Peritoneal macrophages exposed to purified macrophage colony-stimulating factor (M-CSF) suppress mitogen- and antigen-stimulated lymphocyte proliferation

The effect of M-CSF-exposed macrophages on murine splenic lymphocyte responses was determined. Resident peritoneal macrophages incubated with purified M-CSF for 48 hr inhibited lymphocyte proliferation to Con A, PHA, and listerial antigen as determined by [3H]TdR uptake, and inhibited Con A-stimulated lymphocyte IL 2 production. The inhibition was similar to that observed with macrophages from BCG-infected mice. Maximal suppression occurred at M-CSF concentrations of 500 U/ml or greater and when the incubation time with M-CSF was 48 hr or more. M-CSF effect was specific because rabbit anti-M-CSF IgG blocked the suppression whereas control rabbit IgG did not. Secretory products of macrophages could not be implicated in this interaction. Catalase and indomethacin, alone or together, did not reverse the inhibition. In addition, putative suppressive factors were not detected in supernatants of M-CSF-stimulated macrophages. Lymphocytes that were removed from macrophage monolayers and were recultured in medium plus Con A were able to proliferate. Macrophages stimulated by M-CSF therefore appear to have inhibitory activity for proliferating lymphocytes, and may play a role in immunoregulatory mechanisms.     

Lipopolysaccharide inhibits the binding of colony-stimulating factor (CSF-1) to murine peritoneal exudate macrophages

These studies demonstrate the potent effect of bacterial endotoxin (LPS) on the inhibition of iodinated colony-stimulating factor- (125I-CSF-1) binding by murine peritoneal exudate macrophages (PEM) from C3H/An and C57BL/6 mice. As small an amount as 0.1 ng/ml LPS is sufficient to cause a significant inhibitory effect; this effect is temperature-, time- and concentration-dependent. LPS, however, causes minimal or no inhibition of 125I-CSF-1-binding by PEM from LPS-resistant C3H/HeJ mice. Inhibition of 125I-CSF-1-binding does not appear to be a result of a direct occupancy by LPS of CSF-1 receptors present on the cell membrane and is most likely due to a progressive loss of available CSF-1-binding sites. The effect can be neutralized by the addition of the antibiotic polymyxin B, which binds to the lipid A portion of LPS. The action of LPS on PEM is transient; treated cells recover their 125I-CSF-1-binding activity whether or not LPS is later removed. The restoration of 125I-CSF-1-binding activity can be blocked completely by the addition of cyclohexamide. These findings suggest the rapid, LPS-induced disappearance of CSF-1 receptors from the cell surface may be related to the activation of macrophages by LPS.     

Structure-function studies on human macrophage colony-stimulating factor (M-CSF)

M-CSF (CSF-1) can be produced in a variety of structural forms that may affect function in vivo. Truncated, nonglycosylated forms of recombinant M-CSF (rM-CSF) from E. coli have been refolded in vitro in high yield and shown to be functionally equivalent in vitro to glycosylated rM-CSF secreted from mammalian cells. An N-terminal domain of 149 amino acids is produced by all of the known M-CSF mRNA splice variants and is the region responsible for bioactivity observed in vitro. Heterodimeric rM-CSFs from different splice variants containing this domain were produced in pure form by refolding in vitro, and are fully active, but have yet to be observed in vivo. The circulating half-life of truncated M-CSF forms injected intravenously into rats increased with the MW of the M-CSF used. Large increases in half-life in vivo were observed following chemical addition of a single molecule of 10 kD polyethylene glycol to rM-CSF in vitro. The crystal structure of rM-CSF revealed that M-CSF is a member of a family of molecules related by having a distinctive four-helical-bundle structural core. Site-directed mutagenesis showed that residues in or near helix A and helix C are involved in receptor binding, as reflected by decreased bioactivity and receptor binding of certain mutants. A soluble form of the M-CSF receptor, c-fms, was produced in a baculovirus/Sf9 expression system and purified to homogeneity. The MW of rM-CSF saturated with this soluble receptor was determined by molecular sieve chromatography and light scattering. Each dimeric M-CSF molecule appears to bind two soluble receptor molecules in vitro, supporting the observation that M-CSF signaling is linked to receptor dimerization. Mol Reprod Dev 46:31–38, 1997. © 1997 Wiley-Liss, Inc.     

