Identification and characterization of a phospholipase C activity in resident mouse peritoneal macrophages. Inhibition of the enzyme by phenothiazines.
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ABSTRACT:
SUBMITTER: Wightman PD
PROVIDER: S-EPMC1163156 | biostudies-other | 1981 Aug
REPOSITORIES: biostudies-other
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Project description:Resident mouse peritoneal macrophages synthesize and release large amounts of prostaglandins in response to inflammatory stimuli. Release of prostaglandin E2 and 6-oxoprostaglandin F1 alpha occurs at a rate of 1 nmol/h per mg of cell protein. The mechanisms by which substrate arachidonic acid is released have yet to be established. We have therefore initiated studies to characterize those enzymes that can catalyse its release from phospholipid and may be of significance at the cellular level. We report initially the characterization of two phospholipase A2 activities in homogenates of mouse peritoneal macrophages. The first is active at pH 4.5 and is not dependent on Ca2+. The second is Ca2+-dependent and is optimally active at pH 8.5. Either phospholipase A2 activity is capable of hydrolysing [14C] arachidonic acid from [14C] arachidonic acid-labelled phospholipids in quantities sufficient to account for the amounts of prostaglandins by macrophages in culture. Phospholipid substrates are prepared from mouse LM fibroblasts in serum-free Higuchi medium containing radiolabelled phospholipid precursors. Single-labelled phospholipids bear the 14C label in the arachidonic acid moiety. Dual-labelled phospholipids bear a 14C label in the polar head group and a 3H label in the arachidonic acid moiety. Experiments with dual-labelled substrates establish that both phospholipase activities are of the A2 type as indicated by the equimolar recovery of [3H] arachidonic acid and [14C] lysophospholipid. Studies with aqueous sonicated dispersions of purified [14C] arachidonic acid-labelled phospholipid or mixed liposomal substrates formed from mixtures of cellular polar lipids reveal that the pH 4.5 activity hydrolyses phosphatidylethanolamine and phosphatidylcholine more efficiently when they are present in a mixture of other polar lipids. The pH 8.5 activity, however, hydrolyses the purified phospholipids more efficiently.
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Project description:Resident mouse peritoneal macrophages have three phospholipase activities: a phospholipase A2 active at pH 4.5, a Ca2+-dependent phospholipase A2 active at pH 8.5 and a phosphatidylinositol-specific phospholipase C activity. When macrophages are exposed to zymosan in culture, the cellular activity of pH-4.5 phospholipase A2 is diminished in a manner dependent on zymosan concentration and time of exposure, whereas the cellular activities of pH-8.5 phospholipase A2 and phospholipase C remain unchanged. The depletion of pH-4.5 phospholipase A2 activity from the cell is paralleled by a quantitative recovery of this activity in the culture medium in a manner similar to the cellular depletion and extracellular recovery of two lysosomal enzymes. This release is specifically elicited by an inflammatory substance such as zymosan, since macrophages incubated with 6 micrometer latex spheres retain pH-4.5 phospholipase A2 activity and lysosomal enzyme activities intracellularly.
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Project description:Apoptotic cells are swiftly engulfed by macrophages to prevent the release of noxious materials from dying cells. Apoptotic cells expose phosphatidylserine (PtdSer) on their surface, and macrophages engulf them by recognizing PtdSer using specific receptors and opsonins. Here, we found that mouse resident peritoneal macrophages expressing Tim4 and MerTK are highly efficient at engulfing apoptotic cells. Neutralizing antibodies against either Tim4 or MerTK inhibited the macrophage engulfment of apoptotic cells. Tim4-null macrophages exhibited reduced binding and engulfment of apoptotic cells, whereas MerTK-null macrophages retained the ability to bind apoptotic cells but failed to engulf them. The incubation of wild-type peritoneal macrophages with apoptotic cells induced the rapid tyrosine phosphorylation of MerTK, which was not observed with Tim4-null macrophages. When mouse Ba/F3 cells were transformed with Tim4, apoptotic cells bound to the transformants but were not engulfed. Transformation of Ba/F3 cells with MerTK had no effect on the binding or engulfment of apoptotic cells; however, Tim4/MerTK transformants exhibited strong engulfment activity. Taken together, these results indicate that the engulfment of apoptotic cells by resident peritoneal macrophages proceeds in two steps: binding to Tim4, a PtdSer receptor, followed by MerTK-mediated cell engulfment.
