Role of the maleyl-albumin receptor in activation of murine peritoneal macrophages in vitro

It has been previously demonstrated that maley-lated-BSA (maleyl-albumin) induces functional activation in murine peritoneal macrophages. Furthermore, maleyl-albumin has been shown to interact with two distinct sites on human monocytes; one site is the scavenger receptor, a 260-kDa oligomeric protein which recognizes modified forms of low density lipoprotein (LDL), and the second is a lower affinity site which has yet to be structurally characterized. In the present study, we wished to quantitatively assess the number and character of maleyl-albumin-binding sites on murine peritoneal macrophages and to determine which site or sites are involved in signaling the macrophage to undergo extensive functional development. Binding studies. demonstrate at least two distinct receptors for maleyl-albumin on murine peritoneal macrophages. Scatchard analyses of the binding isotherms reveal two sites characterized by dissociation constants (Kd) of 17.6 nM and 4.9 microM and maximal binding of 1.2 x 10(5) and 1 x 10(6) sites/cell, respectively. The contribution of the scavenger receptor, determined by binding analyses of malondialdehyde-LDL, is described by two sites with Kd of 39.4 pM and 9.6 nM, and maximal binding of 2.7 x 10(3) and 1.9 x 10(4) sites/cell, respectively. Maleyl-albumin blocks binding of malondialdehyde-LDL, whereas modified LDL fails to inhibit binding of maleyl-albumin. Maleyl-albumin, at concentrations producing lower affinity binding, stimulates tumor cytolysis, expression of mRNA encoding TNF, and suppression of INF-gamma-induced expression of Ia Ag. Malondialdehyde-LDL fails to elicit these responses. We conclude that macrophage activation produced by maleyl-albumin is mediated by interaction with the low affinity, high capacity binding site for maleyl-albumin rather than the scavenger receptor.     

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.     

The dynamics of cytostatic factor production by inactivated resident peritoneal macrophages from Syrian hamsters]

Results presented indicate that resident peritoneal macrophages (Mph) of intact, or tumor-bearing Syrian hamsters release cytostatic factor(s) (CSF) independently on the presence of serum in culture medium and the degree of Mph adherence. Dynamics of CSF secretion and accumulation depends on the number of Mph and duration of time of CSF production. It has been shown that at later time of Mph cultivation (24 hours) supernatants examined can stimulate proliferation of the target cells.     

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.     

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.     

Production of antitumor cytostatic factors by nonactivated resident macrophages of the peritoneal cavity of Syrian hamsters

The production of soluble cytostatic (CS) factor(s) by nonactivated peritoneal resident macrophages (Mph) of Syrian hamsters was found with the use of susceptible spontaneously transformed in vitro cells of STHE cell strain. The CS factor was determined by two modifications of CS test: 1) incorporation of 3H-TdR to the nuclei of target cells and 2) direct determination of the number of cells in the wells. The selected in vivo highly malignant variant of STHE strain appeared to be resistant to CS factors Mph.     

Response of resident murine peritoneal macrophages to in vivo administration of granulocyte-macrophage colony-stimulating factor

The effect of s.c. inoculation of purified recombinant derived granulocyte-macrophage (GM)-CSF on resident murine peritoneal macrophages was assessed in this study. From 18 to 24 h after s.c. administration of GM-CSF to normal mice, the resident peritoneal macrophages were harvested and the levels of membrane-bound IL-1, FcR, Mac-1 cell-surface Ag, and class II MHC expression were assessed. Peritoneal cells from GM-CSF-inoculated mice had significantly greater levels of membrane-bound IL-1 than did control mice. In addition when resident peritoneal macrophages from normal mice were purified by adherence and grown in the presence of GM-CSF, they produced greater levels of both membrane-bound and secreted IL-1. The peritoneal cells from GM-CSF-inoculated mice did not differ from controls in the expression of class II MHC-encoded Ag. This observation was confirmed by the finding that GM-CSF was unable to induce class II MHC expression on P388D1 cells, whereas a secondary mixed leukocyte culture supernatant was. Peritoneal cells from GM-CSF-inoculated mice also exhibited greater levels of expression of FcR and the Mac-1 cell-surface Ag. This resulted in an increase in their ability to phagocytose opsonized SRBC in vitro.     