Macrophage colony-stimulating factor (CSF-1) induces pro-inflammatory gene expression and enhances antimicrobial responses of goldfish (Carassius auratus L.) macrophages

We report on the regulation of pro-inflammatory functions of goldfish macrophages and induction of gene expression by recombinant goldfish CSF-1 (rgCSF-1). Recombinant goldfish TNFα-2 (rg TNFα-2), rgIFNγ but not rgTGFβ induced time-dependent increase of CSF-1 expression in macrophages. Treatment of goldfish macrophages with rgCSF-1 increased expression of several immune genes including CXCL-8 (= IL-8), CCL-1, TNFα-1, TNFα-2, IL-1β-1, IL-1β-2, IL-12-p35, IL-12-p40, IFN, IL-10 and iNOS A and B. The rgCSF-1 treatment did not significantly alter the mRNA levels of TGFβ and NRAMP in macrophages up to 48 h post treatment. However, at 72 h post treatment, the expression of TGFβ increased whereas that of NRAMP decreased. The treatment of macrophages with rgCSF-1 enhanced their respiratory burst and nitric oxide responses that were abrogated after addition of soluble CSF-1 receptor (sCSF-1R) to cell cultures. Macrophages exhibited a concentration-dependent chemotactic response toward rgCSF-1 as well as an increase in phagocytic activity that was abrogated after addition of sCSF-1R to cell cultures. Our results indicate that in addition to being an important growth factor of goldfish macrophages, rgCSF-1 also plays a central role in the regulation of their pro-inflammatory responses.     

Tumor-promoting phorbol esters inhibit the binding of colony-stimulating factor (CSF-1) to murine peritoneal exudate macrophages

L-cell colony-stimulating factor (CSF-1) is a sialoglycoprotein of molecular weight 70,000 daltons that specifically stimulates macrophage colony formation by single committed cells from normal mouse bone marrow and by various classes of more differentiated tissue-derived mononuclear phagocyte colony-forming cells (Stanley et al., 1978). CSF-1 interacts with target cells by direct and specific binding to membrane receptors (CSF-1 receptors) that are present only on cells of the mononuclear phagocyte series and their precursors. We studied the effect of tumor-promoting phorbol esters on the binding of 125I-labeled CSF-1 (125I-CSF-1) to murine peritoneal exudate macrophages (PEM). Biologically active TPA (12-O-tetradecanoyl phorbol-13-acetate) inhibits the binding of 125I-CSF-1 to its receptor on PEM. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, maximum inhibition occurred at about 10(-7) M; inhibition was 50% at 5 X 10(-9) M. At 0 degrees C, the inhibitory activity of TPA is diminished. The action of TPA on PEM is transient. Treated cells recover their 125I-CSF-1-binding activity whether TPA is later removed or not. The process of recovering CSF-1-binding activity is completely blocked by the addition of cycloheximide. When several phorbol derivatives were tested for their inhibitory activities, only biologically active phorbol esters were found to possess such activities. Furthermore, the inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the total number of available CSF-1 receptors rather than a decrease in receptor affinity.     

Macrophage colony-stimulating factor antagonists inhibit replication of HIV-1 in human macrophages

Macrophages infected with HIV-1 produce high levels of M-CSF and macrophage-inflammatory protein-1alpha (MIP-1alpha). M-CSF facilitates the growth and differentiation of macrophages, while the chemotactic properties of MIP-1alpha attract both T lymphocytes and macrophages to the site of HIV infection. Studies described in this work indicate M-CSF may function in an autocrine/paracrine manner to sustain HIV replication, and data suggest possible therapeutic strategies for decreasing viral load following HIV infection. We show that macrophage infection with measles virus or respiratory syncytial virus, in contrast to HIV-1, results in production of MIP-1alpha, but not M-CSF. Thus, M-CSF appears to be specifically produced upon infection of macrophages with HIV-1. Furthermore, addition of M-CSF antagonists to HIV-1-infected macrophages, including anti-M-CSF monoclonal or polyclonal Abs or soluble M-CSF receptors, dramatically inhibited HIV-1 replication and reduced production of MIP-1alpha. Our results suggest that biologic antagonists for M-CSF may represent novel strategies for inhibiting the spread of HIV-1 by 1) blocking virus replication in macrophages, 2) reducing recruitment of HIV-susceptible T cells and macrophages by MIP-1alpha, and 3) preventing the establishment and maintenance of infected macrophages as a reservoir for HIV.     

