Autoradiographical demonstration of C3b receptor activity on resident peritoneal macrophages

The present study was performed to evaluate the usefulness of 125I-labelled C3b bound to constituents of sheep erythrocyte membranes (125I-C3b-OR) for the demonstration of C3b receptor activity of resident peritoneal macrophages at the electron-microscopical level. The binding of 125I-C3b-OR to the cells was studied in biochemical and autoradiographical experiments. The amount of cell-associated radioactivity was dependent on the presence of unlabelled aggregated C3b (AC3b) in a dose-response manner, and diminished strongly after functional inactivation of the receptor by trypsin treatment. In addition, it was found that at 4 degrees C most of the label was associated with the cell surface. However, when the incubation temperature was raised from 4 degrees C to 37 degrees C, internalization of the label was observed. These results indicate that 125I-C3b-OR is a suitable agent for further characterization of the C3b receptor-function of resident peritoneal macrophages at the electron-microscopical level.     

A thrombin receptor in resident rat peritoneal macrophages.

Resident rat peritoneal macrophages possess 6 x 10(2) high-affinity binding sites per cell for bovine thrombin with a Kd of 11 pM, and 7.5 x 10(4) low-affinity sites with a Kd of 5.8 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labeled bovine thrombin is achieved after 1 min at 37 degrees C, and after 12 min at 4 degrees C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 0.27 and 0.06 min-1 at 4 degrees C. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radio-activity migrates as intact thrombin upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3 treatment, and the receptor does not mediate a quantitatively important degradation of the ligand. The binding is not dependent on the catalytic site of thrombin, since irreversibly inactivated thrombin also binds to the receptor. 125I-labeled thrombin covalently cross-linked to its receptor migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr 160,000, corresponding to an approximate receptor size of Mr 120,000.     

Concanavalin A is mitogenic for resident peritoneal macrophages

Con A, a known T-cell mitogen, is also mitogenic for resident peritoneal macrophages. The stimulated cells morphologically resemble macrophages and are actively phagocytic. The concentration of con A (30 micrograms/ml) required to stimulate 3H-TdR incorporation is ten times that required for T-cell activation. Con A must be present throughout the entire culture period to produce the maximum effect, and con A-depleted supernatant fluids from con A-stimulated cells cannot replace the con A requirement. Stimulation of 3H-TdR incorporation occurs after a 48-hour lag period and is maximal on the fifth to seventh day of culture. At the peak of the response, 20-30% of the macrophages can be stimulated to incorporate 3H-TdR, but little or no increase in the total number of cells present in the culture occurs. This and pulse-chase experiments indicate that only a single cycle of replication occurs in the stimulated cells. Con A-responsive peritoneal macrophages appear to be a distinct subpopulation and might play a different role in the interaction with T cells and B cells in the immune response than the con A-non-responsive cells.     

Recognition of acidic phospholipase A2 activity in plasma membranes of resident peritoneal macrophages

Phospholipase (PLase) activities in the plasma membrane of guinea pig peritoneal macrophages were studied, as these enzymes having such activity may be candidates for the release of arachidonic acid (AA) from phosphatidylcholine (PC). An AA release system operating at acidic pH was identified in the macrophage plasma membrane and characterized. This membrane-bound acidic PLase A2 had an optimum pH at 4.5, and enzyme activation was observed in Ca++-free medium; but the maximum activity was found at 0.5 mM Ca++ concentration. The Km value for PC of acidic PLase A2 was 4.2 microM, and a Michaelis-Menten relationship was evident. Calcium might act as a cofactor at some intermediate step during the activation of acidic PLase A2 in light of the uncompetitive manner of Ca++ action. Furthermore, the release of [3H]-AA from preradiolabelled macrophage plasma membranes occurred with the addition of Ca++ at pH 4.5. These data suggest that the acid PLase A2 is a component of the plasma membrane and is not due to lysosomal contamination since membrane-bound acidic PLase A2 properties are opposite to those found for lysosomal PLase A2.     

