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Fas-Fas ligand (FasL)-dependent pathways exert a suppressive effect on inflammatory responses in immune-privileged organs. FasL expression in hepatic Kupffer cells (KC) has been implicated in hepatic immunoregulation. In this study, modulation of FasL expression of KC by endogenous gut-derived bacterial LPS and the role of reactive oxygen species (ROS) as potential mediators of FasL expression in KC were investigated. LPS stimulation of KC resulted in upstream ROS generation and, subsequently, increased FasL expression and consequent Jurkat cell (Fas-positive) apoptosis. The NADPH oxidase and xanthine oxidase enzymatic pathways appear to be major sources of this upstream ROS generation. Increased FasL expression was blocked by antioxidants and by enzymatic blocking of ROS generation. Exogenous administration of [H.sub.2][O.sub.2] stimulated KC FasL expression and subsequent Jurkat cell apoptosis. Intracellular endogenous ROS generation may therefore represent an important signal transduction pathway for FasL expression in KC. antioxidants; hepatic immunoregulation

Endothelial cell ICAM-1 upregulation in response to TNF-[alpha] is mediated in part by reactive oxygen species (ROS) generated by the endothelial membrane-associated NADPH oxidase and occurs maximally after 4 h as the synthesis of new protein is required. However, thrombin-stimulated P-selectin upregulation is bimodal, the first peak occurring within minutes. We hypothesize that this early peak, which results from the release of preformed P-selectin from within Weibel-Palade bodies, is mediated in part by ROS generated from the endothelial membrane-associated xanthine oxidase. We found that this rapid expression of P-selectin on the surface of endothelial cells was accompanied by qualitatively parallel increases in ROS generation. Both P-selectin expression and ROS generation were inhibited, dose dependently, by the exogenous administration of disparate cell-permeable antioxidants and also by the inhibition of either of the known membrane-associated ROS-generating enzymes NADPH oxidase or xanthine oxidase. This rapid, posttranslational cell signaling response, mediated by ROS generated not only by the classical NADPH oxidase but also by xanthine oxidase, may well represent an important physiological trigger of the microvascular inflammatory response. signal transduction; NADPH oxidase; xanthine oxidase; oxidant signaling

Tumor necrosis factor-alpha (TNF-alpha) is an inflammatory cytokine that upregulates the expression of adhesion molecules at the luminal surface of endothelium. Treatment of cultures of human aortic endothelial cells with TNF-alpha induced the expression of intercellular adhesion molecule 1 and E-selectin and the adhesion of neutrophils. Protein kinase inhibition studies revealed that signal transduction is mediated by a tyrosine kinase.

The role of macrophage-generated reactive oxygen metabolites (ROMs) in the phagocytosis and phagocytic activity of resident murine peritoneal macrophages were quantified by fluorochromatic vital staining assay. Superoxide dismutase activity (SOD) combined with allopurinol did not inhibit the phagocytic activity of murine macrophages incubated with Candida parapsilosis . However, macrophage phagocytic activity was inhibited by treatment of SOD with catalase indicating the role of nicotinamide adenine dinucleotide H2 and xanthine oxydase in ROM generation.

Rat mesenteric venules treated with tumor necrosis factor-alpha (TNF-a) were examined using in vivo microscopy techniques to determine if the reactive oxygen metabolites (ROMs) that mediate TNF-a-induced neutrophil adhesion to postcapillary venules after ischemia-reperfusion come from endothelial xanthine oxidase rather than neutrophil NADPH oxidase. The results show that xanthine oxidase is not the source of ROMs, which were shown to have a key role in TNF-a-induced neutrophil adhesion.

A study of cultured porcine endothelial cells (EC) selected as surrogate targets, representing the microvasculature, revealed that in endothelial cells already exposed to endotoxin, the induction of the heat shock response causes programed cell death (apoptosis). A pattern of DNA fragmentation (nucleolysis), typical of apoptosis, was evident in the in cells which died during the exposure of cultured porcine endothelial cells to a nonlethal level of bacterial endotoxin lipopolysaccharide (LPS), and simultaneously to standard stimuli. The lethal apoptosis that had been obtained by the induction of the heat shock gene expression, was caused by the treatment of LPS-exposed cells with cycloheximide to obstruct protein synthesis.

Owaki, Tetsuhiro, Avedis Meneshian, Kosei Maemura, Sonshin Takao, Dajie Wang, Katherine C. Fuh, Gregory B. Bulkley, and Andrew S. Klein. Endothelial cells potentiate phagocytic killing by macrophages via platelet-activating factor release. Am. J. Physiol. Heart Circ. Physiol. 278: H269-H276, 2000.--The immunomodulatory function of endothelial cells (EC) includes the initiation of leukocyte margination, diapedesis, and activation through the upregulation of various cell surface-associated molecules. However, the effect that EC have on the phagocytic function of neighboring monocytes and macrophages is less well described. To address this issue, microvascular EC were cocultured with murine peritoneal macrophages, first in direct contact, then in a noncontact coculture system, and macrophage phagocytosis and phagocytic killing were assessed. The presence of increasing concentrations of EC resulted in a dose-dependent increase in macrophage phagocytic killing. This stimulatory effect was inhibited in a dose-dependent manner by the pretreatment of macrophage/EC cocultures with WEB-2086 or CV-6209, specific platelet-activating factor (PAF)-receptor antagonists, but not by anti-tumor necrosis factor-[Alpha], anti-interleukin (IL)-1[Alpha], or anti-IL-1[Beta]. Furthermore, the effect was reproduced in the absence of EC by the exogenous administration of nanomolar concentrations of PAF. Microvascular EC potentiate macrophage phagocytic killing via the release of a soluble signal; PAF appears to be an important component of that signal. phagocytosis; endothelium; immunomodulation; cytokines