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PubMed1971

Macrophage-melanocyte heterokaryons. II. The activation of macrophage DNA synthesis. Studies with inhibitors of RNA synthesis.

Mouse peritoneal macrophages, which do not synthesize DNA in vitro, were fused with melanocytes, a mouse cell strain which proliferates rapidly in vitro. DNA synthesis was induced in macrophage nuclei 2-3 hr after fusion and occurred irrespective of the number of macrophage nuclei present per melanocyte nucleus in each heterokaryon. 50-80% of macrophage nuclei initiated DNA synthesis in the 3-7 hr period after fusion. The activation of most 11-12-day chick red cell nuclei in melanocyte cytoplasm took longer than 10 hr. The lag before DNA synthesis may reflect the heterochromatin content of each nucleus. Studies with actinomycin showed that heterokaryon RNA synthesis was essential for subsequent macrophage DNA synthesis. This RNA was synthesized 1-4 hr before the DNA and was unlikely to be ribosomal RNA, since it was insensitive to <0.1 microg/ml actinomycin. Melanocytes and macrophages were treated before fusion with actinomycin and bromotubercidin to bring about a more selective inhibition of RNA synthesis. Macrophages pretreated for 1 hr with 5 microg/ml of actinomycin showed less than 20% of control RNA synthesis in the first 4 hr after fusion, but a normal activation of macrophage DNA synthesis. Pretreatment of melanocytes for 3-7 hr with 5 microg/ml bromotubercidin, a reversible inhibitor of RNA synthesis, prevented macrophage DNA synthesis without affecting macrophage RNA synthesis in the heterokaryons (81% of control). These studies showed that only melanocyte RNA synthesis was essential for the production of macrophage DNA. The exposure of one cell partner to actinomycin before fusion caused cross-toxicity of the untreated nucleus after fusion. Bromotubercidin, an adenosine analogue which is incorporated into RNA, did not give rise to such cross-toxicity after fusion. Once the macrophage nucleus becomes activated in the heterokaryon it becomes less sensitive to the action of actinomycin.

Autoradiography↗

PubMed1980

Degradation of connective tissue matrices by macrophages. I. Proteolysis of elastin, glycoproteins, and collagen by proteinases isolated from macrophages.

We have investigated the ability of neutral and lysosomal enzymes of mouse macrophages to degrade the insoluble extracellular matrices secreted by smooth muscle cells, endothelial cells, and fibroblasts. Matrices produced by smooth muscle cells contained glycoproteins, elastin, and collagens, but matrices of endothelial cells and fibroblasts contained no elastin. Sequential enzyme digestion of residual matrix revealed that plasmin, a product of macrophage plasminogen activation, degraded 50-70% of the glycoprotein in the matrices but did not degrade the elastin or the collagens. Purified macrophage elastase degraded glycoprotein and elastin components but had no effect on the collagens. The rate of elastin degradation by macrophage elastase was decreased in the presence of the glycoproteins. In contrast, human granulocyte elastase effectively degraded the matrix glycoproteins, elastin, and, to a lesser extent, collagens, Mammalian collagenase degraded only collagens. Conditioned medium from resident and inflammatory macrophages, containing mixtures of the secreted proteinases, degraded the glycoprotein and elastin components of the matrices. However, conditioned medium was less effective in degrading matrix than comparable amounts of purified macrophage elastase because > 90% of the elastase in the medium was in a latent form. Inclusion of plasminogen in the assays accelerated degradation. In the presence of plasminogen, glycoproteins were degraded readily by medium from P388D1, pyran copolymer-, thioglycollate-, and periodate-elicited macrophages and, to a lesser extent, by medium from endotoxin-elicited and resident macrophages; medium from P388D1, thioglycollate-, and periodate-elicited macrophages was most effective in elastin degradation, and resident, endotoxin-elicited and pyran copolymer-elicited macrophages degraded almost no elastin. The macrophage cathepsins D and B degraded all the matrix components at an optimum pH of 5.5 and acted with the secreted neutral proteinases to degrade the connective tissue macromolecules to amino acids and oligopeptides. These data indicate that macrophages at inflammatory sites contain and secrete proteolytic enzymes that could degrade the extracellular matrix.