Thrombin binds to murine bone marrow-derived macrophages and enhances colony-stimulating factor-1-driven mitogenesis

The binding and mitogenic properties of thrombin have been established in various transformed cell lines. In such systems, thrombin induces cell division in the absence of exogenous growth factors, and the enzyme is considered to act directly as a mitogen. This study explores thrombin's interaction with nontransformed, growth factor-dependent cells. Binding of 125I-alpha-thrombin to colony-stimulating factor (CSF)-1-dependent bone marrow-derived macrophages is saturable, time-dependent, and displaceable by both unlabeled alpha-thrombin, and esterolytically inactive thrombin. Both dissociation studies of pre-bound radio-labeled thrombin and Scatchard analysis assisted by the program "Ligand" suggest adherence of thrombin-binding data to a multi-site model. There are an estimated 2 x 10(4) high affinity sites (Kd = 7 x 10(-9)M) and 2 x 10(6) low affinity sites (Kd = 9 x 10(-7)M) per cell. Quiescent bone marrow-derived macrophages were cultured with either 10(-8)M thrombin, 1000 units of CSF-1/ml, or both and [3H]thymidine incorporation was determined. Thrombin alone did not induce mitogenesis. CSF-1 induced mitogenesis with peak [3H] thymidine incorporation occurring 24 h after addition of the mitogen. This CSF-1-dependent mitogenic influence was enhanced greater than 2-fold by treatment with thrombin.     

Interferon-induced inhibition of receptor-mediated endocytosis of colony-stimulating factor (CSF-1) by murine peritoneal exudate macrophages

Apart from its characteristic antiviral activity, interferon (IFN) also exerts a variety of biologic effects on macrophages. We have studied the effect of IFN on the expression of the colony-stimulating factor receptors (CSF-1 receptors) by murine peritoneal exudate macrophages (PEM). At 37 degrees C, murine IFN decreased the expression of the CSF-1 receptor activity in a time- and dose-dependent fashion by PEM from both endotoxin-sensitive (C3H/Sn) and endotoxin-resistant strains (C3H/HeJ) of mice. Scatchard analysis from the binding data suggests that the decreased expression of CSF-1 receptors is a result of decreased number of receptors rather than a decreased binding affinity. When IFN was incubated with anti-IFN before the addition to cultures, the effect was completely abolished indicating that this activity resides in the same molecules as IFN. The suppressed CSF-1 receptor activity on PEM by IFN appeared to be stable. Removal of added IFN never resulted in a full recovery of CSF-1 binding activity by PEM even after prolonged incubation (7 days). IFN also inhibited the receptor-mediated uptake and utilization of CSF-1 molecules by treated cells, which appeared to be a direct effect of the decreased number of CSF-1 receptors. Treatment of PEM with dexamethasone, prostaglandin, transferrin, insulin, or dibutyryl cAMP failed to suppress both the expression of CSF-1 receptors and CSF-1 utilization by PEM. These studies suggest that IFN may play a role in the regulation of both macrophage production and differentiation via the modulation of specific membrane receptors and inhibition of receptor-mediated CSF-1 endocytosis.     

Macrophage colony stimulating factor (M-CSF) is essential for osteoclast formation in vitro

The op/op mouse, in which the M-CSF gene is mutated, has greatly reduced numbers of macrophages and osteoclasts. We assessed the ability of M-CSF to induce osteoclast and macrophage formation in op/op hemopoietic cells in vitro. Osteoclast production was undetectable in op/op cell cultures, but was restored by M-CSF at concentrations approximately an order of magnitude higher than those that induced macrophages. In normal hemopoietic tissue M-CSF similarly increased macrophage numbers, but inhibited osteoclast formation. Despite cure of the macrophage defect, neither interleukin 3 nor granulocyte-macrophage CSF were able to induce osteoclastic differentiation in op/op cells. The results suggest that M-CSF induces osteoclastic differentiation but that macrophages, which are also induced by M-CSF, suppress osteoclast differentiation. Macrophages induced by other cytokines seem unable to contribute to osteoclast-formation.     

