Cultured Mouse Peritoneal Macrophages Research Articles

Modulations of functional activity in differentiated macrophages are accompanied by early and transient increase or decrease in c-fos gene transcription.

Marked changes in c-fos proto-oncogene mRNA level and transcription rate were observed upon modulation of the functional activity of cultured mouse peritoneal macrophages. Cholera toxin (CT), dexamethasone (dex), interferon-gamma (IFN-gamma), concanavalin A (Con A), and endotoxin (LPS) induced changes in mRNA levels and transcription rates of both urokinase-type plasminogen activator and tumor necrosis factor/cachectin genes, the products of which are sensitive indices of macrophage activity. All of these agents also caused rapid and transient changes in c-fos gene expression, either enhancement (CT, dex, and LPS) or decrease (IFN-gamma and Con A). Moreover, inhibition of protein synthesis elicited a transient increase in the level of c-fos gene transcription, suggesting that the transcriptional activity of the c-fos gene is controlled by labile protein repressor(s). Taken together, these results suggest a possible role for the c-fos gene product, a nuclear protein, in the modulation of the functional activity of differentiated macrophages.

Degradation and regurgitation of extracellular proteins by cultured mouse peritoneal macrophages and baby hamster kidney fibroblasts. Kinetic evidence that the transfer of proteins to lysosomes is not irreversible.

The uptake and degradation of bovine serum albumin (BSA), bovine liver catalase, and rabbit muscle enolase have been studied in cultured mouse peritoneal macrophages (MPM) and baby hamster kidney fibroblasts (BHK cells). Rates constant for the uptake of the three proteins by MPM were similar. In addition, BSA accumulation was independent of BSA concentration in the uptake medium and was not inhibited by a large excess of serum, suggesting that protein accumulation was by fluid phase pinocytosis. Following an overnight uptake, 20-30% of the accumulated protein was subsequently regurgitated into the medium in a trichloroacetic acid/phosphotungstic acid-precipitable form. This material co-migrated with the authentic protein during molecular sieve chromatography on Sephadex G-50. The rates of appearance of trichloroacetic acid/phosphotungstic acid-insoluble products were greater than expected for cell death and leakage. The observed first order rate constants, kobs, for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products in the culture medium were identical, indicating that both products were released in parallel from MPM and BHK cells. The kobs for intracellular BSA degradation and regurgitation were independent of the initial BSA concentration in the uptake medium, but were decreased about 35% when degradation was allowed to proceed in the presence of high concentrations of serum. Degradation was also inhibited by chloroquine and pepstatin. Inhibition of degradation was accompanied by an increase in the total amount of regurgitated protein appearing in the medium. Remarkably, however, these inhibitors also decreased kobs for regurgitation, thereby preserving the similarity in the observed rate constants for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products. These and other results were inconsistent with desorption of proteins from the surface of the culture dish or the surface of cells as the source of the trichloroacetic acid/phosphotungstic acid-insoluble label appearing in the medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Silver affects viability and structure of cultured mouse peritoneal macrophages and peroxidative capacity of whole mouse liver.

The effects of silver on cultured mouse peritoneal macrophages were examined by estimation of cell survival and by light and electron microscopy. Addition of silver lactate to the culture medium at final concentrations of 40 and 80 microM resulted in coagulation necrosis and rapid cell death. At lower concentrations cell structure appeared normal. However, the rate of cell death at 20 microM silver lactate was increased as compared to controls. Silver, visualized by physical development/autometallography, was invariably located in lysosomes. The production of malondialdehyde in mouse liver of silver-treated mice as compared to controls was also examined. This lipid peroxidation product had increased to the same amount in animals treated with silver for either 3 days or with only one silver injection 4 h before examination. This study has demonstrated that silver affects viability and structure of cultured macrophages, possibly due to induction of lipid peroxidation, as demonstrated to occur in the liver of silver-exposed mice.

Transport of beta-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway.

