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PubMed1991

Macrophage colony-stimulating factor is required for human monocyte survival and acts as a cofactor for their terminal differentiation to macrophages in vitro.

Functional competence as well as phenotype heterogeneity of macrophages depend on the completion of their maturation pathway. Differentiation of committed myeloid progenitor cells is induced by colony-stimulating factors (CSF), but no consistent data exist on which factor(s) induce the terminal maturation from the circulating blood monocyte to the mature macrophage. In vitro, monocyte to macrophage transformation occurs in the presence of serum and can be followed by the expression of the maturation-associated antigens gp65-MAX.1, gp68-MAX.3, and CD51. We describe that the differentiation-inducing activity in serum cannot be replaced by any of the known and available purified recombinant cytokines. In the absence of serum monocytes die in suspension cultures while surviving as non-differentiating cells when cultured adherent to plastic. In serum-free suspension cultures survival can be significantly improved by the addition of recombinant human macrophage (rhM)-CSF whereas other cytokines do not. At any stage of serum-free adherent culture, monocyte to macrophage differentiation can be induced rapidly by the addition of serum, whereas cytokines (rhM-CSF, recombinant human granulocyte macrophage [rhGM]-CSF, recombinant human granulocyte [rhG]-CSF, recombinant human interleukin [rhIL]-1, rhIL-3, rhIL-4, rhIL-6, tumor necrosis factor [TNF]-alpha, interferon [IFN]-alpha, IFN-gamma) alone or in combination are not effective. Serum-induced maturation, however, was suppressed in the presence of neutralizing anti-M-CSF antibodies. In addition to phenotype analysis, the secretory repertoire of rhM-CSF cultured monocytes was analyzed in comparison to serum cultured monocytes which further characterized them to be immature cells, i.e., low release of maturation-associated products such as alpha-2-macroglobulin, neopterin, fibronectin, and TNF-alpha, but high IL-6 secretion, an attribute of blood monocytes. We conclude that for monocyte survival in vitro the presence of endogenous M-CSF and possibly other autocrine factors elicited by cell adherence are required for the induction of macrophage maturation; however, yet undefined additional factor(s) are necessary. They are present in serum and may act in conjunction with M-CSF but are distinct from all known cytokines. Our in vitro system may be useful in the screening and discovery of these serum factor(s).

Antibodies↗

PubMed1995

Elevation of rainbow trout Oncorhynchus mykiss macrophage respiratory burst activity with macrophage-derived supernatants.

A variety of supernatants were prepared by stimulating rainbow trout Oncorhynchus mykiss head kidney macrophages with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), or a leucocyte-derived macrophage-activating factor (I-MAF), individually and in combination. If generated using a 12-h stimulation period, such supernatants were found to elevate significantly the respiratory burst activity of target macrophages; that is, they contained a macrophage-derived MAF (m-MAF), but supernatants generated using a shorter incubation period showed no significant activity. Combinations of these treatments were particularly effective in generating m-MAF-containing supernatants. The elevation of respiratory burst activity by supernatants generated using combined treatments could be partially inhibited by prior treatment of the target macrophages with anti-TNF-alpha receptor 1 (TNFR1) monoclonal antibodies (mAbs). Similarly, treatment of macrophages with combinations of 1-MAF and m-MAF generated supernatants with potent m-MAF activity and this activity was partially inhibited by prior treatment of the target cells with anti-TNFR1 mAb. In addition, the presence of anti-transforming growth factor beta 1 (TGF-beta 1) serum while generating these latter supernatants resulted in significantly increased m-MAF activity. Such data suggest that fish leukocytes secrete a variety of potent macrophage-activating (TNF-alpha) and -deactivating (TGF-beta) factors.

Animals↗

PubMed1986

Mechanism(s) of in vitro macrophage activation with Nocardia rubra cell wall skeleton: the effects on macrophage activating factor production by lymphocytes.