Solubilization and assay of a colony-stimulating factor receptor from murine macrophages

The colony-stimulating factor, CSF-1, selectively stimulates the survival, proliferation, and differentiation of mononuclear phagocytes. The solubilization, assay, and characteristics of the CSF-1 receptor from the J774.2 murine macrophage cell line are described. The recovery of cell-surface receptor in the postnuclear supernatant membrane fraction of hypotonically disrupted cells was 76%. Recovery of the ligand binding activity of the receptor after solubilization of this fraction with 1% Triton X-100 was approximately 150%. The binding of 125I-CSF-1 to intact cells and membrane preparations was consistent with the existence of a single class of high-affinity receptor sites. In contrast, the equilibrium binding of 125I-CSF-1 to the solubilized postnuclear fraction indicated the existence of two distinct classes of binding site (apparent Kds 0.15 nM and 10 nM). A rapid assay was developed for the high-affinity sites, which were shown to be associated with the CSF-1 receptor. The function of the low-affinity sites, which have not been demonstrated on intact cells or cell membranes and which are 13 times more abundant than the high-affinity sites, is unknown. The solubilized high-affinity receptor-CSF-1 complex was stable on storage at 0 degrees C and -70 degrees C but dissociated at 37 degrees C. Dissociation also occurred at 0 degrees C in buffers of low pH (4.0) or high ionic strength (0.7 M NaCl).     

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.     

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.     

Effects of granulocyte-macrophage colony-stimulating factor and colony-stimulating factor-1 on the proliferation and differentiation of murine alveolar macrophages

Murine alveolar macrophages (AM) were shown to have proliferative ability and to form colonies in vitro. The factors in lung-conditioned medium (CM) and L929-CM which stimulate the proliferation of AM were considered to be granulocyte-macrophage colony-stimulating factor (GM-CSF) and CSF-1, respectively, because recombinant murine (rm)GM-CSF and recombinant human (rh)CSF-1 could replace the activities of lung-CM and L929-CM, respectively. The phenotype of the cells in the colonies formed by AM incubated with rmGM-CSF or lung-CM was AM-like; more than 90% of the cells were stained by anti-asialo GM1 but not by FITC-LPS, and had AM-like morphology. Expression of Mac-1 Ag determined by M1/70HL in these cells as well as original AM was low. However, the phenotype of the cells in the colonies formed by AM incubated with rhCSF-1 or L929-CM was peritoneal macrophage (PM)-like; more than 90% of the cells were stained by FITC-LPS and M1/70HL, but not by anti-asialo GM1, and showed PM-like morphology. The cells in the colonies formed by AM incubated with rmGMCSF changed their phenotype after treatment with rhCSF-1; the percentage of cells stained by anti-asialo GM1 decreased, and that of cells stained by FITC-LPS increased. The cells in the colonies formed by AM incubated with rhCSF-1 never changed their phenotype after incubation with rmGM-CSF. In contrast to AM, more than 90% of the cells in all colonies formed by PM incubated with either rmGM-CSF, rhCSF-1, lung-CM, or L929-CM were stained by FITC-LPS but not by anti-asialo GM1. These results show that although AM and PM can proliferate, AM, in contrast to PM, are bipotential cells that can differentiate into two types of macrophages responding to distinct types of CSF, and that one of the molecular mechanisms controlling macrophage heterogeneity may be based on the type of CSF produced at distinct tissues.     

Macrophage colony-stimulating factor enhances phagocytosis and oxidative burst of mononuclear phagocytes against Penicillium marneffei conidia

The responses of rabbit pulmonary alveolar macrophages (PAMs) and elutriated human monocytes (EHMs) to Penicillium marneffei, an emerging dimorphic fungus that may cause fatal disease in human immunodeficiency virus-infected patients, were studied. PAMs and EHMs comparably phagocytosed conidia of two P. marneffei strains in the presence of serum. Electron microscopy showed intraphagosomal destruction of conidia after 12 h. Serum-opsonized conidia elicited significantly more superoxide anion (O(2)(-)) release from EHMs compared to non-opsonized conidia, but equivalent O(2)(-) amounts to that elicited by serum-opsonized Aspergillus fumigatus conidia. Macrophage colony-stimulating factor (M-CSF) significantly enhanced phagocytosis of P. marneffei conidia by PAMs and EHMs, as shown by light microscopy. Moreover, M-CSF enhanced O(2)(-) production by EHMs in response to both serum-opsonized (P<0.001) and non-opsonized (P=0.03) conidia of A. fumigatus as well as conidia of the P. marneffei isolates (P<0.001 and 0.03). We conclude that M-CSF enhances phagocytosis and oxidative metabolism of mononuclear phagocytes suggesting a potential role for this cytokine in host defense against pulmonary and disseminated P. marneffei infection.     

