Lymphokine research最新文献

A material that enhances the glucose consumption of oil-induced peritoneal macrophages of guinea pig, the activity being named MAF-G activity, was purified approximately 780-fold from human T cell hybridoma conditioned medium. A material showing growth-promoting activity was co-purified with that showing the MAF-G activity from the same conditioned medium. The two materials showed an identical isoelectric point of pH 4.7. When the partially purified MAF-G increased glucose consumption, it did not induce glucose carbon-1 oxidation. MAF-G activity was inhibited by mitomycin C and colchicine, which inhibit DNA synthesis and mitosis, respectively, but not by 2-deoxy-D-glucose, an inhibitor of glucose metabolism. The partially purified MAF-G also enhanced the glucose consumption and proliferation of human monocytes. These observations suggest that MAF-G may be identical with the growth-promoting factor for macrophages.

Specific binding of iodinated-interleukin-1 alpha or beta to YT cells could be inhibited by the lectins wheat germ agglutinin (WGA) and concanavalin A (Con A). WGA and Con A inhibition of IL-1 binding was abrogated by previous exposure of these plant proteins to the lectin-specific sugars N-acetylglucosamine (GlcNAc) and methyl glucoside (MG), respectively. Tunicamycin, an inhibitor of glycosylation, decreased interleukin-1 (IL-1) binding to YT cells, but also reduced total protein synthesis. These observations suggest that carbohydrate moieties on or near the interleukin-1 receptor may be important for optimal receptor binding of IL-1 to intact YT cells.

In this study, we have set up and optimized three distinct T cell-independent polyclonal B cell activation assay systems using highly T cell-depleted murine spleen B cells which were either preactivated in vitro with lipopolysaccharide (LPS) or costimulated with anti-IgM antibodies (anti-mu) or dextran sulfate (DxS). Using these assays, we have investigated the effects of recombinant human or murine interleukins, as well as those of a partially purified T cell-derived factor, designated BSF-LPS. Our results show that none of the interleukins, alone or in combination, is able to maintain growth of the LPS-preactivated B cell blasts, even at the highest concentrations tested, whereas the addition of our BSF-LPS preparation to the cultures significantly increases DNA synthesis. As expected, recombinant murine IL-4 (r mu IL-4) causes a substantial proliferation of anti-mu costimulated B cells. Such anti-mu costimulated B cells also respond, to a lower degree, to recombinant human IL-1 alpha (r hu IL-1 alpha), and do not significantly proliferate upon addition of r mu IL-2, r mu IL-5 or BSF-LPS. On the other hand, r mu IL-5 stimulates very efficiently DxS-costimulated B cells proliferation, whereas r hu IL-1 alpha only exerts a marginal effect; r mu IL-2, r mu IL-4 or BSF-LPS did not maintain growth of DxS-costimulated B cells. We believe that such a thorough investigation of the particular behaviour of activated B cell subpopulations towards various lymphokines provides the background for setting up a valuable bioassay method to differentiate the various interleukins acting on B cells through parallel use of the three distinct T cell-independent polyclonal B cell activation assay systems.

Minimal concentration ranges of lipopolysaccharides, natural and synthetic lipid As, and partial structures were established for the induction of release of IL-1 and TNF from human MNC. LPS was more potent than lipid A. Partial structures of lipid A lacking nonhydroxylated fatty acids were inactive, but in combination with LPS or lipid A exhibited time-dependent synergistic or antagonistic effects.

Eight stable hybridoma cell lines producing monoclonal antibodies reactive with rat interferon-gamma have been generated. The antibodies produced belong to three different immunoglobulin isotypes: IgG1 (4 monoclonal antibodies, designated DB-1, DB-10, DB-12 and DB-13), IgG2a (3 monoclonal antibodies, DB-9, DB-14 and DB-16) and IgA (1 monoclonal antibody, DB-2). The antibodies were characterized in terms of epitope specificity and reactivity with rat, mouse and human interferon-gamma. Three antibodies (DB-1, -2 and -14) show high antiviral neutralizing activity against natural and recombinant DNA derived rat interferon-gamma, whereas the others have low (DB-10, -12, -13 and -16) or no (DB-9) detectable activity. Two antibodies (DB-1 and -2) effectively bind and neutralize mouse interferon-gamma, one antibody (DB-14) exhibits some cross-reactivity and the others show no reactivity towards the mouse lymphokine. None of the antibodies reacts with human interferon-gamma. All antibodies recognize immunoblotted recombinant rat interferon-gamma, although substantial variation in the immunoreactivity existed. Competition binding experiments reveal that the antibodies are directed to three spatially distinct sites on the rat lymphokine. Two non-competing monoclonal antibodies were selected and used for the development of a specific enzyme-linked immunosorbent assay for the detection of rat interferon-gamma in biological fluids.

