Aspirin inhibits tumor necrosis factoralpha gene expression in murine tissue macrophages.

Aspirin has been reported to inhibit the activation of nuclear factor-kappaB (NF-kappaB) through stabilization of inhibitor kappaB (IkappaB). This observation led us to investigate the role of aspirin in suppressing the activation of the NF-kappaB-regulated tumor necrosis factor-alpha (TNF-alpha) gene expression in primary macrophages. We now report that therapeutic doses of aspirin suppress lipopolysaccharide-inducible NF-kappaB binding to an NF-kappaB binding site in the TNF-alpha promoter, lipopolysaccharide-induced TNF-alpha mRNA accumulation, and protein secretion. IkappaB is also stabilized under these conditions. The aspirin-initiated stabilization of IkappaB, suppression of induced TNF-alpha mRNA, and NF-kappaB binding to the TNF-alpha promoter are blocked by pretreatment with pertussis toxin. These studies suggest that aspirin may exert significant anti-inflammatory effects by suppressing the production of macrophage-derived inflammatory mediators.

Differential responsivity of monocyte cytokine and adhesion proteins in high- and low-hostile humans.

This study tested the general hypothesis that high- and low-hostile respondents would show different patterns of change in monocyte cytokine and adhesion protein (MCAP) expression in response to pharmacologically induced alterations in sympathetic nervous system (SNS) and parasympathetic nervous system (PNS) balance. On 3 separate days, 4 high- and 4 low-hostile respondents received isoproterenol infusions after saline, atropine (PNS blockade), or neostigmine (PNS stimulation) pre-treatment. Dual color flow cytometry with fluorescently labeled monoclonal antibodies to CD 14 (monocyte market), interleukin-1, leukocyte function activator (LFA-1), Class II major histocompatibility complex (MHC-II), and tumor necrosis factor was used to quantify cytokine and adhesion protein expression on monocytes in blood samples drawn before and after the combination drug infusions on the 3 test days in each respondents. Following PNS stimulation and istoproterenol infusion there was a decrease (compared to saline pretreatment) in MHC II expression in high hostiles that was significantly (p<.02) different from an increase in low hostiles. A similar trend (p = .08) was seen for LFA-1 expression, with high hostiles showing an increase and low hostiles a decrease. These findings support the broad hypothesis that high-and low-hostile respondents will show different MCAP responses to pharmacologically induced alterations in SNS-PNS balance. Such differences could contribute to accelerated atherogenesis among high-hostile individuals.

Maleylated-BSA induces hydrolysis of PIP2, fluxes of Ca2+, NF-kappaB binding, and transcription of the TNF-alpha gene in murine macrophages.

The interaction of altered lipids or proteins with the several scavenger receptors (SR) on macrophages can lead to disparate results in both gene expression and cell function. However, the molecular bases of signaling induced by SR ligation have remained obscure. Here we report that maleylated-bovine serum albumin (maleyl-BSA) binds a low-affinity SR, initiating PIP2 hydrolysis, [Ca2+]i spikes, phospholipase A2 (PLA2) activation, nuclear factor-kappa(B) (NF-kappa(B)) binding to its cognate nucleotide and tumor necrosis factor alpha (TNF-alpha) gene transcription. We recently reported that oxidized low-density lipoprotein (ox-LDL), which binds another macrophage SR, induced pertussis-toxin-sensitive hydrolysis of PIP2 and elevations in [Ca2+]i [J. Biol. Chem. 270, 3475-3478, 1995]. By contrast, maleyl-BSA-initiated events were not pertussis toxin-sensitive and produced less [Ca2+]i spiking than ox-LDL. Furthermore, maleyl-BSA led to binding of NF-kappa(B) to its cognate nucleotide and TNF-alpha gene transcription, whereas ox-LDL suppressed these events. Collectively, this data suggests that maleyl-BSA and ox-LDL bind to distinct SR on murine macrophages, initiate distinct signal transduction pathways, and produce different functional effects.

Sequence elements required for function of the MHC class II A beta gene promoter in murine macrophages.