The receptor-mediated uptake of rat hypercholesterolemic very low density lipoproteins (beta VLDL) and rat chylomicron remnants was studied in monolayer cultures of the J774 and P388D1 macrophage cell lines and in primary cultures of mouse peritoneal macrophages. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was reduced 80-90% in the presence of high concentrations of unlabeled human low density lipoproteins (LDL). Human acetyl-LDL did not significantly compete at any concentration tested. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was also competitively inhibited by specific polyclonal antibodies directed against the estrogen-induced LDL receptor of rat liver. Incubation in the presence of anti-LDL receptor IgG, but not nonimmune IgG, reduced specific uptake greater than 80%. Anti-LDL receptor IgG, 125I-beta VLDL, and 125I-chylomicron remnants bound to two protein components of apparent molecular weights 125,000 and 111,000 on nitrocellulose blots of detergent-solubilized macrophage membranes. Between 70-90% of 125I-lipoprotein binding was confined to the 125,000-Da peptide. Binding of 125I-beta VLDL and 125I-chylomicron remnants to these proteins was competitively inhibited by anti-LDL receptor antibodies. Comparison of anti-LDL receptor IgG immunoblot profiles of detergent-solubilized membranes from mouse macrophages, fibroblasts, and liver, and normal and estrogen-induced rat liver demonstrated that the immunoreactive LDL receptor of mouse cells is of a lower molecular weight than that of rat liver. Incubation of J774 cells with 1.0 micrograms of 25-hydroxycholesterol/ml plus 20 micrograms of cholesterol/ml for 48 h decreased 125I-beta VLDL uptake and immuno- and ligand blotting to the 125,000- and 111,000-Da peptides by only 25%. Taken together, these data demonstrate that uptake of beta VLDL and chylomicron remnants by macrophages is mediated by an LDL receptor that is immunologically related to the LDL receptor of rat liver.

Regulation of lipoprotein lipase secretion by mouse peritoneal macrophages

Resident mouse peritoneal macrophages in culture spontaneously secrete lipoprotein lipase. Secretion of lipoprotein lipase was 10-fold greater in thioglycollate-elicited and 6-fold greater in mineral oil-elicited macrophages. The increase in enzyme secretion was dependent on protein synthesis and glycosylation of the newly synthesized enzyme. The addition of 1 to 500 ng per ml of lipopolysaccharide to the culture medium resulted in a dose-dependent inhibition of lipoprotein lipase secretion. Thus, secretion of macrophage lipoprotein lipase is a regulated process, and may be increased as part of the response to inflammation. This may have important implications in the atherogenic process in the arterial wall.

マクロファージのプラスミノーゲン・アクチベーター分泌に及ぼすリンフォカインの刺激効果

In our previous study, we have demonstrated that plasminogen activators (PAs) with Mr=24, 000 and Mr=48, 000 play an important role in tissue fibrinolysis during the development of hypersensitivity granulomas. In order to investigate the cellular source of these PAs, we cultured mouse peritoneal macrophages in different conditions. C57BL/6N mice were used with or without infection by Mycobacterium lepraemurium. Antigen stimulated peritoneal exudate macrophages were obtained by intraperitoneal injection of sonicated M. lepraemurium to the infected mice. PA activity in conditioned media was assayed by using two-stage colorimetric method and electrophoretic enzymography. Resident peritoneal macrophages secreted PA with Mr=48, 000. However antigen stimulated peritoneal exudate macrophages secreted a high activity of PA with Mr=24, 000 in addition to Mr=48, 000. In order to investigate secretion mechanism, in vitro stimulation of macrophages was carried out by lymphokines which were obtained from culture supernatants of splenic cells exposed with antigen or splenic T cells with Concanavalin A. The results clearly demonstrated that macrophages became secretable of PA with Mr=24, 000 by lymphokine stimulation. Further experiments showed that the effect of lymphokine to induce secretion of PA was partially represented by γ-Interferon. These data indicated that the immunological stimulation induced macrophages to secrete PA, the molecular weight of which was altered from resident macrophages.

Regulation of the secretion of lipoprotein lipase by mouse macrophages

The regulation of the secretion of lipoprotein lipase was studied in primary cultures of mouse peritoneal macrophages and in the murine macrophage cell line J774. As previously reported, both cell types secrete a lipase with the characteristics of lipoprotein lipase. Incubation of macrophages with insulin, insulin-like growth factor, and l-thyroxine had no effect on lipoprotein lipase secretion. Incubation with dexamethasone and with several agents which increase intracellular cyclic AMP led to a decrease in lipoprotein lipase secretion by mouse peritoneal macrophages. These results suggest that the hormonal regulation of lipoprotein lipase in macrophages is different from that in adipose tissue and heart muscle. Incubation of the macrophages with heparin caused a marked increase in the secretion of lipoprotein lipase. Short incubations with heparin (5 min) caused a release of the enzyme into the media, while longer incubations caused a 2–8-fold increase in net lipoprotein lipase secretion which was maximal after 2–16 h depending on cell type, and persisted for 24 h. The effect of heparin was dose-dependent and specific (it was not duplicated by other glycosaminoglycans). The mechanism of heparin-induced increase in lipoprotein lipase secretion was explored. The increase was not caused by the release of a presynthesized intracellular pool of lipoprotein lipase or by the stabilization of lipoprotein lipase by heparin after secretion. The heparin-induced increase in lipoprotein lipase secretion was dependent on protein synthesis. The secretion of lipoprotein lipase by macrophages in response to low levels of heparin may be a significant factor in the formation of atherosclerotic lesions.

