Contrasting requirements for ubiquitylation during Fc receptor-mediated endocytosis and phagocytosis

Fc receptors on leukocytes mediate internalization of antibody-containing complexes. Soluble immune complexes are taken up by endocytosis, while large antibody-opsonized particles are internalized by phagocytosis. We investigated the role of ubiquitylation in internalization of the human FcgammaRIIA receptor by endocytosis and phagocytosis. A fusion of FcgammaRIIA to green fluorescent protein (GFP) was expressed in ts20 cells, which bear a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme. Uptake of soluble IgG complexes mediated by FcgammaRIIA-GFP was blocked by incubation at the restrictive temperature, indicating that endocytosis requires ubiquitylation. In contrast, phagocytosis and phagosomal maturation were largely unaffected when ubiquitylation was impaired. FcgammaRIIA-GFP was ubiquitylated in response to receptor cross-linking. Elimination of the lysine residues present in the cytoplasmic domain of FcgammaRIIA impaired endocytosis, but not phagocytosis. The proteasomal inhibitor clasto-lactacystin beta-lactone strongly inhibited endocytosis, but did not affect phagocytosis. These studies demonstrate a role for ubiquitylation in the endocytosis of immune receptors, and reveal fundamental differences in the mechanisms underlying internalization of a single receptor depending on the size or multiplicity of the ligand complex.     

Internalization and degradation of macrophage Fc receptors during receptor-mediated phagocytosis

Macrophage receptors for the Fc domain of immunoglobulin G (IgG) can mediate the efficient binding and phagocytosis of IgG-coated particles. After internalization, phagocytic vacuoles fuse with lysosomes, initiating the degradation of their contents. Using specific monoclonal and polyclonal antireceptor antibodies, we have now analyzed the internalization and fate of Fc receptors during the uptake of IgG- coated erythrocytes and erythrocyte ghosts by mouse peritoneal macrophages. Receptor-mediated phagocytosis led to the selective and largely irreversible removal of Fc receptors (greater than 50%) from the macrophage plasma membrane. The expression of several other plasma membrane proteins (including a receptor for complement), recognized by a series of antimacrophage monoclonal antibodies, was affected only slightly. Interiorized Fc receptors were rapidly and selectively degraded. This was demonstrated by a series of turnover studies in which Fc receptor was immunoprecipitated from lysates of 125I-labeled macrophages. These experiments were made possible by the development of a polyclonal rabbit antiserum, raised against isolated Fc receptor, which recognized the receptor even in the presence of bound ligand. In control cells, the receptor turned over with a t1/2 of approximately 10 h; after phagocytosis, greater than 50% of the receptors were degraded with a t1/2 of less than 2 h. The turnover of other unrelated plasma membrane proteins was unaffected (t1/2 of 18-23 h) under these conditions.     

Differential involvement of Src family kinases in Fc gamma receptor-mediated phagocytosis

The tyrosine phosphorylation cascade originated from Fc gamma receptors (Fc gamma Rs) is essential for macrophage functions including phagocytosis. Although the initial step is ascribed to Src family tyrosine kinases, the role of individual kinases in phagocytosis signaling is still to be determined. In reconstitution experiments, we first showed that expression in the RAW 264.7 cell line of C-terminal Src kinase (Csk) inhibited and that of a membrane-anchored, gain-of-function Csk abolished the Fc gamma R-mediated signaling that leads to phagocytosis in a kinase-dependent manner. We next tested reconstruction of the signaling in the membrane-anchored, gain-of-function Csk-expressing cells by introducing Src family kinases the C-terminal negative regulatory sequence of which was replaced with a c-myc epitope. Those constructs derived from Lyn and Hck (a-Lyn and a-Hck) that associated with detergent-resistant membranes successfully reconstructed Fc gamma R-mediated Syk activation, filamentous actin rearrangement, and phagocytosis. In contrast, c-Src-derived construct (a-Src), that was excluded from detergent-resistant membranes, could not restore the series of phagocytosis signaling. Tyrosine phosphorylation of Vav and c-Cbl was restored in common by a-Lyn, a-Hck, and a-Src, but Fc gamma RIIB tyrosine phosphorylation, which is implicated in negative signaling, was reconstituted solely by a-Lyn and a-Hck. These findings suggest that Src family kinases are differentially involved in Fc gamma R-signaling and that selective kinases including Lyn and Hck are able to fully transduce phagocytotic signaling.     

