Factors mediating lipopolysaccharide-induced ganglioside expression in murine peritoneal macrophages

The ganglioside composition of endotoxin-responsive C3H/HeN murine peritoneal macrophages is known to undergo dramatic changes in vivo in response to intraperitoneal lipopolysaccharides (LPS), unlike endotoxin-hyporesponsive C3H/HeJ macrophages. To better investigate the mechanism behind LPS-induced macrophage ganglioside changes, resident C3H/HeN peritoneal macrophages were treated in vitro with 0.1-1.0 micrograms/ml LPS for 6-96 hr, but showed no differences in membrane ganglioside patterns. Coincubation of macrophages with lymphocytes and treating with LPS again elicited no ganglioside changes. In contrast, interferon gamma (IFN-gamma)-primed macrophages showed a dramatic shift in intensity of one ganglioside when treated with LPS in vitro; an additional macrophage ganglioside appeared when IFN-gamma-primed, LPS-treated macrophages were coincubated with lymphocytes. Ganglioside expression induced in vitro still did not approach the complex changes seen in vivo. However, transplanting C3H/HeN macrophages intraperitoneally into C3H/HeJ mice, followed by administration of intraperitoneal LPS, did reveal striking changes in ganglioside expression that resembled the pattern seen in vivo. Thus, LPS alone does not provide the necessary direct signal to promote macrophage ganglioside change even though it alters macrophage function. IFN-gamma appears to be one important mediator; however, complex interactions involving other cytokines or migration of independent populations of mononuclear cells may be required for the full manifestation of LPS-induced ganglioside expression in macrophages.     

Human neutrophil-derived CAP37 inhibits lipopolysaccharide-induced activation in murine peritoneal macrophages

Human cationic antimicrobial protein, CAP37, is released from neutrophil granules during infection. CAP37 attracts monocytes, binds Gram-negative endotoxin (lipopolysaccharide, LPS), is bactericidal for a range of Gram-negative bacteria, and reduces mortality in murine polymicrobial sepsis. Here, we report that recombinant CAP37 specifically targets murine peritoneal macrophages. Under steady-state conditions, the bulk of cell-associated CAP37 was localized at the plasma membrane. However, a fraction of CAP37 gained access to the endocytic system, but did not accumulate in recycling endosomes or in the trans-Golgi network (TGN). Instead, CAP37 was internalized by fluid phase endocytosis and accumulated in a prelysosomal compartment. Macrophages that were preexposed to CAP37 exhibited diminished LPS responsiveness, as determined by analysis of c-Jun phosphorylation. Further examination showed that pretreatment with CAP37 reduced the ability of macrophages to bind LPS. Taken together, these observations demonstrate that prolonged exposure to CAP37 desensitizes macrophages to LPS and suggest that this protein plays a novel anti-inflammatory role in polymicrobial sepsis.     

IFN-gamma activates cAMP/PKA/CREB signaling pathway in murine peritoneal macrophages.

Interferon-gamma (IFN-gamma) is a macrophage-activating cytokine that serves critical functions in innate and adaptive immunity and is thought to be mediated by the Jak-Stat signaling pathway. The present study establishes for the first time that cyclic adenosine monophosphate, protein kinase A, and cAMP response element-binding protein (cAMP/PKA/CREB) are coregulators of the IFN-gamma signaling pathway. Experimental data indicate that exogenous IFN-gamma stimulated cAMP accumulation and PKA activation in time-dependent and dose-dependent manners in murine peritoneal macrophages. Moreover, IFN-gamma stimulated CREB phosphorylation and CREB DNA binding, which could be significantly attenuated by PKA inhibition with H89. It appears that a novel cAMP/PKA/CREB signaling pathway is activated by IFN-gamma in macrophages, suggesting that an alternate signaling pathway exists in macrophages in response to IFN-gamma.     

Murine peritoneal macrophages support murine cytomegalovirus replication.

Because there have been different conclusions regarding the susceptibility of murine macrophages to murine cytomegalovirus (MCMV) infection and replication, we have undertaken a detailed comparison of MCMV infection of macrophages with that of a permissive cell line, mouse embryo cells. Although both cell lines undergo productive infections with MCMV, there are marked differences in certain aspects of the viral replication which may account for some of the different conclusions regarding the MCMV cycle in macrophages. Although both cell lines produce MCMV after infection, the time course of the infection differed markedly between the cell types. Similarly, the proportion of infected macrophages that are releasing infection virions is much smaller than the proportion of a comparably infected mouse embryo cell culture. Tissue culture passage of MCMV first enhanced (after one passage) and then reduced the infectivity of the virus for macrophages in vitro. The delayed time course and lesser production at early intervals after infection of macrophage cultures could not be attributed to demonstrable inhibitors or to replication in contaminating fibroblasts in the macrophage cultures.     