Animals↗

PubMed1980

Augmenting effect of exogenous macrophages on the rat graft-versus-host reaction in F1 hybrids depleted of macrophages.

The crucial role of macrophages in the generation of the rat graft-versus-host reaction (GVHR) was investigated in normal and macrophage-depleted F1 hybrid rats. The addition of exogenous macrophages, colloidal carbon, or lipopolysaccharide (LPS) resulted in a great augmentation of GVH reactivity of parental lymph node cells in normal F1 hybrid rats. Uptake of foreign materials by F1 macrophages exerts a biphasic action on the GVHR, activating or blocking the biological activity. This depends on the time of injection of foreign materials. To define the extent of macrophage dependence in the GVHR, the additive effect of exogenous macrophages on the reaction was investigated in F1 hybrid rats depleted of effective macrophages and lymphocytes, which had received preliminary host irradiation and colloidal carbon injection. In F1 hybrids depleted of macrophages and given injections of parental lymph node cells depleted of macrophages, the addition of exogenous F1 macrophages resulted in a much higher reaction than did lymphocytes alone. The data suggest that macrophage-induced augmentation of the reaction may be a reflection of the regulatory function of macrophages in the cellular interactions between parental GVH-reactive lymphocytes and alloantigen-bearing host cells in vivo.

Animals↗

PubMed1995

Rabies virus replication in primary murine bone marrow macrophages and in human and murine macrophage-like cell lines: implications for viral persistence.

To determine whether rabies viruses replicate in macrophage or macrophage-like cells, several human and murine macrophage-like cell lines, as well as primary cultures of murine bone marrow macrophages, were incubated with the Evelyn-Rokitnicki-Abelseth (ERA) virus and several different street rabies viruses (SRV). ERA rabies virus replicated well in human monocytic U937 and THP-1 cells and murine macrophage IC-21 cells, as well as primary cultures of murine macrophages. Minimal replication was detected in murine monocytic WEHI-3BD- and PU5-1R cells, and ERA virus did not replicate in murine monocytic P388D1 or J774A.1 cells. A tissue culture-adapted SRV of bat origin also replicated in IC-21 and U937 cells. Non-tissue culture-adapted SRV isolated from different animal species, particularly bats, replicated minimally in U937, THP-1, IC-21 cells and primary murine bone marrow macrophages. To determine whether rabies virus replication is dependent upon the state of differentiation of the macrophage-like cell, human promyelocytic HL-60 cells were differentiated with 12-O-tetradecanoylphorbol-13-acetate (TPA). ERA rabies virus replicated in the differentiated HL-60 cells but not in undifferentiated HL-60 cells. Persistent infections were established in macrophage-like U937 cells with ERA rabies virus and SRV, and infectious SRV was isolated from adherent bone marrow cells of mice that had been infected 96 days previously. Virus harvested from persistently infected U937 cells and the adherent bone marrow cells had specifically adapted to each cell. This specificity was shown by the inability of the viruses to infect macrophages other than U937 cells and primary bone marrow macrophages, respectively. Virus titers of the persistently infected U937 cells fluctuated with extended cell passage. After 30 passages, virus released from the cells had lost virulence as shown by its inability to kill intracranially inoculated mice. However, the avirulent virus released from the persistently infected cells was more efficient in infecting and replicating in naive U937 cells than the virus which was used to establish the persistent infection. These results suggest that macrophages may serve as reservoirs of infection in vivo, sequestering virus which may subsequently be activated from its persistent state, resulting in clinical infection and death.

Animals↗

PubMed2001

[Calcium oscillations induced by lindane in peritoneal macrophages of mice: control by the maturation stage of the macrophage].