Fc receptor-mediated phagocytosis requires CDC42 and Rac1.

At the surface of phagocytes, antibody-opsonized particles are recognized by surface receptors for the Fc portion of immunoglobulins (FcRs) that mediate their capture by an actin-driven process called phagocytosis which is poorly defined. We have analyzed the function of the Rho proteins Rac1 and CDC42 in the high affinity receptor for IgE (FcepsilonRI)-mediated phagocytosis using transfected rat basophil leukemia (RBL-2H3) mast cells expressing dominant inhibitory forms of CDC42 and Rac1. Binding of opsonized particles to untransfected RBL-2H3 cells led to the accumulation of F-actin at the site of contact with the particles and further, to particle internalization. This process was inhibited by Clostridium difficile toxin B, a general inhibitor of Rho GTP-binding proteins. Dominant inhibition of Rac1 or CDC42 function severely inhibited particle internalization but not F-actin accumulation. Inhibition of CDC42 function resulted in the appearance of pedestal-like structures with particles at their tips, while particles bound at the surface of the Rac1 mutant cell line were enclosed within thin membrane protrusions that did not fuse. These phenotypic differences indicate that Rac1 and CDC42 have distinct functions and may act cooperatively in the assembly of the phagocytic cup. Inhibition of phagocytosis in the mutant cell lines was accompanied by the persistence of tyrosine-phosphorylated proteins around bound particles. Phagocytic cup closure and particle internalization were also blocked when phosphotyrosine dephosphorylation was inhibited by treatment of RBL-2H3 cells with phenylarsine oxide, an inhibitor of protein phosphotyrosine phosphatases. Altogether, our data show that Rac1 and CDC42 are required to coordinate actin filament organization and membrane extension to form phagocytic cups and to allow particle internalization during FcR-mediated phagocytosis. Our data also suggest that Rac1 and CDC42 are involved in phosphotyrosine dephosphorylation required for particle internalization.     

Differential role of actin,clathrin, and dynamin in Fc gamma receptor-mediated endocytosis and phagocytosis

Clustering of macrophage Fc gamma receptors by multimeric immunoglobulin complexes leads to their internalization. Formation of small aggregates leads to endocytosis, whereas large particulate complexes induce phagocytosis. In RAW-264.7 macrophages, Fc gamma receptor endocytosis was found to be dependent on clathrin and dynamin and insensitive to cytochalasin. Clathrin also associates with nascent phagosomes, and earlier observations suggested that it plays an essential role in phagosome formation. However, we find that phagocytosis of IgG-coated large (> or =3 microm) particles was unaffected by inhibition of dynamin or by reducing the expression of clathrin using antisense mRNA but was eliminated by cytochalasin, implying a distinct mechanism dependent on actin assembly. The uptake of smaller particles (< or =1 microm) was only partially blocked by cytochalasin. Remarkably, the cytochalasin-resistant component was also insensitive to dominant-negative dynamin I and to clathrin antisense mRNA, implying the existence of a third internalization mechanism, independent of actin, dynamin, and clathrin. The uptake of small particles occurred by a process distinct from fluid phase pinocytosis, because it was not inhibited by dominant-negative Rab5. The insensitivity of phagocytosis to dominant-negative dynamin I enabled us to test the role of dynamin in phagosomal maturation. Although internalization of receptors from the plasma membrane was virtually eliminated by the K44A and S45N mutants of dynamin I, clearance of transferrin receptors and of CD18 from maturing phagosomes was unaffected by these mutants. This implies that removal of receptors from the phagosomal membrane occurs by a mechanism that is different from the one mediating internalization of the same receptors at the plasma membrane. These results imply that, contrary to prevailing notions, normal dynamin and clathrin function is not required for phagocytosis and reveal the existence of a component of phagocytosis that is independent of actin and Rab5.     