Mcl-1 depletion in apoptosis elicited by ionizing radiation in peritoneal resident macrophages of C3H mice

Remarkably, apoptosis was induced by exposing peritoneal resident macrophages (PRM) of C3H mice, but not other strains of mice, to ionizing radiation. The molecular mechanism of this strain-specific apoptosis in PRM was studied. The apoptosis elicited in C3H mouse PRM 4 h after exposure was effectively blocked by proteasome inhibitors. Irradiation-induced disruption of mitochondrial transmembrane potential and the release of cytochrome c into the cytosol were also suppressed by a proteasome inhibitor but not by a caspase inhibitor. To determine whether the apoptosis occurred due to a depletion of antiapoptotic proteins, Bcl-2 family proteins were examined. Irradiation markedly decreased the level of Mcl-1, but not Bcl-2, Bcl-X(L), Bax, A1, or cIAP1. Mcl-1's depletion was suppressed by a proteasome inhibitor but not by a caspase inhibitor. The amount of Mcl-1 was well correlated with the rate of apoptosis in C3H mouse PRM exposed to irradiation and not affected by irradiation in radioresistant B6 mouse PRM. Irradiation increased rather than decreased the Mcl-1 mRNA expression in C3H mouse PRM. On the other hand, Mcl-1 protein synthesis was markedly suppressed by irradiation. Global protein synthesis was also suppressed by irradiation in C3H mouse PRM but not in B6 mouse PRM. The down-regulation of Mcl-1 expression with Mcl-1-specific small interfering RNA or antisense oligonucleotide significantly induced apoptosis in both C3H and B6 mouse PRM without irradiation. It was concluded that the apoptosis elicited in C3H mouse PRM by ionizing radiation was attributable to the depletion of Mcl-1 through radiation-induced arrest of global protein synthesis.     

Identification and characterization of a phospholipase C activity in resident mouse peritoneal macrophages. Inhibition of the enzyme by phenothiazines

Resident mouse peritoneal macrophages contain a phospholipase C of high activity that is specific for phosphatidylinositol. The activity has a neutral pH optimum, is Ca²⁺-dependent and has a maximum reaction velocity of 525nmol/h per mg of protein. Certain phenothiazines are potent inhibitors of this activity.     

Cytochemical localization of beta-galactosidase in resident and inflammatory peritoneal macrophages from C57BL mice

A cytochemical method for the detection of beta-galactosidase (beta-Gase) in mouse peritoneal macrophages was used to study the ultrastructural localization of this enzyme in these cells. It was found that the reaction product for beta-Gase was localized in the perinuclear cisternae, the endoplasmic reticulum, the Golgi complex, lysosomes, vesicles and on the cell surface of peritoneal macrophages from untreated C57BL mice. When examined by X-ray microanalysis the crystalline reaction product was found to contain bromine, an element present in the indolyl substrate which was used to identify beta-Gase. Injection of Proprionibacterium acnes (P. acnes) intraperitoneally or BCG intravenously caused a visible loss in beta-Gase from all the organelles and from the cell surface of the macrophages.     

Caveolin isoforms in resident and elicited rat peritoneal macrophages

Caveolin--an integral membrane protein--is the principal component of caveolae membranes in vivo. Multiple forms of caveolin have been identified: caveolin-1alpha, caveolin-1beta, caveolin-2 and caveolin-3. They differ in their specific properties and tissue distribution. When we studied the lysate of resident and elicited macrophages isolated from rat peritoneal cavity by Western blot analysis, we identified two different proteins (approximately 29 kDa and approximately 20 kDa) which were labelled with anti-caveolin antibodies. The approximately 20-kDa protein was labelled specifically only by anti-VIP21/caveolin-1, while the approximately 29-kDa protein was labelled by anti-VIP21/caveolin-1 and anti-caveolin-2. The presence of the approximately 29-kDa protein was characteristic of resident macrophages, and only a small amount of the approximately 20-kDa protein was detected in these cells. Elicitation resulted in a significant increase in the amount of the approximately 20-kDa protein labelled by anti-VIP21/caveolin-1 only. According to its molecular mass and antibody-specificity, this protein might be identical with the caveolin-1beta isoform. Our morphological (confocal and electron microscopical) studies have shown that in resident cells caveolin was present in the cytoplasm, in smaller vesicles and multivesicular bodies around the Golgi area. Only a very small amount of caveolae was found on the surface of these cells. In elicited macrophages, caveolae (labelled with the anti-VIP21/caveolin-1 antibody) appeared in large numbers on the cell surface, but caveolin detected by anti-caveolin-2 was also found in small vesicles and multivesicular bodies in the cytoplasm. According to these results, the absence of caveolae in resident cells can be explained by the absence of caveolin-1. The expression of the approximately 29-kDa (caveolin-related) protein in resident macrophages seems to be insufficient for caveolae formation. Elicitation significantly increased the expression of caveolin-1, and the increased amount of caveolin-1 resulted in caveolae formation on the cell surface.     

