Differential effects of copper and zinc on human peripheral blood monocyte cytokine secretion

The addition of copper and zinc salts to human peripheral blood leukocytes cultured in complete medium containing endotoxin and fetal calf serum stimulated tumor necrosis factor (TNF) secretion in a concentration-dependent manner. The secretion of interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) was inhibited by copper under the same culture conditions, while zinc stimulated IL-1 beta secretion in a concentration-dependent manner and had no effect on leukocyte IL-6 release. Both copper and zinc induced increases in TNF mRNA (54 and 14%, respectively) when compared to cells cultured in complete medium alone. In serum-free, low endotoxin medium (less than 6 pg/ml), both copper and zinc failed to stimulate either TNF or IL-1 beta secretion. Under the same conditions the addition of lipopolysaccharide (LPS), at concentrations above 0.01 micrograms/ml, induced a concentration-dependent release of both cytokines. When either copper or zinc were combined with 0.01 micrograms/ml LPS, a synergistic stimulation of TNF secretion resulted. IL-1 beta secretion, unlike TNF, was not synergistically stimulated by combining metals and LPS in serum-free medium. Combining copper and zinc with inhibitors of TNF secretion, transforming growth factor beta, prostaglandin E2, and plasma alpha-globulins, resulted in a reduction of the suppressive effects of each of these agents. This study suggests that the trace metals copper and zinc may play important and possibly distinct roles in regulating leukocyte secretion of TNF, IL-1 beta, and IL-6.     

Suppression of macrophage responses to bacterial lipopolysaccharide (LPS) by secretory leukocyte protease inhibitor (SLPI) is independent of its anti-protease function

Secretory leukocyte protease inhibitor (SLPI), a potent serine protease inhibitor, has been shown to suppress macrophage responses to bacterial lipopolysaccharide (LPS). SLPI contains two topologically superimposable domains. Its C-terminal domain binds and inhibits target proteases. It is not clear whether SLPI's anti-protease function plays a role in the LPS-inhibitory action of SLPI. Four single amino acid substitution mutants of SLPI, M73G, M73F, M73E and M73K, were generated. Wild type SLPI is a potent inhibitor of chymotrypsin and elastase. Mutants M73G and M73F selectively lost inhibitory function towards chymotrypsin and elastase, respectively, whereas mutants M73K and M73E inhibited neither elastase nor chymotrypsin. Macrophage cell lines were established from RAW264.7 cells to stably express each SLPI mutant. Expression of the SLPI protease inhibition mutants suppressed NO and TNF production in response to LPS in a similar fashion as wild type SLPI. Expression of truncated forms of SLPI, containing only its N-terminus or its C-terminus, was similarly sufficient to confer inhibition of LPS responses. Thus, the LPS-inhibitory action of SLPI is independent of its anti-protease function.     

Regulation of tumor necrosis factor (TNF) expression: interferon-gamma enhances the accumulation of mRNA for TNF induced by lipopolysaccharide in murine peritoneal macrophages

The secretion of tumor necrosis factor (TNF) by macrophages is initiated by lipopolysaccharide (LPS); considerable evidence indicates that such secretion can be potentiated by interferon-gamma (IFN-gamma). The present studies show that accumulation of mRNA for tumor necrosis factor, which represents an important regulatory focus for controlling secretion of TNF, is enhanced by physiologic doses of IFN-gamma (20 units/ml of purified recombinant IFN-gamma). mRNA for TNF induced by LPS, which was maximal 2 hr after LPS was applied to the cells, was enhanced 5- to 8-fold by IFN-gamma as determined by Northern blot analysis. Interferon did not change the kinetics of accumulation but did change the dose effects of LPS in that increasing amounts of LPS led to increasing amounts of TNF mRNA in IFN-gamma-treated macrophages. IFN-gamma itself, however, did not induce expression of TNF mRNA. These studies document that IFN-gamma potentiates the cytoplasmic accumulation of mRNA for TNF induced in murine peritoneal macrophages by LPS.     