Mouse resident peritoneal macrophages loaded with Fluo-3 were examined for changes in cytosolic calcium concentration ([Ca2+]i) after stimulation with gamma-hexachlorocyclohexane (Lindane or gamma-HCCH). These studies, realized on macrophage populations, or single cells, by digital imaging microscopy, sought to determine the role of calcium influx on cyclical changes according to maturation stages of macrophages. Single cell analysis of [Ca2+]i changes in macrophages, after gamma-HCCH exposure in 600 microM extracellular calcium, demonstrated that: 1) these [Ca2+]i variations were asynchronous oscillations with the same frequency (1.7 min-1), and 2) these [Ca2+]i variations in macrophages were not at the same [Ca2+]i level. This heterogeneity could be correlated to a cell size partition of the macrophage population (10.1 +/- 0.44 and 11.45 +/- 0.43 microns). In the presence of 100 microM calcium, gamma-HCCH induced a calcium influx into the two subpopulations, but the calcium oscillations appeared only in small macrophages. In the largest ones, [Ca2+]i slowly decreased back down to the basal level. The cell size variation could be correlated to a phenotypic heterogeneity, linked to the differenciation stage of the cell. Peroxydase activity showed that small macrophages were in fact exudate macrophages and the largest ones were resident macrophages. Inhibition of the oscillatory patterns by a decrease in the extracellular calcium concentration ([Ca2+]ext) or by lanthanum chloride (LaCl3) addition is indicative of the important role of calcium influx in the triggering of oscillations. The calcium influx was transient and induced inositol phosphate (InsP3) production in macrophages. The maintainance of these calcium oscillations depended on calcium mobilization from intracellular calcium stores by InsP3, since neomycin and 8-(diethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8) abolished the oscillations. gamma-HCCH induced a transient calcium entry which triggered phospholipase C activation and the associated [Ca2+]i oscillations. However, we showed that differences in cell responses were observed in relationship with the differentiation stage of the mouse peritoneal macrophages, and with the extracellular calcium concentration.

Aniline Compounds↗

PubMed1989

[Inducing effect of periodontopathic bacteria on activation of macrophage functions and production of interleukin-1 by mouse peritoneal macrophages].

Periodontal disease is thought to be initiated by a bacterial infection and subsequently developed by immunopathological mechanisms thorough host-parasite interactions. The macrophage and lymphocyte are the major functional cell types in the lesion of the disease and participate in tissue destruction and alteration of the periodontal connective tissue as well as in host defense mechanisms. However, the detailed implications of macrophages in development of the disease is still unclear. The aim of this study was to gain more understanding of the functional role of macrophages in periodontal disease. In this study, we examined the inducing effects of sonicated extracts from some gram-negative and gram-positive bacteria associated with the pathogenesis of periodontal disease, including Bacteroides gingivalis, Fusobacterium nucleatum, Haemophilus actinomycetemcomitans, and Actinomyces viscosus, on activation of macrophage functions and IL-1 production by the macrophages from the mouse peritoneum. At a dose as low as 1 microgram/ml (dry weight) sonicated extracts from B. gingivalis induced an increase in acid phosphatase activity and in glucose consumption of mouse peritoneal macrophages in vitro. A significant increase in the acid phosphatase and in glucose consumption was observed in the cultures at 24 h and 48 h, respectively, after the addition of the sonicate. Sonicated extracts from A. viscosus, a gram-positive bacterium, as well as B. gingivalis, F. nucleatum, and H. actinomycetemcomitans, gram-negative ones, were able to induce the increase in acid phosphatase activity and in glucose consumption of the macrophages. These periodontopathic bacteria were found to strongly induce IL-1 production by the macrophages as early as 24 h after addition of the sonicates. A significant increase in the IL-1 production was observed at a dose of 1 microgram/ml of the sonicates. The inducing ability was equivalent to 1 microgram/ml Escherichia coli lipopolysaccharide. The highest production of IL-1 was observed in the macrophages treated with H. actinomycetemcomitans among these sonicates. Sonicated extracts from both gram-negative and gram-positive bacteria were able to induce the IL-1 production by macrophages from C3H/HeJ mice, which are LPS low-responders. These results suggest that periodontopathic bacteria have potent ability to induce macrophage activation and IL-1 production and that the activated macrophages may play an important role in development of periodontal disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Actinomyces↗