Enhanced leukotriene C4 synthase activity in thioglycollate-elicited peritoneal macrophages

The utilization of LTA4 by peritoneal macrophages (MO) obtained from untreated rats (control) as well as by those elicited from rats was investigated at designated intervals (on days 3, 7, and 14) following the intraperitoneal injection of thioglycollate (TG). On day 7 following the injection the elicited MO converted LTA4 to LTC4 at the highest rate while the resident MO showed the lowest rate. The conversion of LTA4 to LTC4 and LTB4 was next examined by using each MO lysate. The apparent LTC4 synthase activity was significantly higher in the MO lysate both on day 3 and day 7, with the latter being the highest value obtained. The GSH S-transferase activity in each lysate using as the substrate, DNCB was significantly lower on day 3 but significantly higher on day 7 as compared to control values. However, this elevated activity was less variable than that observed with LTC4 synthase. The possible implication for these observations is discussed.     

Interferon-gamma modulates protein kinase C activity in murine peritoneal macrophages

The content of Ca2+-, phospholipid-dependent protein kinase activity (protein kinase C) in murine peritoneal macrophages treated with recombinant interferon-gamma (IFN-gamma) has been investigated. Protein kinase C activity was solubilized by nonionic detergent extraction of sonicated cells and separated by high performance liquid chromatography on a TSK 4000 SW gel filtration column. The enzyme eluted from the column in a molecular weight range of 60-80 X 10(3) and was identified by virtue of Ca2+ and phospholipid requirements. Macrophages treated with recombinant IFN-gamma exhibited a substantial increase in total protein kinase activity which could be accounted for entirely by increased protein kinase C activity. This activity was enhanced as much as 5-fold over that seen in untreated macrophages and was specific for IFN-gamma in that other agents known to signal changes in macrophage function had no effect. The time required for the elevation of kinase activity was identical to that required for induction of other functions by IFN-gamma in macrophages. These observations suggest that protein kinase C may be a focus of regulatory action in IFN-gamma-mediated macrophage activation.     

Persistent expression of Ia-antigen on a subpopulation of murine resident peritoneal macrophages

The stability of Ia-antigen expression on murine resident peritoneal macrophages was assessed during the course of in vitro culture. Contrary to published findings with radioimmunoassays and immunofluorescence assays, the cultured cells bore Ia-antigen, as shown by their rosetting with sheep erythrocytes coupled with anti-Ia.2 monoclonal antibody. In support of this finding, cultured cells presented the copolymer of glutamine, alanine, and tyrosine (GAT) to GAT-primed T lymphocytes in an Ia-dependent manner. Thus, functional Ia antigen is present on cultured macrophages. Disappearance of the antigen after fixation of macrophages with either glutaraldehyde or paraformaldehyde, a routine procedure in the radioimmunoassays and immunofluorescence assays, explains its presumed absence on cultured cells.     

