Inhibition of macrophage tumoricidal activity by glucocorticoids

In this study, the effect of corticosteroids on the activation of macrophages to a fully tumoricidal state was examined. Thioglycolate-elicited peritoneal exudate macrophages from C3H/HeJ mice were rendered cytolytic for P815 mastocytoma cells in a two-signal tumoricidal assay that used recombinant interferon-gamma (rIFN-gamma; 1 to 10 U/ml) as a "priming" signal and butanol-extracted lipopolysaccharide (But-LPS; 0.1 to 5 micrograms/ml) as a "trigger" signal. Treatment of macrophages with either rIFN-gamma alone or But-LPS alone failed to result in significant cytolytic ability. Tumoricidal activity was markedly inhibited in a dose-dependent fashion when glucocorticoids were added simultaneously to the cultures with rIFN-gamma and But-LPS at concentrations ranging from 1 X 10(-10) to 1 X 10(-5) M. Nonglucocorticoid sex hormones failed to inhibit tumoricidal activity in this system under identical culture conditions. Inhibition was most effective if the glucocorticoids were added simultaneously with the priming and triggering signals (rIFN-gamma and But-LPS); however, if the glucocorticoids were added 24 hr after the signals were provided to the cultures, suboptimal inhibition was observed. Experiments that dissociated the priming phase of activation from the triggering phase showed that glucocorticoids inhibited both the rIFN-gamma-induced priming stage as well as the But-LPS-induced triggering stage of activation. These observations provide evidence that glucocorticoids, but not other steroid hormones, inhibit the activation of macrophages to a fully tumoricidal state by interfering with either the priming or triggering signals in this two-signal model of macrophage activation.     

cAMP inhibits CSF-1-stimulated tyrosine phosphorylation but augments CSF-1R-mediated macrophage differentiation and ERK activation

Macrophage colony stimulating factor (M-CSF) or CSF-1 controls the development of the macrophage lineage through its receptor tyrosine kinase, c-Fms. cAMP has been shown to influence proliferation and differentiation in many cell types, including macrophages. In addition, modulation of cellular ERK activity often occurs when cAMP levels are raised. We have shown previously that agents that increase cellular cAMP inhibited CSF-1-dependent proliferation in murine bone marrow-derived macrophages (BMM) which was associated with an enhanced extracellular signal-regulated kinase (ERK) activity. We report here that increasing cAMP levels, by addition of either 8-bromo cAMP (8BrcAMP) or prostaglandin E(1) (PGE1), can induce macrophage differentiation in M1 myeloid cells engineered to express the CSF-1 receptor (M1/WT cells) and can potentiate CSF-1-induced differentiation in the same cells. The enhanced CSF-1-dependent differentiation induced by raising cAMP levels correlated with enhanced ERK activity. Thus, elevated cAMP can promote either CSF-1-induced differentiation or inhibit CSF-1-induced proliferation depending on the cellular context. The mitogen-activated protein kinase/extracellular signal-related protein kinase kinase (MEK) inhibitor, PD98059, inhibited both the cAMP- and the CSF-1R-dependent macrophage differentiation of M1/WT cells suggesting that ERK activity might be important for differentiation in the M1/WT cells. Surprisingly, addition of 8BrcAMP or PGE1 to either CSF-1-treated M1/WT or BMM cells suppressed the CSF-1R-dependent tyrosine phosphorylation of cellular substrates, including that of the CSF-1R itself. It appears that there are at least two CSF-1-dependent pathway(s), one MEK/ERK dependent pathway and another controlling the bulk of the tyrosine phosphorylation, and that cAMP can modulate signalling through both of these pathways.     