BALB/c mouse peritoneal macrophages prepared from WPC which had been treated with N. CWS demonstrated potent cytostatic activity against syngeneic Meth A fibrosarcoma cells. The maximum cytostatic activity developed in the macrophages when WPC were incubated with 25 micrograms/ml N. CWS for 3 days. NAPC from BALB/c mice given an i.p. injection with 100 micrograms N. CWS 7 days previously (N. CWS-NAPC) or supernatants from N. CWS-NAPC also activated peritoneal macrophages in vitro. However, when peritoneal macrophages were incubated with N. CWS in the absence of NAPC, or when T cells were depleted from WPC by treatment with anti-Thy 1.2 antibody and complement, N. CWS failed to enhance the cytostatic activity of the macrophages. Furthermore, thioglycollate-elicited peritoneal macrophages from C3H/HeN mice increased their cytolytic properties by incubation with supernatant fluids from N. CWS-treated spleen cells. These findings suggest that in vitro macrophage activation with N. CWS depends on MAF secreted from T lymphocytes.

Animals↗

PubMed1995

Macrophage depletion in the rat after intraperitoneal administration of liposome-encapsulated clodronate: depletion kinetics and accelerated repopulation of peritoneal and omental macrophages by administration of Freund's adjuvant.

The purpose of this study was to develop a method for the depletion of macrophages from the peritoneal cavity and the omentum of the rat. Rats received two intraperitoneal injections (at days 0 and 3) with liposome-encapsulated clodronate (dichloromethylene bisphosphonate: Cl2MBP-liposomes). This treatment resulted in complete elimination of mature tissue macrophages (ED2-positive macrophages) from the peritoneal cavity and the omentum within 2 days. The elimination included the strongly ED2-positive spindle-shaped cells of the omental membrane. Repopulation of the omental ED2-positive macrophages was not seen within the next 23 days. Whereas ED2-positive macrophages were completely depleted, few ED1-positive cells remained and repopulation of ED1-positive cells was faster. The treatment further depleted macrophages from the spleen, especially from the red pulp, parathymic lymph nodes and liver. Freund's incomplete adjuvant administered one day after the last injection of Cl2MBP-liposomes considerably accelerated repopulation in the omentum. The protocol described might be used to investigate the contribution of mature tissue macrophages to the induction of immune responses, drug metabolism and the elimination of intestinal tumours.

Animals↗

PubMed1990

Whole-cell currents in macrophages: II. Alveolar macrophages.

Although an outwardly rectifying K+ conductance has been described in murine peritoneal macrophages and a murine macrophage cell line, the expression of this conductance in human monocyte-derived macrophages (HMDMs) is rare. Whole-cell current recordings in this study were obtained from HMDMs differentiated in adherent culture for varying periods of time following isolation and compared to currents obtained in human alveolar macrophages (HAMs) obtained from bronchoalveolar lavage. These studies were undertaken to compare ionic current expression in the in vitro differentiated macrophage to that of a human tissue macrophage. HAMs are the major population of immune and inflammatory cells in the normal lung and are the most readily available source of human tissue macrophages. Of the 974 HMDMs in the study obtained from a total of 36 donors, we were able to observe the presence of the inactivating outward current (IA) which exhibited voltage-dependent availability in only 49 (or 5%) of the cells. In contrast, whole-cell current recordings from HAMs, revealed a significantly higher frequency of IA expression (50% in a total of 160 cells from 26 donors). In the alveolar cell, there was no correlation observed between cell size and peak IA amplitude, nor was there a relationship between peak IA amplitude and time in culture. The current in both cell types was K+ selective and 4-aminopyridine (4-AP) sensitive. IA in both cell types inactivated with a time course which was weakly voltage-dependent and which exhibited a time constant of recovery from inactivation of approximately 30 sec. The time course of current inactivation was dependent upon the external K+ concentration. An increase in the time constant describing current decay was observed in elevated K+. Current activation was half-maximal at approximately -18 mV in normal bathing solution. Steady-state inactivation was half-maximal at approximately -44 mV. The presence of the outwardly rectifying K+ conductance may alter the potential of the mononuclear phagocyte to respond to extracellular signals mediating chemotaxis, phagocytosis, and tumoricidal functions.