Interferon-gamma and lipopolysaccharide stimulation increases matrix metalloproteinase-9 expression and enhances invasion activity in ras/myc-transformed serum-free mouse embryo cells

Ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells were treated with interferon-gamma (IFN-gamma, 100 units/ml) and/or lipopolysaccharide (LPS, 0.5 microg/ml) for 24 h to investigate the effects of these ligands on the expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Aminoguanidine (AG, 1mM; a nitric oxide synthase [NOS] inhibitor) was also added along with IFN-gamma and LPS to analyze a possible association of NO with invasiveness. Treatment of cells with IFN-gamma alone did not alter MMP-9 mRNA expression or pro-MMP-9 production, but LPS alone and IFN-gamma+LPS co-treatment enhanced them significantly. TIMP-1 mRNA expression remained unchanged with or without treatment and the mRNA expression of MMP-9 exceeded that of TIMP-1 in LPS- or IFN-gamma+LPS-treated cells. Co-treatment of cells with IFN-gamma and LPS up-regulated invasiveness and indicated a possible involvement of NO in the enhancement of invasiveness. These results suggest that ras/myc SFME cells respond to inflammatory and infectious conditions and that they may possibly modulate their characteristics as cancer cells due to their increase in MMP-9 expression and invasion activity.     

Murine macrophages produce secretory leukocyte protease inhibitor during clearance of apoptotic cells: implications for resolution of the inflammatory response

Macrophage-derived secretory leukocyte protease inhibitor (SLPI) can be induced locally as well as systemically in response to microbial products such as LPS and lipotechoic acid. It is not known whether phagocytosis of apoptotic cells, an essential function of macrophages, can regulate expression and secretion of SLPI. In this study, we report that exposure of peritoneal macrophages of BALB/c mice or murine macrophage cell lines RAW264.7 and J774.1 to apoptotic target cells induced an elevation in SLPI secretion. Secreted SLPI retained its antichymotrypsin activity. SLPI expression in thymuses from BALB/c mice that had been injected with anti-CD3 Ab to induce apoptosis of thymocytes was also elevated both at the mRNA and protein levels. Colchicine, a microtubular inhibitor, blocked the internalization of apoptotic cells by macrophages but not SLPI secretion, suggesting that surface recognition of apoptotic cells is sufficient for the induction of SLPI. Exposure of RAW264.7 cells to apoptotic CTLL-2 cells induced both SLPI and TNF-alpha, and addition of IFN-gamma inhibited SLPI but augmented TNF-alpha production. Transfection of either the secreted or a nonsecreted form of SLPI into RAW264.7 cells led to suppression of TNF-alpha production in response to apoptotic cells. Thus, macrophages secrete an increased amount of SLPI when encountering apoptotic cells, which may help to attenuate potential inflammation during clearance of these cells.     

Effect of L-arginine on the retention of macrophage tumoricidal activity

It has been reported that the tumoricidal activity of macrophages (M phi) depends on L-arginine and that L-arginine metabolites such as reactive nitrogen intermediates alter M phi physical capacities. The aim of this report is to investigate the dose-related effect of L-arginine on the expression and retention of M phi tumoricidal activity. Cytotoxicity of M phi activated by IFN-gamma plus LPS was detected in the presence of about 0.1 mM or more of L-arginine. This paralleled the NO2- production in the presence, but not in the absence, of L-arginine. On the other hand, activated M phi were destined to die and lost their tumoricidal activity with time in the presence of 0.3 mM or more L-arginine. They retained, however, considerable activity in the absence or presence of 0.15 mM L-arginine. This retention of M phi cytotoxicity was longer when M phi were preactivated by 100 ng/ml than 10 ng/ml of LPS in combination with IFN-gamma. Addition of indomethacin, an inhibitor of prostaglandin production, did not prevent the decay of M phi cytotoxicity but rather facilitated it even in the absence of L-arginine. Regardless of indomethacin, consecutive stimulation with LPS or LPS plus IFN-gamma during culture was effective in maintaining the tumoricidal activity at a high level. In addition, we found that M phi which had lost tumoricidal activity during culture in L-arginine deficient medium could be reactivated by LPS to attack tumor target cells.     

Distinct modulatory effects of LPS and CpG on IL-18-dependent IFN-gamma synthesis

Innate cellular production of IFN-gamma is suppressed after repeated exposure to LPS, whereas CpG-containing DNA potentiates IFN-gamma production. We compared the modulatory effects of LPS and CpG on specific cellular and cytokine responses necessary for NK-cell dependent IFN-gamma synthesis. C3H/HeN mice pretreated with LPS for 2 days generated 5-fold less circulating IL-12 p70 and IFN-gamma in response to subsequent LPS challenge than did challenged control mice. In contrast, CpG-pretreated mice produced 10-fold more circulating IFN-gamma without similar changes in IL-12 p70 levels, but with 10-fold increases in serum IL-18 relative to LPS-challenged control or endotoxin-tolerant mice. The role of IL-18 in CpG-induced immune potentiation was studied in splenocyte cultures from control, LPS-conditioned, or CpG-conditioned mice. These cultures produced similar amounts of IFN-gamma in response to rIL-12 and rIL-18. However, only CpG-conditioned cells produced IFN-gamma when cultured with LPS or CpG, and production was ablated in the presence of anti-IL-18R Ab. Anti-IL-18R Ab also reduced in vivo IFN-gamma production by >2-fold in CpG-pretreated mice. Finally, combined pretreatment of mice with LPS and CpG suppressed the production of circulating IFN-gamma, IL-12 p70, and IL-18 after subsequent LPS challenge. We conclude that CpG potentiates innate IFN-gamma production from NK cells by increasing IL-18 availability, but that the suppressive effects of LPS on innate cellular immunity dominate during combined LPS and CpG pretreatment. Multiple Toll-like receptor engagement in vivo during infection can result in functional polarization of innate immunity dominated by a specific Toll-like receptor response.     

