Macrophage colony-stimulating factor enhances the expression of Fc receptors on murine peritoneal macrophages

Experiments were performed to test the postulate that macrophage colony-stimulating factor (M-CSF) modulates the expression of specific membrane structures on mature murine macrophages. Resident peritoneal macrophages were incubated with M-CSF for 48-72 hr and analysed for Fc receptor and Ia antigen expression. M-CSF treatment of macrophages increased the expression of Fc receptors two- to three-fold over that of unstimulated macrophages. The effect was detected at 1 U/ml of M-CSF, with maximal expression between 100 and 500 U/ml. The specificity of the enhancement was indicated by two sets of experiments. Purified rabbit anti-M-CSF IgG, but not normal rabbit IgG, inhibited the M-CSF-mediated Fc receptor enhancement. Also, a highly purified M-CSF preparation, which was essentially free of endotoxin and interferon, was active in these assays. M-CSF augmented both major types of IgG Fc receptors, FcR I (recognizing IgG 2a) and FcR II (recognizing IgG 2b/IgG1). A second membrane marker, the Ia antigen, was induced by recombinant IFN-gamma (rIFN-gamma) but not by M-CSF. These results indicate that M-CSF is an inducer of two classes of IgG Fc receptors on mature tissue macrophages.     

Evidence for two distinct Fc receptors on guinea pig peritoneal macrophages

Fcreceptors of guinea pig peritoneal macrophages for homologous IgG1 and IgG2 antibodies were found to be solubilized by treatment of the cells with Nonidet P-40, since the soluble fraction obtained inhibited the binding reactions of complexes of IgG1 and IgG2 antibodies with ovalbumin to the intact cells. The solubilized Fc receptors for IgG1 and IgG2 antibodies were indistinguishable from one another by means of gel filtration on a column of Sepharose 6B in the presence or absence of the detergent. Both of the Fc receptors were almost completely removed by affinity chromatography on a column of IgG2-bound Sepharose 6B. Affinity chromatography on a column of IgG1-bound Sepharose 6B, however, showed that the Fc receptor for IgG1 antibody was also almost completely adsorbed, though about 50% of the Fc receptor for IgG2 antibody remained unbound. These results demonstrate the existence of two distinct Fc receptors on guinea pig macrophages; one binds to IgG2 antibody alone and the other bind IgG2 as well as IgG1 antibodies.     

The expression of Fc receptors on guinea-pig peritoneal macrophages and neutrophils.

The equilibrium binding of soluble complexes of guinea-pig anti-dinitrophenyl IgG1 or IgG2 and dinitrophenylated bovine serum albumin (DNPBSA) to homologous peritoneal macrophages and neutrophils has been compared. The immunoglobulin receptors on these phagocytes were found to differ in two major respects. Macrophages express specific binding activity for both IgG1 and IgG2 complexes whereas neutrophils possess specificity only for the IgG2 subclass. Furthermore, the number of receptors for IgG2 on macrophages (0.8-1 x 10(6)) is fifty- to seventy-fold greater than the number on neutrophils (1.3-2.6 x 10(4)). The phagocytes also displayed differences in their avidity for soluble IgG2-containing complexes which could either reflect the disparity in receptor densities on their membranes or indicate differences in the structure of their Fc receptors. Inhibition of complex binding by immunoglobulin fragments indicated that, at least, the macrophages and neutrophils recognize the same portion of the IgG2 molecule.     

The effects of malnutrition on murine peritoneal macrophages

Differences were detected between peritoneal macrophages (both resident and elicited) from mice on a low protein diet and from normal animals. The concentration of resident peritoneal macrophages was lower in animals on low protein diets than in normal controls. Although total protein (and therefore cell mass) of resident macrophages from malnourished mice was increased, their contents of thiamine pyrophosphatase, succinate dehydrogenase, and non-specific esterase were disproportionately reduced. In addition they did not ingest as many glutaraldehyde-fixed sheep erythrocytes or attach to as many adherent C3b sensitized sheep red blood cells as those from normal animals, although reduction of nitroblue tetrazolium was unaffected. Initially (24 hr after thioglycollate), elicited macrophages from malnourished mice did not divide as frequently as those from normal mice but by 48 hr the differences were insignificant. The elicited macrophage possessed lower levels of total protein (indicating a reduced cell mass); the levels of acid phosphatase, thiamine pyrophosphatase, succinate dehydrogenase, and nonspecific esterase and nitroblue reducing activity were also proportionately reduced. They ingested fewer glutaraldehyde-fixed erythrocytes and reacted with fewer C3b sensitised sheep red blood cells than those from normal mice; ingestion of IgG-coated sheep erythrocytes, on the other hand, was somewhat increased. These abnormalities may influence adversely the efficiency of early phlogistic responses and favor the establishment of infection in malnourished animals.     