In vitro modulation of macrophage tumoricidal activity

Summary NaIO₄ treatment of mouse adherent peritoneal cells or lymphocyte-free cloned macrophages enhances their cytotoxic and tumoricidal activity. 5×10^–3 M NaIO₄ treatment of nontumoricidal BCG-activated macrophages renders them completely tumoricidal, whereas the same treatment of stimulated (peptone-normal) macrophages renders them weakly tumoricidal. Addition of LPS in nanogram quantities too low to enhance tumor cell killing by untreated peptone-normal macrophages causes NaIO₄-treated peptone-normal macrophages to be maximally tumoricidal. The activating action of NaIO₄, MAF, or LPS can be potently, but inconsistently, blocked or reversed by the reducing agent NaBH₄ or the aldehyde-reacting agent dimedone. NaIO₄ treatment of lymphocyte-free macrophage colonies does not make them cytotoxic, but NaIO₄-treated colony macrophages are cytotoxic for tumor cells when cultured in 10 ng/ml LPS (an amount of LPS inadequate to render untreated colony macrophages cytotoxic). Supernatants of NaIO₄-treated adherent peritoneal cells contain MAF activity. Thus, the NaIO₄-induced enhancement of peritoneal cell tumoricidal activity may result from both direct NaIO₄ activating effects on macrophages and indirect NaIO₄ effects through NaIO₄-induced MAF production.     

Stimulation of macrophage colony-stimulating factor synthesis by interleukin-1.

Interleukin-1 (IL-1), which plays an important role in the inflammatory response, was found to induce colony-stimulating factor-1 (CSF-1) expression in the MIA PaCa-2 cells. IL-1-induced CSF-1 production was markedly suppressed (70%) by pertussis toxin. This inhibition by pertussis toxin was reversed by benzamide, an inhibitor of ADP-ribosylation reactions. Similarly, IL-1-induced CSF-1 production was inhibited by cholera toxin and this inhibition was reversed by an arginine analog, p-methoxy-benzylaminodecamethylene guanidine sulfate. Dibutyryl-cAMP as well as other cAMP elevating agents such as theophylline and forskolin also suppressed IL-1-induced CSF-1 production, suggesting that cAMP concentrations inversely regulate the biosynthesis of CSF-1. Measurement of cAMP concentration indicated that IL-1 treatment of MIA PaCa-2 cells did not change the cAMP level. IL-1-induced CSF-1 production was not suppressed by the protein kinase C (PKC) inhibitor, H7, under conditions in which 12-O-tetradecanoylphorbol-13-acetate-induced CSF-1 production was completely abolished. These data suggest that IL-1-induced CSF-1 production is not mediated via the activation of PKC. Analysis of oncogene c-fos and c-jun expression has shown the enhancement of expression of both protooncogenes prior to CSF-1, suggesting that the expression of these two oncogenes may be the mechanism which triggers CSF-1 gene expression.     

Inhibition of macrophage tumoricidal activity by immune complexes and altered erythrocytes

Engagement of the macrophage membrane by biologic particles including insoluble immune complexes inhibited the development of lymphokine-mediated nonspecific tumoricidal activity by murine macrophages. The degree of inhibition was dependent on the dose of particles and the lymphokine concentration. Inhibition was not due to macrophage cell death or to diminution of cell adherence after ingestion of the immune complexes. Soluble immune complexes were not inhibitory, although approximately 10% of the complexes became cell-associated. Monomeric or heat-aggregated IgG was also not inhibitory. IgG-opsonized erythrocytes (EA) were inhibitory and inhibition was dependent on the degree of opsonization. In contrast, nonopsonized erythrocytes (E), which did not bind to macrophages, were not inhibitory. Phagocytosis of glutaraldehyde-treated E or E carrying IgM antibody and complement (EAC) also led to a reduction of tumorilytic activity. Insoluble immune complexes were inhibitory when added either before or after lymphokine. Phagocytosis was neither sufficient nor necessary to cause inhibition because 1) ingestion of polystyrene latex beads did not diminish tumoricidal activity, and 2) macrophages plated on IgG-coated surfaces were inhibited with respect to the tumoricidal function. Inhibition was not affected when indomethacin (10(-6) M) was included in the assay, which indicated that prostaglandins were not involved in the process. Thus, macrophage tumoricidal responsiveness may be compromised by interaction of biologic substances with macrophage plasma membranes. This process may thereby inactivate an important host defense mechanism against neoplastic cells.     

