Despite the established role of dendritic cells (DCs) in regulating T lymphocyte activation, intracellular mechanisms responsible for controlling DC function are largely undefined. Here, we have studied DCs from mice deficient in the p50, RelA, and cRel subunits of the immunomodulatory NF-kappaB transcription factor. Although DC development and function was normal in mice lacking individual NF-kappaB subunits, development of doubly deficient p50(-/-)RelA(-/-) DCs was significantly impaired. In contrast, DCs from p50(-/-)cRel(-/-) mice developed normally, but CD40L- and TRANCE-induced survival and IL-12 production was abolished. Surprisingly, no significant impairment in MHC and costimulatory molecule expression was seen, despite significantly reduced kappaB site binding activity. These results therefore indicate essential, subunit-specific functions for NF-kappaB proteins in regulating DC development, survival, and cytokine production.

Tissue damage induced by infection or injury can result in necrosis, a mode of cell death characterized by induction of an inflammatory response. In contrast, cells dying by apoptosis do not induce inflammation. However, the reasons for underlying differences between these two modes of cell death in inducing inflammation are not known. Here we show that necrotic cells, but not apoptotic cells, activate NF-κB and induce expression of genes involved in inflammatory and tissue-repair responses, including neutrophil-specific chemokine genes KC and macrophage-inflammatory protein-2, in viable fibroblasts and macrophages. Intriguingly, NF-κB activation by necrotic cells was dependent on Toll-like receptor 2, a signaling pathway that induces inflammation in response to microbial agents. These results have identified a novel mechanism by which cell necrosis, but not apoptosis, can induce expression of genes involved in inflammation and tissue-repair responses. Furthermore, these results also demonstrate that the NF-κB/Toll-like receptor 2 pathway can be activated both by exogenous microbial agents and endogenous inflammatory stimuli.

Serum IgE concentrations and the expression of the low-affinity receptor for IgE (FceRHl/CD23) Nitric oxide (NO) generated by activated murine macrophages is cytostatic or cytotoxic for a variety of pathogens, including Leishmania major, Plasmodium falciparum, Schistosoma mansoni, Trypanosoma cruzi, and Toxoplasma gondii (1-6). NO is generated from the oxidation of the terminal guanido nitrogen atom(s) of L-arginine by an NADPH-dependent enzyme, the NO synthase (NOS) (7-9). In murine macrophages, NOS activity is induced by lipopolysaccharide (LPS) (9) IgE-IC) or by a specific monoclonal antibody (mAb) to CD23 (CD23 mAb) promotes the generation of cGMP. Simultaneous measurement of the generation of nitrite (NO-j) indicated that the enhanced production of cGMP is consequent to activation of the L-arginine:NO pathway.The low-affinity receptor for IgE (FceRII/CD23) is also expressed on normal human monocytes/macrophages after their activation in vivo (22,23). Studies in patients infected with Leishmania braziliensis or in disease-free, immunoreactive donors have shown both in situ CD23 expression and serum IgE concentrations to be increased in these conditions (23). We have, therefore, studied whether cell activation through ligation of the membrane receptor CD23 induces the L-arginine:NO pathway and results in leishmanicidal activity in human macrophages. MATERIALS AND METHODSReagents. The following reagents were used: recombinant human interleukin 4 (IL-4; a gift from J. Banchereau, Schering-Plough); IFN--y (a gift from J. M. Mencia-Huerta, Institut Beaufour, Paris); TNF-a and anti-TNF-a mAb (Genzyme); human IgE (Stallergene, Paris), and goat anti-human IgE (Nordic, Tilburg, The Netherlands). Polymyxin B, LPS (Escherichia coli 055, L-2880), NG-monomethyl-L-arginine (L-NMMA), D-NMMA, L-arginine, D-arginine, superoxide dismutase (SOD), and catalase were all obtained from Sigma.Cells. Human mononuclear cells were obtained by Ficoll gradient separation of peripheral blood leukocytes from healthy volunteers. Monocytes were separated from lymphocytes by adherence to plastic dishes coated with fetal calf serum as described (24). After this procedure, >90% of cells expressed CD14 antigen and displayed cytochemical characteristics of monocytes (24). The cells were then incubated in Dulbecco's modified Eagle's medium (DMEM) supplemented with nonessential L-amino acids, sodium pyruvate, glutamine, penicillin, streptomycin, and 10% (vol/vol) fetal calf serum (all from GIBCO). The culture medium was routinely controlled for the absence of a direct activation effect on human monocytes (CD23 expression and TNF-a production as activation Abbreviations: mAb, monoclonal antibody; CD23 mAb, anti-CD23 mAb; IgE-IC, IgE-anti-IgE immune complexes; L-NMMA, NA3-monomethyl-L-arginine; LPS, lipopolysaccharide; NOS, NO synthase; iNOS, inducible NOS; IFN-y, interferon y; IL, interleukin; TNF-a, tumor necrosis factor ai; SOD, superoxide dismutase; HPRT, hypoxanthine phosphoribosyltransferase. tTo whom reprint requests should be ad...

