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Free cells arising in organ-cultured embryonic rat and hamster lungs share ultrastructural, lysosomal enzyme, and cell membrane properties with typical alveolar macrophages, expressing the developmental potential of the earliest-macrophage precursors resident in the lungs. In the lung culture environment cell proliferation is supported and macrophage attributes are developed despite absence of lymphocytes from the system. We have shown previously that among these attributes, the cells respond with increased phagocytosis of erythrocytes if these are opsonized with immunoglobulin G. Attention has now been turned to the question of nonimmune-mediated phagocytosis by the same population. Living macrophages that emerged from lung cultures bound rhodamine-coupled soybean and wheat germ agglutinins to a greater degree than concanavalin A (Con A), which nevertheless promoted lateral translocation of occupied receptors in the cell membrane. Emerged cells also phagocytosed living bacteria and native yeast cells (Y). The percentage of macrophages ingesting 3 or more yeast cells increased 400 (hamsters) to 500% (rats) when yeast was preincubated with Con A (200 micrograms/ml). Pretreatment of macrophages with Tuftsin (100 microM) enhanced uptake of Y by 100 (hamster) to 200% (rat). Pretreatment of macrophages with macrophage-inhibitory peptide (500 microM) appeared to inhibit phagocytosis of Y by 60% in hamsters but had no significant effect on cells from rat lung cultures.
OBJECTIVE: To further investigate the effects of interleukin-1 (IL-1) in immune-mediated joint inflammation, we examined the role of IL-2, Th1 interferon-gamma (IFNgamma), and Th2 (IL-4) cytokines, joint macrophages, and macrophage-derived cytokines (IL-12 p40, IL-6, leukemia inhibitory factor [LIF], oncostatin M [OSM], and granulocyte-macrophage colony-stimulating factor [GM-CSF]) in a CD4+ T lymphocyte-dependent model of acute arthritis. METHODS: Methylated bovine serum albumin (mBSA)/IL-1-induced arthritis was elicited in wild-type, gene-knockout, and monoclonal antibody-treated mice. Synovial lining macrophages were selectively depleted by intraarticular injection of clodronate liposomes prior to disease induction. The severity of arthritis was assessed histologically. RESULTS: Mice deficient in IL-2 were almost completely protected from arthritis, and neutralization of IL-4 reduced the severity of disease. In contrast, arthritis severity and resolution appeared to be independent of IFNgamma. Synovial lining macrophage depletion markedly reduced arthritis severity. IL-6 or LIF deficiency was only modestly protective, although as previously reported, GM-CSF deficiency conferred profound disease resistance. IL-12 p40-deficient mice (which lack IL-12 and IL-23) and OSM receptor-deficient mice were susceptible to mBSA/IL-1-induced arthritis. CONCLUSION: Acute mBSA/IL-1-induced arthritis is dependent on IL-2 and IL-4, but not IFNgamma. In vivo, the Th1/Th2 paradigm may be distorted by the presence of macrophage-derived cytokines such as IL-1. Synovial lining macrophages are essential in mBSA/IL-1-induced arthritis. However, the requirement for macrophage-derived cytokines is selective; that is, IL-6, LIF, and especially GM-CSF are necessary, but IL-12, IL-23, and OSM are dispensable. IL-1 may therefore influence both adaptive and innate immune mechanisms in acute inflammatory arthritis.
Tumor necrosis factor alpha (TNF-alpha) more than doubles tritiated thymidine ([3H]TdR) uptake in mouse macrophages stimulated by macrophage colony-stimulating factor (CSF-1). However, nothing is known of how TNF-alpha affects this increase or even whether it is manifested by increased cellular proliferation. Here we characterize the effects of TNF-alpha on CSF-1-stimulated proliferation of both primary cells (bone marrow-derived macrophages, BMMs) and a cloned growth factor-dependent macrophage cell line (S1). We show that the TNF-alpha-induced increase in [3H]TdR uptake of CSF-1-stimulated macrophages is directly proportional to an increase in the DNA content of the culture and that the effects of TNF-alpha are direct and independent of cell number. TNF-alpha decreases the population doubling time of log-phase growing macrophages having quite different growth rates to the same (approximately 30%) extent: the doubling time of BMMs decreases from 24 to 17 h and that of S1 cells from 17 to 13 h. TNF-alpha exerts its effects on log-phase growth by increasing to the same proportion CSF-1-stimulated proliferation at all concentrations of CSF-1; that is, TNF-alpha does not shift, but rather amplifies, the CSF-1 dose-response curve. Although TNF-alpha alone does not stimulate macrophage proliferation, its presence in S1 cell cultures coming to quiescence after withdrawal of CSF-1 greatly increases subsequent CSF-1-stimulated [3H]TdR uptake as the cells reenter the cycle. Finally, we show that both human and mouse TNF-alpha increase CSF-1-stimulated log-phase growth and reentry of quiescent cells into the cycle equally on a molar basis (half-maximal stimulation of approximately 0.3 nM). The latter observation argues that the growth-stimulatory effects of TNF-alpha are mediated via the 55-60-kd TNF receptor. We conclude that TNF-alpha acts directly on growth-competent macrophages to decrease significantly the population doubling time in a manner that enhances the mitogenic effects of CSF-1.