The regulation of macrophage protein turnover by a colony stimulating factor (CSF-1)

CSF-1 is a hemopoietic growth factor that specifically regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells. A homogeneous population of mononuclear phagocytes, bone marrow derived macrophages (BMM), were used to study the regulation of protein turnover by CSF-1. Removal of CSF-1 (approximately 0.4 nM) from exponentially growing BMM cultured in 15% fetal calf serum containing medium decreases the rate of DNA synthesis by more than 100-fold. Addition of CSF-1 to these cells causes them to resume DNA synthesis within 12 h. More immediate effects of CSF-1 were observed on BMM protein metabolism. BMM cultured for 24 h in the absence of CSF-1 reduce their protein synthetic rate by 50-60%. The protein synthetic rate commences to decrease at 2-3 h after CSF-1 removal. Readdition of CSF-1 to BMM previously incubated in its absence causes a return to the protein synthetic rate of exponentially growing cells within 2 h. In the presence of CSF-1, BMM synthesize protein at a rate of approximately 8.7%/h and degrade it at a rate of approximately 0.9%/h. Removal of CSF-1 results in a decrease in the protein synthetic rate to approximately 3.4%/h and an increase in the rate of protein degradation to approximately 3.4%/h. The rate of protein synthesis by BMM increases linearly with CSF-1 concentration over the range of concentrations stimulating both survival and proliferation, while the rate of protein degradation decreases exponentially over the range of concentrations stimulating survival without proliferation. Therefore, it appears that the stimulation of the rate of protein synthesis and inhibition of the rate of protein degradation are two distinct effects of CSF-1, both part of the pleiotropic response to this growth factor. The inhibition of the rate of protein degradation by CSF-1 may be most significant for its survival inducing effect.     

Effect of L-arginine on the retention of macrophage tumoricidal activity

It has been reported that the tumoricidal activity of macrophages (M phi) depends on L-arginine and that L-arginine metabolites such as reactive nitrogen intermediates alter M phi physical capacities. The aim of this report is to investigate the dose-related effect of L-arginine on the expression and retention of M phi tumoricidal activity. Cytotoxicity of M phi activated by IFN-gamma plus LPS was detected in the presence of about 0.1 mM or more of L-arginine. This paralleled the NO2- production in the presence, but not in the absence, of L-arginine. On the other hand, activated M phi were destined to die and lost their tumoricidal activity with time in the presence of 0.3 mM or more L-arginine. They retained, however, considerable activity in the absence or presence of 0.15 mM L-arginine. This retention of M phi cytotoxicity was longer when M phi were preactivated by 100 ng/ml than 10 ng/ml of LPS in combination with IFN-gamma. Addition of indomethacin, an inhibitor of prostaglandin production, did not prevent the decay of M phi cytotoxicity but rather facilitated it even in the absence of L-arginine. Regardless of indomethacin, consecutive stimulation with LPS or LPS plus IFN-gamma during culture was effective in maintaining the tumoricidal activity at a high level. In addition, we found that M phi which had lost tumoricidal activity during culture in L-arginine deficient medium could be reactivated by LPS to attack tumor target cells.     

Stimulation of nuclear sphingosine kinase activity by platelet-derived growth factor

Subcellular fractionation revealed that a significant fraction of total sphingosine kinase, the enzyme that phosphorylates sphingosine to form the bioactive lipid metabolite sphingosine-1-phosphate, resides in the nuclei of Swiss 3T3 cells, localized to both the nuclear envelope and the nucleoplasm. Platelet-derived growth factor, in addition to rapidly stimulating cytosolic sphingosine kinase, also induced a large increase in nucleoplasm-associated activity after 12-24 h that correlated with progression of cells to the S-phase of the cell cycle and translocation of sphingosine kinase-green fluorescent protein fusion protein to the nuclear envelope. Our results add sphingosine kinase to the growing list of lipid-metabolizing enzymes associated with the nucleus, and suggest that sphingosine-1-phosphate may also play a role in signal transduction in the nucleus.     

Partial primary structures of human and murine macrophage colony stimulating factor (CSF-1)

Approximately 40 amino-terminal residues and 20 internal residues of CSF-1 purified from the media of cultured human pancreatic carcinoma (MIA PaCa) cells and of cultured murine L cells have been identified. Results indicated that the two subunits in each molecule of biologically active CSF-1 are identical in their amino-terminal portions. The twelve amino-terminal residues of MIA PaCa CSF-1 were found to be identical to those of human-urinary CSF-1, suggesting that the polypeptide portions of the two human proteins may be identical. Approximately 75% of the amino acids identified in both MIA PaCa CSF-1 and murine CSF-1 were found to be common to both. No homology to other proteins was observed. This study suggests a subunit polypeptide Mr nearer to 17K than to 26K predicted from cDNA.     