The LPS induced synthesis of tumor necrosis factor in macrophage cultures, as determined in a fibroblast cytolysis assay was found to be effectively blocked by inhibitors of lipoxygenases. Likewise, the presence of tumor necrosis factor in serum of D-galactosamine sensitized mice after challenge with endotoxin was suppressed by the lipoxygenase inhibitors. Indomethacin, a blocker of cycclooxygenase was neither in vivo nor in vitro effective in the prevention of the endotoxin-induced synthesis of TNF. From LPS-treated macrophages we were able to isolate 13-hydroxylinoleic acid, a lipoxygenase product, which is significantly increased after LPS treatment of the cells, covalently bound to cellular constituents and may, therefore, be possibly involved in the formation of TNF.

Since monocytes are the major source of tumor necrosis factor-alpha (TNF) in whole blood, we have developed a new method for stimulating TNF production using heparinized human whole blood instead of isolated monocytes. Test agents were dissolved in endotoxin-free buffer and 25 microliters aliquots were added directly to 225 microliters of whole blood. Following incubation at 37 degrees C, TNF levels were measured directly from diluted plasma (10%) by enzyme-linked immunoassay or L929 bioassay. Unstimulated whole blood released no detectable TNF (less than 150 pg/ml; less than 40 U/ml) over a 24 hour incubation period, but significant TNF release could be detected following a 6 hour incubation with 10 ng/ml of LPS (2163 pg/ml; 390 +/- 240 U/ml). In contrast to methods using isolated monocytes, the measurement of TNF production in whole blood ex vivo avoids monocyte activation by an adherence step, reduces the risk of contamination by endotoxin during isolation, and eliminates potentially confounding exogenous serum factors. More importantly, this method examines monocyte TNF release in response to stimuli in the relevant physiologic milieu.

We investigated the expression of several cytokine genes known to be differentially expressed in human mononuclear cells depending on the way of stimulation. In order to investigate cytokine gene expression in cell lines we stimulated peripheral blood mononuclear cells, the human cell lines UBC 5637, Wil-2, K562, U937 and Jurkat with PHA or a phorbol ester. The patterns of cytokine expression were assessed at 8 and 20 hours after stimulation by Northern blot analysis. Our data show that after stimulation each cell line expressed its own pattern of cytokine mRNA levels. Additionally, superinduction with cycloheximide revealed no change of a stimulation dependent pattern. Furthermore, we found that each cytokine has its own kinetic suggesting that signal transduction may be followed by cell type specific intracellular mechanisms.

We have previously shown that interleukin-1 (IL-1) rapidly stimulates the hydrolysis of phosphatidylcholine in the human T lymphocyte cell line, Jurkat (Rosoff, et al., Cell 54: 73-81, 1988). This was apparently mediated by a phospholipase-C catalyzed mechanism, occurring initially at the outer plasma membrane. In this report, I have further characterized this activity of IL-1. The hydrolysis of phosphatidylcholine was dependent upon extracellular Ca2+, although it appeared to be relatively independent of Mg2+. The activity was totally inhibited by prior treatment of intact Jurkat cells with trypsin. In addition, treatment of Jurkat cells with a phosphatidylinositol-specific phospholipase C, which selectively removes proteins anchored by glycosyl-phosphatidylinositol linkages, completely blocked the ability of IL-1 to stimulate the hydrolysis of phosphatidylcholine. These data suggest that the initial activity of IL-1 is to stimulate a Ca2+-dependent, glycosyl-PI-anchored phospholipase C, the active site of which is on the extracellular surface.

Human cord blood (CB) mononuclear cells were fractionated into peanut agglutinin positive (PNA+) and PNA negative (PNA-) subsets. The PNA+ subset was enriched for T6+(CD1a)Ia+ cells, which we have previously shown to resemble the Langerhans cells (LCs) of the skin, and therefore described as circulating LCs precursors. Supernatants of PNA+ and PNA- cells, and of FACS purified populations of T6+ CB cells, cultured with and without LPS, were tested for IL-1 activity. It was found that cord blood PNA+ mononuclear cells as well as purified populations of T6+ CB cells produce significant amounts of, both extracellular and cell associated, IL-1 as compared to PNA- and T6- cells, and comparable to those produced by macrophages. LPS stimulation mainly affected T6+ cells. It can be concluded that cord blood T6+ cells, presumably LCs precursors, are capable of IL-1 production.