Functional alterations of murine peritoneal macrophages during pregnancy.

We have studied some functional characteristics of murine peritoneal macrophages (MOp) related to hormonal changes produced during pregnancy. Maximal expression of Ia antigen and release of interleukin 1 (IL1) were observed during the first week of pregnancy (implantation), when the highest peak of estradiol was produced. Both Ia antigen expression and IL1 levels progressively decreased as gestation advanced. Inversely, MOp capability to phagocyte and reduce nitro blue tetrazolium (NBT) was diminished at the beginning of pregnancy but returned to normal values in the last week. Sexual steroid levels (estradiol and progesterone) were inversely related, and the release of prostaglandin E2 (PGE2) by MOp decreased when progesterone levels increased. Although PGE2 production had no evident relation with Ia antigen expression and IL1 activity during the first and second weeks of pregnancy, the increment in PGE2 values and percentages of NBT+ cells could indicate a different stage of macrophage activation. These results suggest a possible hormonal regulation of macrophage functionality.     

Zinc L-carnosine suppresses inflammatory responses in lipopolysaccharide-induced RAW 264.7 murine macrophages cell line via activation of Nrf2/HO-1 signaling pathway

Context: Zinc L-carnosine (ZnC) is a chelate of Zn and L-carnosine and is used clinically in the treatment of peptic ulcer.

Objective: In this study, we aim to investigate the involvement of heme oxygenase-1 (HO-1) in the anti-inflammatory effects of ZnC in lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages.

Materials and methods: We used immunoblotting analysis to evaluate the involvement of HO-1 in the anti-inflammatory effects of ZnC and the signaling pathway involved was measured using Dual luciferase reporter assay.

Results: Results from immunoblotting analysis demonstrated that pretreatment of cells with ZnC enhanced the expression of HO-1 in RAW 264.7 cells. Pretreatment of cells with HO-1 inhibitor (tin protoporphyrin IX dichloride) significantly attenuated the inhibitory effects of ZnC on nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression and NF-κB activation in LPS-induced RAW 264.7 cells, suggesting that HO-1 play an important role in the suppression of inflammatory responses induced by ZnC. Furthermore, results from co-immunoprecipitation of Nrf2 and Keap1 and dual luciferase reporter assay showed that pretreatment of ZnC was able to activate the Nrf2 signaling pathway. Treatment of cells with p38 inhibitor (SB203580), c-Jun N-terminal kinase inhibitor (SP600125), and MEK 1/2 inhibitor (U0126) did not significantly suppress the induction of HO-1 by ZnC. Moreover, our present findings suggest that the effects of ZnC on NO production, HO-1 expression, and Nrf2 activation were attributed to its Zn subcomponent, but not l-carnosine.

Conclusion: Pretreatment with ZnC was able to activate Nrf2/HO-1 signaling pathway, thus suppressing the expression of inflammatory mediators, such as NO and iNOS in LPS-induced RAW 264.7 cells.     

脂多糖诱导的小鼠腹腔巨噬细胞基因表达谱分析

目的　利用基因芯片技术分析脂多糖 (LPS)活化的小鼠腹腔巨噬细胞基因表达谱 ,以更全面地了解LPS诱导的巨噬细胞反应。方法　以未刺激的和用 1mg/LLPS刺激的小鼠腹腔巨噬细胞制备3 3 P标记的cDNA探针 ,分别与含有 1176个已知基因的小鼠cDNA芯片杂交。结果　活化组和未刺激组间的 2倍差异表达基因为 118个 ,3倍差异表达基因为 6 9个 ,其中 4 4个上调 ,2 5个下调。转录因子、细胞内信号调节蛋白、炎症细胞因子和细胞凋亡相关基因的转录发生明显的调节变化。结论提供了LPS活化的巨噬细胞综合基因表达信息 ,并筛选出一些新的可能与LPS活化相关的基因。     

Trypanosoma cruzi-induced tyrosine phosphorylation in murine peritoneal macrophages