Phorbol myristate acetate (PMA) treatment of an EL-4 thymoma cell line (EL-4FARRAR) induced secretion of a factor that inhibited intracellular killing of Leishmania major amastigotes by activated macrophages. Analysis of the cytokines produced by EL-4 cells after PMA stimulation identified interleukin-2 (IL-2, 2500 U/ml), IL-4 (1280 U/ml), interferon-gamma (IFN-gamma; 100 U/ml), and granulocyte-macrophage colony-stimulating factor (GM-CSF; 50 U/ml). Neither tumor necrosis factor nor transforming growth factor beta (TGF-beta) was detected. Each of the cytokines present in EL-4 fluids was assessed for capacity to activate macrophages for destruction of parasites or to suppress intracellular killing. IFN-gamma and GM-CSF both activated macrophages to kill Leishmania; IL-2 and IL-4 had no activity for induction of this antimicrobial effector function. IL-2 and IL-4 were tested for their capacity to inhibit lymphokine- or IFN-gamma-induced destruction of L. major by macrophages: IL-4 was ineffective, but IL-2 markedly suppressed the activation of macrophages for intracellular killing. Addition of > or = 10 U/ml of IL-2 at the time of infection, or up to 4 h before, blocked up to 100% of the capacity of activated macrophages to kill intracellular amastigotes. Immunoaffinity treatment of EL-4 fluids with anti-IL-2 antibody resulted in > 80% reduction in suppression of intracellular killing. The suppressive effects of IL-2 were not direct, but mediated by TGF-beta. IL-2 induced resident peritoneal macrophages to secrete > 5000 pg/ml TGF-beta 1, a quantity that is > 500-fold higher than constitutive background levels (20-40 pg/ml) and is sufficient to block intracellular killing activities. This increase in secretion of TGF-beta was not dependent increases in TGF-beta 1 mRNA. Treatment of cultures with EL-4 fluids or recombinant IL-2 in the presence of antibody to TGF-beta 1 blocked the suppressive activity of both. Thus, IL-2 was the major suppressor factor in EL-4 fluids, and it acted indirectly through the induction and autocrine action of TGF-beta.
The specific function of interstitial macrophages (IM) in the lung is poorly understood because of difficulties in isolating these cells in high purity or large number. In the present studies, a pure population of enzymatically isolated IM and lung macrophages obtained mechanically from the lung were compared functionally with alveolar macrophages recovered by lavage (AM). Macrophages isolated mechanically from the tissue and AM displayed similarly high levels of Fc-receptor mediated phagocytosis. In contrast, IM phagocytized significantly fewer opsonized sheep red blood cells per macrophage than AM. In addition, although some variations in the amounts of nitric oxide and superoxide anion produced by AM and macrophages obtained by mechanical tissue disruption were observed, these subpopulations released significantly more of these mediators than IM. These data support the concept that macrophages isolated by mechanical disruption of the tissue represent a subpopulation of AM. We also found that, in contrast to AM, IM did not respond synergistically to combinations of IFN-gamma and lipopolysaccharide (LPS) or tumor necrosis factor alpha in terms of nitric oxide production. Furthermore, regulation of superoxide anion release in AM and IM by LPS and/or IFN-gamma was distinct. Taken together, these studies demonstrate that IM are functionally different from other macrophage subpopulations which might reflect their unique location within the lung.