PubMed1983

Chemotactic macrophage subpopulations defined by macrophage chemotactic factors from delayed hypersensitivity reaction sites.

The chemotactic specificity of ia-positive and -negative macrophages was studied by using three macrophage chemotactic factors (MCF), -a, -b, and -c, isolated from delayed hypersensitivity reaction (DHR) skin sites in guinea pigs. Listeria-elicited macrophages migrated toward MCF-a, -b, and -c. The chemotactic responses suggested responsive subpopulations to MCF. The electronic programmable individual cell sorter (EPICS) was used to separate macrophages with anti-la monoclonal antibodies. Ia-positive subpopulations responded to MCF-c, although they did not migrate toward MCF-a and -b. In contrast, Ia-negative subpopulations migrated toward MCF-a and -b, but not toward MCF-c. Furthermore, MCF-c attracted Ia-positive macrophages, whereas MCF-a and -b were Ia-negative in vitro; MCF did not induce Ia-negative macrophages to express surface Ia-antigens in vitro. MCF-c was able to produce massive Ia-positive macrophage accumulations when injected i.p., whereas MCF-a accumulated Ia-negative macrophages. The data suggest that MCF-a and -b, which mediate initial macrophage reactions, attract Ia-negative macrophages, and that MCF-c, which mediates predominant macrophage reactions, attract Ia-positive macrophages in the DHR.

Animals↗

PubMed1993

Macrophages and multicellular tumor spheroids in co-culture: a three-dimensional model to study tumor-host interactions. Evidence for macrophage-mediated tumor cell proliferation and migration.

In a new co-culture model involving multicellular tumor spheroids and different phenotypes of human macrophages, we studied the effects of the latter on migration and proliferation of the human colon carcinoma cell line, HRT-18. The macrophage phenotypes are detectable with monoclonal antibodies and are inducible in culture. 12-O-tetradecanoyl-phorbol-13-acetate-activated macrophages are associated with the phenotype 27E10, which is an acute inflammatory macrophage. The glucocorticoid-induced macrophage phenotype RM3/1 is associated with the down-regulation of inflammation. The phenotype resembling the mature resident macrophage termed 25F9 arises spontaneously in prolonged culture. It could be shown that inflammatory macrophages are localized at invasive areas of the tumor-host interface of colorectal carcinoma, whereas resident and anti-inflammatory macrophages were found in the central tumor region or at well-bordered areas of the tumor-host interface. The results obtained with this co-culture model show that 27E10-associated macrophages stimulate tumor cell migration and inhibit tumor cell proliferation. RM3/1 had only a slight inhibiting effect on proliferation and a slight promoting effect on migration. The 25F9-positive macrophage-stimulated tumor cell proliferation and inhibited migration completely. This investigation indicates that this in vitro system is useful for studying different macrophage effects on tumor cells and that indeed proliferation and migration of tumor cells could be influenced in an opposite manner by different types of macrophages.

Carcinoma↗

PubMed1996

A monoclonal antibody to human macrophage gangliosides inhibits macrophage migration.