IgG-binding sites on macrophage cell membrane. I. Identification of two distinct Fc receptors on mouse peritoneal macrophages

Evidence is presented concerning the existence on mouse peritoneal macrophages of two separate and distinct Fc receptors, one for cytophilic monomeric IgG (mIgG) and the other for polymeric IgG. The latter Fc receptor recognizes both heat-aggregated IgG and antigen-complexed IgG. The major findings of our studies are: (a) the different susceptibility of the two Fc receptor types by pronase, trypsin or phospholipase C; (b) the independent modulation of these two binding sites on the cell membrane; (c) the inability of mIgG to inhibit the binding of particulate antigen-complexed IgG ligand; (d) the ability of mIgG molecules which are devoid of the cytophilic property to attach to the macrophage surface upon their polymerization induced by heating or antigen. The results are discussed in terms of "cytophilic" and "opsonic" Fc receptor types which may provide different functional abilities for normal macrophages.     

Binding properties and expression of the Fc gamma receptors on guinea pig peritoneal macrophages treated with inhibitors of glycosylation.

The effect of inhibition of glycosylation in guinea pig peritoneal macrophages on interaction of their surface Fc gamma receptors with guinea pig and rabbit IgG was studied. The inhibitors used were tunicamycin and monensin. The cells treated with tunicamycin incorporated markedly less [3H]mannose, bound less peanut (PNA) and wheat germ (WGA) lectins and showed diminished ability of binding IgG in comparison with control cells. Treatment of the cells with monensin resulted in an increased incorporation of [3H]mannose, increased binding of PNA but a decreased binding of WGA. Monensin affected binding of guinea pig IgG2 and rabbit IgG to macrophages and had no effect on guinea pig IgG1 binding. Analysis of binding parameters showed that although the number of IgG-binding sites on treated cells was significantly lower, especially in the case of tunicamycin, the apparent association constants were 2-4 times higher than in control cells. The effect of tunicamycin and monensin on parameters of binding of guinea pig IgG2 or rabbit immunoglobulins was much more pronounced than in the case of binding guinea pig IgG1. The results showed that glycosylation modulates expression and IgG binding ability of the Fc gamma receptors on the surface of guinea pig peritoneal macrophages.     

Functional heterogeneity of macrophages: subclasses of peritoneal macrophages with different antigen-binding activities and immune complex receptors

Cell suspensions prepared from 24-hour cultures of adherent peritoneal exudate cells from rabbits were separated into density subclasses on discontinuous gradients of Ficoll. Of the five subclasses obtained, macrophages comprised over 95 per cent of the cells in the two least dense subclasses and over 90 per cent of the cells in the subclasses of intermediate density. The most dense subclass contained approximately 80 per cent macrophages and 20 per cent lymphocytes.The antigen-binding properties of the subclasses were studied with a selected number of ¹²⁵I-labelled antigens in the presence and absence of added antibodies. In the absence of antibody the subclasses differed from one another in antigen-binding activities; these differences were independent of both the test antigens and the state of antigen aggregation. In the presence of specific antibody, two macrophage subclasses bound substantially more antigen than the other subclasses. The enhanced responses of these subclasses were confirmed by studying the cells'' capacity to form rosettes when incubated with sensitized chicken erythrocytes. The results indicated that macrophages in these two subclasses differed quantitatively and/or qualitatively from other macrophage subclasses with regard to immunoglobulin receptor sites.These studies demonstrate the feasibility of using gradient separation procedures to obtain functionally distinct subclasses of cells rich in macrophages. The availability of well-defined macrophage populations should permit more precise studies of macrophage functions in immunity.     

