Tumor necrosis factor alpha (TNF-alpha) is a multifunctional cytokine that has an important role in the pathogenesis of inflammation, cachexia, and septic shock. Although TNF-alpha is mainly produced by macrophages, there is evidence regarding TNF-alpha production by cells that are not derived from bone marrow. TNF-alpha production by normal and inflamed human liver was assessed at both mRNA and protein levels. Using a wide panel of novel anti-TNF-alpha monoclonal antibodies and a specific polyclonal antiserum, TNF-alpha immunoreactivity was found in hepatocytes from patients chronically infected with either hepatitis B virus (HBV) or hepatitis C virus. Minimal TNF-alpha immunoreactivity was detected in the mononuclear cell infiltrate and Kupffer cells. In situ hybridization experiments using a TNF-alpha RNA probe showed a significant expression of TNF-alpha mRNA in hepatocytes, Kupffer cells, and some infiltrating mononuclear cells. By contrast, TNF-alpha was detected at low levels in liver biopsies from normal individuals or patients with alcoholic liver disease and low expression of TNF-alpha mRNA was observed in these specimens. Transfection of HepG2 hepatoblastoma cells with either HBV genome or HBV X gene resulted in induction of TNF-alpha expression. Our results demonstrate that viral infection induces, both in vivo and in vitro, TNF-alpha production in hepatocytes, and indicate that the HBV X protein may regulate the expression of this cytokine. These findings suggest that TNF-alpha may have an important role in human liver diseases induced by viruses.

The activation of the endothelial cells by extravascular stimuli is the key event in the extravasation of circulating leukocytes to target tissues. L-selectin, a member of the selectin family, is constitutively expressed by white cells, and is the molecule involved in the initial binding of leukocytes to activated endothelium. After activation, leukocytes rapidly release L-selectin from the cell surface, -suggesting that the functional activity of this molecule is controlled in large part by its appearance and disappearance from cell surface. We have studied in a neutrophil-activated endothelial cell binding assay, the effect of different antiinflammatory drugs (steroidal and nonsteroidal) in the L-selectin-mediated interaction of neutrophils with activated endothelial cells. Some nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin, diclofenac, ketoprofen, and aspirin, but not steroids, strongly inhibited the neutrophil-endothelial cell attachment. Furthermore, we also investigated the underlying mechanism of this functional effect. The expression of L-selectin on the neutrophil surface rapidly decreased in the presence of different NSAIDs, in a dose-and timedependent manner, whereas no changes in the expression of other adhesion molecules such as CD11a, CD11b, CD31, or ICAM-3 (CD50) were observed. Interestingly, studies in vivo on healthy volunteers treated with physiological doses of indomethacin showed a significant decrease of L-selectin neutrophil expression. Only diclofenac induced an upregulation of CD11b expression, suggesting an activating effect on neutrophils. No enzyme release was observed upon treatment of neutrophils with different NSAIDs, indicating a lack of degranulatory activity of NSAIDs, with the exception of diclofenac. The downregulation of L-selectin expression was due to the rapid cleavage and shedding of the membrane L-selectin, as determined by both immunoprecipitation from "2I-labeled neutrophils, and quantitative estimation in cell-free supernatants. These results suggest that NSAIDs exert a specific action on adhesion receptor expression in neutrophils, which might account, at least in part, for the antiinflammatory activities of NSAIDs. (J. Clin. Invest. 1995.

A screen for natural products bearing pharmacological properties has yielded a secretion of the mollusk Cryptomphalus aspersa (SCA), which possesses skin-regenerative properties. In this report, we outline some of the cellular and molecular effects underlying this observation. First, we found that SCA contained antioxidant SOD and GST activities. In addition, SCA stimulated fibroblast proliferation and rearrangement of the actin cytoskeleton. Additional mechanisms involved in the regenerative effect of SCA included the stimulation of extracellular matrix assembly and the regulation of metalloproteinase activities. Together, these effects provide an array of molecular mechanisms underlying SCA-induced cellular regeneration and postulate its use in regeneration of wounded tissue.

Adhesion of human neutrophils to endothelial cells is a crucial step during migration to the extravascular sites of inflammation. A large number of molecules, including the CD44 and LAM-1 antigens, have been described to participate in this process. We have investigated the regulation by human recombinant tumor necrosis factor-alpha (TNF-alpha) of human neutrophil plasma membrane expression of both CD44 and LAM-1 adhesion molecules, as well as that of CD43 sialophorin, which has been involved in adhesion and activation of leukocytes. The expression of these three antigens was down-regulated in neutrophils upon TNF-alpha treatment, as determined by immunofluorescence and immunoprecipitation experiments. However, the expression of other cell surface molecules, such as CD45 or CD11b, was up-regulated. Similar regulatory effects were also observed upon neutrophil treatment with other activating agents such as the chemoattractant peptide formyl-Met-Leu-Phe, the calcium ionophore A23187, or the phorbol ester phorbol 12-myristate 13-acetate. Protease inhibitors virtually abrogated the TNF-alpha-induced down-regulation of CD43 and CD44 expression, but not that of LAM-1, suggesting the involvement of a protease activity in this process. These results underline the role of TNF-alpha on the differential regulation of cell surface expression of neutrophil adhesion molecules, thus implying modifications in the neutrophil adhesive properties.

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