Stimulation of human neutrophil Fc receptor-mediated phagocytosis by a low molecular weight cytokine

Human neutrophil Fc receptor-mediated phagocytosis can be markedly enhanced by a low m.w. (less than 10,000) heat-labile cytokine(s) derived from specifically stimulated human mononuclear cells and from a human T cell line, MO(t). PMN incubated with supernatant from control mononuclear leukocyte (MNL) culture bound EIgG (percentage of rosettes = 73.7% +/- 7.1) but did not ingest the attached targets (phagocytic index, PI = 40.7 +/- 9.5) as efficiently as PMN incubated with supernatant from adherent MNL, which had ingested EIgG and were then cocultured with nonadherent MNL (PI = 264.3 +/- 46.3). Cytokine-containing supernatants were fractionated on YM-10 Centricon microconcentrators, and the effluent (YM-10E) was found to contain the phagocytosis-enhancing activity. Optimal Fc receptor-mediated ingestion by YM-10E-stimulated PMN required a critical level of target-bound IgG; stimulation was dose dependent and detectable after 5 min at 37 degrees C with a maximal response by 15 min. Monoclonal antibody 3G8 (anti-PMN Fc receptor) inhibited in a dose-dependent fashion both Fc receptor-mediated rosette formation and ingestion by nonstimulated and YM-10E-stimulated PMN. Solid-phase 3G8 Fab had the same effect. A previously undescribed monoclonal antibody, 1C2, exhibited a different pattern of inhibition. It had no effect on rosetting or ingestion of EIgG by nonstimulated PMN; however, it inhibited EIgG phagocytosis by YM-10E-stimulated PMN down to the level of nonstimulated ingestion without affecting rosette formation. Solid-phase 1C2 had the same effect. These data indicate that phagocytosis mediated by 3G8-positive Fc receptors may be enhanced by cytokine(s) stimulation in a manner requiring the molecule recognized by 1C2. Monoclonal antibodies to the alpha-chain of CR3 had only minimal effects on YM-10E-stimulated ingestion. Fluorescence flow cytometry of YM-10E-stimulated PMN, indirectly stained with 3G8 or 1C2, indicated that cytokine enhancement of EIgG ingestion occurred without an increase in either 3G8 or 1C2 binding sites. These data show that the low avidity Fc receptor, which binds immune complexes, may be functionally modulated at sites of inflammation where PMN and macrophages mediate clearance and destruction of immune complexes and opsonized particles.     

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.     

Effects of inhibitors of C1q biosynthesis on macrophage Fc receptor subclass-mediated antibody-dependent cellular cytotoxicity and phagocytosis

We examined the effects of the inhibitors of C1q or collagen biosynthesis, 2,2'-dipyridyl (DP), and 3,4-dehydro-DL-proline (DHP) on murine macrophage (M phi) FcR subclass-mediated antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis of sheep erythrocyte targets. Oil-elicited peritoneal M phi from C3HeB/FeJ mice which were cultured for 24 hr with DP (0.08 or 0.10 mM) or DHP (0.8 or 1.0 mM) showed a significant decrease in FcR subclass-mediated ADCC for murine monoclonal IgG2a (FcRI) and IgG2b/IgG1 (FcRII) as well as for heterologous polyclonal IgG. These collagen inhibitors also blocked phagocytosis mediated by both IgG2a- and IgG2b-opsonized erythrocytes. DP was more potent than DHP in blocking FcR effector functions in a reversible fashion and neither inhibitor affected M phi C3b receptor function. Pretreatment of M phi with collagenase resulted in significant reduction in FcR-mediated ADCC and phagocytosis. The inhibition of M phi FcR subclass-mediated ADCC and phagocytosis by collagen C1q synthetic inhibitors or by collagenase treatment further confirms a functional relationship between cell-associated C1q and FcR-dependent functions.     