Melatonin reduces both basal and bacterial lipopolysaccharide-induced lipid peroxidation in vitro

The protective effect of melatonin against lipopolysaccharide (LPS)-induced oxidative damage was examined in vitro. Lung, liver, and brain malonaldehyde (MDA) plus 4-hydroxyalkenals (4-HDA) concentrations were measured as indices of induced membrane peroxidative damage. Homogenates of brain, lung, and liver were incubated with LPS at concentrations of either 1, 10, 50, 200, or 400μg/ml for 1 h and, in another study, LPS at a concentration of 400 μg/ml for either 0, 15, 30, or 60 min. Melatonin at increasing concentrations from 0.01–3 mM either alone or together with LPS (400μg/ml) was used. Liver, brain, and lung MDA + 4-HDA levels increased after LPS at concentrations of 10, 50, 200 or 400 μg/ml; this effect was concentration-dependent. The highest levels of lipid peroxidation products were observed after tissues were incubated with an LPS concentration of 400 μg/ml for 60 min; in liver and lung this effect was totally suppressed by melatonin and partially suppressed in brain in a concentration-dependent manner. In addition, melatonin alone was effective in brain at concentrations of 0.1 to 3 mM, in lung at 2 to 3 mM, and in liver at 0.1 to 3 mM; in all cases, the inhibitory effects of melatonin on lipid peroxidation were always directly correlated with the concentration of melatonin in the medium. The results show that the direct effect of LPS on the lipid peroxidation following endotoxin exposure is markedly reduced by melatonin.     

Involvement of tumor necrosis factor in cytotoxicity mediated by human monocytes

Human monocytes cultured in a specially prepared medium free of lipopolysaccharide (LPS) constitutively produced a small, though significant, amount of tumor necrosis factor (TNF). Upon addition of LPS, the amount produced remained constant until the LPS concentration reached 1-10 ng/ml, whereupon the production of TNF dramatically increased, eventually becoming 100-fold greater than when the LPS concentration was below 1 ng/ml. Priming the monocytes with recombinant interferon-gamma (rIFN-gamma) before LPS exposure resulted in a 2- to 10-fold increase in TNF production, the highest relative increase being obtained at lower LPS concentrations and in the absence of LPS. Monocyte-produced TNF appears to be the effector molecule in monocyte-mediated killing of some target cell types, since antiserum against recombinant TNF inhibited killing of both actinomycin D-treated and untreated WEHI 164 cells by human monocytes. However, it also appears that TNF may not in all cases be an effector molecule in monocyte-mediated killing, since cytolysis of K562 cells mediated by IFN-gamma/LPS-activated monocytes was not inhibited by antiserum against recombinant TNF. Antiserum which was raised against a monocyte-derived cytotoxic factor and which neutralized recombinant TNF did, however, inhibit monocyte-mediated cytolysis of K562 cells, suggesting that an extracellular factor, perhaps related to TNF, was also involved in monocyte-mediated killing of K562 cells. A TNF-like activity was associated with the monocyte surface membrane, since paraformaldehyde-fixed monocytes expressed cytotoxic activity which was neutralized by antiserum against recombinant TNF. Fixed monocytes activated with rIFN-gamma in addition to LPS before fixation were generally more cytotoxic than those exposed to LPS alone, and those exposed to LPS were much more cytotoxic than those not exposed to LPS. Thus it is possible that high local TNF concentrations may be generated near the target cell upon direct contact between effector and target cells, and that also monocyte-associated TNF may in this way be involved in monocyte-mediated cytotoxicity.     