Characterization of Fc receptors for IgE on human alveolar macrophages.

Human alveolar macrophages (aM phi) isolated from lung lavages performed during bronchoscopy and after surgical removal of pulmonary lobes were analysed for Fc receptors for IgE (Fc epsilon R) and IgG (Fc gamma R) by rosette assays. A mean+/-s.d. of 8.0+/-2.6% of aM phi formed rosettes with fixed ox erythrocytes coated with an IgE myeloma protein (Eo''-IgE). The Eo''-IgE rosettes were inhibited by two IgE myeloma proteins and by IgE Fc fragments but not by myeloma proteins of the other Ig classes or by IgE denatured by heating or reduction and alkylation. Fresh ox erythrocytes sensitized with rabbit IgG antibodies (EoA) formed rosettes with 64.1+/-20.3% of the aM phi. Peripheral blood monocytes formed 10.6+/-1.2% Eo''-IgE and 90.2+/-6.0% EoA rosettes. Incubation of the aM phi with a goat antiserum to human lymphocyte Fc epsilon R inhibited Eo''-IgE rosette formation on aM phi by 80% but did not affect the percentage of EoA rosettes. The antiserum also inhibited Eo''-IgE rosettes formed by peripheral blood monocytes and cultured macrophage-like U937 cells but not those formed by basophilic granulocytes obtained from a patient with chronic myelogenous leukaemia. There was no relationship between age, sex, diagnosis or smoking history of the patients and the percentage of aM phi forming Eo''-IgE rosettes. These studies demonstrate that a subpopulation of human aM phi bear Fc epsilon R that share antigenic determinants with Fc epsilon R on lymphocytes and monocytes. Fc epsilon R(+) aM phi may play an important role in allergic and inflammatory pulmonary diseases by inducing the release of mediators of inflammation after interaction with IgE immune complexes.     

Activation of murine peritoneal macrophages by Salmonella typhimurium

The results of these studies indicate that the interaction between activated macrophages and S. typhimurium depends on the characteristics of the micro-organism and the kind of activation.     

Expression of IgA and IgM Fc receptors on murine T lymphocytes

Fc receptors are induced on T cells following activation via the TCR. T cells that express Fc receptors transiently have the ability to use two different cognate systems: the TCR and immunoglobulins bound to the Fc receptors. The studies discussed in this article are focused on the Fc alpha and Fc mu receptors that can be induced on certain subsets of murine T lymphocytes. The article emphasizes the role of the T cell receptor for antigen in the expression of Fc alpha and Fc mu receptors on murine T cells and reviews experimental observations that suggest significant molecular heterogeneity of these Fc receptors. The finding that regulation of expression of Fc alpha receptors and Fc mu receptors on T lymphocytes is linked to cellular activation via the CD3/TCR complex implies that these Fc receptors might mediate important functions in the biology and pathology of T cells.     

Trypanosoma cruzi-induced tyrosine phosphorylation in murine peritoneal macrophages

 Trypanosoma cruzi, the etiological agent of Chagas’disease, binds to and invades macrophages and other cells (fibroblasts, muscle cells) via a complicated set of interactions, but the changes induced by parasite-to-cell interactions are largely unknown. This report investigates the ability of T. cruzi to elicit a tyrosine kinase pathway in immature and mature resident murine peritoneal macrophages (MPM) that differ in their susceptibility to parasite infection. T. cruzi stimulated the phosphorylation of tyrosine residues in several endogenous substrates (proteins of 40–42, 53–56, 66, 75, 80, 90, 95, 100, and 112 kDa), but only in immature MPM. Mature MPM had high levels of spontaneous tyrosine phosphorylation. Upstream tyrosine kinases, such as src-like tyrosine kinases, were not responsible for the differential patterns of tyrosine phosphorylation since they were present in both mature and immature MPM. We suggest that the tyrosinephosphorylation patterns stimulated by T. cruzi reflect most of the biochemical events that occur in parasite host-cell interactions. Received: 20 October 1995 / Accepted: 23 January 1996     

Murine peritoneal macrophages support murine cytomegalovirus replication.

