Development of the appropriate CD4+ T helper (TH) subset during an immune response is important for disease resolution. With the use of naïve, ovalbumin-specific alpha beta T cell receptor transgenic T cell, it was found that heat-killed Listeria monocytogenes induced TH1 development in vitro through macrophage production of interleukin-12 (IL-12). Moreover, inhibition of macrophage production of IL-12 may explain the ability of IL-10 to suppress TH1 development. Murine immune responses to L. monocytogenes in vivo are of the appropriate TH1 phenotype. Therefore, this regulatory pathway may have evolved to enable innate immune cells, through interactions with microbial pathogens, to direct development of specific immunity toward the appropriate TH phenotype.

Listeriosis in mice with the severe combined immunodeficiency (SCID) mutation is an established model in vivo and in vitro of interferon y(IFN-y)-dependent macrophage activation by natural killer (NK) cells during the development of natural immunity. We demonstrate that IFN-y production from SCID splenocytes is stimulated by interleukin (IL) 12, tumor necrosis factor a (TNF-a), and IL-2 but is inhibited by

NF-B-induced gene expression contributes significantly to the pathogenesis of inflammatory diseases such as arthritis. I B kinase (IKK) is the converging point for the activation of NF-B by a broad spectrum of inflammatory agonists and is thus a novel target for therapeutic intervention. We describe a small molecule, selective inhibitor of IKK-2, SC-514, which does not inhibit other IKK isoforms or other serine-threonine and tyrosine kinases. SC-514 inhibits the native IKK complex or recombinant human IKK-1/IKK-2 heterodimer and IKK-2 homodimer similarly. IKK-2 inhibition by SC-514 is selective, reversible, and competitive with ATP. SC-514 inhibits transcription of NF-B-dependent genes in IL-1␤-induced rheumatoid arthritis-derived synovial fibroblasts in a dosedependent manner. When the mechanism of NF-B activation was evaluated in the presence of this inhibitor, several interesting observations were found. First, SC-514 did not inhibit the phosphorylation and activation of the IKK complex. Second, there was a delay but not a complete blockade in I B␣ phosphorylation and degradation; likewise there was a slightly slowed, decreased import of p65 into the nucleus and a faster export of p65 from the nucleus. Finally, both I B␣ and p65 were comparable substrates for IKK-2, with similar K m and K cat values, and SC-514 inhibited the phosphorylation of either substrate similarly. Thus, the effect of SC-514 on cytokine gene expression may be a combination of inhibiting I B␣ phosphorylation/degradation, affecting NF-B nuclear import/ export as well as the phosphorylation and transactivation of p65.

Objective To examine cyclooxygenase‐2 (COX‐2) enzyme expression, its regulation by interleukin‐1β (IL‐1β), and the role of prostaglandin E2 (PGE2) in proteoglycan degradation in human osteoarthritic (OA) cartilage. Methods Samples of human OA articular cartilage, meniscus, synovial membrane, and osteophytic fibrocartilage were obtained at knee arthroplasty and cultured ex vivo with or without IL‐1β and COX inhibitors. COX expression was evaluated by immunohistochemistry and Western blot analysis. The enzymatic activity of COX was measured by conversion of arachidonic acid to PGE2. Cartilage degradation was evaluated by measuring the accumulation of sulfated glycosaminoglycans in the medium. Results IL‐1β induced robust expression of COX‐2 and PGE2 in OA meniscus, synovial membrane, and osteophytic fibrocartilage explants, whereas low levels were produced in OA articular cartilage. IL‐1β also induced cartilage proteoglycan degradation in OA synovial membrane‐cartilage cocultures. Increased proteoglycan degradation corresponded to the induction of COX‐2 protein expression in, and PGE2 production from, the synovial membrane. Dexamethasone, neutralizing IL‐1β antibody, or the selective COX‐2 inhibitor, SC‐236, attenuated both the IL‐1β‐induced PGE2 production and cartilage proteoglycan degradation in these cocultures. The addition of PGE2 reversed the inhibition of proteoglycan degradation caused by SC‐236. Conclusion IL‐1β‐induced production of COX‐2 protein and PGE2 was low in OA articular cartilage compared with that in the other OA tissues examined. IL‐1β‐mediated degradation of cartilage proteoglycans in OA synovial membrane‐cartilage cocultures was blocked by the selective COX‐2 inhibitor, SC‐236, and the effect of SC‐236 was reversed by the addition of exogenous PGE2. Our data suggest that induction of synovial COX‐2‐produced PGE2 is one mechanism by which IL‐1β modulates cartilage proteoglycan degradation in OA.

The cyclooxygenase isoforms, COX-1 and COX-2, are involved in the biosynthesis of prostaglandin E2, a major prostaglandin involved in epidermal homeostasis and repair. Cancer originating in the epidermis can develop when keratinocyte proliferation and apoptosis become dysregulated, resulting in sustained epidermal hyperplasia. COX-2 inhibitors, which demonstrate significant in vivo selectivity relative to COX-1, suppress both ultraviolet-induced epidermal tumor development and progression, suggesting that prostaglandin regulation of keratinocyte biology is involved in the pathogenesis of epidermal neoplasia. In this study, we characterized the expression of COX-1 and COX-2, as well as keratinocyte proliferation, differentiation, and apoptosis, following acute ultraviolet irradiation in the hairless SKH-1 mouse. Following acute ultraviolet exposure, COX-2 expression was predominantly induced in the basal keratinocyte layer coincident with an increase in keratinocyte proliferation and apoptosis. The role of COX-2 was further evaluated using a selective COX-2 inhibitor, SC-791, as well as the traditional nonsteroidal COX inhibitor, indomethacin. Following acute ultraviolet irradiation, inhibition of COX-2 with either inhibitor decreased epidermal keratinocyte proliferation. Likewise, keratinocyte apoptosis was increased with COX-2 inhibition, particularly in the proliferating basal keratinocyte layer. There was also a modest inhibition of keratinocyte differentiation. These data suggest that COX-2 expression is probably necessary for keratinocyte survival and proliferation occurring after acute ultraviolet irradiation. We hypothesize that selective COX-2 inhibition, as described herein, may lead to enhanced removal of ultraviolet-damaged keratinocytes, thereby decreasing malignant transformation in the epidermis.