We have studied the DNA sequence elements of the murine MHC class II A beta gene involved in transcriptional regulation in macrophages. For this study, the A beta promoter was fused to the human growth hormone gene and transfected into bone marrow-derived macrophages. These macrophages were stimulated by interferon-gamma (IFN-gamma), after which cellular RNA was assayed for the amount of human growth hormone transcripts. A -146 to +14 fragment of the A beta promoter was found to be sufficient to confer positive regulation by IFN-gamma. Induction was suppressed by bacterial lipopolysaccharide, tumor necrosis factor (TNF-alpha), or maley-lated bovine serum albumin. Substitution mutations were made within each of the conserved sequence elements of the promoter as well as within the spacer regions between these elements. All four conserved sequences found in class II promoters, the H, X1, X2, and Y elements, were found to be essential for promoter activation in macrophages.

Selective sensitivity of macrophages to cytotoxicity by inhibitors of macromolecular synthesis: induction of apoptosis.

Initial studies designed to measure the effect of inhibiting RNA synthesis by dactinomycin on macrophage functions revealed that the cells were uniformly killed at concentrations that have been routinely used to inhibit RNA synthesis in other cell types. We, thus, determined the dose curve for the cytotoxicity of dactinomycin for macrophages and two other cell types, L929 cells and splenic lymphocytes. Macrophages were extremely sensitive to the cytotoxicity of dactinomycin compared to the other cell types. Submicromolar concentrations that induced 100% cytotoxicity in macrophages caused little death in L929 cells or lymphocytes. Concentrations of dactinomycin that inhibited RNA synthesis by 40% in macrophages induced almost complete cell death but inhibition of over 80% of RNA synthesis in L929 cells or lymphocytes induced no measurable cytotoxicity. Macrophages did take up more dactinomycin than other cells but the amount was not sufficient to account for the large differences in cytotoxicity. We next tested the effects of doxorubicin and cycloheximide and found that macrophages were also extremely sensitive to killing by these compounds, and there was a very close association between the amount of inhibition of protein synthesis and the amount of toxicity. The morphology of macrophages exposed to these agents was consistent with death by apoptosis. This was further supported by assays measuring membrane integrity and DNA fragmentation. These data demonstrate that inhibition of macromolecular synthesis in macrophages, by different mechanisms, causes macrophages to undergo apoptosis. They further suggest that, in contrast to other cell types that require protein synthesis for apoptosis, macrophages require the synthesis of certain proteins to avoid apoptosis.

Effects of oxidized LDL on mononuclear phagocytes: inhibition of induction of four inflammatory cytokine gene RNAs, release of NO, and cytolysis of tumor cells.

A critical step in development of atherosclerosis is the interaction of oxidized low-density lipoprotein (LDL) with mononuclear phagocytes. Oxidized LDL, as well as acetyl-LDL, is rapidly taken up into macrophages via a family of scavenger receptors. We report that macrophages treated with oxidized LDL have markedly lower levels of mRNA specific for the genes MCP-1, TNF-alpha, IL-1 alpha, and KC as measured by Northern blot analyses of lipopolysaccharide (LPS)-stimulated macrophages. By contrast, acetyl-LDL does not inhibit these genes at the doses at which oxidized-LDL is effective. Similar effects are observed whether the LDL is oxidized in the presence of Cu2+ or of Fe2+. Such inhibition also occurs when maleylated bovine serum albumin (BSA), which also clears by one or more scavenger receptors on macrophages, is used as the stimulant. Fe2+ or Cu2+ oxidized LDL inhibits release of nitric oxide when triggered by LPS and direct cytolysis of tumor cells when triggered by maleylated BSA or LPS. Taken together, the data presented indicate that oxidized LDL inhibits induction of several important gene RNAs as well as functional markers that characterize the development of inflammatory and fully activated macrophages.

IFN-gamma and lipopolysaccharide induce DNA binding of transcription factor PU.1 in murine tissue macrophages.