 Trypanosoma cruzi, the etiological agent of Chagas’disease, binds to and invades macrophages and other cells (fibroblasts, muscle cells) via a complicated set of interactions, but the changes induced by parasite-to-cell interactions are largely unknown. This report investigates the ability of T. cruzi to elicit a tyrosine kinase pathway in immature and mature resident murine peritoneal macrophages (MPM) that differ in their susceptibility to parasite infection. T. cruzi stimulated the phosphorylation of tyrosine residues in several endogenous substrates (proteins of 40–42, 53–56, 66, 75, 80, 90, 95, 100, and 112 kDa), but only in immature MPM. Mature MPM had high levels of spontaneous tyrosine phosphorylation. Upstream tyrosine kinases, such as src-like tyrosine kinases, were not responsible for the differential patterns of tyrosine phosphorylation since they were present in both mature and immature MPM. We suggest that the tyrosinephosphorylation patterns stimulated by T. cruzi reflect most of the biochemical events that occur in parasite host-cell interactions. Received: 20 October 1995 / Accepted: 23 January 1996     

African trypanosomiasis alters prostaglandin production by murine peritoneal macrophages.

Sera from 39 patients with systemic sclerosis were examined for a cytotoxic effect on human umbilical vein endothelium. Although none of the sera produced direct cytotoxicity of 51Cr-labelled endothelial cells, even with added complement, nine sera did produce increased 51Cr release when co-cultured with endothelial cells and normal human peripheral blood mononuclear cells. The effector cells involved in this cytotoxicity possessed Fc receptors but were non-T and non-adherent while the responsible serum factor(s) was present in IgG containing fractions. This cytotoxicity tended to occur in patients with both circulating immune complexes and precipitating antibodies to nuclear and cytoplasmic antigens who, as a group, had more severe and extensive visceral disease than those without such serological abnormalities. Control studies using sera from both 27 normal controls and 19 patients with either diabetes or extensive athero-sclerotic vascular disease failed to reveal any similar cytotoxicity.     

Activation of murine peritoneal macrophages by Salmonella typhimurium

The results of these studies indicate that the interaction between activated macrophages and S. typhimurium depends on the characteristics of the micro-organism and the kind of activation.     

The effects of malnutrition on murine peritoneal macrophages

Differences were detected between peritoneal macrophages (both resident and elicited) from mice on a low protein diet and from normal animals. The concentration of resident peritoneal macrophages was lower in animals on low protein diets than in normal controls. Although total protein (and therefore cell mass) of resident macrophages from malnourished mice was increased, their contents of thiamine pyrophosphatase, succinate dehydrogenase, and non-specific esterase were disproportionately reduced. In addition they did not ingest as many glutaraldehyde-fixed sheep erythrocytes or attach to as many adherent C3b sensitized sheep red blood cells as those from normal animals, although reduction of nitroblue tetrazolium was unaffected. Initially (24 hr after thioglycollate), elicited macrophages from malnourished mice did not divide as frequently as those from normal mice but by 48 hr the differences were insignificant. The elicited macrophage possessed lower levels of total protein (indicating a reduced cell mass); the levels of acid phosphatase, thiamine pyrophosphatase, succinate dehydrogenase, and nonspecific esterase and nitroblue reducing activity were also proportionately reduced. They ingested fewer glutaraldehyde-fixed erythrocytes and reacted with fewer C3b sensitised sheep red blood cells than those from normal mice; ingestion of IgG-coated sheep erythrocytes, on the other hand, was somewhat increased. These abnormalities may influence adversely the efficiency of early phlogistic responses and favor the establishment of infection in malnourished animals.     

Ceroid accumulation by murine peritoneal macrophages exposed to artificial lipoproteins.

Murine resident peritoneal macrophages were maintained in cell culture in a medium containing 10% lipoprotein-deficient fetal calf serum to which various artificial lipoproteins (lipid-bovine serum albumin complexes) had been added. Ceroid accumulated in cells exposed to artificial lipoproteins containing cholesteryl esters or acylglycerols possessing polyunsaturated fatty acid residues, but not in cells exposed to lipoproteins containing less readily oxidized lipids. Oxidation of cholesteryl linoleate before its incorporation into artificial lipoprotein accelerated ceroid production. Incorporation of free radical scavengers into cholesteryl linoleate-containing artificial lipoproteins impaired ceroid formation. The results are discussed in terms of the mechanisms by which the ceroid might have been produced and its significance for human atherogenesis.     

Immunomodulation by Blastomyces dermatitidis: functional activity of murine peritoneal macrophages.

