Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages

Studies with populations of macrophages have produced conflicting results concerning the possibility that the concentration of intracellular ionized calcium [( Ca2+]i) may act as an important mediator for phagocytosis. Since asynchronous changes in [Ca2+]i in individual cells undergoing phagocytosis may be averaged to undetectability in population studies, we studied single adhering murine macrophages using fura-2 and our previously described digital imaging system. The proportion of macrophages phagocytosing IgG-coated latex beads was greater than for uncoated beads (percent phagocytosing cells: 71 +/- 7 vs. 27 +/- 7, P less than 0.01). Phagocytosis of IgG-coated and uncoated beads was always associated with a calcium transient that preceded the initiation of phagocytosis. No calcium transients were detected in cells that bound but did not phagocytose beads. Four major differences between Fc receptor-mediated and nonspecific phagocytosis were detected: (a) the duration of calcium transients was longer for nonspecific phagocytosis compared with Fc receptor-mediated phagocytosis (69.9 +/- 10.2 vs. 48.7 +/- 4.7 s, P less than 0.05) and the magnitude of calcium transients was less for nonspecific phagocytosis (178 +/- 43 vs. 349 +/- 53 nM, P less than 0.05); (b) removal of extracellular calcium abolished the calcium transients associated with nonspecific phagocytosis but had no effect on those associated with receptor-mediated phagocytosis; (c) in the absence of extracellular calcium, buffering intracellular calcium with a chelator reduced Fc receptor-mediated phagocytosis but had no additive inhibitory effect on nonspecific phagocytosis; and (d) inhibition of protein kinase C (PKC) with staurosporine inhibited nonspecific phagocytosis but had no effect on receptor-mediated phagocytosis. Our observations suggest that despite both types of phagocytosis being associated with intracellular calcium transients, the role played by intracellular calcium in the signaling pathways may differ for Fc receptor-mediated and nonspecific phagocytosis by elicited murine macrophages.     

Yeast mannans inhibit binding and phagocytosis of zymosan by mouse peritoneal macrophages

We have examined the effects of various mannans, glycoproteins, oligosaccharides, monosaccharides, and sugar phosphates on the binding and phagocytosis of yeast cell walls (zymosan) by mouse peritoneal macrophages. A phosphonomannan (PO(4):mannose ratio = 1:8:6) from kloeckera brevis was the most potent inhibitor tested; it inhibited binding and phagocytosis by 50 percent at concentrations of approximately 3-5 μg/ml and 10 μg/ml, respectively. Removal of the phosphate from this mannan by mild acid and alkaline phosphatase treatment did not appreciably reduce its capacity to inhibit zymosan phagocytosis. The mannan from saccharomyces cerevisiae mutant LB301 inhibits phagocytosis by 50 percent at 0.3 mg/ml, and a neutral exocellular glucomannan from pichia pinus inhibited phagocytosis by 50 percent at 1 mg/ml. Cell wall mannans from wild type S. cervisiae X2180, its mnn2 mutant which contains mannan with predominantly 1(arrow)6- linked mannose residues, yeast exocellular mannans and O-phosphonomannans were less efficient inhibitors requiring concentrations of 1-5 mg/ml to achieve 50 percent reduction in phagocytosis. Horseradish peroxidase, which contains high-mannose type oligosaccharides, was also inhibitory. Mannan is a specific inhibitor of zymosan binding and phagocytosis. The binding and ingestion of zymosan but not of IgG- or complement-coated erythrocytes can be obliterated by plating macrophages on substrates coated with poly-L-lysin (PLL)-mannan. Zymosan uptake was completely abolished by trypsin treatment of the macrophages and reduced by 50-60 percent in the presence of 10 mM EGTA. Pretreatment of the macrophages with chloroquine inhibited zymosan binding and ingestion. These results support the proposal that the macrophage mannose/N-acetylglucosamine receptor (P. Stahl, J.S. Rodman, M.J. Miller, and P.H. Schlesinger, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1399-1403, mediates the phagocytosis of zymosan particles.     

Phosphatidylserine-mediated phagocytosis of influenza A virus-infected cells by mouse peritoneal macrophages

Influenza virus induces apoptosis in cultured cell lines as well as in animal tissues. HeLa cells were infected with influenza virus A/Udon/72 (H3N2) under conditions resulting in almost 100% infection. Such cells underwent typical caspase-dependent apoptosis and were efficiently phagocytosed by macrophages prepared from peritoneal fluids of thioglycolate-treated mice. The membrane phospholipid phosphatidylserine appeared on the surfaces of virus-infected cells at around the time efficient phagocytosis became detectable. In fact, the phagocytosis was almost completely inhibited in the presence of liposomes containing phosphatidylserine, which did not influence the antibody-dependent uptake of zymosan particles by the same macrophages. These results indicate that macrophages phagocytose influenza virus-infected HeLa cells in a manner mediated by phosphatidylserine that appears on the surfaces of infected cells during the process of apoptosis.     