4-Aminopyridine↗

PubMed1995

Cholesterol metabolism in monocyte-derived macrophages from macrophage colony-stimulating factor administered rabbits.

The metabolism of beta-very-low-density lipoproteins (beta-VLDL) in macrophages from the blood monocytes of rabbits, which had been administered macrophage colony-stimulating factor (M-CSF) in vivo, was investigated in order to clarify the mechanism of the suppressive effect of M-CSF on cholesterol accumulation in macrophages. Cholesterol ester content after incubation with beta-VLDL, and [3H]cholesterol oleate-beta-VLDL incorporation remarkably increased in cultured macrophages from blood monocytes in the high cholesterol diet control group compared to those in the normal diet control group. Those in macrophages from M-CSF-treated groups, both normal diet and high cholesterol diet, were the same as in the normal diet control group. The ratio of released [3H]cholesterol to incorporated [3H]cholesterol oleate-beta-VLDL in macrophages from control was smaller than that from the M-CSF group. The acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity was enhanced in the high cholesterol diet groups, and the activity in M-CSF-treated groups decreased. The neutral cholesterol esterase activity was enhanced in the M-CSF-treated groups compared with that in the non-treated control groups. These results suggest that high cholesterol diet induced further cholesterol esterification and that M-CSF treatment enhanced cholesterol clearance in macrophages.

Animals↗

PubMed1991

Cytolytic activities of activated macrophages versus paraformaldehyde-fixed macrophages; soluble versus membrane-associated TNF.

Membrane-associated tumor necrosis factor (TNF) and soluble TNF were compared as to their lytic activities, and as to the kinetics of their expression by macrophages activated with LPS and/or IFN-gamma in the presence or absence of cycloheximide. EL 4 tumor cells, resistant and sensitive to lysis by recombinant TNF or membrane-associated TNF (paraformaldehyde (PF)-fixed activated macrophages) were used as targets. In the presence of cycloheximide the TNF-resistant S-EL4 cells were lysed by both TNFs. PF-fixed macrophages was cytolytic after 1 hr activation but not after 3 or more hours of activation. Their activity was totally inhibited by anti-TNF antibodies and was a composite of transmembrane (integral) TNF and soluble TNF conjugated to macrophage membrane TNF receptors. Treatment of the macrophages with glycine pH 3.0 buffer dissociated the conjugated TNF without affecting the integral membrane TNF. When macrophages were activated with LPS +/- IFN-gamma in the presence of cycloheximide or activated just with IFN-gamma their activity after fixation with paraformaldehyde was no longer detected. Nonfixed macrophages under these conditions still remained cytotoxic. Tumor cell susceptibility to membrane-associated TNF activity, in contrast to recombinant (soluble) TNF, was greatly reduced in the presence of nicotinamide, an inhibitor of ADP-ribosyltransferase, suggesting that the mechanisms of lysis by these TNFs may be different. The lytic activity of both TNFs was found to be receptor-dependent in that tumor cells, whose TNF binding sites were "down-regulated" by TPA, were rendered resistant to lysis by both membrane-associated and soluble TNFs.

Animals↗

PubMed1991

Differential regulation of interleukin-6, macrophage inflammatory protein-1, and JE/MCP-1 cytokine expression in macrophage cell lines.