Possible role of factor XIII subunit A in Fcgamma and complement receptor-mediated phagocytosis

Besides its traditional role in hemostasis, factor XIII subunit A (FXIII-A) is supposed to function as a cellular transglutaminase and to be involved in certain intracellular processes, including cytoskeletal remodeling. To investigate its intracellular role, the aim of the present study was to follow changes in FXIII-A production in combination with the receptor-mediated phagocytic activities of monocytes/macrophages and to examine the phagocytic functions of monocytes in patients with FXIII-A deficiency. Human blood monocytes were isolated from the buffy coats of healthy volunteers and cultured for 4 days. The FcgammaR-mediated phagocytosis of sensitized erythrocytes (EA) and the complement receptor (CR)-mediated phagocytosis of complement-coated yeast particles were studied during monocyte/macrophage differentiation. Changes in the gene expression of FXIII-A were detected by real-time quantitative RT-PCR. FXIII-A protein production was investigated with fluorescent image analysis at single cell level and Western immunoblot analysis. Both the FcgammaR and CR-mediated phagocytosis increased during culturing, which peaked on day 3. The phagocytic activity of the cells could be markedly inhibited with monodansylcadaverine, an inhibitor of the transglutaminase-induced crosslinking of proteins. The phagocytosis of EA, complement-coated and uncoated yeast particles was found to be strongly diminished in monocytes of FXIII-A deficient patients. The phagocytic functions of cultured cells showed a change in parallel with the alterations in FXIII-A mRNA expression, as well as with that in FXIII-A in protein synthesis detected by image and Western immunoblot analyses in concert. Our results suggest that FXIII-A plays a role in the Fcgamma and complement receptor-mediated phagocytic activities of monocytes/macrophages.     

Signaling pathways required for macrophage scavenger receptor-mediated phagocytosis: analysis by scanning cytometry

Background

Scavenger receptors are important components of the innate immune system in the lung, allowing alveolar macrophages to bind and phagocytose numerous unopsonized targets. Mice with genetic deletions of scavenger receptors, such as SR-A and MARCO, are susceptible to infection or inflammation from inhaled pathogens or dusts. However, the signaling pathways required for scavenger receptor-mediated phagocytosis of unopsonized particles have not been characterized.

Methods

We developed a scanning cytometry-based high-throughput assay of macrophage phagocytosis that quantitates bound and internalized unopsonized latex beads. This assay allowed the testing of a panel of signaling inhibitors which have previously been shown to target opsonin-dependent phagocytosis for their effect on unopsonized bead uptake by human in vitro-derived alveolar macrophage-like cells. The non-selective scavenger receptor inhibitor poly(I) and the actin destabilizer cytochalasin D were used to validate the assay and caused near complete abrogation of bead binding and internalization, respectively.

Results

Microtubule destabilization using nocodazole dramatically inhibited bead internalization. Internalization was also significantly reduced by inhibitors of tyrosine kinases (genistein and herbimycin A), protein kinase C (staurosporine, chelerythrine chloride and Gö 6976), phosphoinositide-3 kinase ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin), and the JNK and ERK pathways. In contrast, inhibition of phospholipase C by U-73122 had no effect.

Conclusion

These data indicate the utility of scanning cytometry for the analysis of phagocytosis and that phagocytosis of unopsonized particles has both shared and distinct features when compared to opsonin-mediated phagocytosis.     

CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens

The complement system serves an important role in clearance of pathogens, immune complexes, and apoptotic cells present in the circulation. Complement fragments deposited on the particle surface serve as targets for complement receptors present on phagocytic cells. Although Kupffer cells, the liver resident macrophages, play a dominant role in clearing particles in circulation, complement receptors involved in this process have yet to be identified. Here we report the identification and characterization of a Complement Receptor of the Immunoglobulin superfamily, CRIg, that binds complement fragments C3b and iC3b. CRIg expression on Kupffer cells is required for efficient binding and phagocytosis of complement C3-opsonized particles. In turn, Kupffer cells from CRIg-deficient mice are unable to efficiently clear C3-opsonized pathogens in the circulation, resulting in increased infection and mortality of the host. CRIg therefore represents a dominant component of the phagocytic system responsible for rapid clearance of C3-opsonized particles from the circulation.     