The augmentation of human natural killer cell activity by interferon-gamma is not associated with the induction of the interferon-alpha-inducible proteins

Treatment of partly purified large granular lymphocytes (LGL) with either IFN-alpha or IFN-gamma for 2 hr augmented their NK cell activity. This augmentation was completely inhibited by the addition of 10 micrograms/ml of cycloheximide. In contrast, when the effects of IFN-gamma on the synthesis of specific proteins in these cells was directly studied by use of two-dimensional gel electrophoresis, we found that IFN-gamma was unable to induce any of the earlier detected, IFN-alpha/IFN-beta-inducible proteins within 18 hr of incubation. No additional, IFN-gamma-induced proteins were detected in either the partly purified LGL or purified T cells. In contrast, the effects of the two factors were comparable in the glioma cell line 251 MG. This shows i) that the effects of IFN-alpha and IFN-gamma are dependent on the responder cell type, ii) that there exists at least one mechanism that can augment NK cell activity that is not dependent on the increased synthesis of the IFN-alpha-inducible proteins, and iii) that either the nine IFN-alpha-inducible proteins are not involved in any leukocyte function that is augmentable by both IFN-alpha and IFN-gamma, or that the two factors exert their actions in leukocyte through different mechanisms.     

Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-gamma-stimulated macrophages by induction of tumor necrosis factor-alpha

Macrophages exposed to IFN-gamma and infected with amastigotes of Leishmania major develop the capacity to eliminate the intracellular pathogen. This antimicrobial activity of activated macrophages correlates with the initiation of nitrogen oxidation of L-arginine, yet other reports suggest that two signals are required for induction of this biochemical pathway for effector activity. In the present studies, macrophages treated with up to 100 U/ml IFN-gamma, or 100 ng LPS, or 10(7) amastigotes produced minimal quantities (less than 9 microM) of NO2- and failed to develop cytotoxic effector activities. In contrast, the combination of IFN-gamma and either LPS (greater than 0.1 ng) or amastigotes (10(6) induced high concentrations (much greater than 30 microM) of NO2- and macrophage cytotoxicity against intra- and extracellular targets. The induction of nitrogen oxidation by amastigotes could be dissociated from LPS-induced events by 1) performing the assays in the presence of polymyxin B (which blocked LPS effects, but not amastigote effects), 2) determining the threshold of IFN-gamma required to prime cells for subsequent trigger (1 U/ml for LPS trigger effects; 10-fold higher for amastigotes), and 3) determining the heat sensitivity of the two trigger agents (amastigote effects abolished at 100 degrees C; LPS effects unaffected at this temperature). Further, culture fluids from amastigote-infected macrophages did not contain detectable LPS (less than 6 pg/ml). Possible parasite and cell-associated factors that could contribute to the induction of nitrogen oxidation and cytotoxic activity of IFN-gamma treated macrophages were examined: only certain intact microorganisms, LPS from a variety of bacteria, and the cytokine TNF alpha were effective. Both NO2- production and intracellular killing were abolished by the addition of anti-TNF-alpha mAb in the assay. TNF-alpha was produced by amastigote-infected macrophages and IFN-gamma dramatically enhanced secretion of this cytokine; IFN-gamma alone had no effect. Endogenous TNF-alpha produced during infection of macrophages with L. major acted in an autocrine fashion to trigger the production of L-arginine-derived toxic nitrogen intermediates that killed the intracellular parasites.     

Evidence that protease action is not specifically involved in the hatching of rabbit blastocysts caused by commercial bovine serum albumin in culture

Commercial samples of bovine serum albumin (BSA) in a complex medium caused growth of 1-cell rabbit embryos to completely hatched blastocysts. Heat treatment of the BSA at 65 or 80 degrees C significantly decreased blastocyst formation and expansion and destroyed the ability to cause blastocyst hatching. Addition of trypsin at levels down to 20 ng/ml caused the formation of hatched blastocysts which degenerated rapidly. The effects of 5 protease inhibitors (ovomucoid trypsin inhibitor, alpha-1-antitrypsin, TAME, TLCK and soybean) were tested. Ovomucoid trypsin inhibitor, TAME and TLCK significantly inhibited blastocyst hatching but only at the highest concentration used. These inhibitors also reduced blastocyst formation and expansion, indicating that their effect was not specifically on blastocyst hatching in vitro. It is concluded that hatching of rabbit blastocysts is probably not dependent on protease action.     

Evidence against a possible involvement of the serine, and thiol proteases in the exocytotic mechanism of catecholamine secretion in cultured bovine adrenal medullary cells

The effects of protease inhibitors on the secretion of catecholamines were studied in cultured bovine adrenal medullary cells. Although the inhibitors of serine proteases could inhibit the carbamylcholine-induced secretion, they failed to inhibit the secretion evoked by either high K+ or A23187. The thiol protease inhibitor had no effect on the secretion. These results therefore seem to indicate that the serine protease inhibitors may inhibit the receptor-mediated secretion probably through their effects on the plasma membrane, thus suggesting that a possible involvement of the serine, and thiol proteases in exocytosis may be unlikely.     