Because there have been different conclusions regarding the susceptibility of murine macrophages to murine cytomegalovirus (MCMV) infection and replication, we have undertaken a detailed comparison of MCMV infection of macrophages with that of a permissive cell line, mouse embryo cells. Although both cell lines undergo productive infections with MCMV, there are marked differences in certain aspects of the viral replication which may account for some of the different conclusions regarding the MCMV cycle in macrophages. Although both cell lines produce MCMV after infection, the time course of the infection differed markedly between the cell types. Similarly, the proportion of infected macrophages that are releasing infection virions is much smaller than the proportion of a comparably infected mouse embryo cell culture. Tissue culture passage of MCMV first enhanced (after one passage) and then reduced the infectivity of the virus for macrophages in vitro. The delayed time course and lesser production at early intervals after infection of macrophage cultures could not be attributed to demonstrable inhibitors or to replication in contaminating fibroblasts in the macrophage cultures.     

Regulation of Fc mu receptor-mediated functions of resident and provoked peritoneal macrophages

Modifying and/or regulating effects on Fc mu receptor (R) mediated phagocytic and ADCC activity of both resident (r) and provoked (p) peritoneal macrophages (PM) was studied applying drugs affecting the cytoskeleton and cation transport. In addition, the effects of exogenous PGE2 and cyclic nucleotides were also examined. Fc mu-receptors appear to be functionally less significant in provoked PMs than in resident ones since both Fc mu-receptor mediated phagocytosis and ADCC were lower in the formers. The cytoskeletal system is important in the regulation of both Fc mu-receptor-mediated functions. Phagocytosis through Fc mu-receptor is decreased by Cytochalasin B and by Vinblastine treatment, whereas ADCC is enhanced by Cytochalasin B. Extracellular PGE2 and cAMP induced a higher phagocytic activity and suppressed ADCC, whereas cGMP displayed an opposite effect. The sensitivity of Fc mu R-mediated activities to ionophoric and to cytoskeleton-damaging drugs was lower in provoked than in resident PMs. In addition, the regulatory role of PGE2 and of cyclic nucleotides on the same activities was less marked on provoked PMs. In contrast, ouabain inhibits Fc mu R-dependent antigen incorporation and ADCC on provoked PM monolayers only. These findings suggest differing regulatory mechanisms for Fc mu R-mediated functions in provoked PMs as compared with resident ones.     

Low affinity Fc gamma receptors on murine macrophages: mitogen-activated protein kinase activation and AP-1 DNA binding activity.

Mouse macrophage cell lines such as J774 express Fc receptors for IgG2a immune complexes, which upon binding of the proper ligand, trigger several signal transduction pathways. A surface to nucleus signaling through these receptors has been demonstrated. We describe here the activation of the mitogen-activated protein kinase (MAPK) and an increase in the binding of the activator protein 1 (AP-1) to DNA upon receptor stimulation. The described effects are only partially blocked by inhibitors of the Ca2+/diacylglycerol-dependent protein kinase (PKC), suggesting that differential signaling pathways are activated upon receptor cross-linking and that they converge at or above the MAPK level. These results pave the way to our understanding of Fc gammaR cross-linking induced gene expression regulation.     

Functional alterations of murine peritoneal macrophages during pregnancy.

We have studied some functional characteristics of murine peritoneal macrophages (MOp) related to hormonal changes produced during pregnancy. Maximal expression of Ia antigen and release of interleukin 1 (IL1) were observed during the first week of pregnancy (implantation), when the highest peak of estradiol was produced. Both Ia antigen expression and IL1 levels progressively decreased as gestation advanced. Inversely, MOp capability to phagocyte and reduce nitro blue tetrazolium (NBT) was diminished at the beginning of pregnancy but returned to normal values in the last week. Sexual steroid levels (estradiol and progesterone) were inversely related, and the release of prostaglandin E2 (PGE2) by MOp decreased when progesterone levels increased. Although PGE2 production had no evident relation with Ia antigen expression and IL1 activity during the first and second weeks of pregnancy, the increment in PGE2 values and percentages of NBT+ cells could indicate a different stage of macrophage activation. These results suggest a possible hormonal regulation of macrophage functionality.     

