Role of CrkII in Fcgamma receptor-mediated phagocytosis

Phagocytosis of IgG-opsonized pathogens by Fcgamma receptors requires extensive remodeling of the actin cytoskeleton, a process regulated by the small GTPase Rac. Vav was thought to be the guanine nucleotide exchange factor responsible for the activation of Rac, but recent evidence indicates that Fcgamma receptor-mediated phagocytosis is unaffected in macrophages lacking all three isoforms of Vav. We therefore tested whether another GEF, DOCK180, participates in Fcgamma receptor-initiated phagocytosis. DOCK180 associates with the adaptor protein Crk, which mediates recruitment of the GEF to sites of tyrosine phosphorylation. CrkII and DOCK180 were found to accumulate at the phagocytic cup. Knockdown of Crk or DOCK180 in murine macrophages using small interfering RNA inhibited phagocytosis of IgG-opsonized particles. Moreover, transfection of dominant negative CrkII prevented both recruitment of DOCK180 and the activation of Rac at the phagocytic cup. This is the first report of a role for either Crk or DOCK180 in Fcgamma receptor-mediated phagocytosis. The Crk-DOCK180 complex is involved in the clearance of apoptotic cells, which unlike the ingestion of IgG-opsonized particles, is an anti-inflammatory process. The finding that CrkII-DOCK180 is also responsible, at least in part, for the effects of Fcgamma receptors implies that additional, parallel pathways must account for the associated pro-inflammatory effect.     

Dissociation of recruitment and activation of the small G-protein Rac during Fcgamma receptor-mediated phagocytosis

Rho-family proteins play a central role in most actin-dependent processes, including the control and maintenance of cell shape, adhesion, motility, and phagocytosis. Activation of these GTP-binding proteins is tightly regulated spatially and temporally; however, very little is known of the mechanisms involved in their recruitment and activation in vivo. Because of its inducible, restricted signaling, phagocytosis offers an ideal physiological system to delineate the pathways linking surface receptors to actin remodeling via Rho GTPases. In this study, we investigated the involvement of early regulators of Fcgamma receptor signaling in Rac recruitment and activation. Using a combination of receptor mutagenesis, cellular, molecular, and pharmacological approaches, we show that Src family and Syk kinases control Rac and Vav function during phagocytosis. Importantly, both the immunoreceptor tyrosine-based activation motif within Fcgamma receptor cytoplasmic domain and Src kinase control the recruitment of Vav and Rac. However, Syk activity is dispensable for Vav and Rac recruitment. Moreover, we show that Rac and Cdc42 activities coordinate F-actin accumulation at nascent phagosomes. Our results provide new insights in the understanding of the spatiotemporal regulation of Rho-family GTPase function, and of Rac in particular, during phagocytosis. We believe they will contribute to a better understanding of more complex cellular processes, such as cell adhesion and migration.     

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.     

A phosphatidylinositol-3-kinase-dependent signal transition regulates ARF1 and ARF6 during Fcgamma receptor-mediated phagocytosis

Fcgamma receptor (FcgammaR)-mediated phagocytosis of IgG-coated particles is regulated by 3'-phosphoinositides (3'PIs) and several classes of small GTPases, including ARF6 from the ADP Ribosylation Factor subfamily. The insensitivity of phagocytosis to brefeldin A (BFA), an inhibitor of certain ARF guanine nucleotide exchange factors (GEFs), previously indicated that ARF1 did not participate in phagocytosis. In this study, we show that ARF1 was activated during FcgammaR-mediated phagocytosis and that blocking normal ARF1 cycling inhibited phagosome closure. We examined the distributions and activation patterns of ARF6 and ARF1 during FcgammaR-mediated phagocytosis using fluorescence resonance energy transfer (FRET) stoichiometric microscopy of macrophages expressing CFP- or YFP-chimeras of ARF1, ARF6, and a GTP-ARF-binding protein domain. Both GTPases were activated by BFA-insensitive factors at sites of phagocytosis. ARF6 activation was restricted to the leading edge of the phagocytic cup, while ARF1 activation was delayed and delocalized over the phagosome. Phagocytic cups formed after inhibition of PI 3-kinase (PI-3K) contained persistently activated ARF6 and minimally activated ARF1. This indicates that a PI-3K-dependent signal transition defines the sequence of ARF GTPase activation during phagocytosis and that ARF6 and ARF1 coordinate different functions at the forming phagosome.     

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.     