Kinetic analysis of superoxide anion production by activated and resident murine peritoneal macrophages

Using a continuous spectrophotometric assay, we have monitored the formation of superoxide anion (O₂^?) by activated and resident murine peritoneal macrophages. Macrophages elicited by injection with Corynebacterium parvum, as well as resident macrophages from untreated mice, were kept in suspension culture overnight to eliminate short-lived, contaminating neutrophils. Cytochemical analysis of the cultured macrophages disclosed that essentially all of the activated macrophages reduced nitroblue tetrazolium (NBT) dye vigorously. In contrast, only 18% of the resident macrophages demonstrated vigorous NBT reduction; the remainder of the resident macrophages reduced NBT very weakly. Kinetic analysis of macrophage O₂^? formation revealed that activated macrophages exposed to phorbol myristate acetate (PMA) produced O₂^? at a 13-fold greater maximum rate than resident macrophages. The decline in the rate of O₂^? production with time by activated macrophages was also greater than that of resident macrophages. The data indicate that the greater O₂^? production by activated macrophage populations is due to (i) the presence of an increased percentage of macrophages that respond to PMA with vigorous O₂^? production, and (ii) an increased maximum rate of O₂^? formation by these macrophages.     

Role of Corynebacterium parvum in the activation of peritoneal macrophages: I. Association between intracellular C. parvum and cytotoxic macrophages

We have investigated the association between intracellular C. parvum (CP) and macrophage (Mφ) cytotoxicity. Mouse peritoneal Mφs were activated by ip administration of CP and were subjected to a combination of fractionation techniques to study this. Velocity sedimentation demonstrated that only the largest cells were cytotoxic. These same cells contained CP and suggested an association between the two variables. Further separation of the largest Mφs using a BSA equilibrium buoyant density gradient demonstrated that cytotoxicity was due to Mφs and further substantiated the strong correlation between intracellular CP and cytotoxicity. Various fluorochrome tagged CP preparations were also used to activate Mφs and to isolate CP-containing Mφs using fluorescence-activated cell sorting. When velocity-enriched Mφs were sorted on the basis of the presence or absence of fluorescent CP, only the Mφ fractions which contained CP were cytotoxic. The results indicate that most cytolytic macrophages present at the peak of the response contain CP. Thus, a convenient probe with which to follow macrophage activation at the single cell level was provided.     

Susceptibility of cells with different stage of malignancy to cytostatic action of resident peritoneal macrophages of Syrian hamsters

Syrian hamster non-activated resident peritoneal cells (PC) and peritoneal macrophages (Mph) was demonstrated. The in vivo selection of highly tumourigenic and highly metastatic variants of this strain correlated with their resistance to CSA PC and Mph in four cell variants out of five examined. The highly tumourigenic Syrian hamster embryo cells in vitro transformed by Rous sarcoma virus were highly resistant to CSA PC without selection in vivo. The resistance of highly malignant cells to CSA PC appeared to be unrelated to their ability to produce immunosuppressing prostaglandins of E type.     

Colony stimulating factor-1 stimulates diacylglycerol generation in murine bone marrow-derived macrophages, but not in resident peritoneal macrophages

Colony stimulating factor-1 (CSF-1) stimulates DNA synthesis in murine bone marrow-derived macrophages (BMM); however, unlike BMM, murine resident peritoneal macrophages (RPM) undergo a poor proliferative response. It has previously been shown that phosphatidylinositol-4,5-bisphosphate hydrolysis is not associated with CSF-1 action in BMM. In this report we demonstrate that, despite a lack of inositol trisphosphate generation, CSF-1 transiently elevated both [3H]myristoyl- and [3H]arachidonyl-diacylglycerol (DAG) in BMM in a dose-dependent fashion. CSF-1 failed, however, to stimulate an increase in either species of DAG in RPM. Thus, DAG could be a second messenger for the proliferative action of CSF-1 in macrophages. Other mitogenic agents, 12-0-tetradecanoyl phorbol 13-acetate (TPA) and exogenous phospholipase C, also increased BMM levels of [3H]myristoyl- and [3H]arachidonyl-DAG. The nonmitogenic agents, lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) and zymosan, had different effects on the generation of either species of DAG in BMM. LPS failed to elevate either form, TNF-alpha increased only [3H]arachidonyl-DAG, while zymosan stimulated levels of both species of DAG. It therefore appears that increased diacylglycerol generation may be necessary, but perhaps not sufficient, for macrophage proliferation.     

Cyclosporin A inhibits phorbol ester-induced activation of superoxide production in resident mouse peritoneal macrophages.

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.     