Activated macrophages produce a number of proinflammatory cytokines including IL-6, JE, MIP-1 alpha and MIP-1 beta. The induction requirements for production of either IL-6 or the MIP-1 related inflammatory proteins (MIP-1 alpha, MIP-1 beta, and JE) have been analyzed independently using fibroblasts, monocytes, or endothelial cells. However, little is known about the regulation of these cytokines in macrophages. Since activated macrophages produce prostaglandins (PGE2) which may participate in the autoregulation of cytokine production by stimulation of adenylate cyclase and the induction of cAMP-dependent signal pathways, we determined the effects of PGE on the production of IL-6 and MIP-1-related proteins. Murine macrophage cell lines were incubated with PGE1, PGE2, cholera toxin, or dibutyryl cAMP in the presence of absence suboptimal doses of LPS. Pharmacologic agents alone did not induce IL-6 production but incubation of macrophages with combinations of adenylate cyclase stimulators and LPS or dcAMP and LPS led to the dose-dependent enhancement of IL-6 secretion and mRNA expression. In contrast, PGE1 inhibits LPS-induced JE, MIP-1 alpha, and MIP-1 beta mRNA expression and this inhibition is partially dependent on a cAMP-mediated pathway of signal transduction. In previous work we demonstrated that IFN-gamma and PMA do not stimulate the production of IL-6 by macrophages. Here we show that incubation of macrophages with either IFN-gamma or PMA induces the expression of JE, MIP-1 alpha and MIP-1 beta mRNA expression. JE mRNA expression is much more responsive to the stimulatory effects of IFN-gamma than are the MIP-1 genes. Finally, PGE inhibits PMA and IFN-gamma-induced JE and MIP-1-related mRNA expression.

Alprostadil↗

PubMed1991

Granules of human CD3+ large granular lymphocytes contain a macrophage regulating factor(s) that induces macrophage H2O2 production and tumoricidal activity but decreases cell surface Ia antigen expression.

CTL (cytotoxic T lymphocytes) and LGL (large granular lymphocytes) exocytose cytoplasmic granules on activation after recognition of their target, releasing granule-associated molecules. We have previously suggested that this process could release immunoregulatory molecules. In this study we investigated whether normal human LGL granules contained a factor regulating different macrophage activity. Human CD3+ LGL cells were generated by activating peripheral blood lymphocytes (PBL) for 10-12 days with recombinant human IL-2 (rhIL-2), and granules were isolated from disrupted cell homogenate by Percoll gradient fractionation. Solubilized granules were tested for macrophage-activating factor (MAF) activity in three different macrophage assays. When M-CSF-differentiated murine bone marrow-derived macrophages were incubated 9 hr with human LGL granules, they were fully activated to lyse the TNF-resistant P815 tumor cells. The granule-MAF showed a synergistic effect with rhIL-1 beta, rmTNF-alpha, and rmIFN-tau in the cytolytic assay. In addition, proteose-peptone-elicited murine peritoneal macrophages profoundly increased H2O2 production after activation with human LGL granules. However, unlike IFN-tau, no increase in peritoneal macrophage Ia antigen expression was detected after incubation with granules. Moreover, granule-MAF suppressed Ia induction by IFN-tau. These results confirm that human CD3+ LGL granules contain a molecule(s) capable of regulating macrophage function.

Animals↗

PubMed1992

TNF-alpha differentially regulates Ia antigen expression and macrophage tumoricidal activity in two murine macrophage cell lines.

We have assessed tumor necrosis factor-alpha (TNF-alpha) production and its autocrine effects on activation in two murine macrophage cell lines which have distinct responses to the activation stimuli interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS), and compared these responses to those observed in thioglycollate-elicited peritoneal macrophages. IFN-gamma induced TNF-alpha production in RAW 264.7 cells and this induction was regulated at the transcriptional level. IFN-gamma did not stimulate TNF-alpha production in either WEHI-3 cells or peritoneal macrophages, although MHC class II antigen expression was induced. LPS stimulated TNF-alpha production in the RAW 264.7 cell line and peritoneal macrophages; however, no TNF-alpha was detected in WEHI-3 cells activated with LPS. We also assessed the ability of endogenous TNF-alpha to serve as an autocrine regulator of two aspects of IFN-gamma-mediated macrophage activation, namely, induction of antibody-independent tumoricidal activity and induction of MHC class II antigen expression. These studies revealed that TNF-alpha could act synergistically or antagonistically with IFN-gamma in the regulation of these two functions, depending on both the macrophage population used and the function assessed. The results of our experiments suggest that the mechanism of induction of TNF-alpha production by IFN-gamma or LPS, and the ultimate autocrine contribution of such TNF-alpha to a given activation response, is dependent on the activated macrophage target population under analysis. The WEHI-3 and RAW 264.7 cell lines provide a model system for comparative exploration of the mechanistic basis of this differential regulation.