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Project description:Peritoneal resident macrophages from mice are sensitive to inhibition by cyclosporin A (CsA) of phorbol 12-myristate 13-acetate (PMA)-stimulated oxidative burst. Inhibition was assessed in terms of superoxide anion (O2.-) and H2O2 production. Key findings were as follows. (a) CsA inhibited in a dose-dependent manner the production of O2.- when cells were stimulated with PMA. CsA did not alter the respiratory burst induced by other stimuli (zymosan, concanavalin A and fMet-Leu-Phe). It was verified that CsA itself had no scavenger effect. (b) A concomitant decrease in H2O2 liberation following CsA exposure was found. This inhibition was observed both in the initial rate of synthesis and in the accumulation after 15 min of incubation. (c) NADPH oxidase activity in the crude supernatant was unaffected by the previous incubation of macrophages with CsA. CsA does not inhibit glucose transport measured as 14CO2 production. (d) The production of O2.- was strongly dependent on the glucose concentration. Sodium oleate also stimulated O2.- production in resident macrophages. These data might be correlated with the inhibitory effect of CsA upon other functions of macrophages.
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Project description:Post-operative adhesions are a leading cause of abdominal surgery-associated morbidity. Exposed fibrin clots on the damaged peritoneum, in which the mesothelial barrier is disrupted, readily adhere to surrounding tissues, resulting in adhesion formation. Here we show that resident F4/80HighCD206- peritoneal macrophages promptly accumulate on the lesion and form a 'macrophage barrier' to shield fibrin clots in place of the lost mesothelium in mice. Depletion of this macrophage subset or blockage of CD11b impairs the macrophage barrier and exacerbates adhesions. The macrophage barrier is usually insufficient to fully preclude the adhesion formation; however, it could be augmented by IL-4-based treatment or adoptive transfer of this macrophage subset, resulting in robust prevention of adhesions. By contrast, monocyte-derived recruited peritoneal macrophages are not involved in the macrophage barrier. These results highlight a previously unidentified cell barrier function of a specific macrophage subset, also proposing an innovative approach to prevent post-operative adhesions.
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Project description:The transport of cystine has been investigated in mouse peritoneal macrophages cultured in vitro. The transport activity for cystine was very low in freshly isolated macrophages but was potently induced during culture in the presence of bacterial lipopolysaccharide (LPS) at concentrations as low as 0.1 ng/ml. The transport activity for cystine was enhanced when the cells were incubated with tumour necrosis factor-alpha (TNF-alpha), but not with interferon-gamma (IFN-gamma) or interleukin-1. IFN-gamma was rather repressive in the induction of the activity by LPS or TNF-alpha. The transport activity for cystine induced by LPS has been characterized. Cystine was transported mainly by Na(+)-independent system and the uptake of cystine was inhibited by extracellular glutamate and homocysteate, but not by aspartate, indicating that the transport of cystine in macrophages treated with LPS is mediated by System xc-. Glutathione content of the macrophages increased when they were exposed to LPS, and this increase was, at least in part, attributable to the induced activity of the cystine transport.
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Project description:Apoptotic cells must be cleared efficiently by macrophages (Mø) to prevent autoimmunity, yet their ingestion impairs Mø microbicidal function. The principal murine resident lung phagocyte, the alveolar Mø (AMø), is specifically deficient at apoptotic cell ingestion, both in vitro and in vivo, compared with resident peritoneal Mø (PMø). To further characterize this deficiency, we assayed static adhesion in vitro using apoptotic thymocytes and resident AMø and PMø from normal C57BL/6 mice. Adhesion of apoptotic thymocytes by both types of Mø was rapid, specific, and cold-sensitive. Antibody against the receptor tyrosine kinase MerTK (Tyro12) blocked phagocytosis but not adhesion in both types of Mø. Surfactant protein A increased adhesion and phagocytosis by AMø, but not to the levels seen using PMø. Adhesion was largely cation-independent for PMø and calcium-dependent for AMø. Adhesion was not inhibited in either Mø type by mAbs against beta1 or beta3 integrins or scavenger receptor I/II (CD204), but AMø adhesion was inhibited by specific mAbs against CD11c/CD18. Thus, resident murine tissue Mø from different tissues depend on qualitatively disparate receptor systems to bind apoptotic cells. The decreased capacity of murine AMø to ingest apoptotic cells is only partially explained by reduced initial adhesion.
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