Proteomic analysis of macrophage differentiation. p46/52(Shc) Tyrosine phosphorylation is required for CSF-1-mediated macrophage differentiation

Macrophage colony stimulating factor (M-CSF or CSF-1) acts to regulate the development and function of cells of the macrophage lineage. Murine myeloid FDC-P1 cells transfected with the CSF-1 receptor (FD/WT) adopt a macrophage-like morphology when cultured in CSF-1. This process is abrogated in FDC-P1 cells transfected with the CSF-1 receptor with a tyrosine to phenyalanine substitution at position 807 (FD/807), suggesting that a molecular interaction critical to differentiation signaling is lost (Bourette, R. P., Myles, G. M., Carlberg, K., Chen, A. R., and Rohrschneider, L. R. (1995) Cell Growth Differ. 6, 631--645). A detailed examination of lysates of CSF-1-treated FD/807 cells by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE) revealed a number of proteins whose degree of tyrosine phosphorylation was modulated by the Y807F mutation. Included in this category were three phosphorylated proteins that co-migrated with p46/52(Shc). Immunoprecipitation, Western blotting, and in vitro binding studies suggest that they are indeed p46/52(Shc). A key regulator of differentiation in a number of cell systems, ERK was observed to exhibit an activity that correlated with the relative degree of differentiation induced by CSF-1 in the two cell types. Transfection of cells with a non-tyrosine-phosphorylatable form of p46/52(Shc) prevented the normally observed CSF-1-mediated macrophage differentiation as determined by adoption of macrophage-like morphology and expression of the monocyte/macrophage lineage cell surface marker, Mac-1. These results are the first to suggest that p46/52(Shc) may play a role in CSF-1-induced macrophage differentiation. Additionally, a number of proteins were identified by two-dimensional SDS-PAGE whose degree of tyrosine phosphorylation is also modulated by the Y807F substitution. This group of molecules may contain novel signaling molecules important in macrophage differentiation.     

Macrophage growth factor CSF-1 stimulates human monocyte production of interferon, tumor necrosis factor, and colony stimulating activity

CSF-1, a macrophage colony stimulating factor that causes proliferation and differentiation of progenitor cells, may also have effects on mature cells. Human peripheral blood monocytes were used to examine this possibility. Monocytes, separated from normal blood by density centrifugation and adherence, were incubated for 3 days with or without CSF-1 (1,000 U/ml, purified from the MIA PaCa pancreatic carcinoma line). The two groups of cells were then washed and tested for the ability, when induced, to produce several factors. When induced for 2 days with LPS and PMA, the monocytes produced a factor that was cytotoxic to L929 cells, and this factor was completely neutralized by polyclonal antibody to tumor necrosis factor. The cells preincubated with CSF-1 consistently produced an average of 12 times more of this factor than cells not exposed to CSF-1. Monocytes induced with LPS and PMA also produced a colony stimulating activity, as measured by colony formation when using mouse bone marrow. Cells preincubated with CSF-1, washed, and induced with LPS and PMA produced more than three times as much activity compared with control monocytes. When monocytes were induced with poly-I.C, 22-fold higher levels of interferon were produced by the cells exposed to CSF-1. These results show that CSF-1 has direct stimulating effects on mature human monocytes, and suggest that the macrophage growth factor may have clinical application in the treatment of infectious diseases and cancer.     