Target ablation by olfactory bulbectomy synchronizes the degenerative cell death of olfactory receptor neurons (ORNs), infiltration of macrophages, and proliferation of progenitor cells, leading to neurogenesis, ORN replacement, and regeneration of the sensory epithelium. Although macrophages participate in the degenerative and regenerative events, little is known of the molecular and cellular mechanisms associated with their recruitment during the earliest period following target ablation. Macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemoattractant protein-1 (MCP-1), which are members of the CC or beta-chemokine subfamily, are chemoattractants for monocytes/macrophages. Shortly after target ablation, the protein and mRNA levels for MIP-1alpha and MCP-1 were up-regulated, showing peak expression levels from 16 hr to 3 days post-OBX; this coincided with the pattern of infiltration of activated F4/80(+) macrophages. The mRNAs for MIP-1alpha and MCP-1, as well as their cognate receptors CCR1 and CCR2, respectively, were localized in resident and infiltrating macrophages in numbers commensurate with those of F4/80-immunopositive macrophages in adjacent tissue sections. The mRNA(+) macrophages were localized within olfactory epithelial compartments that corresponded with their proposed functions associated with phagocytosis, proliferation, and infiltration. Our data support the hypothesis that MIP-1alpha and MCP-1 are chemoattractant chemokines associated with the recruitment of macrophages into the olfactory epithelium shortly after target ablation.
We have examined species differences in the mobilization of arachidonic acid and generation of prostacyclin in non-activated and activated peritoneal macrophages in vitro. Mobilization of 3H-arachidonic acid was reduced in rat activated macrophages compared with that in non-activated macrophages, but a similar difference was not observed in guinea-pig macrophages. In guinea-pig peritoneal macrophages, exposure to formyl-Methionyl-Leucyl-Phenylalanine (fMLP), platelet-activating factor (Paf), zymosan and A23187 increased the generation of prostacyclin. In contrast, in rat peritoneal macrophages, fMLP and Paf did not stimulate the mobilization of arachidonic acid or the generation of prostacyclin, whereas both zymosan and A23187 were effective stimuli. Pretreatment of either rats or guinea-pigs by intraperitoneal injection of C. Parvum reduced prostacyclin generation by peritoneal macrophages in vitro. We conclude that there may be species differences in receptor populations between guinea-pig and rat peritoneal macrophages. However, the reduction in eicosanoid generation induced by the inflammatory stimulus, C. Parvum is not species-dependent.
MA158.2, a rat monoclonal antibody with binding specificity for cells of the monocyte-macrophage lineage, reacts with an antigen (158.2) whose expression is enhanced on mononuclear cells activated to the tumoricidal phenotype by treatment with lymphokine supernatant containing macrophage activating factor (MAF). The functional relevance of enhanced expression of this antigen has been examined in mouse peritoneal macrophages treated with a variety of immunomodulatory agents and assayed for augmented macrophage-mediated defense reactions, including O-2 production, microbicidal, and tumoricidal activity. An interferon-gamma (IFN-gamma) preparation produced by recombinant DNA technology induced a dose-dependent increase in expression of the 158.2 antigen in inflammatory macrophages which was accompanied by acquisition of microbicidal activity against Listeria monocytogenes. However, these cells did not express tumoricidal activity and induction of this property required concomitant exposure to lipopolysaccharide (LPS). Similar results were obtained using macrophages elicited with pyran copolymer. Exposure to LPS alone induced enhanced expression of antigen 158.2 but did not elicit microbicidal activity. Macrophages challenged with IFN-alpha, IFN-beta, MDP, and bestatin did not exhibit increased 158.2 and also failed to acquire tumoricidal activity when treated concomitantly with LPS. Collectively, these data indicate that the MA 158.2 antibody recognizes an antigen expressed by macrophage populations displaying the so-called primed phenotype in which microbicidal activity is expressed but in which induction of tumoricidal activity requires the addition of a second signal such as LPS.