Class I phosphoinositide 3-kinase p110beta is required for apoptotic cell and Fcgamma receptor-mediated phagocytosis by macrophages

Phosphoinositide 3-kinases (PI3Ks) play an important role in a variety of cellular functions, including phagocytosis. PI3Ks are activated during phagocytosis induced by several receptors and have been shown to be required for phagocytosis through the use of inhibitors such as wortmannin and LY294002. Mammalian cells have multiple isoforms of PI3K, and the role of the individual isoforms during phagocytosis has not been addressed. The class I PI3Ks consist of a catalytic p110 isoform associated with a regulatory subunit. Mammals have three genes for the class IA p110 subunits encoding p110alpha, p110beta, and p110delta and one gene for the class IB p110 subunit encoding p110gamma. Here we report a specific recruitment of p110beta and p110delta (but not p110alpha) isoforms to the nascent phagosome during apoptotic cell phagocytosis by fibroblasts. By microinjecting inhibitory antibodies specific to class IA p110 subunits, we have shown that p110beta is the major isoform required for apoptotic cell and Fcgamma receptor-mediated phagocytosis by primary mouse macrophages. Macrophages from mice expressing a catalytically inactive form of p110delta showed no defect in the phagocytosis of apoptotic cells and IgG-opsonized particles, confirming the lack of a major role for p110delta in this process. Similarly, p110gamma-deficient macrophages phagocytosed apoptotic cells normally. Our findings demonstrate that p110beta is the major class I catalytic isoform required for apoptotic cell and Fcgamma receptor-mediated phagocytosis by primary macrophages.     

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.     

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.     

Dynamics of cytoskeletal proteins in developing pine ectomycorrhiza

 Mycorrhizal short roots of Pinus contorta Dougl. ex Loud colonized by Suillus variegatus (Sow. ex Fr.) O. Kuntze or Paxillus involutus (Batsch) Fr. were collected 1–>60 days after fungal contact. The proteins of the inoculated roots were extracted, electrophoretically separated, blotted and immunostained for α-tubulin and actin. The development of the mycorrhiza was also followed microscopically. The signal of plant α-tubulin was stronger than the signal of fungal α-tubulin during the first 5 days in S. variegatus mycorrhiza and was then exceeded by fungal α-tubulin. This correlated well with the increase of fungal mycelium in the mycorrhiza. A transient drop in both plant and fungal α-tubulin signals was observed 20 days after fungal contact, suggesting a change in the metabolism of the mycorrhiza. The signals for plant and fungal actins in the mycorrhiza increased steadily during early infection and then remained at a high level as the mycorrhiza matured. Similar trends were observed in P. contorta–P. involutus mycorrhiza. The data from P. contorta–S.variegatus mycorrhizas suggests that α-tubulin is a growth-related protein, subject to changes, while the amount of actin reflects the general metabolic activity of the mycorrhiza. In both mycorrhizal systems clear α-tubulin and actin signals were detected 60 days after colonization, which indicates that the mycorrhizas were metabolically active in spite of their withered appearance. Accepted: 6 May 1996     

Identification of cytoskeletal regulatory proteins required for efficient phagocytosis in Drosophila

Phagocytosis is a complex and apparently evolutionarily conserved process that plays a central role in the immune response to infection. By ultrastructural and functional criteria, Drosophila hemocyte (macrophage) phagocytosis resembles mammalian phagocytosis. Using a non-saturated forward genetic screen for larval hemocyte phagocytosis mutants, D-SCAR and profilin were identified as important regulators of phagocytosis in Drosophila. In both hemocytes ex vivo and the macrophage-like S2 cell line, lack of D-SCAR significantly decreased phagocytosis of Escherichia coli and Staphylococcus aureus. In contrast, profilin mutant hemocytes exhibited increased phagocytic activity. Analysis of double mutants suggests that D-SCAR and profilin interact during phagocytosis. Finally, RNA interference studies in S2 cells indicated that the D-SCAR homolog D-WASp also participates in phagocytosis. This study demonstrates that Drosophila provides a viable model system in which to dissect the complex interactions that regulate phagocytosis.     