The transcription factor PU.1, the Spi-1 oncogene product, is found principally in B cells and macrophages. PU.1 binds to a purine-rich sequence of DNA with a core consensus sequence of 5'-GAGGAA-3'. By using an electrophoretic mobility shift assay, we have been able to identify PU.1 in nuclear extracts of murine tissue macrophages. When macrophages are stimulated with IFN-gamma, an increase in PU.1 binding is observed. This increase is gradual, peaking at 6 to 9 h and decaying to basal levels 24 h after stimulation. The increase in PU.1 binding is unaffected by inhibiting protein synthesis, but is attenuated by either an inhibitor of the NA+/H+ antiporter or elevations in cAMP. In addition, we have found that low doses (1 ng/ml) of bacterial LPS induce PU.1 binding.

Oxidized low density lipoprotein suppresses activation of NF kappa B in macrophages via a pertussis toxin-sensitive signaling mechanism.

The interaction of oxidized low density lipoprotein (ox-LDL) and macrophages is generally believed to be a significant inductive step in atherogenesis. Endocytosis of ox-LDL by scavenger receptors (SR) on macrophages is one result of this interaction, as is suppressed expression of several lipopolysaccharide (LPS)-stimulated, inflammatory genes such as tumor necrosis factor-alpha (TNF-alpha). Events subsequent to SR ligation, including intracellular signaling events if any, have not been established. We report here that ox-LDL initiates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate 2 (PIP2) and intracellular fluxes of Ca2+ in macrophages, both of which are sensitive to pertussis toxin. ox-LDL also suppresses the LPS-induced binding of macrophage extracts to an NF kappa B sequence oligonucleotide and the LPS-initiated accumulation of RNA specific for TNF-alpha. These latter two effects are pertussis toxin-sensitive. Ligation of SR by ox-LDL thus initiates a pertussis toxin-sensitive signaling pathway in macrophages, which involves hydrolysis of PIP2 and which can suppress expression of the TNF-alpha gene by modulating activation of NF kappa B.

Molecular biology of macrophage activation: a pathway whereby psychosocial factors can potentially affect health.

Over the past decade, numerous links between the mind, the immune system, and the nervous system have been clearly elucidated in regard to the lymphoid elements of the immune system. Recently, attention has been given to the suggestion that such links exist to mononuclear phagocytes. The fundamental biology and physiology of these cells are clearly consonant with such a role. Mononuclear phagocytes bear receptors on their surface for numerous ligands, including neuroendocrine peptides and hormones. Furthermore, macrophages can be modulated by a variety of life-style choices such as smoking, ingestion of alcohol, and dietary lipid. Mononuclear phagocytes are key cells in the development of atherosclerosis and play significant roles in host protection against neoplasia and in the development of certain autoimmune diseases. In regard to atherogenesis, stress can potentiate the effects of a high lipid diet in initiating formation of the macrophage-laden lesions of early atherosclerosis. We have been able to show that oxidized lipids, acting in concert with stress as modeled pharmacologically in terms of catecholamines, induces a macrophage phenotype which would likely promote the development of atherosclerosis but lower host resistance to neoplasia. We are currently testing this hypothesis critically. Over the coming decade, it will be important for numerous investigators to examine the broad hypothesis that mononuclear phagocytes are one key element in the induction of psychosocially produced diseases. A clear understanding of the biology and molecular biology of these cells will provide a potent tool for analyzing these problems.

Effects of benzene metabolites on receptor-mediated phagocytosis and cytoskeletal integrity in mouse peritoneal macrophages.

