In vivo production of heat shock protein in mouse peritoneal macrophages by administration of lipopolysaccharide.

The in vivo production of heat shock protein was studied by administration of bacterial lipopolysaccharide (LPS) into mice. Heat shock protein 70 was detected in the extract of adherent peritoneal cells from mice injected intraperitoneally with LPS by using the immunoblotting method. The expression of heat shock protein 70 was found 2 days after injection of LPS and reached its peak 4 days after injection. The intraperitoneal injection of LPS induced the expression of heat shock protein 70, whereas its subcutaneous injection did not. The in vivo production of heat shock protein 70 was inhibited by administration of LPS together with quercetin, an inhibitor of accumulation of heat shock protein 70 mRNA. Tumor necrosis factor alpha enhanced LPS-induced heat shock protein production in vivo. There was a decrease of gamma delta T cells in the peritoneal cavity of mice injected intraperitoneally with LPS. It was suggested that bacterial LPS is a stressful agent which induces the in vivo heat shock protein response, and its administration leads to the production of heat shock protein 70 in peritoneal macrophages.     

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.     

Differential protein synthesis by murine peritoneal macrophages elicited by various stimuli

Protein synthetic patterns of murine peritoneal macrophages were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) of 35S methionine-labeled proteins. While the protein synthetic patterns exhibited by resident, inflammatory, and activated macrophages had numerous common features that distinguished them from the other normal non-macrophage cell types examined, unique proteins also characterized each macrophage population from the others. The accumulation by resident macrophages of proteins 23, 25, and 37 distinguished them from elicited cells, as did the former's more abundant synthesis of proteins 54 and 52. The protein synthetic patterns of inflammatory thioglycollate- and proteose peptone-elicited macrophages were strikingly similar, save for the former's greater levels of accumulation of proteins 14 and 28, and the latter's more pronounced expression of p23.5. Peritoneal macrophages elicited by treatment with heat-killed Propionibacterium acnes, the live, attenuated Mycobacterium bovis strain BCG, Listeria monocytogenes, and the protozoan flagellate Trypanosoma rhodesiense, all exhibited tumoricidal activity in 16-h or 72-h functional assays. They shared a common protein synthetic profile that differentiated them from the synthetic patterns characteristic of the non-tumoricidal resident and inflammatory macrophages. These tumoricidal macrophages were unique in synthesizing a protein(s) of approximate molecular weight 26,000 daltons. A time-course study employing P. acnes-activated peritoneal macrophages indicated that p26 accumulation decayed with tumoricidal capacity as a function of time in culture, although no direct correlation between lytic activity and p26 expression could be definitively established. Peritoneal macrophages elicited with proteose peptone were not directly tumoricidal but were rendered so upon in vitro treatment with nanogram amounts of bacterial lipopolysaccharide. The accumulation of low levels of p26 by the newly explanted proteose peptone-elicited macrophages suggests the possibility that this protein characterizes macrophage populations primed as well as triggered for tumoricidal activity.     

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.     

Induction of interferon synthesis and cytotoxicity by murine peritoneal macrophages exposed to glycoprotein ligands.

Thioglycollate-induced murine C57BL/6 and C3H/HeN peritoneal macrophages synthesized interferon-beta (IFN-beta) in response to exposure to glycoproteins such as horseradish peroxidase (HRP) or mannosyl or fucosyl bovine serum albumin (BSAman of BSAfuc, respectively), but not glucosylated or galactosylated BSA (BSAglu or BSAgal, respectively). These results suggest participation of the mannosyl-fucosyl receptor (MFR) in this response. IFN synthesis was augmented by culturing macrophages in L cell-conditioned medium prior to exposure to these substances. Macrophages obtained from lipopolysaccharide (LPS)-resistant C3H/HeJ mice did not produce IFN in response to HRP. Furthermore, IFN-induction by HRP was blocked by polymyxin B. In addition, exposure of macrophages to HRP or BSAman induced cytotoxicity against NIH 3T12 cells. Cytotoxicity was not inhibited by the presence of anti-IFN-alpha/beta. In contrast to IFN induction, however, macrophages activation was LPS-independent, since this activity was demonstrated in macrophages from C3H/Hej mice. The carbohydrate specificity of these responses suggests that the MFR or an another scavenger receptor may be involved in the responses to these substances, and that cytotoxicity and IFN-induction by glycoproteins follow unique pathways.     