Determination of quantitative parameters of Escherichia coli phagocytosis by mouse peritoneal macrophages

The fluorometric method was used to study guantitative parameters of phagocytosis of fluorescein-labeled Escherichia coli cells by mouse peritoneal macrophages. E. coli cells were conjugated with fluoresceinisothiocyanate (FITC) and then incubated with macrophages. At the end of the assay phagocytosis was arrested with a lysing solution (0.5% Triton X-100 in 0.01 M phosphate-buffered 0.15 M saline, pH 7.4). Trypan blue at a concentration of 0.04% was used as a quenching agent to differentiate between attachment and ingestion of E. coli cells. The time course analysis within this method showed that phagocytosis of E. coli cells was temperature and opsonin dependent. The number of E. coli cells ingested by macrophages increased rapidly during the initial 60 min of incubation at 37 degrees C. E. coli cells required opsonization with 5% native serum to achieve their optimal uptake. The uptake of nonopsonized bacteria by macrophages was significantly lower that that of opsonized ones (P < 0.05). It was demonstrated that sodium azide inhibited phagocytosis of E. coli cells by mouse peritoneal macrophages in a dose-dependent manner.     

Sustained depolarization and ADP-ribose activate a common ionic current in rat peritoneal macrophages

Phagocytosis is associated with large changes in the membrane potential of macrophages, but the functional significance of this is unknown. Whole cell recordings were made from rat peritoneal macrophages. Sustained (>30 s) depolarization of the cells progressively activated a conductance that remained high (several nanoSeimens) for several tens of seconds. This current: 1) was linearly dependent on potential between -100 and +50 mV; 2) reversed close to 0 mV in a physiological external solution; 3) could also be carried in part by N-methyl-D-glucamine (P(NMDG)/P(Na) 0.7), chloride (P(Cl)/P(Na) 0.5), or calcium (P(Ca)/P(Na) 1.3); and 4) was blocked by intracellular ATP (5 mM) or ADP (10 mM) and by extracellular lanthanum (half-maximal concentration 1 mM). A current with all the same properties was recorded in cells when the intracellular solution contained ADP-ribose (10-300 micro M) or beta-NAD (1 mM) (but not any other nucleotide analogs tested). The results suggest that prolonged depolarization leads to an increased intracellular level of ADP-ribose, which in turn activates this nonselective conductance(s).     

Determination of quantitative parameters of Escherichia coli phagocytosis by mouse peritoneal macrophages

Quantitative parameters of phagocytosis of fluorescein-labeled Escherichia coli cells by mouse peritoneal macrophages were studied using a fluorimetric method. E. coli cells were conjugated with fluoresceinisothiocyanate (FITC) and then incubated with macrophages. At the end of incubation, phagocytosis was stopped by the addition of a lysing solution (0.5% Triton X-100 in 0.01 M phosphate buffer in 0.15 M saline, pH 7.4). Trypan blue at a concentration of 0.04% was used as a quenching agent to differentiate between attached and ingested E. coli cells. It was shown that phagocytosis of E. coli cells depended on temperature and opsonization of bacteria. The number of E. coli cells ingested by macrophages increased rapidly for the initial 60 min of incubation at 37°C. To achieve optimal uptake of E. coli cells, their opsonization with 5% native serum was needed. The uptake of nonopsonized bacteria by macrophages was significantly lower than that of the opsonized ones (p < 0.05). Sodium azide was shown to produce a dose-dependent suppression of phagocytosis of E. coli cells by mouse peritoneal macrophages.     

Positive regulation of phagocytosis by SIRPbeta and its signaling mechanism in macrophages

SIRPbeta (signal-regulatory protein beta) is a transmembrane protein that is expressed in hematopoietic cells but whose functions are unknown. We have now cloned mouse SIRPbeta cDNA and have shown that the gene is expressed in various tissues in addition to cells of the macrophage lineage. Engagement of SIRPbeta by specific monoclonal antibodies promoted Fcgamma receptor-dependent or -independent phagocytosis in mouse peritoneal macrophages. It also induced marked activation of MAPK and the upstream kinase MEK but weak activation of Akt. MEK inhibitors markedly blocked the promotion of phagocytosis by SIRPbeta, whereas an inhibitor of phosphoinositide 3-kinase partly blocked such response. In addition, inhibitors of myosin light chain kinase or of myosin ATPase blocked the promotion of phagocytosis by SIRPbeta. Furthermore, SIRPbeta induced the formation of filopodia and lamellipodia in macrophages as well as the translocation of activated MAPK to these structures. It also elicited tyrosine phosphorylation of DAP12, Syk, and SLP-76, and a Syk inhibitor blocked the promotion of phagocytosis and activation of MAPK by SIRPbeta. Our results suggest that engagement of SIRPbeta promotes phagocytosis in macrophages by inducing the tyrosine phosphorylation of DAP12, Syk, and SLP-76 and the subsequent activation of a MEK-MAPK-myosin light chain kinase cascade.     