Accumulation of cholesteryl esters and triglycerides in cultured macrophages incubated with plasma very low density lipoproteins from rats fed on casein or soybean protein diets containing moderate levels of cholesterol

Even when administered at a comparatively low level, dietary cholesterol produces significant changes in the properties of plasma lipoproteins in rats, particularly the d < 1.006 g/ml fraction (VLDL). The occurrence of these changes is promoted by dietary casein. To test the hypothesis that these dietary-induced perturbations might include properties influencing lipoprotein-cell interactions of relevance to atherogenesis, cultured mouse peritoneal macrophages were incubated with VLDL isolated from male rats fed on diets containing either 0, 0.25 or 0.5% cholesterol with casein or soybean protein, respectively, as the sole source of protein. No increase in cholesteryl ester content, and a comparatively small rise in triglyceride content, was observed in macrophages incubated with VLDL from rats fed on cholesterol-free diets. In contrast, a significant and apparently saturable cellular accumulation of cholesteryl esters as well as triglycerides was produced by VLDL from cholesterol-fed rats. The curves showing cellular lipid accumulation versus VLDL-protein (or VLDL-cholesterol) content in the cell medium indicated different cellular affinity for VLDL from casein-fed rats in comparison with VLDL from soybean protein-fed rats. The apoprotein composition of VLDL differed between groups of rats fed on different types of dietary protein with higher proportions of apo C's in the casein-fed rats. In addition, cholesterol feeding resulted in increased proportions of apo A-I and apo A-IV in the plasma VLDL fraction.

Effect of gamma interferon on hydrogen peroxide production by cultured mouse peritoneal macrophages.

Macrophage activation is thought to be mediated via a number of T-lymphocyte products, including gamma interferon (IFN-gamma). However, our studies indicate that IFN-gamma acts as a regulator molecule rather than solely as an activator. This depends upon the status of the macrophage. IFN-gamma treatment of resting macrophages and those activated or elicited by sodium caseinate, lipopolysaccharide, or Mycobacterium bovis BCG did not result in activation, as measured by hydrogen peroxide production; however, when thioglycolate was used as an eliciting agent, incubation with IFN-gamma resulted in a dramatic increase in hydrogen peroxide production compared with that by untreated controls.

Effects of activation on lipoprotein lipase secretion by macrophages. Evidence for autoregulation.

Lipoprotein lipase (LPL) activity was measured in the media of cultured mouse peritoneal macrophages that were isolated after the intraperitoneal injection of inflammatory agents in order to yield a variety of states of activation. Fully activated macrophages obtained from Corynebacterium parvum-injected mice secreted very low levels of LPL when compared to unstimulated macrophages, while inflammatory and primed macrophages had increased LPL secretion. When inflammatory macrophages were incubated with conditioned medium obtained from fully activated macrophages, LPL secretion decreased in a time- and dose-dependent fashion. The factor(s) secreted by fully activated macrophages that inhibited LPL secretion was shown to be thermolabile and distinct from tumor necrosis factor. These results demonstrate that activation dramatically alters macrophage LPL secretion.

Effect of proteolysis of low-density serum lipoproteins on their interaction with macrophages

125I-labelled human serum low density lipoproteins (LDL) were incubated with cultured mouse peritoneal macrophages at 37 degrees C, with the following study of cellular uptake and 125I-LDL degradation by measuring the content of TCA-soluble products of LDL hydrolysis in the cultural medium. It was shown that limited pepsin proteolysis of LDL (10%) led to a more effective LDL uptake and degradation by macrophages. The data suggest that enzyme-induced modification of LDL may increase their atherogenicity.