Fc receptor-mediated phagocytosis occurs in macrophages at exceedingly low cytosolic Ca2+ levels

Cytosolic free Ca2+ ([Ca2+]i) homeostasis was investigated in mouse peritoneal macrophages and in the macrophage-like cell line J774. [Ca2+]i measurements were performed in both cells in suspension and cells in monolayers loaded with either quin2 or fura-2. Resting [Ca2+]i was 110-140 and 85-120 nM for cell suspensions and monolayers, respectively. There were no significant differences in [Ca2+]i between the two macrophage populations whether quin2 or fura-2 were used as Ca2+ indicators. Addition of heat-aggregated IgG, IgG-coated erythrocyte ghosts, or a rat monoclonal antibody (2.4G2) directed against mouse Fc receptor II induced a rise in [Ca2+]i. This [Ca2+]i increase was consistently observed in J774 and peritoneal macrophage suspensions and in J774 macrophage monolayers; in contrast it was observed inconsistently in peritoneal macrophages in monolayer cultures. The increase in [Ca2+]i induced by ligation of Fc receptors was inhibited totally in macrophages in suspension and by 80% in macrophages in monolayers by a short preincubation of macrophages with PMA; however, phagocytosis itself was unaffected. The effect of reducing cytosolic Ca2+ to very low concentrations on Fc receptor-mediated phagocytosis was also investigated. By incubating macrophages with high concentrations of quin2/AM in the absence of extracellular Ca2+, or by loading EGTA into the cytoplasm, the [Ca2+]i was buffered and clamped to 1-10 nM. Despite this, the phagocytosis of IgG-coated erythrocytes proceeded normally. These observations confirm the report of Young et al. (Young, J. D., S. S. Ko, and Z. A. Cohn. 1984. Proc. Natl. Acad. Sci. USA. 81:5430-5434) that ligation of Fc receptors causes Ca2+ mobilization in macrophages. However, these results confirm and extend the findings of McNeil et al. (McNeil, P. L., J. A. Swanson, S. D. Wright, S. C. Silverstein, and D. L. Taylor. 1986. J. Cell Biol. 102:1586-1592) that a rise in [Ca2+]i is not required for Fc receptor-mediated phagocytosis; and they provide direct evidence that Fc receptor-mediated phagocytosis occurs normally even at exceedingly low [Ca2+]i.     

Rab20 regulates phagosome maturation in RAW264 macrophages during Fc gamma receptor-mediated phagocytosis

Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es) in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.     

Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc gamma receptor-mediated phagocytosis.

Phagocytosis is a highly localized and rapid event, requiring the generation of spatially and temporally restricted signals. Because phosphatidylinositol 3-kinase (PI3K) plays an important role in the innate immune response, we studied the generation and distribution of 3' phosphoinositides (3'PIs) in macrophages during the course of phagocytosis. The presence of 3'PI was monitored noninvasively in cells transfected with chimeras of green fluorescent protein and the pleckstrin homology domain of either Akt, Btk, or Gab1. Although virtually undetectable in unstimulated cells, 3'PI rapidly accumulated at sites of phagocytosis. This accumulation was sharply restricted to the phagosomal cup, with little 3'PI detectable in the immediately adjacent areas of the plasmalemma. Measurements of fluorescence recovery after photobleaching were made to estimate the mobility of lipids in the cytosolic monolayer of the phagosomal membrane. Stimulation of phagocytic receptors induced a marked reduction of lipid mobility that likely contributes to the restricted distribution of 3'PI at the cup. 3'PI accumulation during phagocytosis was transient, terminating shortly after sealing of the phagosomal vacuole. Two factors contribute to the rapid disappearance of 3'PI: the dissociation of the type I PI3K from the phagosomal membrane and the persistent accumulation of phosphoinositide phosphatases.     