Exposure to benzene can induce a number of hematotoxicities and decrease host resistance to microorganisms and tumors. Several studies have shown that metabolism of benzene to reactive intermediates is required for myelotoxicity. Since receptor-mediated phagocytosis by macrophages is an important host defense, we have examined the effects of benzene metabolites on receptor-mediated phagocytosis in cultured murine peritoneal macrophages. 1,4-Benzoquinone (BQ) was the most potent of the metabolites examined. Ten-minute exposures to a 12.5 microM concentration inhibited Fc and complement receptor-mediated phagocytosis by > or = 90%. Macrophage viability was largely unaffected by BQ treatment. Exposure to 50 and 100 microM 1,2,4-benzenetriol (BT) inhibited Fc receptor-mediated phagocytosis by 70 and 95%, respectively. Hydroquinone (HQ) elicited a major decrease (50%) only at 100 microM. The comparative inhibitory potencies of BT and HQ correlate with previously published data on their relative facility for autooxidation to quinones at physiological pH. Catechol had no effect at the concentrations employed. Macrophages treated with BQ and BT failed to recover their Fc receptor-mediated phagocytic capacity when incubated overnight in the absence of the xenobiotics. Only small differences in the inhibition of Fc receptor-mediated phagocytosis were observed between macrophages exposed to BQ at 4 versus 37 degrees C. BQ also had little effect on the Fc receptor binding of target cells. Fluorescent digital imaging microscopy demonstrated that BQ treatment markedly decreased the filamentous actin content of macrophages. However, BQ bound in low amounts to purified actin and did not affect its assembly. Our findings suggest that a mechanism for inhibition of Fc receptor-mediated phagocytosis by BQ is disruption of filamentous actin via an effect(s) other than the direct alkylation of actin by BQ.

Quantification of contributions of phospholipid precursors to diradylglycerols in stimulated mononuclear phagocytes.

Phosphatidylcholine (PC) hydrolysis has been shown to occur in hormone-stimulated cells and represents a potential metabolic source, in addition to phosphoinositides, for the generation of diradylglycerols (DG). We performed studies in order to quantify the importance of this pathway in DG formation. We incubated murine peritoneal macrophages with platelet-activating factor (PAF), ionomycin, phorbol myristate acetate (PMA) or no stimulus in a series of timed incubations ranging from 15 s to 20 min. We quantified the profiles of the molecular species in the accumulated DG after extraction, specific radiolabelling to give [32P]phosphatidic acid by DG kinase, and conversion to the dimethyl derivative. We used two independent methods for molecular species analysis: (1) reversed-phase h.p.l.c. separation with in-line beta-radiation detection of peaks, and (2) an argentation-t.l.c. separation with scintillation counting of bands. Our results showed a clearly biphasic sequence in the composition of accumulated DG. The molecular species composition of early DG (up to 1 min stimulation time) was very similar to that of unstimulated DG, whereas the proportions of the species present in later DG were substantially altered. In the same experiments, we extracted native phospholipids from unstimulated macrophages, separated phosphatidylinositol (PI), PC, phosphatidylethanolamine (PE) and phosphatidylserine (PS), converted them to the corresponding DGs by using phospholipase C, and determined their molecular species compositions as above. In comparison with the diradyl compositions of stimulated DG, the diradyl composition of PI closely matched that of early DG, the differences between the PC and PI compositions matched the differences between early and late DG very closely, and the compositions of PE and PS were unique and unrelated. We quantified these relationships more precisely by multilinear regression analysis to calculate the theoretical best mix of five molecular species compositions (PI, PC, PE, PS and unstimulated DG) that would most closely replicate the early and late accumulated DG compositions. We found that by both h.p.l.c. and t.l.c. analyses, 15-30% (PAF) or 25-50% (ionomycin and PMA) of the later DG could be accounted for by PC hydrolysis. These results represent quantifications of phospholipid class contributions to stimulated DG formation, and demonstrate the potential importance of PC hydrolysis in phagocytic leucocytes.

Inhibition of prostaglandin E2-stimulated cAMP accumulation by lipopolysaccharide in murine peritoneal macrophages.