Activation of macrophages either in vivo or in vitro can modulate the capacity to generate and secrete reactive oxygen intermediates including H2O2 and O2-. Thus, the cellular and biochemical components requisite for execution of the respiratory burst must be regulated during the activation process. In the present report, we have examined murine peritoneal macrophages in different stages of activation for their sensitivity to stimulants of respiratory burst known to activate protein kinase c (i.e., phorbol dibutyrate or diacylglycerol). The results demonstrated that more highly activated macrophages showed, in addition to greater magnitude of H2O2 or O2- production, a two- to fourfold greater sensitivity to these stimuli. While more active macrophages also exhibited a higher rate of H2O2 secretion, the time at which secretion was measured did not account for or modulate the heightened sensitivity. The increased sensitivity to stimulation was dependent upon the stage of activation and not on the agent used to elicit the macrophages. Increased sensitivity of the more active macrophage populations was also seen when physiologic stimuli (i.e., insoluble immune complexes or unopsonized zymosan) were used. These findings indicate that macrophage activation for H2O2 secretion modulates the sensitivity to stimulation such that more H2O2 is produced in a shorter time and at a lower concentration of stimulus, thereby heightening the inflammatory response in several independent ways. Because all the stimuli employed in the present study have in common the ability to activate protein kinase c (either directly or indirectly), the data also suggest that this form of macrophage activation may involve, at least in part, modulation of the stimulus-response coupling mechanisms which utilize this enzyme.
Progesterone and estrogen induce uterine epithelial cells to produce macrophage colony stimulating factor (CSF-1), and mouse uterine epithelial cells produce large amounts of CSF-1 during embryo development. The present study demonstrated that estrogen and progesterone each induce uterine CSF-1 gene transcription and translation detectable by Northern blotting and bioassay. Intrauterine CSF-1 production was greater in the presence of both estrogen and progesterone than following exposure to either hormone alone. CSF-1 mRNA was detectable in the uterus throughout the estrous cycle while CSF-1 bioactivity was detected only during proestrus. CSF-1 production was directly related to the presence of macrophages in the uterus. Ovariectomy, which was rapidly followed by a loss of uterine CSF-1 gene transcription, also was followed by a dramatic decrease in the number of uterine macrophages. Both uterine CSF-1 and uterine macrophages were reconstituted in a dose-dependent manner by systemic administration of estrogen or progesterone. High concentrations of circulating estrogen and progesterone increased the number of macrophages in the uterus and increased their accumulation near epithelial surfaces. Similar relationships were observed in the uterus of cycling mice. Macrophages accumulated in the uterus following intraluminal injection of recombinant human CSF-1 to ovariectomized mice, directly demonstrating the ability of CSF-1 to recruit macrophages from peripheral blood into the uterus. These studies demonstrated that estrogen and progesterone stimulation of CSF-1 production by mouse uterine epithelial cells controls recruitment and distribution of macrophages in the uterus during the estrous cycle.
Recent studies in our laboratory demonstrated that fish macrophages produce nitric oxide. To elucidate the mechanisms which regulate nitric oxide production in teleosts, we examined whether macrophage activating factors (MAFs) secreted by mitogen stimulated leukocytes, induced nitric oxide production in a long-term cultured macrophage cell line and in primary cultures of kidney macrophages from the goldfish. The results indicate that both primary and long term cultured goldfish macrophages produce nitric oxide in response to MAF or bacterial lipopolysaccharide (LPS), and co-stimulation with both factors results in a synergistic induction of nitric oxide production. MAF that induced nitric oxide production were present in leukocyte supernatants as early as 24 h after addition of mitogens to cell cultures. The production of MAF was dependent upon the incubation temperature, presence of serum in the culture medium and duration of incubation: maximal MAF activity was detected in 72-96 h supernatants raised in media with serum at 30 degrees C. MAF-induced nitric oxide production by long term cultured macrophages was inhibited by 1000 microM NG-monomethyl-L-arginine or amino-guanidine, indicating an L-arginine-dependent metabolic pathway for the production of the reactive nitrogen intermediates in teleosts. The biochemical events of cytokine induced nitric oxide production by teleost macrophages appear to be similar to those of mammalian macrophages.
A sensitive assay was developed for in vitro evaluation of anti-HIV agents in monocyte-macrophage cells (M/M) (a crucial target of HIV in the body). Monocyte-macrophage cells are usually poorly sensitive to the cytopathic effect induced by HIV. However, when fresh adherent monocyte-macrophage cells are cultured at relatively high density in the presence of macrophage-colony stimulating factor (M-CSF), they undergo cytolysis and die in 2-3 weeks. HIV-mediated cell-killing can thus be assessed with a method based on the reduction of the yellow colored 3-(4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by metabolically active cells to a blue formazan, which can be measured spectrophotometrically. HIV-mediated cytopathic effect of M-CSF-exposed monocyte-macrophage cells was consistently achieved in all experiments performed under the conditions described herein. Anti-HIV activity of zidovudine (AZT) was also comparatively evaluated in M-CSF- and normal monocyte-macrophage cells both using the MTT assay and by measuring HIV-p24 antigen production in supernatants of monocyte-macrophage cells cultures, and similar results obtained with both methods. These results support the use of this colorimetric assay for broad screening of anti-HIV agents in monocyte-macrophage cells.