MTOC reorientation occurs during FcgammaR-mediated phagocytosis in macrophages

Cell polarization is essential for targeting signaling elements and organelles to active plasma membrane regions. In a few specialized cell types, cell polarity is enhanced by reorientation of the MTOC and associated organelles toward dynamic membrane sites. Phagocytosis is a highly polarized process whereby particles >0.5 microm are internalized at stimulated regions on the cell surface of macrophages. Here we provide detailed evidence that the MTOC reorients toward the site of particle internalization during phagocytosis. We visualized MTOC proximity to IgG-sRBCs in fixed RAW264.7 cells, during live cell imaging using fluorescent chimeras to label the MTOC and using frustrated phagocytosis assays. MTOC reorientation in macrophages is initiated by FcgammaR ligation and is complete within 1 h. Polarization of the MTOC toward the phagosome requires the MT cytoskeleton and dynein motor activity. cdc42, PI3K, and mPAR-6 are all important signaling molecules for MTOC reorientation during phagocytosis. MTOC reorientation was not essential for particle internalization or phagolysosome formation. However Golgi reorientation in concert with MTOC reorientation during phagocytosis implicates MTOC reorientation in antigen processing events in macrophages.     

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.     

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.     

EPR studies on Fcgamma receptor-mediated changes in lymphocyte membrane

Biophysical evidence has been presented for the interaction of human lymphocyte membrane Fc receptors with aggregated IgG by severely restricting the rotational mobility of the cell surface proteins, as well as membrane lipids. Decrease in membrane fluidity was more prominent with aggregated IgG since the multivalency of Fc regions in aggregated IgG cross-linked cell surface Fc receptor.     

Transmembrane potential changes during phagocytosis in rat alveolar macrophages

Studies were carried out to measure changes in the transmembrane potential of rat alveolar macrophages during exposure of the cells to zymosan particles or to the membrane perturbant, phorbol-12-myristate-13-acetate (PMA), and to determine if changes in membrane potential are related to superoxide anion release. Exposure of the cells to either zymosan or PMA leads to membrane depolarization, which precedes superoxide anion release. Furthermore, the magnitude of the depolarization is dependent upon the concentration of either zymosan or PMA. During exposure of the alveolar macrophages to increasing levels of zymosan, there is an increase in the amount of superoxide released as well as an increase in the magnitude of the depolarization. Incubation of the cells in medium containing 150 mM K+, a medium which causes membrane depolarization, leads to superoxide release from resting cells and a decrease in the amount of superoxide released from cells exposed to zymosan. These results indicate that release of superoxide anion from rat alveolar macrophages is related to membrane depolarization and suggest that the transmembrane potential change may act as a signal to initiate the phagocytotic responses of the cells.     

Interferon suppresses pinocytosis but stimulates phagocytosis in mouse peritoneal macrophages: related changes in cytoskeletal organization

Treatment of thioglycolate-elicited macrophages with mouse beta-interferon markedly reduces pinocytosis of horseradish peroxidase and fluorescein isothiocyanate (FITC)-dextran but stimulates phagocytosis of IgG-coated sheep erythrocytes. Experiments with FITC-dextran have revealed that the overall decrease in pinocytosis is due to a nearly complete inhibition of pinocytosis in a large fraction of interferon-treated macrophages. In the remaining cells pinocytosis continues at a rate similar to that in untreated control cells. A considerable reduction in the number of cells pinocytosing FITC-dextran was observed within 12 h from the beginning of interferon treatment. Measurement of the overall level of pinocytic activity with horseradish peroxidase showed a progressive decline through 72 h of treatment. In the interferon-sensitive subpopulation, there were marked changes in cytoskeletal organization. Microtubules and 10-nm filaments were aggregated in the perinuclear region while most of the peripheral cytoplasm became devoid of these cytoskeletal structures as observed by fluorescence and electron microscopy. In addition, interferon treatment of macrophages appeared to disrupt the close topological association between bundles of 10-nm filaments and organelles such as mitochondria, lysosomes, and elements of the Golgi apparatus and endoplasmic reticulum. Such alterations in the distribution of microtubules and 10-nm filaments were not seen in the interferon-insensitive subpopulation. We have investigated the mechanism of the interferon-induced enhancement of phagocytic activity by binding IgG-coated sheep erythrocytes to mouse peritoneal macrophages at 4 degrees C and then initiating a synchronous round of ingestion by warming the cells to 37 degrees C. Thioglycolate-elicited macrophages that had been treated with mouse beta-interferon ingested IgG-coated erythrocytes faster and to a higher level than control cells in a single round of phagocytosis. In interferon-treated cultures, phagocytic cups became evident within 30 s of the shift of cultures from 4 degrees to 37 degrees C, whereas in control cultures, they appeared in 2 min. Cytochalasin D, an inhibitor of actin assembly and polymerization, abolished phagocytic activity in both control and beta-interferon-treated macrophages. However, to inhibit phagocytosis completely in thioglycolate-elicited interferon-treated macrophages, twice as much cytochalasin D was required in the treated as in control cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.