IL-4 pretreatment selectively enhances cytokine and chemokine production in lipopolysaccharide-stimulated mouse peritoneal macrophages

Although well recognized for its anti-inflammatory effect on gene expression in stimulated monocytes and macrophages, IL-4 is a pleiotropic cytokine that has also been shown to enhance TNF-alpha and IL-12 production in response to stimulation with LPS. In the present study we expand these prior studies in three areas. First, the potentiating effect of IL-4 pretreatment is both stimulus and gene selective. Pretreatment of mouse macrophages with IL-4 for a minimum of 6 h produces a 2- to 4-fold enhancement of LPS-induced expression of several cytokines and chemokines, including TNF-alpha, IL-1alpha, macrophage-inflammatory protein-2, and KC, but inhibits the production of IL-12p40. In addition, the production of TNF-alpha by macrophages stimulated with IFN-gamma and IL-2 is inhibited by IL-4 pretreatment, while responses to both LPS and dsRNA are enhanced. Second, the ability of IL-4 to potentiate LPS-stimulated cytokine production appears to require new IL-4-stimulated gene expression, because it is time dependent, requires the activation of STAT6, and is blocked by the reversible protein synthesis inhibitor cycloheximide during the IL-4 pretreatment period. Finally, IL-4-mediated potentiation of TNF-alpha production involves specific enhancement of mRNA translation. Although TNF-alpha protein is increased in IL-4-pretreated cells, the level of mRNA remains unchanged. Furthermore, LPS-stimulated TNF-alpha mRNA is selectively enriched in actively translating large polyribosomes in IL-4-pretreated cells compared with cells stimulated with LPS alone.     

Scavenger receptor-mediated uptake and metabolism of lipid vesicles containing acidic phospholipids by mouse peritoneal macrophages

We studied the mechanism of uptake and metabolism of exogenous phospholipids in mouse peritoneal macrophages using vesicles composed of various phospholipids and cholesterol. Macrophages in culture were found to actively incorporate and metabolize phosphatidylcholine/cholesterol vesicles containing small amounts of acidic phospholipids such as phosphatidylserine, phosphatidylinositol, or phosphatidic acid and to store the fatty acyl chains and cholesterol in triacylglycerol and cholesteryl ester form in their cytosol. These cells exhibited massive amounts of oil red O-positive lipid droplets, a typical feature of foam cells. The metabolism of exogenous phospholipid vesicles was completely inhibited by chloroquine and cytochalasin B, suggesting that vesicle uptake occurs by endocytosis. A similar type of metabolism was observed in guinea pig peritoneal macrophages, macrophage cell line J774.1, but not in Swiss 3T3 fibroblasts. Competition studies using various ligands for the scavenger receptor showed that acetylated low density lipoprotein (acetyl-LDL), dextran sulfate, or fucoidan was able to compete for up to 60% of the binding of phosphatidylserine-containing vesicles, and that copper-oxidized LDL (oxidized LDL) competed for more than 90% of the vesicle binding. On the other hand, phosphatidylserine-containing vesicles was able to compete for more than 90% of the binding of acetyl-LDL. These results indicate that acidic phospholipids are recognized by the scavenger receptors on the surface of macrophages and that more than one scavenger receptor exists on mouse peritoneal macrophages, i.e. one capable of recognizing acetyl-LDL, oxidized LDL, and an array of acidic phospholipids on membranes, and the other recognizing both acidic phospholipids and oxidized LDL but not acetyl-LDL.     

Enhancement of phagocytosis by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages

Calcitonin gene-related peptide (CGRP) is widely distributed in sensory neurons and nerve fibers. To clarify the function of CGRP on the immune system, the effect of CGRP on phagocytosis by peritoneal mactophages was examined by means of flow cytofluorometry. CGRP enhanced phagocytosis of latex beads in a dose-dependent manner. Because the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) enhanced the CGRP-induced enhancement of phagocytosis, the enhancement might be mediated by cAMP. In the presence of mannan, the phagocytosis was suppressed and the CGRP-induced enhancement was also blocked, suggesting that mannose receptors on macrophages were involved in mediating the phagocytosis of latex beads, and CGRP enhanced the mannose receptor-mediated phagocytosis. The present results indicate that CGRP can modulate the function of macrophages in nerve terminals of sensory neurons during the development and maintenance of inflammation.     

Utilization of gammalinolenic acid by mouse peritoneal macrophages.

