine balance that shapes the type and extent of immune responses. To investigate the relationship between TNF-α and IL-12 production, we analyzed the effects of exposure of human monocyte-derived macrophages to TNF-α on LPS- or Staphylococcus aureus-induced IL-12 production in the presence or absence of IFN-γ. TNF-α is a potent inhibitor of IL-12 p40 and p70 secretion from human macrophages induced by LPS or S. aureus. IL-10 is not responsible for the TNF-α-mediated inhibition of IL-12. TNF-α selectively inhibits IL-12 p40 steady-state mRNA, but not those of IL-12 p35, IL-1α, IL-1β, or IL-6. Nuclear run-on analysis identified this specific inhibitory effect at the transcriptional level for IL-12 p40 without down-regulation of the IL-12 p35 gene. The major transcriptional factors identified to be involved in the regulation of IL-12 p40 gene expression by LPS and IFN-γ, i.e., c-Rel, NF-κB p50 and p65, IFN regulatory factor-1, and ets-2, were not affected by TNF-α when examined by nuclear translocation and DNA binding. These data demonstrate a selective negative regulation on IL-12 by TNF-α, identifying a direct negative feedback mechanism for inflammation-induced suppression of IL-12 gene expression.

Cutaneous neurogenic inflammation is a complex biological response of the host immune system to noxious stimuli. Present evidence suggests that zinc metalloproteases may play an important role in the regulation of neurogenic inflammation by controlling the local availability of neuropeptides, such as substance P (SP), that are capable of initiating or amplifying cutaneous inflammation after release from sensory nerves. To address the hypothesis that the dipeptidyl carboxypeptidase angiotensin-converting enzyme (ACE) is capable of modulating skin inflammation, we have analyzed murine allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD) using wild-type C57BL/6J (ACE+/+) or genetically engineered mice with a heterozygous deletion of somatic ACE (ACE+/−). In 2,4-dinitro-1-fluorobenzene-sensitized ACE+/− mice, ACD was significantly augmented in comparison to ACE+/+ controls as determined by the degree of ear swelling after exposure to hapten. Likewise, systemic treatment of ACE+/+ mice with the ACE inhibitor captopril before sensitization or elicitation of ACD significantly augmented the ACD response. In contrast, local damage and neuropeptide depletion of sensory nerves following capsaicin, injection of a bradykinin B2, or a SP receptor antagonist before sensitization significantly inhibited the augmented effector phase of ACD in mice with functionally absent ACE. However, in contrast to ACD, the response to the irritant croton oil was not significantly altered in ACE+/− compared with ACE+/+ mice. Thus, ACE by degrading bradykinin and SP significantly controls cutaneous inflammatory responses to allergens but not to irritants, which may explain the frequently observed exacerbation of inflammatory skin disease in patients under medication with ACE inhibitors.

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