Gangliosides have diverse immunoregulatory properties. The gangliosides endogenous to macrophages may have immunoregulatory properties that distinguish them from other gangliosides. Gangliosides have been indirectly implicated in macrophage migration as putative cell surface receptors for migration inhibitory factor (MIF). In this study, a monoclonal antibody to human macrophage gangliosides (antibody 25F4) was developed and characterized. This is the first report of the development of monoclonal antibodies to gangliosides of macrophages of any species. Thin-layer chromatographic immunostaining indicated that antibody 25F4 recognized major gangliosides of human macrophages but did not recognized those previously identified as containing fucose. Immunofluorescent surface labeling of viable human macrophages indicated that antibody 25F4 recognized a surface-accessible epitope, present on all cells, and that this was abolished with lipid depletion of macrophage membranes. This epitope was not present on several human nonmacrophage cells. Finally, human macrophages pretreated with antibody 25F4 demonstrated striking inhibition of migration of an agarose droplet assay, whereas an irrelevant monoclonal antibody or monoclonal antibodies to nonganglioside surface epitopes of human macrophages had no effect on migration. Migration inhibition occurred even though antibody 25F4 was removed from the extracellular milieu and was not due to formation of cellular aggregates. These studies support a role for human macrophage gangliosides in macrophage migration.

Antibodies, Monoclonal↗

PubMed2001

Macrophage myeloperoxidase regulation by granulocyte macrophage colony-stimulating factor in human atherosclerosis and implications in acute coronary syndromes.

Inflammation and oxidative stress contribute to the pathogenesis of many human diseases including atherosclerosis. Advanced human atheroma contains high levels of the enzyme myeloperoxidase that produces the pro-oxidant species, hypochlorous acid (HOCl). This study documents increased numbers of myeloperoxidase-expressing macrophages in eroded or ruptured plaques causing acute coronary syndromes. In contrast, macrophages in human fatty streaks contain little or no myeloperoxidase. Granulocyte macrophage colony-stimulating factor, but not macrophage colony-stimulating factor, selectively regulates the ability of macrophages to express myeloperoxidase and produce HOCl in vitro. Moreover, myeloperoxidase-positive macrophages in plaques co-localized with granulocyte macrophage colony-stimulating factor. Pro-inflammatory stimuli known to be present in human atherosclerotic plaque, including CD40 ligand, lysophosphatidylcholine, or cholesterol crystals, could induce release of myeloperoxidase from HOCl production by macrophages in vitro. HOCl-modified proteins accumulated at ruptured or eroded sites of human coronary atheroma. These results identify granulocyte macrophage colony-stimulating factor as an endogenous regulator of macrophage myeloperoxidase expression in human atherosclerosis and support a particular role for the myeloperoxidase-expressing macrophages in atheroma complication and the acute coronary syndromes.

Arteriosclerosis↗

PubMed2002

Morphine-induced macrophage apoptosis modulates migration of macrophages: use of in vitro model of urinary tract infection.

BACKGROUND: Morphine has been reported to alter immune function. Morphine-induced macrophage apoptosis has been shown to contribute to altered immune status in an opiate milieu. We studied the effect of morphine-induced macrophage apoptosis on the migration of macrophages. Because urinary tract infection (UTI) is one of the commonest infections to evoke an inflammatory response; i.e., migration of neutrophils and monocytes to the site of infection, we used an in vitro model of UTI to test our hypothesis. MATERIALS AND METHODS: We carried out both in vivo and in vitro studies. Mice of the FVB/N strain were treated with morphine for short (three doses, 24 hours) and long (11 doses, 96 hours) durations, and their bone marrow cells were isolated. In addition, apoptotic macrophages were prepared by heat treatment. To simulate the in vitro model of UTI, E. coli-activated tubular cell (TC)-conditioned medium containing transforming growth factor-beta (TGF-beta) and macrophage-monocyte chemoattractant protein-1 (MCP-1) was used to test migration of macrophages across a filter in a modified Boyden chamber. In addition, migration of macrophages into the peritoneal cavity was evaluated in both control and morphine-treated states. The effect of morphine on apoptosis as well as migration was studied in murine macrophages and bone marrow cells. RESULTS: Morphine not only promoted apoptosis of bone marrow cells (20% apoptotic cells) but also inhibited their migration across the filter. Control cells showed minimal apoptosis but displayed greater migration. Similarly, heat-treated (apoptotic) cells showed minimal migration. In peritoneal macrophage studies, morphine treatment retarded migration. CONCLUSION: Morphine inhibits macrophage migration both in vivo and in vitro. This attenuated transmigration of macrophages seems to be secondary to the apoptotic effect of morphine.