Treatment of murine peritoneal macrophages with 100 nM prostaglandin E2 (PGE2) produced a rapid biphasic increase in intracellular cAMP that was maximal at 1 min and sustained through 20 min. Pretreatment of macrophages with 100 ng/ml of lipopolysaccharide (LPS) for 60 min prior to PGE2 decreased the magnitude of cAMP elevation by 50%, accelerated the decrease of cAMP to basal levels, and abolished the sustained phase of cAMP elevation. The effect of LPS was concentration-dependent, with maximal effect at 10 ng/ml in cells incubated in the presence of 5% fetal calf serum and at 1 microgram/ml in the absence of fetal calf serum. LPS also inhibited cAMP accumulation in cells treated with 100 microM forskolin, but the decrease was about half that seen in cells treated with PGE2. LPS concentrations that inhibited cAMP accumulation produced a 30% increase in soluble low Km cAMP phosphodiesterase activity while having no effect on particulate phosphodiesterase activity. The nonspecific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, as well as the more specific inhibitors rolipram and Ro-20-1724 were effective in inhibiting soluble phosphodiesterase activity in vitro, producing synergistic elevation of cAMP in PGE2-treated cells, and blocking the ability of LPS to inhibit accumulation of cAMP. Separation of the phosphodiesterase isoforms in the soluble fraction by DEAE chromatography indicated that LPS activated a low Km cAMP phosphodiesterase. The enzyme(s) present in this peak could be activated 6-fold by cGMP and were potently inhibited by low micromolar concentrations of Ro-20-1724 and rolipram. Using both membranes from LPS-treated cells and membranes incubated with LPS, no decrease in adenylylcyclase activity could be attributed to LPS. Although effects of LPS on the rate of synthesis of cAMP cannot be excluded, the present evidence is most consistent with a role for phosphodiesterase activation in the inhibitory effects of LPS on cAMP accumulation in murine peritoneal macrophages.

Effects of TNF alpha on the expression of class II MHC molecules in macrophages induced by IFN gamma: evidence for suppression at the level of transcription.

Tumor necrosis factor-alpha (TNF-alpha) induces surface expression of class II major histocompatibility (MHC) molecules (la molecules) in many cells, including macrophage-like cell lines. When we tested the effects of this cytokine on murine peritoneal macrophages, TNF alpha had little effect on surface expression of la. The strong expression of such molecules induced by interferon-gamma (IFN gamma) was, however, suppressed moderately by TNF alpha. These effects were reflected at the level of specific messenger RNA (mRNA) as detected by Northern blot analysis. Furthermore, the locus of control appears to be transcriptional; in nuclear run-on assays, TNF alpha suppressed the IFN gamma-induced enhancement of transcription for the murine beta-chain of I-A (I-A beta.). Taken together the data suggest that TNF alpha has little effect on class II MHC genes and surface expression in murine peritoneal macrophages, that TNF alpha is a modest suppressant of such molecules when their levels are raised by IFN gamma, and that these suppressive effects are mediated at the level of transcription.

Gene regulation in macrophage activation: differential regulation of genes encoding for tumor necrosis factor, interleukin-1, JE, and KC by interferon-gamma and lipopolysaccharide.

Although macrophage activation is induced in a complex manner by signals such as interferon-gamma (IFN gamma) and bacterial lipopolysaccharide (LPS) and depends on alterations in levels of specific proteins due to differences in gene expression, the complexity of gene regulation during macrophage activation in regard to multiple signals is not fully appreciated. To probe this question, we selected four model genes encoding for tumor necrosis factor (TNF), interleukin-1 (IL-1), and the immediate early genes JE and KC. After analyses of Northern blots for specific mRNA, LPS was found to enhance levels of mRNA for IL-1, TNF, JE, and KC. IFN gamma initiated heightened mRNA levels for JE but did not alter specific mRNA for IL-1, TNF, or KC. When IFN gamma and LPS were combined, additive effects on levels of specific mRNA for JE, enhancement of mRNA for TNF, suppressed mRNA for KC, and no effect on mRNA for IL-1 were observed. When transcription of these genes was assessed by nuclear "run on" experiments, LPS increased transcription of KC and TNF but not of IL-1 or JE, implying that the increased levels of mRNA for JE and IL-1 were attributable to increased stability of mRNA. Likewise, IFN gamma did not initiate transcription of JE. When IFN gamma and LPS were given together, IFN gamma enhanced the LPS-induced transcription of TNF and KC, suggesting decreased stability of mRNA for KC. A distinct pattern of regulation for each of the four genes was thus observed. Taken together, the data suggest that gene regulation in macrophage activation represents a complex response of enhanced and suppressed transcription and mRNA stability, the precise pattern of which depends on the stimuli given to the macrophages and the gene examined.