Macrophage colony-stimulating factor rapidly enhances beta-migrating very low density lipoprotein metabolism in macrophages through activation of a Gi/o protein signaling pathway

Previous studies have examined lipoprotein metabolism by macrophages following prolonged exposure (>24 h) to macrophage colony-stimulating factor (M-CSF). Because M-CSF activates several signaling pathways that could rapidly affect lipoprotein metabolism, we examined whether acute exposure of macrophages to M-CSF alters the metabolism of either native or modified lipoproteins. Acute incubation of cultured J774 macrophages and resident mouse peritoneal macrophages with M-CSF markedly enhanced low density lipoproteins (LDL) and beta-migrating very low density lipoproteins (beta-VLDL) stimulated cholesteryl [(3)H]oleate deposition. In parallel, M-CSF treatment increased the association and degradation of (125)I-labeled LDL or beta-VLDL without altering the amount of lipoprotein bound to the cell surface. The increase in LDL and beta-VLDL metabolism did not reflect a generalized effect on lipoprotein endocytosis and metabolism because M-CSF did not alter cholesterol deposition during incubation with acetylated LDL. Moreover, M-CSF did not augment beta-VLDL cholesterol deposition in macrophages from LDL receptor (-/-) mice, indicating that the effect of M-CSF was mediated by the LDL receptor. Incubation of macrophages with pertussis toxin, a specific inhibitor of G(i/o) protein signaling, had no effect on cholesterol deposition during incubation with beta-VLDL alone, but completely blocked the augmented response promoted by M-CSF. In addition, incubation of macrophages with the direct G(i/o) protein activator, mastoparan, mimicked the effect of M-CSF by enhancing cholesterol deposition in cells incubated with beta-VLDL, but not acetylated LDL. In summary, M-CSF rapidly enhances LDL receptor-mediated metabolism of native lipoproteins by macrophages through activation of a G(i/o) protein signaling pathway. Together, these findings describe a novel pathway for regulating lipoprotein metabolism.     

Transforming growth factor-beta enhances the M-CSF and GM-CSF-stimulated proliferation of macrophages.

Transforming growth factor-beta (TGF-beta) has been shown to regulate the proliferation and function of several different cell types in the immune system. We have examined the effect of TGF-beta on the proliferation of murine macrophages in liquid culture. TGF-beta by itself did not induce proliferation of differentiated (7 days in culture) bone marrow-derived macrophages (BMM). In the presence of M-CSF, TGF-beta enhanced the proliferation of differentiated BMM and elicited peritoneal macrophages but had an inhibitory effect on the proliferation of nonadherent BMM (3 days in culture). The effect of TGF-beta was not restricted to M-CSF-dependent proliferation but was also observed for GM-CSF-dependent proliferation. The autocrine production of TGF-beta appeared to contribute to the proliferation of BMM. The addition of antibody against TGF-beta inhibited M-CSF- and GM-CSF-dependent proliferation 32% and 28%, respectively. In bone marrow, TGF-beta may be an important negative regulator of macrophage proliferation; whereas, in the tissues, TGF-beta may enhance macrophage proliferation.     

Transfection of immature murine bone marrow-derived dendritic cells with the granulocyte-macrophage colony-stimulating factor gene potently enhances their in vivo antigen-presenting capacity.

Ag presentation by dendritic cells (DC) is crucial for induction of primary T cell-mediated immune responses in vivo. Because DC culture from blood or bone marrow-derived progenitors is now clinically applicable, this study investigated the effectiveness of in vitro-generated murine bone marrow-derived DC (Bm-DC) for in vivo immunization protocols. Previous studies demonstrated that GM-CSF is an essential growth and differentiation factor for DC in culture and that in vivo administration of GM-CSF augments primary immune responses, which renders GM-CSF an attractive candidate to further enhance the effectiveness of DC-based immunotherapy protocols. Therefore, immature Bm-DC were transiently transfected with the GM-CSF gene and tested for differentiation, migration, and Ag-presenting capacity in vitro and in vivo. In vitro, GM-CSF gene-transfected Bm-DC were largely unaltered with regard to MHC and costimulatory molecule expression as well as alloantigen or peptide Ag-presenting capacity. When used for in vivo immunizations, however, the Ag-presenting capacity of GM-CSF gene-transfected Bm-DC was greatly enhanced compared with mock-transfected or untransfected cells, as determined by their effectiveness to induce primary immune reactions against hapten, protein Ag, and tumor Ag, respectively. Increased effectiveness in vivo correlated with the better migratory capacity of GM-CSF gene-transfected Bm-DC. These results show that GM-CSF gene transfection significantly enhances the capacity of DC to induce primary immune responses in vivo, which might also improve DC-based vaccines currently under clinical investigation.