人参皂苷Rg1对小鼠腹腔巨噬细胞的影响

目的研究人参皂苷Rg1对小鼠腹腔巨噬细胞的影响,并探讨其作用机制。方法无菌分离小鼠腹腔巨噬细胞,制备单细胞悬液,加入不同终浓度的人参皂苷Rg1 4 h后,细菌脂多糖LPS(10μg/ml)作用24 h,直径1μm的微球(1×1010/L)结合流式细胞术分析Rg1对小鼠腹腔巨噬细胞吞噬作用的影响;Griess试剂盒检测NO的释放;H2DCFDA染色检测ROS的含量;Fluo-4/AM染色检测Rg1对Ca2+超载的影响;Sytox R Green染色,荧光酶标仪检测Rg1对CHX、CTX诱导的细胞凋亡的影响。结果 10、20μmol/L的Rg1时能明显抑制LPS诱导的巨噬细胞NO和ROS的产生以及吞噬微球的能力;10μmol/L的Rg1能显著抑制Ion诱导的的巨噬细胞Ca2+的超载以及CHX、CTX诱导的细胞的凋亡。结论 Rg1具有显著的抗炎作用,并可抵抗多种因素诱导的细胞凋亡,为进一步开发为免疫治疗药物提供了依据。     

Effect of rat surfactant lipids on complement and Fc receptors of macrophages.

Murine resident alveolar macrophages have very low numbers of complement receptors detectable by rosetting with erythrocyte complexes. These macrophages are relatively inactive in tests of complement- or antibody-mediated bacterial phagocytosis in vitro. It is not known whether these characteristics are intrinsic or are environmentally modulated. We found previously that rat bronchoalveolar lavage fluid or isolated rat surfactant lipid causes a unique and marked reduction in the ability of peritoneal macrophages to form rosettes with immunoglobulin G or complement containing erythrocyte complexes in vitro. In this study the antirosetting activity of rat surfactant was found to be due to its neutral lipid component and, specifically, to the free fatty acid (FFA) fraction of the neutral lipids. Rat surfactant contained a higher level of FFA than has been reported for canine, guinea pig, or human surfactant. Studies with pure FFA showed that activity in blocking macrophage Fc and complement receptors correlated with increasing unsaturation and chain length. A mixture of eight commercially available FFAs, representing the most abundant FFA in rat surfactant and consisting mostly of saturated FFA, had much less effect on rat macrophage receptors than the naturally occurring FFA mixture. These findings suggest that long-chain-unsaturated FFAs in rat surfactant are the most important for the antireceptor activity. The antireceptor activity of rat surfactant or FFA on peritoneal macrophage receptors in vitro and known intrinsic properties of murine alveolar macrophages could not be precisely correlated, suggesting that impaired binding of immune complexes by murine alveolar macrophages and a high level of FFAs in rat surfactant may be independent rather than causally related phenomena.     

Factors mediating lipopolysaccharide-induced ganglioside expression in murine peritoneal macrophages

The ganglioside composition of endotoxin-responsive C3H/HeN murine peritoneal macrophages is known to undergo dramatic changes in vivo in response to intraperitoneal lipopolysaccharides (LPS), unlike endotoxin-hyporesponsive C3H/HeJ macrophages. To better investigate the mechanism behind LPS-induced macrophage ganglioside changes, resident C3H/HeN peritoneal macrophages were treated in vitro with 0.1-1.0 micrograms/ml LPS for 6-96 hr, but showed no differences in membrane ganglioside patterns. Coincubation of macrophages with lymphocytes and treating with LPS again elicited no ganglioside changes. In contrast, interferon gamma (IFN-gamma)-primed macrophages showed a dramatic shift in intensity of one ganglioside when treated with LPS in vitro; an additional macrophage ganglioside appeared when IFN-gamma-primed, LPS-treated macrophages were coincubated with lymphocytes. Ganglioside expression induced in vitro still did not approach the complex changes seen in vivo. However, transplanting C3H/HeN macrophages intraperitoneally into C3H/HeJ mice, followed by administration of intraperitoneal LPS, did reveal striking changes in ganglioside expression that resembled the pattern seen in vivo. Thus, LPS alone does not provide the necessary direct signal to promote macrophage ganglioside change even though it alters macrophage function. IFN-gamma appears to be one important mediator; however, complex interactions involving other cytokines or migration of independent populations of mononuclear cells may be required for the full manifestation of LPS-induced ganglioside expression in macrophages.