Activation of the cAMP cascade inhibits an early event involved in murine macrophage Ia expression.

The ability of interferon-gamma (IFN gamma) to increase class II major histocompatibility complex (class II MHC) gene products in murine macrophages involves activation of Na+/H+ exchange (Prpic V., Yu, S. F., Figueiredo, F., Hollenbach, P. W., Gawdi, G., Herman, B., Uhing, R. J., and Adams, D. O. (1989) Science 244, 469-471). The ability of IFN gamma to increase class II MHC gene product expression is inhibited by a variety of agents. In the present studies, the involvement of cAMP-dependent protein kinase in modulating IFN gamma-induced expression of MHC gene products and the mechanism of regulation were assessed in macrophages treated with agents which activated cAMP-dependent protein kinase by different molecular mechanisms. Prostaglandin E2 (PGE2) produced a rapid (within 30 s) dose-dependent elevation of cAMP which was paralleled by the activation of cAMP-dependent protein kinase. The elevation of cAMP by PGE2 was still evident at 1 h and maintained through a 4-h incubation. Concentrations of PGE2 which activated the protein kinase produced a dose-dependent inhibition of surface expression of I-A and transcription of class II MHC genes. Inhibition of IFN gamma-induced class II MHC gene product expression was also observed in macrophages treated with agents which activated cAMP-dependent protein kinase by postreceptor mechanisms. Dibutyryl-cAMP (0.01-1 mM), 25 microM forskolin, 0.1 micrograms/ml cholera toxin, and 3-isobutyl-1-methylxanthine (0.1-1 mM) each suppressed IFN gamma-induced cell surface I-A expression, class II MHC gene transcription, and 22Na+ influx. The results are consistent with the suggestion that activation of cAMP-dependent protein kinase regulates an early transductional event initiated by IFN gamma, perhaps Na+/H+ exchange, which is involved in regulating transcription of class II MHC genes and their subsequent expression.

IFN-gamma potentiates the accumulation of diacylglycerol in murine macrophages.

Activation of mononuclear phagocytes for a variety of functional responses is potentiated by prior exposure to IFN-gamma. Inasmuch as protein kinase C has been suggested to mediate several of these responses, we have examined the effects of IFN-gamma exposure on subsequent accumulation of sn-1,2-diacylglycerol (DAG). Exposure of murine macrophages to IFN-gamma (greater than 4 h) results in an increase in basal DAG as well as potentiating DAG accumulation in response to the macrophage chemoattractant, platelet-activating factor (PAF). An increased DAG accumulation was similarly observed in response to PMA and ionomycin. Our results further indicate that the increased DAG accumulation during activation of macrophages is unlikely to involve alterations in phosphatidylinositol metabolism. PAF-stimulated production of [3H]inositol phosphates was not altered by the prior exposure of macrophages to IFN-gamma. Similarly, IFN-gamma did not potentiate the ability of PAF to cause an increase in cytosolic calcium. Our data indicate that phosphatidylcholine metabolism may be involved in IFN-gamma-regulated DAG accumulation. Exposure of [3H]choline-labeled macrophages to IFN-gamma resulted in an increase in the basal level of aqueous [3H]choline metabolites as well as potentiating the production of [3H]choline in response to PAF, PMA, and ionomycin. Our results thus suggest that the potentiated protein kinase C-mediated responses occurring during macrophage activation may be due to potentiated DAG accumulation independent of potentiated phosphatidylinositol metabolism.

Effects of exposure to benzene in vivo on the murine mononuclear phagocyte system.