Animals↗

PubMed1986

Effect of lentinan on pinocytosis in mouse peritoneal macrophages and the murine macrophage cell line C4M phi in vitro.

Lentinan, an immunopotentiating polysaccharide, stimulated the pinocytosis of horseradish peroxidase (HRP) or FITC-dextran by resident or thioglycollate-elicited mouse macrophages from 10 to 50% in a dose dependent manner. Pinocytosis of HRP and FITC-dextran by C4M phi cells, a murine macrophage cell line, exhibiting a lower basic pinocytic activity than peritoneal cells, was augmented up to 310 and 120%, respectively, by lentinan. Mannan inhibited the HRP uptake by peritoneal macrophages via specific mannose receptors. This inhibitory effect was partly abolished, when lentinan was also added to the cells. Mannan was not able to inhibit pinocytosis of HRP by C4M phi macrophages, indicating little or no mannose receptor activity on these cells. Pinocytosis of FITC-dextran was not affected by mannan. Lentinan, opsonized in mouse sera inhibited the uptake of HRP by peritoneal macrophages by 30-35%. Opsonized lentinan and mannan added together caused 60% inhibition of HRP uptake in peritoneal macrophages indicating a possible functional relationship between the mannose and C3b receptors. The results demonstrate that lentinan activates the pinocytic function of macrophages predominantly via specific beta-glucan receptors. These mechanisms may contribute to the antitumor and immunopotentiating action of lentinan and other glucan-type polysaccharides.

Adjuvants, Immunologic↗

PubMed1994

Effect of macrophage colony-stimulating factor (M-CSF) on macrophage morphology, phagocytosis, and intracellular multiplication of Histoplasma capsulatum.

M-CSF induced dramatic morphological changes in resident peritoneal macrophages (R-PM) in a time and dose dependent manner. Macrophages increased 2-3-fold in size and developed a dendritic morphology. Compared to macrophages cultured in medium alone M-CSF-treated macrophages were less phagocytic (67 versus 82%) for serum opsonized yeast cells of Histoplasma capsulatum. However, in M-CSF macrophages the intracellular multiplication of ingested yeast cells were significantly inhibited compared to growth in control macrophages. The fungistatic activity of M-CSF macrophages persisted for at least 48 h after infection.

Animals↗

PubMed1996

Acetylcholinesterase-induced respiratory burst in macrophages: evidence for the involvement of the macrophage mannose-fucose receptor.

It has long been suggested that acetylcholinesterase is capable of functioning in a non-cholinergic manner. However, very little is known about the molecular structures which mediate the interaction between this protein and the cellular membrane. Previously it was demonstrated that acetylcholinesterase interacted in a carbohydrate-specific manner with peritoneal macrophages and induced the 'respiratory burst' [1]. This study aimed to establish whether a carbohydrate-binding site exists on the acetylcholinesterase molecule itself, or alternatively, whether the macrophage carbohydrate-binding receptor is involved. No carbohydrate binding properties intrinsic to acetylcholinesterase were detected using affinity chromatography with immobilised monosaccharides, erythrocyte agglutination and gel-diffusion techniques. The interaction between acetylcholinesterase and several monosaccharide columns observed in this study appeared to be due to ionic interactions. Moreover, it was shown that a specific inhibitor of the enzymatic activity of AChE, BW284C51, could inhibit the peritoneal cell response not only to acetylcholinesterase, but also to several other stimuli, thus exhibiting a non-specific effect on macrophages. However, the inhibitory effects of specific ligands of the macrophage mannose-fucose receptor and the inability of non-glycosylated acetylcholinesterase to interact with macrophages suggested that the effect of acetylcholinesterase on peritoneal cells is most probably mediated by the macrophage mannose-fucose receptor. The role of the mannose-fucose receptor in triggering the respiratory burst response was supported by the fact that several ligands of these receptors were capable of inducing the functional response of macrophages.