Regulation of cytoplasmic pH in resident and activated peritoneal macrophages

Cytoplasmic pH (pHi) has been shown to be an important determinant of the activity of the NADPH oxidase in phagocytic cells. We hypothesized that a difference in pHi and/or its regulation existed between activated and resident macrophages (RES MOs) which might explain the increased NADPH oxidase activity observed in the former. The pHi of RES and lipopolysaccharide (LPS)-elicited MOs was examined using the fluorescent dye BCECF. Resting pHi did not differ between resident (RES) and elicited (ELI) MOs (7.16 +/- 0.05 and 7.20 +/- 0.05, respectively). pHi recovery after intracellular acid loading was partially dependent on the presence of Na+ in the extracellular medium, and was partially inhibited by the Na+/H+ antiport inhibitor, amiloride. At comparable pHi, the rate of acid extrusion during recovery was not different in RES and ELI MOs (1.48 +/- 0.12 and 1.53 +/- 0.06 mM/min, respectively). In both RES and ELI MOs, approx. 40% of total pHi recovery was insensitive to amiloride and independent of extracellular Na+. In both RES and ELI MOs, stimulation with TPA resulted in a biphasic pHi response: an initial acidification followed by a sustained alkalinization to a new steady-state pHi. This alkalinization was Na(+)-dependent and amiloride-sensitive, consistent with a TPA-induced increase in Na+/H+ antiport activity. The new steady-state pHi attained after TPA stimulation was equivalent in RES and ELI MOs (7.28 +/- 0.04 and 7.31 +/- 0.06, respectively), indicating comparable stimulated Na+/H+ antiport activity. However, the initial acidification induced by TPA was greater in ELI than in RES MOs (0.18 +/- 0.02 vs. 0.06 +/- 0.02 pH unit, respectively, P less than 0.05). The specific NADPH oxidase inhibitor diphenylene iodonium (DPI) completely inhibited the respiratory burst but reduced the magnitude of this pHi reduction by only about 50%. This suggested that the TPA-induced pHi reduction was due in part to acid produced via the respiratory burst, and in part to other acid-generating pathways stimulated by TPA.     

Stimulatory effects of purified macrophage colony-stimulating factor on murine resident peritoneal macrophages

A purified preparation of macrophage colony-stimulating factor (M-CSF) free of interferon and endotoxin activity was studied for its effects on resident murine peritoneal macrophages. M-CSF was found to induce profound morphologic alterations in resident macrophages. These changes included a marked increase in cell size, membrane ruffling, and cytoplasmic vacuolization. Further, after 72 hr of incubation with 1000 U/ml of M-CSF, there were significant increases in macrophage DNA synthesis as measured by autoradiography (P less than 0.001), and in macrophage monolayer protein content (P less than 0.01). None of these changes was seen in control macrophages or those exposed to recombinant interferon-gamma (IFN). Low activity levels of the ectoenzymes 5'-nucleotidase (5'NTD) and alkaline phosphodiesterase I (APD) have been associated with certain macrophage functions, particularly the expression of tumor cytotoxicity. Macrophage monolayers exposed to M-CSF demonstrated an unaltered level of 5'NTD activity from controls and a significantly increased level of APD activity (P less than 0.01) and did not demonstrate an increased ability to kill tumor cells, as measured by the 51Cr-release assay. On the other hand, IFN caused significant decreases in both 5'NTD (P less than 0.05) and APD (P less than 0.01) and also induced marked tumoricidal activity in macrophage monolayers. These results indicate that purified M-CSF induces highly specific alterations in the functional activity and morphologic appearance of resident macrophages and these changes are distinct from those induced by IFN.     

Differences in the arginase activity produced by resident and stimulated murine and rat peritoneal macrophages.

1. Murine macrophages showed a considerably higher in vitro arginase production in short time cultures than rat peritoneal cells. 2. The in vivo stimulation with casein or thioglycollate resulted in an enhanced in vitro enzyme production in mice. 3. The adherence is not the condition of the enzyme production. 4. The difference between the two species cannot be explained by the lack of bivalent ions, the absence of energy supply, proteolysis, the low number of macrophages or by the different cell types of the peritoneal exudate of mouse and rat. 5. The lysozyme production of murine and rat peritoneal macrophages was also investigated and no difference was observed between the two species.     