Exposure to benzene has been shown to decrease resistance to challenges by Listeria monocytogenes or tumor cells in mice. Alterations of T-lymphocytes have been suggested as one probable cause. Macrophages are also critical participants in resistance to Listeria and to tumor cells. We have previously shown that exposure of macrophages in vitro to benzene metabolites, but not to benzene, inhibited several functions of macrophages that are critical for host resistance. The present studies were conducted to determine the effects of exposure to benzene in vivo on the murine mononuclear phagocyte system. Treated animals received daily subcutaneous injections of benzene (800 mg/kg) for 5 days. Macrophages were obtained by lavage from the peritoneal cavity after ip injection of sterile eliciting agents. Enumeration, peroxidase histochemistry, and a series of functional assays were performed. Animals exposed to benzene displayed a decreased number of macrophages elicited by peptone injection. Specific alterations of macrophage functions included a 50% decrease of Fc-receptor-mediated phagocytosis and 70% inhibition of tumor cell cytolysis but an enhancement of TPA-stimulated H2O2 release. There was no effect on interferon-gamma stimulated expression of Ia antigen. The observations that the elicited macrophage populations were composed of newly immigrated cells and that benzene treatment was terminated 3 days before the functional analyses were performed suggest that benzene was affecting monocytes in the blood and precursor cells in the bone marrow. Alterations of macrophage functions in injection controls suggested that determining the primary effect of exposure to benzene may be complicated by the inflammation induced by treatment at the site of injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular mechanisms regulating the expression of class II histocompatibility molecules on macrophages. Effects of inductive and suppressive signals on gene transcription.

The surface expression of class II MHC molecules (immune associated or Ia Ag) is an acquired and important property of macrophages. Recent evidence from several laboratories demonstrates that mRNA levels for class II genes reflect levels of surface expression. We have investigated the effects of agents that regulate Ia expression, either positively as IFN-gamma or negatively as bacterial LPS or maleylated proteins, on transcription of these genes. By using probes for I-A beta and I-E beta as representative class II MHC genes, we found that IFN-gamma induces transcription in murine peritoneal macrophages of I-A beta and of I-E beta as determined in nuclear run-on assays. The measured transcription peaked 6 to 10 h after administration of IFN-gamma to the macrophages. This augmented expression of transcription was markedly suppressed by administration of either LPS or maleylated protein to the macrophages. Collectively, the data indicate that regulation of transcription by either positive or negative stimuli, acting through surface receptors and binding sites, is a major mechanism for controlling the expression of class II MHC molecules in macrophages.

Involvement of protein kinase C in platelet-activating factor-stimulated diacylglycerol accumulation in murine peritoneal macrophages.

Incubation of murine peritoneal macrophages with platelet-activating factor (PAF; 1-O-alkyl(C16 + C18)-2-acetyl-sn-glycerol-3-phosphorylcholine) results in the rapid accumulation of [3H]inositol phosphates and sn-1,2-diacylglycerol (DAG) and mobilization of intracellular calcium (Prpic, V., Uhing, R. J., Weiel, J. E., Jakoi, L., Gawdi, G., Herman, B., and Adams, D. O. (1988) J. Cell Biol. 107, 363-372). We have further investigated the relationship of phosphoinositide metabolism to accumulation of DAG and the possible involvement of protein kinase C in the accumulation of DAG in response to PAF. DAG accumulation proceeds at a slower rate than the accumulation of either [3H] inositol 1,4,5-trisphosphate or total [3H]inositol phosphates. Accumulation of DAG from additional precursors is suggested from both an estimation of the mass of total inositol phosphates produced and the accumulation of [3H]choline in response in PAF. Down-regulation of protein kinase C by prolonged pretreatment with phorbol ester or inhibition of the enzyme with sphingosine inhibited the PAF-generated accumulation of DAG at 10 min by approximately 80%. Under the same conditions, no inhibition of PAF-stimulated generation of [3H]inositol 1,4,5-trisphosphate was observed. Similar inhibition was observed when 10 microM ionomycin or 0.1 microM phorbol 12-myristate 13-acetate were used to stimulate accumulation of DAG. The results suggest that PAF stimulates the accumulation of DAG from source other than phosphatidylinositol metabolism in peritoneal macrophages and that this occurs subsequent to the activation of protein kinase C.