Animals↗

PubMed1982

Dengue virus multiplication in cultures of mouse peritoneal macrophages: effects of macrophage activators.

Dengue-2 virus multiplied in cultures of methylcellulose-induced peritoneal macrophages of BALB/c mice. The in vitro-cultivated macrophages from dengue-1 virus-immune mice produced larger amounts of dengue-2 virus than did those from nonimmune controls. The effect of macrophage activators was examined by using nonimmune macrophages. Enhanced virus production was demonstrated in cultures of macrophagges pretreated with phytohemagglutinin (PHA) or bacterial lipopolysaccharide (LPS). The number of virus-infected cells in the pretreated cultures was estimated to be about 0.01% or less of the total macrophages. Continuous treatment of macrophages with PHA before and after virus inoculation brought about the most marked enhancement of dengue-2 virus multiplication. On the other hand, treatment with concanavalin A or pokeweed mitogen showed little effect on the multiplication of the same virus. Treatment with carrageenan, a specific macrophage blocking agent, markedly suppressed dengue-2 virus production in both dengue-1 virus-immune macrophages and LPS-treated macrophages. The indirect fluorescent-antibody (FA) technique revealed dengue-2 viral antigen in the cytoplasm of infected macrophages, and the FA-positive macrophages were more numerous in PHA-treated cultures than in untreated controls. The results obtained are discussed in relation to a possible role of activated monocytes/macrophages in the pathogenesis of dengue hemorrhagic fever.

Animals↗

PubMed1990

Prostaglandin E1 suppresses macrophage infiltration and ameliorates injury in an experimental model of macrophage-dependent glomerulonephritis.

Prostaglandin E1 (PGE1) suppresses macrophage infiltration and ameliorates injury in an experimental model of macrophage dependent glomerulonephritis. Macrophages are mediators of glomerular injury in models of proliferative glomerulonephritis. We have recently shown that macrophages in glomerulonephritis have low prostaglandin E2 (PGE) generation, and other evidence implicates eicosanoids as regulators of macrophage activation. Here we have studied in rats the effect of 15(s)-15-methyl PGE1 (M-PGE1) on accelerated nephrotoxic nephritis, a model of acute macrophage-dependent glomerular injury. M-PGE1 ameliorated proteinuria (day 4; 61 +/- 13 mg/24 h, n = 9; vehicle treated, 164 +/- 17 mg/24 h, n = 11; P less than 0.002) and glomerular hypercellularity; quantification of infiltrating macrophages by isolating glomerular cells showed reduction in the numbers of macrophages (44 +/- 9/glomerulus; vehicle treated, 119 +/- 15/glomerulus; P less than 0.02) with inhibition of Ia antigen expression on infiltrating macrophages (8 +/- 5%; vehicle treated 25 +/- 4% P less than 0.05). Glomerular binding of nephrotoxic globulin and levels of autologous antibodies were not affected by M-PGE1. Thus the mechanism of suppression involves inhibition of macrophage accumulation and activation. M-PGE1 administered to normal rats did not affect numbers of resident leucocytes (12.6 +/- 1.5/glomerulus; vehicle treated, 13.2 +/- 1.3/glomerulus) or alter Ia antigen expression (4.1 +/- 0.2 Ia + cells/glomerulus; vehicle treated, 3.9 +/- 0.6/glomerulus). This study suggests a therapeutic role for PGE1 in this type of glomerulonephritis, and has implications for the pathophysiology of macrophage-mediated inflammation.