Increased secretion of tumour necrosis factor and interleukin 6 from isolated, perfused liver of rats after partial hepatectomy

The present study explored the changes in hepatic secretion of tumour necrosis factor (TNF) and interleukin 6 (IL-6) during the regenerative process of the liver, focusing on the role of Kupffer cells. The secretions of TNF and IL-6 from the perfused rat liver were increased after 67% partial hepatectomy, reaching a maximum at 48 h. The response of cytokine secretion induced by lipopolysaccharide (LPS: 1 microgram/ml) was also potentiated in regenerating liver. The secretion of TNF, but not that of IL-6, induced by LPS was almost totally suppressed by pretreatment of rats with gadolinium chloride, which depletes Kupffer cells. These results indicate that hepatic secretions of TNF and IL-6 are increased during the regenerative process of the liver. Kupffer cells play an important role in hepatic secretion of TNF, whereas the production of IL-6 can be achieved by other cells of the liver.     

Processing and secretion of tumor necrosis factor alpha in endotoxin-treated Mono Mac 6 cells are dependent on phorbol myristate acetate.

Lipopolysaccharide (LPS, endotoxin) is a potent stimulator of tumor necrosis factor alpha (TNF alpha) synthesis and secretion in mouse macrophage tumor cells (Golenbock, D. T., Hampton, R. Y., Qureshi, N., Takayama, K., and Raetz, C. H. R. (1991) J. Biol. Chem. 266, 19490-19498). In contrast, addition of LPS (10 ng/ml) to human monomyelocytic (Mono Mac 6) cells induces very little production of TNF alpha, as judged by immunoassay of the growth medium. When 30 ng/ml 4-beta-phorbol-12-myristate 13-acetate (PMA) is added together with LPS, large amounts of TNF alpha are secreted. PMA alone is inactive. Maximal TNF alpha levels in the medium are achieved at 1 ng/ml of LPS. Protein kinase C inhibitors, such as H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine), staurosporine, and sphingosine, reduce TNF alpha secretion stimulated by PMA. The effect of PMA has been investigated at each stage of TNF alpha biogenesis. Treatment of Mono Mac 6 cells with LPS alone results in rapid, transient, and full expression of TNF alpha mRNA. Concomitant addition of PMA does not increase TNF alpha mRNA synthesis any further, but it prolongs the half-life of TNF alpha mRNA about 3-fold. However, mRNA stabilization does not account for the striking effect of PMA on TNF alpha secretion. Analysis of TNF alpha synthesis and secretion by immunoprecipitation indicates that LPS alone is fully effective in stimulating the formation of the intracellular 26-kDa TNF alpha precursor. LPS alone is not sufficient to allow processing of the precursor and secretion of mature 17-kDa TNF alpha. The rate of TNF alpha secretion observed immediately after the addition of PMA to LPS-pretreated cells is similar to the maximum rate from LPS/PMA-treated cells, but without the lag observed in cells after being exposed to LPS and PMA simultaneously. In summary, PMA is required for the completion of TNF alpha precursor processing and secretion in LPS-treated human Mono Mac 6 cells, whereas murine RAW cells are able to complete the terminal steps of TNF alpha processing in the absence of PMA.     

Duodenase activates rat peritoneal mast cells via protease-activated receptors of type 1

It was found that duodenase, a serine protease from the bovine duodenum, activates rat peritoneal mast cells (PMC) in vitro presumably via protease-activated receptors (PARs). Like thrombin (a serine protease from the blood coagulation system) and the PAR1 agonist peptide (PAR1-AP), duodenase was shown to accelerate the secretion of β-hexosaminidase (a marker of cell degranulation) by PMC in a dose-dependent manner. The blockage of the proteolytic activity of duodenase toward the substrate Tos-Gly-Pro-Lys-pNA by the soybean Bauman-Birk protease inhibitor substantially reduced (by 40%) the ability of duodenase to stimulate the secretory activity of PMC. Pretreatment of PMC with duodenase decreased the β-hexosaminidase secretion induced by thrombin and PAR1-AP by 35 and 41.7 %, respectively, and abolished the antiinflammatory effect of activated protein C. At the same time, pretreatment of PMC with duodenase did not affect the secretion of β-hexosaminidase induced by compound 48/80, a nonspecific degranulator of mast cells. Duodenase, unlike PAR1-AP (30–100 μM), in a broad concentration range (10–100 nM) did not induce aggregation of human platelets, but suppressed the platelet aggregation elicited by PAR1-AP.