Peripheral blood monocyte derived macrophages obtained from breast cancer patients are non-cytotoxic towards human tumor cells. However, when indomethacin, a prostaglandin synthetase inhibitor, was added to the macrophage tumor cell incubation mixture, the breast cancer patient's macrophages became capable of killing the tumor cells. Furthermore, the macrophage preparations obtained from breast cancer patients demonstrated a 64% increase in the secretion of PGE2, when compared with macrophages obtained from normal donors. It is already known that prostaglandin can inhibit both natural lymphocyte cytotoxicity and antibody dependent cell-mediated cytotoxicity against human tumor cells in vitro. These results indicate that increased synthesis of prostaglandin can also inhibit macrophage mediated cytotoxicity. Therefore, it is possible that manipulation of this mechanism might enhance the effectiveness of the macrophage response and should be a consideration in assessing macrophage-tumor cell interaction in vitro and in vivo.
Macrophage activation factors (MAF), induced maximal priming of the respiratory burst response in GMCL after 6 h of stimulus, but by 24 or 48 h no priming effect was observed. Bacterial lipopolysaccharide (LPS) also primed the respiratory burst of goldfish macrophages, but the kinetics of priming were different from that induced by MAF. LPS induced a gradual increase in priming potential over 48 h of cultivation. Co-stimulation of macrophages with MAF and LPS resulted in enhanced priming of respiratory burst activity compared to either factor alone; however, the kinetics of priming were similar to those induced by MAF only. The MAF antagonized the ability of LPS to prime the respiratory burst over extended cultivation. The priming kinetics of the respiratory burst induced by MAF and/or LPS were not unique to GMCL, but were also similar for primary cultures of IVDKM. Respiratory burst deactivated macrophages-mounted potent nitric oxide response, indicating that this deactivation event was selective for respiratory burst activity. Autocrine factors produced by MAF-activated macrophages augmented priming of the respiratory burst, suggesting that deactivation of primed respiratory burst responses was not due to cytokine mediators produced by activated macrophages, but was most likely an intracellular deactivation event. Furthermore, production of reactive intermediates by activated fish macrophages was biphasic; with maximal ROI production occurring 6 h after stimulus, and maximal RNI occurring 72 h after stimulus. Our results indicate that activated fish macrophages mount sequential antimicrobial responses that are selectively deprogrammed once maximal induction has occurred. The ability to selectively deactivate ROI production without affecting subsequent RNI production may play an important role in host defense: regulating the duration of ROI production, and thus minimizing host tissue damage in an otherwise futile attempt to eliminate ROI resistant pathogens.
UNLABELLED: We are interested in identifying a suitable model for investigating mycobacteria interactions with alveolar macrophages. MH-S, a murine alveolar macrophage cell line, is a possible candidate. OBJECTIVE: To compare the receptor mediated interactions of mycobacteria with primary murine macrophages and MH-S. DESIGN: The association of MH-S monolayers with Mycobacterium tuberculosis (MTB) and other defined particles was compared to that of resident Day 1 peritoneal macrophage (PM) and Day 4 alveolar macrophage (AM) monolayers. RESULTS: In the absence of serum, the association of MTB with MH-S was comparable to that of AM, with approximately 35% of each macrophage type binding at least one bacterium. In contrast, almost 80% of PM bound at least one bacterium. MTB binding was enhanced for all macrophage types by a heat-labile component of normal mouse serum. Antibodies recognising CR3 inhibited the serum-mediated enhanced binding of MTB by MH-S. Binding of latex, immunoglobulin coated or complement coated SRBC by MH-S, AM and PM was comparable. Binding of zymosan by MH-S was greatly inferior to AM and PM. CONCLUSION: The receptor expression and particle binding properties of MH-S are similar to AM in many, but not all, ways. MH-S, therefore, has the potential to be used as a model for investigating MTB-macrophage interactions.