Faculty Bibliography

Tumor necrosis factor (TNF) promotes certain immune and inflammatory responses, whereas glucocorticoids exert immunosuppressive and anti-inflammatory actions. We show that TNF treatment produced a modest inhibition of glucocorticoid receptor (GR)-mediated transcriptional activation of a mouse mammary tumor virus (MMTV) promoter-driven luciferase construct in HeLa cells. The mitogen-activated protein (MAP) kinases, p38 and c-Jun N-terminal kinase (JNK), are important mediators of target gene activation by TNF, and JNK activation was earlier shown to inhibit GR-mediated transcriptional activation by direct phosphorylation of GR at Ser-246. Transfection of HeLa cells with MKK6b(E), a constitutively active specific upstream activator of p38, led to a potent inhibition of GR activation of the MMTV promoter-driven luciferase construct. A similar inhibition of activation of the MMTV promoter-driven luciferase construct was seen in HeLa cells transfected with MKK7(D), a constitutively functional activator of JNK. Data from 'domain swap' experiments using GR chimeras indicated that the main target of the p38-mediated (but not JNK-mediated) inhibition is the ligand-binding domain of GR (spanning amino acids 525-795), whereas the constitutively active N-terminal AF-1 region (spanning amino acids 106-237) is dispensable for the inhibitory effect of p38. We also demonstrate that activated p38 targets the GR ligand-binding domain indirectly. Suppression of GR function by activated p38 and JNK MAP kinases may be physiologically important as a mechanism of resistance to glucocorticoids seen in many patients with chronic inflammatory conditions

Tumor necrosis factor-stimulated gene 14 (TSG-14)/PTX3 was identified originally as a TNF-alpha and IL-1beta-stimulated gene from normal, human foreskin fibroblasts and vascular endothelial cells, respectively. TSG-14 gene encodes a 42-kDa-secreted glycoprotein with a carboxy-terminal half that shares homology with the entire sequence of C-reactive protein (CRP) and serum amyloid P component (SAP), acute-phase proteins of the pentraxin family. Some experimental evidence suggests that TSG-14 plays a role in inflammation, yet its function and mechanism of action remain unclear. We have generated transgenic mice that overexpress the murine TSG-14 gene under the control of its own promoter. From eight transgenic founders, two lineages were derived and better characterized: Tg2 and Tg4, carrying two and four copies of the transgene, respectively. TSG-14 transgenic mice were found to be more resistant to the endotoxic shock induced by LPS and to the polymicrobial sepsis caused by cecal ligation and puncture (CLP). Moreover, macrophages derived from the transgenic mice produced higher amounts of nitric oxide in response to IFN-gamma, TNF-alpha, and LPS as compared with macrophages from wild-type animals, and the augmented response appears to be the consequence of a higher responsiveness of transgenic macrophages to IFN-gamma. The data shown here are the first in vivo evidence of the involvement of TSG-14 in the inflammatory process and suggest a role for TSG-14 in the defense against bacterial infections

Many Fas-expressing cells do not undergo cell death upon Fas stimulation. In the normal human diploid cell line GM6112, the addition of soluble Fas ligand (sFasL) leads to morphological signs of cell death in less than 1% of cells. Treatment of serum-starved GM6112 fibroblasts with sFasL resulted in a rapid and transient phosphorylation of ERK1/2 without a significant increase in JNK and p38 activities. Unless co-treated with the protein synthesis inhibitor anisomycin, sFasL did not show gene-inducing activity in cells maintained in complete medium. However, when cells were serum-starved for 4 days, treatment with sFasL alone induced interleukin-6 gene expression and, less strongly, interleukin-8 gene expression. Sensitization of the gene-inducing activity by serum starvation correlated with NF-kappaB activation by sFasL. Furthermore, we found that the expression of FADD and caspase-8 was significantly reduced in serum-starved cells, whereas the level of cFLIP remained unchanged. Transfection of GM6112 cells with the antisense caspase-8 expression construct sensitized cells toward sFasL-induced NF-kappaB-dependent reporter activation. Our results support the notion that a change in the ratio of cFLIP and caspase-8 may be responsible for turning on the Fas-activated NF-kappaB pathway, which otherwise is supplanted by the death-inducing pathway

OBJECTIVE: To examine the effect of recombinant TSG-6 on collagen-induced arthritis (CIA) in DBA/1J mice. TSG-6 is a tumor necrosis factor (TNF)/ interleukin-1 (IL-1)-inducible hyaluronan-binding protein produced by synovial cells and chondrocytes that is present in synovial fluids of patients with rheumatoid arthritis. METHODS: To determine the effect of TSG-6 on chronic inflammatory joint disease, we induced CIA in DBA/1J mice by immunization with bovine type II collagen. Animals were treated with 12 intraperitoneal doses of 200 microg of recombinant TSG-6, beginning 3 days before the expected onset of disease symptoms. Progression of arthritis was monitored by determining the disease incidence, arthritis index, and footpad swelling. Levels of IgG1, IgG2a, and IgG2b antibodies against bovine and murine type II collagen and serum concentrations of IL-6 were determined at various time points. Histologic examination of affected joints was performed approximately 20 days after the onset of arthritis. RESULTS: Treatment with recombinant TSG-6 protein had a potent ameliorative effect, manifested by decreases in the disease incidence, arthritis index, and footpad swelling. Histologic examination of affected joints in TSG-6-treated animals revealed little pannus formation and cartilage erosion, features which were conspicuous in control mice. Animals treated with recombinant TSG-6 developed significantly reduced levels of IgG1, IgG2a, and IgG2b antibodies against bovine and murine type II collagen. CONCLUSION: The antiinflammatory effect of the TNF/IL-1-inducible TSG-6 protein in murine CIA suggests a role for this protein as an endogenous regulator of the inflammatory process

Sodium salicylate inhibits activation of the transcription factor NF-kappaB by blocking the phosphorylation and degradation of the NF-kappaB inhibitor IkappaBalpha. We previously demonstrated that salicylate inhibits IkappaBalpha degradation induced by tumor necrosis factor (TNF) but not by interleukin-1 (IL-1) and implicated p38 mitogen-activated protein kinase activation by salicylate in the inhibition of TNF-induced IkappaBalpha phosphorylation. Both TNF and IL-1 rapidly activate the IkappaB kinase (IKK) complex, containing the catalytic subunits IKKalpha and IKKbeta, which directly phosphorylates IkappaB proteins. Others have recently suggested that salicylate inhibits NF-kappaB activation by directly binding to IKKbeta. To clarify the mechanism whereby salicylate inhibits IKK activity, we examined its effects upon cytokine-induced IKK activity in intact cells and in vitro. Treatment of intact cells with salicylate inhibited TNF-induced but not IL-1-induced IKK activity, and this inhibition was prevented by the p38 inhibitor SB203580. In contrast, inhibition of IKK activity by salicylate in vitro was neither selective for TNF nor affected by SB203580. In vitro, salicylate treatment comparably inhibited the kinase activity of overexpressed IKKalpha and IKKbeta and also decreased p38 kinase activity. Therefore, direct inhibition of IKK activity in vitro does not reflect the inhibitory mechanism of salicylate in intact cells, which involves interference with TNF signaling