Augmentation of macrophage complement receptor function in vitro. V. Studies on the mechanisms of ligation of macrophage Fc receptors required to trigger macrophages to signal T lymphocytes to elaborate the lymphokine that activates macrophage C3 receptors for phagocytosis

We previously described a unique lymphokine that activates macrophage C3 receptors for phagocytosis. The lymphokine is generated when T lymphocytes receive a signal from macrophages that have ingested IgG-coated material. In the present work, we examined the mechanisms by which macrophage Fc receptors must be engaged for macrophages to signal lymphocytes to elaborate the lymphokine. We found that ingestion mediated by any of the three classes of murine macrophage Fc receptors was sufficient to trigger macrophages, and that engagement of macrophage Fc receptors by immobilized immune complexes was effective as well. We also found that ligation of Fc receptors by an anti-Fc receptor IgG antibody or by its F(ab')2 or Fab fragments also triggered macrophages. The ability of monovalent ligation of the receptor to elicit biologic activity suggests that this system may be of value in elucidating general mechanisms by which ligand binding of receptors is transduced into biologic effects.     

Flow cytometric assay for phagocytosis of human monocytes mediated via Fc gamma-receptors and complement receptor CR1 (CD35).

We developed a simple flow cytometric assay for phagocytosis by human monocytes that is mediated via Fc gamma receptors and the complement receptor CR1 (CD35), using fluorescent latex beads carrying IgG and complement components C4b and C3b. To prepare fluorescent latex beads carrying IgG(BA), BSA-coated latex beads (B) were incubated with diluted rabbit anti-BSA IgG. To bind complement components, BA-particles were incubated with whole human serum pretreated with K-76 monocarboxylic acid (K-76COOH). K-76COOH inhibits the activities of C5 and factor I (12,13), resulting in the deposition of C1,4b,2a,3b on BA-particles (BAC1,4b,2a,3b). Further incubation of BAC1,4b,2a,3b with EDTA-GVB at 37 degrees C gave particles carrying IgG and C4b,C3b (BAC4b,3b). The C3 fragment, C3b, was confirmed to present on BAC1,4a,2a,3b particles by SDS-PAGE and immunoblot, and these particles were calculated to have approximately 25,000-30,000 C3b molecules per particle. To evaluate the particle attachment, the phagocytic assay was performed with 3 microM cytochalasin D treated cells. The percent cells with ingested particles and the number of ingested particles/100 cells for 60 min were estimated, being 5.1% and 5.4 for B, 12.3% and 26.7 for BA, 42.5% and 108.7 for BAC4b,3b, and 42.6% and 112.5 for BAC1,4b,2a,3b, respectively.     

Ca(2+)-independent F-actin assembly and disassembly during Fc receptor- mediated phagocytosis in mouse macrophages

Phagocytosis of IgG-coated particles by macrophages is presumed to involve the actin-based cytoskeleton since F-actin accumulates beneath forming phagosomes, and particle engulfment is blocked by cytochalasins, drugs that inhibit actin filament assembly. However, it is unknown whether Fc receptor ligation affects the rate or extent of F-actin assembly during phagocytosis of IgG-coated particles. To examine this question we have used a quantitative spectrofluorometric method to examine F-actin dynamics during a synchronous wave of phagocytosis of IgG-coated red blood cells by inflammatory mouse macrophages. We observed a biphasic rise in macrophage F-actin content during particle engulfment, with maxima at 1 and 5 min after the initiation of phagocytosis. F-actin declined to resting levels by 30 min, by which time particle engulfment was completed. These quantitative increases in macrophage F-actin were reflected in localized changes in F-actin distribution. Previous work showed that the number of IgG-coated particles engulfed by macrophages is unaffected by buffering extracellular calcium or by clamping cytosolic free calcium concentration ([Ca2+]i) to very low levels (Di Virgilio, F., B. C. Meyer, S. Greenberg, and S. C. Silverstein. 1988. J. Cell Biol. 106: 657-666). To determine whether clamping [Ca2+]i in macrophages affects the rate of particle engulfment, or the assembly or disassembly of F-actin during phagocytosis, we examined these parameters in macrophages whose [Ca2+]i had been clamped to approximately less than 3 nM with fura 2/AM and acetoxymethyl ester of EGTA. We found that the initial rate of phagocytosis, and the quantities of F-actin assembled and disassembled were similar in Ca(2+)-replete and Ca(2+)-depleted macrophages. We conclude that Fc receptor-mediated phagocytosis in mouse macrophages is accompanied by an ordered sequence of assembly and disassembly of F-actin that is insensitive to [Ca2+]i.