Some effects of lignocaine on cultured mouse peritoneal macrophages—II

Previous studies have shown that pretreatment of macrophages with lignocaine modifies the pattern of enzyme release when the cells are exposed to toxic particulates. Incorporation of lignocaine in the lavage medium used to enhance the yield of peritoneal macrophages does not significantly affect the outcome of cytotoxicity assays of particulates, provided calf serum is present in the medium and the cells are cultured for 3–24 hr prior to the particulate exposure.

Biosynthesis of component C1q of complement in mouse peritoneal macrophage cultures

Biosynthesis of component C1q of complement in mouse peritoneal macrophage cultures

Regulation of macrophage eicosanoid production by hydroperoxy-and hydroxy-eicosatetraenoic acids.

Resident mouse peritoneal macrophages when exposed to zymosan during the first day of cell culture synthesize and secrete large amounts of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), the respective products of cyclo-oxygenase- and 5-lipoxygenase-catalysed oxygenations of arachidonic acid. Under these conditions of cell stimulation only small amounts of hydroxyeicosatetraenoic acids (HETEs) are concomitantly produced. However, exogenously added arachidonic acid is metabolized to large amounts of 12- and 15-HETE and only relatively small amounts of PGE2. No LTC4 is formed under these conditions. In contrast, resident mouse peritoneal macrophages in cell culture for 4 days synthesized less PGE2 and LTC4 when exposed to zymosan. However, these macrophage populations continue to synthesize 12-HETE from exogenously added arachidonic acid. Zymosan induced the secretion of a lysosomal enzyme, N-acetyl-beta-glucosaminidase, equally in both 1- and 4-day cultures. Both 12- and 15-hydroperoxyeicosatetraenoic acids (HPETEs), the precursors of 12- and 15-HETE, were found to be irreversible inhibitors of the cyclo-oxygenase pathway and reversible inhibitors of the 5-lipoxygenase pathway in macrophages. 15-HETE were found to be reversible inhibitors of both pathways. Thus the oxidation of arachidonic oxidation of arachidonic acid to both prostaglandins and leukotrienes may be under intracellular regulation by products of 12- and 15-lipoxygenases.

Lipoproteins of the extravascular space: enhanced macrophage degradation of low density lipoproteins from interstitial inflammatory fluid

Current evidence has demonstrated that cholesteryl ester-loaded macrophages are important components of the atherosclerotic lesion. Additional studies have implicated low density lipoproteins (LDL) and circulating monocytes as central to the origin of lipid-laden foam cells found in the arterial wall. This is a result of the finding of accelerated macrophage uptake of LDL chemically modified by reaction with malondialdehyde (MDA-LDL), acetic anhydride (Ac-LDL), or incubation with arterial cells in vitro. In concert with these chemical modifications, we have previously demonstrated selective in vivo modification of LDL isolated from interstitial inflammatory fluid (IF) of the rabbit. Utilizing the polyvinyl sponge implant model, we reported that IF-LDL had an altered chemical composition, electrophoretic mobility, and particle size distribution when compared to LDL isolated from homologous plasma (WP-LDL). In this study reported herein, we examined the metabolism of IF-LDL by resident mouse peritoneal macrophages (MPM) in culture. IF-LDL was degraded substantially faster by MPM, and resulted in a substantial increase in cellular cholesteryl ester when compared to cells incubated with WP-LDL. IF-LDL binding to MPM was inhibited by Ac-LDL derived from WP-LDL, but only minimally by unmodified WP-LDL. Transmission electron microscopy of MPM revealed extensive lipid deposition in cells incubated with Ac-LDL and IF-LDL. These results implicate LDL from interstitial inflammatory fluid as an in vivo modified lipoprotein that can enhance uptake via the acetyl LDL receptor pathway in resident macrophages.

Some effects of lignocaine on cultured mouse peritoneal macrophages.

Macrophages were exposed to lignocaine in perifused and non-perifused cultures using media with or without foetal calf serum. Effects on morphology, viability, phagocytosis and the release of enzymes were assessed. During the period of contact with lignocaine there was a selective release of beta-glucuronidase. After washing, enzyme release continued over a period of 7 hours and, in the absence of foetal calf serum, a decrease in the total beta-glucuronidase content was found in non-perifused cultures. Although lignocaine-treated cells phagocytosed particulates the rate of enzyme release was reduced compared with normal cells when subsequently exposed to quartz.