The Fas death receptor plays a key role in the killing of target cells by NK cells and CTLs and in activation-induced cell death of mature T lymphocytes. These cytotoxic pathways are dependent on induction of Fas expression by cytokines such as TNF-α and IFN-γ or by signals generated after TCR engagement. Although much of our knowledge of the Fas death pathway has been generated from murine studies, little is known about regulatory mechanisms important for murine Fas expression. To this end, we have molecularly cloned a region of the murine Fas promoter that is responsible for mediating TNF-α and PMA/PHA-induced expression. We demonstrate here that induction of Fas expression by both stimuli is critically dependent on two sites that associate with RelA-containing NF-κB complexes. To determine whether RelA and/or other NF-κB subunits are also important for regulating Fas expression in primary T cells, we used CD4 T cells from RelA−/−, c-Rel−/−, and p50−/− mice. Although proliferative responses were significantly impaired, expression of Fas and activation-induced cell death was unaffected in T cells obtained from these different mice. Importantly, we show that unlike fibroblasts, which consist primarily of RelA-containing NF-κB complexes, T cells have high levels of both RelA and c-Rel complexes, suggesting that Fas expression in T cells may be dependent on redundant functions of these NF-κB subunits.

Binding sites for the nuclear factor (NF)-κB transcription factor have been identified within control regions of many genes involved in inflammatory and immune responses. Such κB sites are often found adjacent to those of interferon (IFN)-γ–inducible transcription factors, suggesting a requirement for multiple signaling pathways for gene regulation. Using fibroblasts from RelA (p65)-deficient mice generated by gene targeting, we have investigated the role of this subunit of NF-κB in gene activation by microbial lipopolysaccharide, tumor necrosis factor α, and in possible synergism with the IFN-γ–signaling pathway. Our results indicate not only that RelA is required for activation of key genes involved in adaptive (acquired) immune responses, including major histocompatibility complex class I, CD40, and the Fas death receptor, but also that both NF-κB–inducing signals and IFN-γ are necessary for maximal activation. In contrast, neutrophil-specific chemokine genes KC and MIP-2, which can function as nonspecific mediators in innate immune responses, were strongly induced by RelA in the absence of IFN-γ. Our results show that RelA plays a critical role in activation of immune system genes in response to nonspecific stimuli and demonstrate a novel proapoptotic function for this protein in Fas-induced cell death.

Elevated IgE levels are commonly observed during the inflammatory responses in allergy and a variety of infections. This Ig activates the release of multiple mediators from monocytes/macrophages. In the present work, we attempted to clarify the IgE-dependent events involved in the activation of monocyte functions. IgE-anti-IgE immune complexes induce the production of tumor necrosis factor-alpha, oxygen radicals, IL-6 and thromboxane B2 from normal human purified monocytes. Expression and cross-linkage of Fc epsilon RII/CD23 were essential for these IgE-mediated effects. Cytokine production following CD23 ligation depended on nitric oxide transduction pathway, as it was inhibited by NG-monomethyl-L-arginine, a competitive inhibitor of the conversion of L-arginine to L-citroline by nitric oxide synthase. Furthermore, addition of the nitric oxide chemical donator, Sin-1, enhanced IgE-induced monokine release. CD23-ligation also induced the production of nitrites by these cells. This work linked CD23 to the L-arginine-dependent transduction pathway and shows their involvement in IgE-mediated stimulation of human monocytes.

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Human keratinocytes (HK) generate nitric oxide (NO) and proinflammatory mediators following activation with either IgE/anti-IgE immune complexes or a combination of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Recently, interleukin-10 (IL-10) has been shown to down-regulate various inflammatory responses and to be secreted by lymphocytes and dendritic cells during skin inflammatory reactions. We show here that IL-10 down-regulates the production of tumor necrosis factor (TNF)-alpha and IL-6 by activated HK. Also, induction of inducible nitric oxide synthase (iNOS) expression in HK by IgE/anti-IgE or LPS/IFN-gamma is significantly reduced by the addition of IL-10. This effect is dose dependent and correlates with reduction of iNOS mRNA production and enzyme level. Therefore, IL-10 down-regulates NO-mediated HK inflammatory responses and may thus participate in the regulation of the skin immune network.