Alprostadil↗

PubMed2007

Monocyte chemoattractant protein 1, macrophage inflammatory protein 1 alpha, and RANTES recruit macrophages to the kidney in a mouse model of hemolytic-uremic syndrome.

The macrophage has previously been implicated in contributing to the renal inflammation associated with hemolytic-uremic syndrome (HUS). However, there is currently no in vivo model detailing the contribution of the renal macrophage to the kidney disease associated with HUS. Therefore, renal macrophage recruitment and inhibition of infiltrating renal macrophages were evaluated in an established HUS mouse model. Macrophage recruitment to the kidney was evident by immunohistochemistry 2 h after administration of purified Stx2 and peaked at 48 h postinjection. Mice administered a combination of Stx2 and lipopolysaccharide (LPS) showed increased macrophage recruitment to the kidney compared to mice treated with Stx2 or LPS alone. Monocyte chemoattractants were induced in the kidney, including monocyte chemoattractant protein 1 (MCP-1/CCL2), macrophage inflammatory protein 1alpha (MIP-1alpha/CCL3), and RANTES (CCL5), in a pattern that was coincident with macrophage infiltration as indicated by immunohistochemistry, protein, and RNA analyses. MCP-1 was the most abundant chemokine, MIP-1alpha was the least abundant, and RANTES levels were intermediate. Mice treated with MCP-1, MIP-1alpha, and RANTES neutralizing antibodies had a significant decrease in Stx2 plus LPS-induced macrophage accumulation in the kidney, indicating that these chemokines are required for macrophage recruitment. Furthermore, mice exposed to these three neutralizing antibodies had decreased fibrin deposition in their kidneys, implying that macrophages contribute to the renal damage associated with HUS.

Animals↗

PubMed1989

Intracellular multiplication of Paracoccidioides brasiliensis in macrophages: killing and restriction of multiplication by activated macrophages.

The effect of coculturing yeast-form Paracoccidioides brasiliensis with murine cells was studied. Coculture of resident peritoneal or pulmonary macrophages with P. brasiliensis for 72 h dramatically enhanced fungal multiplication 19.3 +/- 2.4- and 4.7 +/- 0.8-fold, respectively, compared with cocultures with lymph node cells or complete tissue culture medium alone. Support of P. brasiliensis multiplication by resident peritoneal macrophages was macrophage dose dependent. Lysates of macrophages, supernatants from macrophage cultures, or McVeigh-Morton broth, like complete tissue culture medium, did not support multiplication of P. brasiliensis in 72-h cultures. Time course microscopic studies of cocultures in slide wells showed that macrophages ingested P. brasiliensis cells and that the ingested cells multiplied intracellularly. In sharp contrast to resident macrophages, lymphokine-activated peritoneal and pulmonary macrophages not only prevented multiplication but reduced inoculum CFU by 96 and 100%, respectively, in 72 h. Microscopic studies confirmed killing and digestion of P. brasiliensis ingested by activated macrophages in 48 h. These findings indicate that resident macrophages are permissive for intracellular multiplication of P. brasiliensis and that this could be a factor in pathogenicity. By contrast, activated macrophages are fungicidal for P. brasiliensis.

Animals↗

PubMed1996

Effect of amiodarone on cytokine release and on enzyme activities of mouse alveolar macrophages, bone marrow macrophages, and blood monocytes.