Kinetics of the phenotype and function of murine peritoneal macrophages following acute inflammation

This study was undertaken to have a better understand for the process and the underlying mechanisms to limitmacrophage activation and population of activated macrophages.A comprehensive kinetics of cytokineproduction was performed in murine peritoneal macrophages recovered from Balb/c mice at various timeduring the course of an intraperitoneal injection with thioglycollate (TG).The expression of cell surfacemolecules such as MHC-Ⅰ,MHC-Ⅱ,B7-1 and B7-2 of these macrophages were also determined by flowcytometry.The present findings of our research suggested that the population of activated macrophages and theactivation of macrophages (including cytokines production and expression of cell surface functional molecules)were strictly controlled during inflammation process.This is one of the important mechanisms to retain the hosthomeostasis.Cellular & Molecular Immunology.2004;1(1):57-62.     

Modification of the surface structure of murine peritoneal macrophages following chemotherapy. A scanning electron microscopic study.

The effects on peritoneal macrophages of some chemotherapeutic agents administered subcutaneously to conventional male AB/Jena mice were studied. beta-[1-ethyl-(5-bis-(beta-chloroethyl)-aminobenzimidazolyl-(2)]-DL-alanine (IMET 2925), beta-[1-phenyl-5-bis-(beta-chloroethyl)-amino-benzimidazolyl-(2)]-DL-alanine (IMET 3164), and gamma-[1-phenyl-5-bis-(beta-chloroethyl)-amino-benzimidazolyl-(2)]-butyric acid (IMET 37/70) evoked a pronounced variation of the cell surface consisting in a shortening, coarsening and reduction in number of the microvili and other projections. Likewise, 1.3-bis(piperidinomethyl)-5-ethyl-5-phenyl-barbituric acid induced a flattening of the surface, but the structures appeared to be increased in number per area. Chlorambucil, 6-mercaptopurine, and 5-fluoro-uracil failed to provoke any obvious change in the threedimensional cell surface structure. These morphological findings are consistent with functional results reported previously.     

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.     

Spontaneous monokine release by alveolar macrophages in chronic sarcoidosis.

In pulmonary sarcoidosis an activation of alveolar T lymphocytes and alveolar macrophages (AM) has been demonstrated. There is evidence that in contrast to acute disease a heightened T-cell response cannot be observed in the chronic phase of sarcoidosis. The role of AM in the inflammatory process of chronic sarcoidosis is not yet intensively evaluated. To address this question we measured the release of tumor necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1) by AM of 39 patients with chronic sarcoidosis (duration greater than 4 years; 30 active, 9 inactive diseases) without therapy and correlated the monokine release with parameters of T-cell alveolitis and the course of the disease. The T4/T8 ratio was higher in the active than in the inactive group without reaching statistical significance. TNF alpha as well as IL-1 is spontaneously released by AM of the active group 2,099 +/- 518 pg/ml TNF alpha/10(6) cells/24 h and 8/13 (IL-1+/total) respectively. In the inactive group the AM release 375 +/- 246 pg/ml TNF alpha/10(6) cells/24 h which is in the range of the control and 1 out of 5 patients was IL-1-positive. There was no correlation between the monokine release and any parameter of T-cell alveolitis. These data support the hypothesis that the inflammatory process in chronic sarcoidosis is dominated by the activity of AM and that this activity determines the course of the disease.     

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.     

Mycobacterial 65-kilodalton heat shock protein induces tumor necrosis factor alpha and interleukin 6, reactive nitrogen intermediates, and toxoplasmastatic activity in murine peritoneal macrophages.