Cell-mediated immunity plays the dominant role in the immune response of mice to Blastomyces dermatitidis infections. Since macrophages play an important role in cell-mediated immunity, the interactions between sensitized murine peritoneal macrophages and the yeast phase of B. dermatitidis were investigated. Scanning electron microscopy showed that the sensitized macrophages readily phagocytized B. dermatitidis yeast cells. In addition, there appeared to be activation of metabolic pathways within the sensitized macrophages, as indicated by increased chemiluminescence activity during phagocytosis. Sensitized macrophages were significantly better at controlling intracellular proliferation of the yeast cells when compared to nonsensitized cells. This was determined by disruption of macrophages and plating for viable yeasts. Scanning electron microscope observations offered further substantiation. Experiments with Candida albicans indicated that B. dermatitidis non-specifically activated macrophages. At 2 h postphagocytosis, 30% fewer C. albicans in B. dermatitidis-activated macrophages were able to form germ tubes. These studies demonstrated the multiple potential of activated macrophages with regard to their functional activity.     

Transient expression of virus-specific promoters in murine resident peritoneal macrophages

Monocyte-macrophages (MO), being non-permissive for most viruses, play an important role in resistance to virus infection. In order to establish the mechanism of abortive infection of murine resident peritoneal MO (ResPMO) by herpes simplex virus type 1 (HSV-1), it is desirable to transfect these cells with viral promoters linked to an assayable gene, for example, the bacterial chloramphenicol acetyl transferase (CAT) gene. This will facilitate studies designed to measure levels of promoter activation or repression in these specialized cells. Transient expression of CAT in ResPMO was achieved with DEAE-dextran, but not using either calcium phosphate precipitate or lipofectin. CAT expression driven by various virus-specific promoters was less efficient in ResPMO compared with Vero cells and approximately 50% of input plasmid DNA remained in Vero cells at 48 h post transfection, but only 9% was detectable in ResPMO. However, approximately 6% of ResPMO and 9% of Vero cells contained CAT-specific DNA at 24 h post transfection. In addition, 2% of cells of either cell type contained CAT-specific polypeptide at 48 h. This is therefore the first report that the non-replicating murine ResPMO can be transfected in vitro and more importantly, that these cells express the transfected gene products.     

Time course of antilisterial activity by immunologically activated murine peritoneal macrophages.

Murine peritoneal macrophages were rapidly rendered listericidal after exposure to lymphokine-rich supernatants (LRSs) derived from antigen-pulsed Listeria monocytogenes-immune spleen cells. A 6-h incubation period with LRSs was sufficient to induce microbicidal activity in resident macrophages. In vitro induction of macrophage listericidal activity by constant exposure to LRSs persisted for 18 h, after which time spleen cell factors were no longer capable of modifying intracellular inactivation of Listeria. Results obtained by utilizing a short assay indicated that the killing kinetics is extremely rapid, with large numbers of bacteria destroyed during the first 15 min of infection. Intracellular killing at this time appeared to be greatly dependent upon the stage of growth from which the microorganisms were harvested. Induction of bactericidal macrophages by infection of mice with a sublethal dose of virulent Listeria cells and subsequent intraperitoneal elicitation with heat-killed homologous bacteria was similarly a transient event. Macrophages harvested 18 h after antigenic challenge displayed dramatic antibacterial activity during the first 22 h in culture. After 22 h, activity was lost, and stasis was observed during the ensuing 23 h. At 68 h, macrophages were devoid of antilisterial action. Activity, however, could be recalled after incubation with LRSs.     

Protein phosphorylation in murine peritoneal macrophages induced by infection with Salmonella species.

Infection of peritoneal macrophages from C3H/HeN and C3H/HeJ mice with Salmonella typhimurium or S. enteritidis induced extensive phosphorylation in a set of proteins with molecular masses of 85, 72, 35, 30, and 23 kDa, which were different from those induced by bacterial lipopolysaccharide. The phosphorylated proteins of 35, 30, and 23 kDa (pp35, pp30, and pp23, respectively) originated from the infecting bacteria, because living bacteria could induce these phosphorylated proteins themselves, and no induction of the proteins occurred in macrophages after phagocytosis of heat-killed or UV-irradiated organisms. When the infected macrophages were disrupted and separated into bacterial and macrophage debris fractions, pp85 and pp72 remained in the macrophage debris fraction, with none in the bacterial fraction. Induction of pp85 and pp72 in infected macrophages was inhibited in the presence of chloramphenicol but not cytochalasin D, suggesting that bacterial growth in the macrophages is necessary for induction of both proteins. Neither of these proteins could be detected in macrophages infected with Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, or Listeria monocytogenes. These results support the view that phosphorylation of the 85- and 72-kDa proteins occurs in the macrophages during the early phases of the interaction between Salmonella organisms and macrophages. The functions of specific proteins remain to be clarified.