Acetylcholinesterase↗

PubMed2002

Spontaneous and stimulated secretion of monocyte chemotactic protein-1 and macrophage migration inhibitory factor by peritoneal macrophages in women with and without endometriosis.

OBJECTIVE: To assess spontaneous and stimulated secretion of monocyte chemotactic protein-1 (MCP-1) and macrophage migration inhibitory factor (MIF) by peritoneal macrophages in women with and without endometriosis. DESIGN: Macrophages were isolated from the peritoneal fluid and cultured for different periods of time (6, 20, and 44 hours) without any stimulation to determine spontaneous secretion of MCP-1 and MIF. Macrophages were also exposed to 1 microg/mL lipopolysaccharide for 6 hours to evaluate the stimulated secretion of these cytokines. SETTING: Gynecology clinic and human reproduction research laboratory. PATIENT(S): Twelve fertile women and 11 women with endometriosis. INTERVENTION(S): Peritoneal fluid obtained at laparoscopy. MAIN OUTCOME MEASURE(S): Monocyte chemotactic protein-1 and MIF concentrations in the culture medium using ELISA. RESULT(S): Peritoneal macrophages of women with endometriosis demonstrated an increased capacity to secrete MCP-1 either spontaneously or after stimulation with lipopolysaccharide. They also showed a marked tendency for an increased secretion of MIF, but no statistically significant difference was found. CONCLUSION(S): Monocyte chemotactic protein-1 and MIF production by peritoneal macrophages may contribute to paracrine and autocrine activation and to macrophage accumulation in the peritoneal cavity of women with endometriosis. These mechanisms may exacerbate peritoneal inflammation and favor the growth of endometrial implants.

Adult↗

PubMed2001

Low serum conditions for in vitro generation of human macrophages with macrophage colony stimulating factor.

Animal serum is often used to generate human macrophages in vitro. Since fetal calf serum (FCS) may complicate antigen uptake, processing and presentation on HLA molecules, we tested the ability of M-CSF to generate macrophages at low fetal calf serum conditions. Peripheral blood monocytes from 12 individuals were cultured 1-4 days with 0-100 ng/ml macrophage colony stimulating factor (M-CSF) at either 1 (low) or 5% (v/v) FCS. Regardless of number of days in culture, maximal (50-100 ng/ml) M-CSF stimulation and low FCS induced 65+/-5% esterase positive cells in all individuals compared to 52+/-7% without M-CSF (P<0.001). M-CSF increased the mean proportion of esterase positive cells after 24 or 96 h by 13% (P<0.005) and 13% (P<0.005), respectively, in 1% FCS, and 8% (P<0.05) and 2% (NS), respectively, in 5% FCS, indicating a slight negative interaction between 5% FCS and M-CSF (P<0.05). All cells were positive for CD14 and HLA class II, but cell number did not increase, confirming that M-CSF promote macrophage differentiation also at low FCS. M-CSF increased the average cell size after 24 or 96 h by 5.9+/-1.0 (P<0.05) and 8.6+/-0.5 (P<0.001) microm, respectively, without an increase in 5% FCS, further demonstrating the efficiency of M-CSF to promote macrophage generation at low FCS. The culture supernatants were negative for IL-1beta and TNF-alpha, which demonstrates that M-CSF did not activate the macrophages. The generation of human macrophages by M-CSF at low FCS should prove useful in studies where higher FCS concentrations may interfere with the assay.

Cell Differentiation↗

PubMed1999

Macrophages that kill glioma cells expressing the membrane form of macrophage colony stimulating factor are resistant to prostaglandin E2 and interleukin-10.