The 65-kDa heat shock protein (Hsp65) is supposed to play a role in host defense against infections with various microbial pathogens and in autoimmune inflammatory disorders. These effects are thought to result mainly from an Hsp65-specific T-lymphocyte-mediated immune response that recognizes conserved epitopes. The aim of the present study was to assess whether mycobacterial Hsp65 has a direct effect on resident murine peritoneal macrophages, independent of Hsp65-sensitized T lymphocytes. Exposure of peritoneal macrophages from naive C57BL/6 mice to the mycobacterial Hsp65 in vitro induced an enhanced release of tumor necrosis factor alpha (TNF-alpha) and interleukin 6. These cells also produced large amounts of reactive nitrogen intermediates (RNI) and inhibited the intracellular proliferation of Toxoplasma gondii. Small amounts of gamma interferon acted synergistically with Hsp65. Thus, exposure of murine macrophages to Hsp65 results in activation of these cells. The acquisition of these characteristics by peritoneal macrophages occurred in the absence of sensitized T lymphocytes. Addition of anti-TNF-alpha antiserum resulted in an attenuation of the Hsp65-induced release of RNI and toxoplasmastatic activity, indicating that endogenous TNF-alpha is involved in the Hsp65-induced macrophage activation. The conclusion of this study is that in vitro exposure of peritoneal macrophages to the mycobacterial Hsp65 induces the release of proinflammatory cytokines and RNI and results in inhibition of the intracellular proliferation of T. gondii. These effects on murine macrophages occur independently of Hsp65-specific T lymphocytes. The proinflammatory effect of Hsp65 demonstrated in this study suggests that this heat shock protein may play a role in the initiation of inflammation that adds to a non-species-specific resistance in the early stages of infections.     

Murine peritoneal macrophages activated by the mycobacterial 65-kilodalton heat shock protein express enhanced microbicidal activity in vitro.

After an intraperitoneal (i.p.) injection of purified protein derivative, peritoneal macrophages from mice infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) show an enhanced respiratory burst, inhibit the intracellular proliferation of Toxoplasma gondii, and kill Listeria monocytogenes more efficiently than peritoneal macrophages from normal mice. One of the immunodominant antigens of Mycobacterium spp. is the 65-kDa heat shock protein (Hsp 65), and in the present study, we determined whether injection of this protein into mice leads to activation of their peritoneal macrophages. After an i.p. injection of Hsp 65, peritoneal macrophages from BCG-infected CBA/J mice also released more H2O2, inhibited the proliferation of T. gondii, and killed L. monocytogenes faster than peritoneal macrophages from normal mice, although Hsp 65 was less effective than purified protein derivative. When normal mice were injected with Hsp 65 suspended in saline after a booster injection with Hsp 65, their macrophages did not display enhanced antimicrobial activity, indicating that an adjuvant was required for a cellular immune response against Hsp 65. In the present study, the adjuvant dimethyl dioctadecylammonium bromide (DDA) was preferred because it contains no endotoxin or mycobacterial antigens and because it has been reported that DDA does not induce the production of gamma interferon. Peritoneal macrophages from C57BL/6 and CBA/J mice that had received a subcutaneous injection of Hsp 65 suspended in DDA followed by an i.p. booster injection of Hsp 65 suspended in saline were activated, as indicated by the enhanced production of H2O2, inhibition of the intracellular proliferation of T. gondii, and increased rate of intracellular killing of L. monocytogenes in vitro relative to that by resident peritoneal macrophages and peritoneal macrophages obtained from mice that had received ovalbumin instead of Hsp 65. The rate of phagocytosis of L. monocytogenes was not affected by Hsp 65 treatment. Despite the in vitro expression of enhanced microbicidal activity of peritoneal macrophages, no difference in the growth of L. monocytogenes in the liver and spleen between Hsp 65-treated and control mice was found.     

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.     

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.     

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.