Stimulation of B cell growth and differentiation by murine recombinant interleukin 1

Purified splenic B cells from C57BL/6 mice were separated into high-density (resting) and low-density (activated) B cells. Separated B cell populations were cultured at low cell densities (1 X 10(4) cells/well) with recombinant interleukin 1 (r-IL 1) alone or in combination with dextran sulfate (DXS) or anti-IgM monoclonal antibodies (alpha IgM mab), respectively, and proliferative responses were determined. R-IL 1 alone, as well as in synergy with alpha IgM mab or DXS, respectively, stimulated the growth of low-density B cells. Moreover, r-IL 1 and alpha IgM mab costimulated replication of high-density B cells. Separated B cell populations (1 X 10(5) cells/well) were cultured with r-IL 1 alone or in combination with DXS or alpha IgM mab, respectively, and the generation of plaque-forming cells was determined. R-IL 1 alone, as well as in synergy with DXS, stimulated the differentiation of low-density B cells into Ig-secreting cells. These findings suggest that r-IL 1 has B cell growth and differentiation factor activity and is operative on high- and low-density B cells. Thus, IL 1 may play an important role in B cell growth and maturation.     

Stimulation of macrophage growth and multinucleated cell formation in rat bone marrow cultures by insulin-like growth factor I

In this study the effects of rhIGF-I on macrophage differentiation and growth have been studied using liquid suspension cultures of rat bone marrow cells. IGF-I stimulated macrophage growth in a dose-dependent manner, a maximum response was found at a concentration of 20 ng/ml. IGF-I effects could be ascribed to stimulation of both postmitotic and proliferating cells. A remarkable finding was that IGF-I induced formation of multinucleated cells (MNC). The MNC resembled macrophage-like cells (AcP, NSE positive). A monoclonal antibody to rhIGF-I significantly inhibited IGF-stimulated macrophage growth and MNC formation. A specific antibody to mouse CSF-1 reduced IGF-stimulated macrophage growth in mouse bone marrow cultures indicating that IGF-I effects could, at least in part, be ascribed to endogenous production of CSF-1. These findings indicate that IGF-I in concert with locally induced CSF-1 can influence the differentiation and growth of bone marrow-derived macrophages.     

Purification of human macrophage colony stimulating factor (CSF-1) from medium conditioned by pancreatic carcinoma cells

Colony Stimulating Factor-1 has been purified to apparent homogeneity from the serum-free medium conditioned by cultured human pancreatic carcinoma cells which had been induced with phorbol myristate acetate. The purification scheme consisted of sequential steps of batchwise adsorption to calcium phosphate gel, adsorption to lentil lectin-Sepharose, binding to immobilized antibodies, hydrophobic interaction chromatography, and reversed-phase high-performance liquid chromatography. The purified glycoprotein was found to have a subunit molecular weight corresponding to the smallest of four species (approximately 40,000, 33,000, 28,000 and 23,000) which were observed when less purified preparations were examined.     

Stimulation of macrophage plasminogen activator activity by colony-stimulating factors

Colony-stimulating factors (CSFs) stimulate granulocyte-macrophage production from single hemopoietic progenitor cells. Various preparations of purified CSFs of two different subclasses have been shown here to stimulate a plasminogen-dependent fibrinolytic (plasminogen activator) activity from resident and starch-induced mouse peritoneal macrophages. Lymphocyte supernatants also stimulate macrophage plasminogen activator (PA) activitty. Since they contain colony stimulating activity, it is possible that one or more sublcasses of CSF in these supernatants is responsible for this effect. Since both colony-stimulating and macrophage growth activities have been detected at inflammatory sites, these findings could reflect a role for CSF in inflammatory processes.     

Prevention of macrophage tumoricidal activity by agents known to increase cellular cyclic AMP.

The concept of T-T cell interaction which was first suggested during cell-mediated immune response to alloantigens was evaluated in a syngeneic tumor system. The combination of lymph node and thymus cells from BALB/c mice immune against syngeneic tumor cells, mKSA, was shown to exhibit collaboration with respect to in vitro generation of effector cells capable of preventing growth of corresponding tumor cells in the tumor cell neutralization assay. While each cell population of either anatomical site did not prevent tumor growth when tested alone, combinations of both did. The antigen specificity of effector cells generated by synergizing cultures was similar to that of effectors derived from cultures containing optimal numbers of responding lymph node cells. The lymph node and thymus cell populations participating in synergy were found to be thymus dependent. These results suggest that we may be dealing with the same or similar T₁- and T₂-cell populations described before as displaying synergy in response to alloantigens in the graft versus host, mixed lymphocyte, and cell-mediated cytotoxicity reactions.