Endocytic pathways and time sequence of lysosomal transfer of macromolecules in cultured mouse peritoneal macrophages. Double-labeling experiments with horseradish peroxidase and ferritin.

A double-labeling protocol was used to study endocytic pathways and lysosomal transfer of exogenous macromolecules in cultured mouse peritoneal macrophages. After pulse-chase labeling of lysosomes with horseradish peroxidase (visualized cytochemically), the cells were exposed to native, anionic ferritin for 0-45 min at 37 degrees C and then analysed by transmission electron microscopy. The results show that ferritin binds to the plasma membrane, accumulates in coated pits, and is rapidly taken up in small, smooth-surfaced endocytic vesicles. The latter carry the ferritin molecules directly to lysosomes, recognized by their peroxidase labeling, or fuse with each other to form larger endocytic vacuoles (endosomes) which in turn fuse with and empty their content into lysosomes. The first signs of transfer of ferritin into the lysosomes were seen after 5-10 min of exposure and after 25-30 min most of the lysosomes were labeled. Union of ferritin-labeled and other lysosomes was also noted, suggesting that the contents of the lysosomes were spread within the lysosomal compartment by fusion-fission processes. It is concluded that a multiplicity of structures is involved in the uptake and intracellular transport of exogenous macromolecules in macrophages and that the time sequence of lysosomal transfer of the interiorized material is highly variable.

Increased multiplication of dengue virus in mouse peritoneal macrophage cultures by treatment with extracts of Ascaris-Parascaris parasites.

Methylcellulose-elicited peritoneal macrophages from BALB/c mice were cultivated in vitro and inoculated with dengue virus (DV). At intervals thereafter portions of the culture fluids were taken and titrated for viral infectivity. Extracts from Ascaris suum and Parascaris equorum, either crude or Sephadex G-100 fractionated, were examined for effects on the multiplication of DV. The macrophage cultures treated with the above substances produced larger amounts of DV compared with untreated control cultures. The enhancing effect of the substances depended on doses added and duration of treatment and was suppressed by co-treatment with carrageenan, a specific macrophage-inhibiting agent, but was not related to the viability of cultured cells. In fluorescent antibody (FA) as well as infectious center assay experiments, it was shown that the DV-infected cells were found more frequently in treated cultures than in untreated control cultures. In the treated cultures phagocytosis by cultured cells was also of a higher magnitude than that in untreated cultures. In cocultures of macrophages and splenocytes from the same line of mice, no additive effect of splenocytes was noted. The limulus amebocyte lysate clotting enzyme reaction (Limulus test) indicated that involvement of bacterial lipopolysaccharides in the enhancement phenomena was negligible. The data so far obtained suggest that the enhancing effect was due to direct action of the parasitic extracts on macrophages. Four Sephadex G-100 fractions from the crude extracts showed similar activities; however, the effects of fractions I and III appeared to be comparatively strong. Significance of the findings in relation to the pathogenesis of DV infection was discussed.

Lipopolysaccharide-treated RAW 264.7 cells produce a mediator that inhibits lipoprotein lipase in 3T3-L1 cells.

RAW 264.7 cells upon stimulation with lipopolysaccharide secrete a protein mediator(s) that suppresses lipoprotein lipase activity in differentiated 3T3-L1 cells. The mediator(s), which is absent from unstimulated culture supernatants, is nondialyzable and thermolabile. Preliminary characterization suggests that this mediator(s) may be the same as that previously found in medium from lipopolysaccharide-treated thioglycollate-elicited mouse peritoneal macrophage cultures.

Topography and dynamics of receptors for acetylated and malondialdehyde-modified low-density lipoprotein in the plasma membrane of mouse peritoneal macrophages as visualized by colloidal gold in conjunction with surface replicas.

The topography and dynamics of receptors for acetylated (acetyl) and malondialdehyde-modified (MDA) low-density lipoprotein (LDL) in the plasma membrane of cultured mouse peritoneal macrophages were investigated using a new technique. Modified LDL labeled with gold particles was used to visualize LDL receptors in the plane of the plasma membrane in platinum-carbon surface replicas of critical point-dried cells. It was found that the native distribution of unoccupied acetyl-LDL receptors is diffuse, whereas unoccupied MDA-LDL receptors are preclustered in the plasma membrane. Competition and double labeling experiments suggest the existence of two distinct classes of receptor sites for acetyl-LDL and MDA-LDL.