The effect off amiodarone, a cationic amphiphilic drug, on cytokine release from, and on protein kinase C (PKC) activity of, mouse alveolar macrophages, bone marrow macrophages, and blood monocytes was examined. In addition, its effect on three enzymes in these cells was also determined. Amiodarone suppressed the growth of all cell types at high doses. As regards cytokine release, amiodarone caused an increase in interleukin-1 (IL-1) alpha, IL-1 beta, and tumor necrosis factor (TNF) alpha release from alveolar macrophages but not from marrow macrophages and monocytes. PKC activity was increased by amiodarone only in alveolar macrophages. And the treatment with amiodarone severely suppressed the H(+)-ATPase, sphingomyelinase, and phospholipase A2 activities in alveolar macrophages. But these enzyme activities in bone marrow macrophages and monocytes were not suppressed so much as in alveolar macrophages. This current study indicated that mouse alveolar macrophages treated with amiodarone undergo suppression of H(+)-ATPase, resulting in suppression of sphingomyelinase and phospholipase A2 activity, and in activation of PKC activity and release of cytokines. It also showed that changes in activities of all three enzymes in alveolar macrophages are different from those in bone marrow macrophages and monocytes with respect to reactivity toward amiodarone.

Amiodarone↗

PubMed1999

Adenosine inhibits macrophage colony-stimulating factor-dependent proliferation of macrophages through the induction of p27kip-1 expression.

Adenosine is produced during inflammation and modulates different functional activities in macrophages. In murine bone marrow-derived macrophages, adenosine inhibits M-CSF-dependent proliferation with an IC50 of 45 microM. Only specific agonists that can activate A2B adenosine receptors such as 5'-N-ethylcarboxamidoadenosine, but not those active on A1 (N6-(R)-phenylisopropyladenosine), A2A ([p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamido adenosine), or A3 (N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide) receptors, induce the generation of cAMP and modulate macrophage proliferation. This suggests that adenosine regulates macrophage proliferation by interacting with the A2B receptor and subsequently inducing the production of cAMP. In fact, both 8-Br-cAMP (IC50 85 microM) and forskolin (IC50 7 microM) inhibit macrophage proliferation. Moreover, the inhibition of adenylyl cyclase and protein kinase A blocks the inhibitory effect of adenosine and its analogues on macrophage proliferation. Adenosine causes an arrest of macrophages at the G1 phase of the cell cycle without altering the activation of the extracellular-regulated protein kinase pathway. The treatment of macrophages with adenosine induces the expression of p27kip-1, a G1 cyclin-dependent kinase inhibitor, in a protein kinase A-dependent way. Moreover, the involvement of p27kip-1 in the adenosine inhibition of macrophage proliferation was confirmed using macrophages from mice with a disrupted p27kip-1 gene. These results demonstrate that adenosine inhibits macrophage proliferation through a mechanism that involves binding to A2B adenosine receptor, the generation of cAMP, and the induction of p27kip-1 expression.

Adenosine↗

PubMed1990

Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages.

We examined the cytolytic mechanisms of activated macrophages by using proteose peptone- or thioglycollate broth-induced mouse peritoneal macrophages or mouse macrophage hybridomas as effector cells, L.P3 cells, a clone of L929 cells, and P815 cells as target cells, and IFN-gamma and LPS as activators. It was determined that TNF is the main cytolytic molecule against L.P3 cells from the following results: 1) activated macrophages can produce TNF; 2) TNF shows cytotoxic activity against L.P3 cells; 3) the addition of anti-TNF antibody inhibited most of the cytolytic activity of activated macrophages against L.P3 cells. On the other hand, it was concluded that the main cytolytic mechanism against P815 cells is the production of NO2-/NO3- from L-arginine, from the following results: 1) activated macrophages can produce NO2-; 2) NaNO2 shows high cytotoxic activity against P815 cells; 3) the depletion of L-arginine from the medium inhibited most of the cytolytic activity of activated macrophages against P815 cells and NO2- production by activated macrophages. In this study, however, cytostatic effects of L-arginine-dependent effector mechanism were not studied. Thus, these results show that activated macrophages can express at least two cytolytic mechanisms independently, namely, the one that appears to be mediated by the L-arginine-dependent effector mechanism and the second that appears to be mediated directly by TNF. Furthermore, it was demonstrated that TNF and L-arginine-dependent NO2- production act synergistically as killing mechanisms of activated macrophages. These mechanisms can explain the cytolytic activity of activated macrophages against a variety of target cells.

Animals↗