To delineate the metabolism of gammalinolenic acid (18:3(n-6] by macrophages, primary cultures of resident mouse peritoneal macrophages were incubated with [14C]18:3(n-6). At 3, 6 or 20 h, the majority (greater than 85%) of the radiolabel was recovered in cell phospholipids. With increasing time of incubation, a relative reduction of 14C in glycerophosphocholine (ChoGpl, 58.1% to 46.2%) was noted. This was offset by a corresponding increase in glycerophosphoethanolamine (EtnGpl) labeling (from 8.8% to 18.9%). There was also a time-dependent redistribution of 14C from diacyl to ether-containing phospholipid subclasses in ChoGpl and EtnGpl. Analysis of cell extracts by reverse-phae HPLC following transmethylation demonstrated that 18:3(n-6) was extensively elongated (greater than 80%) to dihomogammalinolenic acid (20:3(n-6] by 3 h. The major radiolabeled phospholipid molecular species in the diacyl (PtdCho) and alkylacylglycerophosphocholine (PakCho) subclasses was 16:0-20:3(n-6). In contrast, diacyl (PtdEtn) and alkenylacylglycerophosphoethanolamine (PlsEtn) subclasses contained primarily [14C]18:0-20:3(n-6) and 16:0-20:3(n-6), respectively. Macrophages prelabeled with [14C]18:3(n-6) for 20 h and stimulated with calcium ionophore A23187 or zymosan synthesized [14C]prostaglandin E1 (PGE1). These data demonstrate that macrophages possess an active long chain polyunsaturated fatty acid elongase capable of converting 18:3(n-6) to 20:3(n-6) which can, upon stimulation, be converted to PGE1.     

Interactions of liposomes with the reticuloendothelial system: II. Nonspecific and receptor-mediated uptake of liposomes by mouse peritoneal macrophages

Liposomes are taken up as intact vesicles by mouse peritoneal macrophages in a process which is temperature sensitive and is affected by inhibitors of glycolytic metabolism and of microfilament activity. Macrophages take up negatively charged vesicles more readily than positively charged vesicles (2-fold) or neutral vesicles (4-fold). Macrophages take up similar amounts of multilamellar liposomes, reversed phase liposomes and small unilamellar liposomes in terms of lipid, however this corresponds to vastly different numbers of particles and amounts of trapped volume. Coating the liposomes with macromolecular ligands capable of interacting with macrophage surface receptors can markedly promote liposome uptake. Thus, formation of an IgG-antigen complex on the liposome surface results in a 10²-fold enhancement of liposome uptake, while coating the vesicles with fibronectin results in a 10-fold augmentation of uptake. Uptake via IgG-mediated and fibronectin-mediated processes seem to be independent since excess unlabelled, IgG-coated liposomes will inhibit the uptake of radioactively-labelled IgG-coated liposomes much more effectively than the uptake of radioactively-labelled fibronectin-coated liposomes. Cell-bound liposomes can readily be visualized on and inside of the macrophages using fluorescence microscopy techniques.     

Negative regulation of class IA phosphoinositide 3-kinase by protein kinase Cdelta Limits Fcgamma receptor-mediated phagocytosis in macrophages

Stimulation of macrophages by various ligands results in the activation of both phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC). Here, we showed that PKCdelta selectively inhibits class IA PI3K. Prior exposure of macrophages to a PKC activator, phorbol 12-myristate 13-acetate (PMA) inhibited the PI3K activation induced by the Fcgamma receptor (FcgammaR) ligation but not that induced by C5a. Prolonged PKC inhibition by GF109203X increased the basal PI3K activity of quiescent macrophages. The effect of the PKC inhibitor can be observed in macrophages from mice lacking class IB PI3K (p110gamma). Thus PKC was suggested to selectively attenuate the class IA activity. Chronic PKC activation by PMA induced PKCdelta degradation and Akt activation. Enhancement of the basal Akt actvity was also observed in cells stably deficient in PKCdelta prepared by shRNA technique. FcgammaR-mediated phagocytosis was dramatically increased in these cells. Thus it is suggested that inactivation of class IA PI3K by PKCdelta is functioning in regulation of FcgammaR-mediated phagocytosis.     

Uptake and degradation of mast-cell granules by mouse peritoneal macrophages.

35S-labelled mast-cell granules isolated from mouse mastocytomas were added to mouse macrophages in vitro. The granules were avidly phagocytosed, and subsequently the radioactivity was released to the medium as inorganic [35S]sulphate. After pulse-labelling, a total of about 80% of the cell-associated radioactivity was thus released in the course of 24 h, indicating an extensive breakdown of the sulphated polysaccharides, mainly heparin, present in the granules. The uptake of the mast-cell granules caused pronounced, but reversible, spreading of the macrophages.