Phenotypic and functional analysis of murine resident and induced peritoneal macrophages

Primary macrophages from the peritoneal cavities of mice are commonly used ex vivo to produce inflammatory cytokines and test anti-inflammatory agents. Although approximately 1 million peritoneal macrophages can be obtained from an untreated mouse, more than twice that number can be collected 48 to 72 h after intraperitoneal injection of sterile inducing agents such as Brewer thioglycollate broth, casein, and proteose peptone. However, whether 'induced' macrophages are functionally equivalent to 'resident' peritoneal macrophages has been unclear. Flow cytometric analysis revealed significant phenotypic differences between these 2 macrophage types. Resident and induced peritoneal macrophages also demonstrated markedly different capacities to produce the inflammatory cytokines interleukins 6 and 1beta in response to lipopolysaccharide stimulation in vitro. Increased understanding of the differences between resident and induced peritoneal macrophages likely will help investigators decide which macrophage type is appropriate for their in vitro assay needs.     

Conditional macrophage ablation demonstrates that resident macrophages initiate acute peritoneal inflammation

The role played by resident macrophages (Mphi) in the initiation of peritoneal inflammation is currently unclear. We have used a conditional Mphi ablation strategy to determine the role of resident peritoneal Mphi in the regulation of neutrophil (PMN) recruitment in experimental peritonitis. We developed a novel conditional Mphi ablation transgenic mouse (designated CD11bDTR) based upon CD11b promoter-mediated expression of the human diphtheria toxin (DT) receptor. The murine DT receptor binds DT poorly such that expression of the human receptor confers toxin sensitivity. Intraperitoneal injection of minute (nanogram) doses of DT results in rapid and marked ablation of F4/80-positive Mphi populations in the peritoneum as well as the kidney, and ovary. In experimental peritonitis, resident Mphi ablation resulted in a dramatic attenuation of PMN infiltration that was rescued by the adoptive transfer of resident nontransgenic Mphi. Attenuation of PMN infiltration was associated with diminished CXC chemokine production at 1 h. These studies indicate a key role for resident peritoneal Mphi in sensing perturbation to the peritoneal microenvironment and regulating PMN infiltration.     

Disappearance and reappearance of resident macrophages: importance in C. parvum-induced tumoricidal activity

We have investigated the role of resident macrophages in the early tumoricidal response to C. parvum. The bacteria were labeled with FITC and resident cells were labeled in situ with blue fluorescent covaspheres to enable subsequent monitoring of cellular changes by flow cytometry. Macrophages disappeared within 5 hr of administration of bacteria. At 24 hr, fibrinous adhesions containing double labeled macrophages were observed at numerous sites on the peritoneum. Macrophages associated with large numbers of bacteria, levels of beads similar to control animals, and elevated plasminogen activator-like activity did not reappear in washings in significant numbers until 72 hr. Thus, the large bacteria-containing cells that account for the majority of the early tumoricidal activity are likely to be derived from resident macrophages.     

Contribution of complement component C3 and complement receptor type 3 to carbohydrate-dependent uptake of oligomannose-coated liposomes by peritoneal macrophages

Peritoneal macrophages (PEMs) preferentially and rapidly take up oligomannose-coated liposomes (OMLs) and subsequently mature to induce a Th-1 immune response following administration of OMLs into the peritoneal cavity. Here, we examine the contributions of complement component C3 and complement receptor type 3 (CR3) to carbohydrate-dependent uptake of OMLs by PEMs. Effective uptake of OMLs into PEMs in vitro was observed only in the presence of peritoneal fluid (PF), and OMLs incubated with PF were incorporated by PEMs in vitro in the absence of PF. These phenomena were inhibited by methyl-alpha-mannoside, N-acetylglucosamine or EDTA, but not by galactose. Pull-down analysis followed by peptide mass fingerprinting of PF-treated OMLs indicated that the OMLs were opsonized with complement fragment iC3b. In vivo uptake of OMLs by PEMs was inhibited by intraperitoneal injection of an antibody against CR3, a receptor for iC3b, and OML uptake by PEMs in the peritoneal cavity was not observed in C3-deficient mice. Thus, our results indicate that OMLs are opsonized with iC3b in a mannose-dependent manner in the peritoneal cavity and then incorporated into PEMs via CR3.