Cyclooxygenase-1-dependent prostaglandin synthesis modulates tumor necrosis factor-alpha secretion in lipopolysaccharide-challenged murine resident peritoneal macrophages

Comprehensive studies of prostaglandin (PG) synthesis in murine resident peritoneal macrophages (RPM) responding to bacterial lipopolysaccharide (LPS) revealed that the primary PGs produced by RPM were prostacyclin and PGE(2). Detectable increases in net PG formation occurred within the first hour, and maximal PG formation had occurred by 6-10 h after LPS addition. Free arachidonic acid levels rose and peaked at 1-2 h after LPS addition and then returned to baseline. Cyclooxygenase-2 (COX-2) and microsomal PGE synthase levels markedly increased upon exposure of RPM to LPS, with the most rapid increases in protein expression occurring 2-6 h after addition of the stimulus. RPM constitutively expressed high levels of COX-1. Studies using isoform-selective inhibitors and RPM from mice bearing targeted deletions of ptgs-1 and ptgs-2 demonstrated that COX-1 contributes significantly to PG synthesis in RPM, especially during the initial 1-2 h after LPS addition. Selective inhibition of either COX isoform resulted in increased secretion of tumor necrosis factor-alpha (TNF-alpha); however, this effect was much greater with the COX-1 than with the COX-2 inhibitor. These results demonstrate autocrine regulation of TNF-alpha secretion by endogenous PGs synthesized primarily by COX-1 in RPM and suggest that COX-1 may play a significant role in the regulation of the early response to endotoxemia.     

Expression of a 120,000 dalton protein during tumoricidal activation in murine peritoneal macrophages

Modulation of protein expression during interferon-gamma (IFN-gamma)-lipopolysaccharide (LPS)-mediated macrophage tumoricidal activation has been examined by metabolic radiolabeling of various murine peritoneal macrophage populations with [35S]methionine followed by SDS-PAGE analysis. Although both IFN-gamma and LPS are capable of stimulating the expression of several proteins when used independently, combined treatment induced the enhanced or de novo expression of a 120,000 dalton polypeptide. The expression of this protein was synergistically regulated by both IFN-gamma and LPS in a manner strongly reminiscent of the functional synergism that these two agents exhibit with respect to induction of tumoricidal activity. p120 expression could be seen first at approximately 3 hr after the addition of both agents, reached optimal expression by 6 hr, and maintained elevated synthesis for up to 24 hr. This time course corresponds closely to that seen for the acquisition of tumoricidal competence. Macrophages elicited in the primed state of activity in vivo with methyl vinyl ether co-polymer II (MVE-II) did not express p120, but could be induced to do so when treated with low doses of LPS. Under similar conditions, MVE-II-elicited cells also acquire tumoricidal activity. Macrophages obtained from mice chronically infected with bacillus Calmette-Guerin constitutively expressed both p120 and cytolytic activity. If such macrophages were cultured for 24 hr, the expression of both events decayed and was lost, but could be restored by treatment with low doses of LPS. Thus the data support a strong correlation between the expression by macrophages of a novel 120,000 dalton protein and the expression of tumor cytotoxicity.     

Oxidative and non-oxidative activation of murine peritoneal macrophages by histone H1

The present study aimed at assessing the role of histone H1 in activating macrophages. Histone H1, injected intraperitoneally at a dose of 1 mg/kg body weight as multiple regimens weekly, significantly increased the number of peritoneal macrophages post 21 days of injection. The oxidative and non-oxidative activation of peritoneal macrophages by histone H1 was assessed. For the assessment of oxidative activation the levels of superoxide radical and nitric oxide radical were assessed. The oxidative activation was evident from release of significantly high levels of superoxide and nitric oxide radicals liberated by macrophages of animals treated with histone H1 (P < 0.001) than in untreated animals. In addition, the higher activities of superoxide dismutase indicated protective effect of histone H1, to keep away the macrophages from noxious effects of superoxide. The catalase activity was decreased significantly in macrophages of histone H1 treated animals. The levels of reduced glutathione were significantly (P < 0.001) lowered in treated animals, whereas the levels of lipid peroxides generated were non-significant. The non-oxidative activation was assessed from the activities of lysosomal enzymes released and also from cytolysis of NO-insensitive L929 cells. The activities of lysosomal enzymes-acid phosphatase and beta-glucuronidase released were significantly high in treated animals than in untreated animals (P < 0.001). Histone H1 stimulated the cytolysis of macrophages in L929 cells than in untreated animals. These results suggest that histone H1 stimulates macrophages by oxidative and non-oxidative mechanisms, which favor its future therapeutic prospects.     

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.     

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.     

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.     

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.