Malignant rat T9 glioma cells retrovirally transduced with the membrane form of macrophage colony stimulating factor (mM-CSF) were killed by bone marrow derived macrophages in 24 h cytotoxicity assays. Prostaglandin E2 (PGE) and interleukin-10 (IL10) were tested for their ability to block this tumoricidal reaction. Only at very high nonphysiological concentrations of PGE (10(-5) and 10(-6) M) was this cytotoxicity inhibited. Use of high doses of theophylline, a phosphodiesterase inhibitor, also prevented macrophages from killing the mM-CSF transduced target cells. IL10 did not alter the killing potential of the mM-CSF tumoricidal macrophages, even though IL10 reduced the production of nitric oxide by macrophages in response to tumor necrosis factor and lipopolysaccharide. IL10 enhanced the growth of bone marrow macrophages suggesting that IL10 has a complex role in the regulation of tumoricidal macrophages. Thus, the mM-CSF may be an ideal agent to treat tumors that utilize either of these two immunosuppressive defense mechanisms that may block other forms of treatment.

Animals↗

PubMed2001

Granulocyte-macrophage colony-stimulating factor ensures macrophage survival and generation of the superoxide anion: a study using a monocytic-differentiated HL60 subline.

A large number of constituents, such as growth factors, cytokines, and vasoregulatory molecules, contribute a network of cellular interactions to atherosclerotic lesions, and current evidence suggests that granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of these constituents. We conducted this study to determine whether GM-CSF has an effect on the fate and function of macrophages. We examined the effect of GM-CSF on macrophages in vitro with a highly inducible HL60 subclone (HL60/DU-1) that we recently established. HL60 cells have been reported to preserve functional GM-CSF receptors, but a GM-CSF allele was rearranged and partially deleted. HL60/DU-1 cells were devoid of GM-CSF immunoreactivity and of autocrine stimulation of GM-CSF. HL60/DU-1 cells fated to die soon after terminal differentiation of macrophages by 1, 25-dihydroxy vitamin D(3) treatment. We found cell death to be mediated mainly by necrosis, not apoptosis, as confirmed by DNA fragmentation in agarose gel electrophoresis, morphological observation under a fluorescence microscope, and assay of lactate dehydrogenase release. Exogeneously administered GM-CSF rescued cells from necrotic death and caused them to survive and generate superoxide anions. We also conducted immunohistochemical analysis on an atherosclerotic human artery. Macrophages, endothelial cells, and smooth muscle cells were found to be GM-CSF positive in an atherosclerotic lesion. In summary, GM-CSF, which is produced by macrophages, endothelial cells, and smooth muscle cells, is thought to act in an autocrine and a paracrine fashion as a necrosis-inhibiting factor against arterial macrophages. This unique function may play an important role in ensuring survival and promoting function in atherosclerotic lesions.

Aorta↗

PubMed2004

Modulation of the activity of sea bass (Dicentrarchus labrax) head-kidney macrophages by macrophage activating factor(s) and lipopolysaccharide.

The aim of this study was to establish the requirements for macrophage activating factor (MAF) production by sea bass head-kidney leucocytes and the kinetics of macrophage activation when exposed to MAF-containing supernatants and/or lipopolysaccharide (LPS), a known macrophage stimulant. MAF activity was found in culture supernatants of total head-kidney leucocytes pulsed with 5 microg ml(-1)Con A, 5 or 10 ng ml(-1)PMA and 100 ng ml(-1)calcium ionophore, or 10 microg ml(-1)Con A alone, as assessed by the capacity to prime macrophages for enhanced production of reactive oxygen intermediates (ROI). Mixed leucocyte cultures from two or eight fish showed higher MAF activity after stimulation, indicating that a mixed leucocyte reaction was also important for MAF production. MAF-induced activation of macrophage cultures was highest at 18 h of exposure and was lost by 72 h except for MAF induced by Con A-stimulation alone. LPS primed macrophages for increased ROI production at early incubation times and down-regulated ROI production after 24 h. LPS had no effect in further stimulating the MAF-induced priming effect on production of ROI and down-regulated the MAF-priming by 48 h. Sea bass head-kidney macrophages did not show increased nitrite production when exposed to MAF and/or LPS, which may be related to their differentiation status.

Animals↗