Fc Receptor-mediated Phagocytosis Research Articles

Neutrophil Function in Septic Dogs

Leukocytes appear to enter a hypo-inflammatory state in human patients with a severe bacterial infection, whereas, in swine, intra-abdominal sepsis produces an initial increase and subsequent decrease in neutrophil Fc receptor mediated phagocytosis. Impaired neutrophil function (hypo-inflammatory state) develops in dogs with sepsis. Thirteen adult client-owned dogs that developed clinical signs consistent with sepsis were assessed for evidence of neutrophil dysfunction. These results were compared with those of 12 healthy dogs. Flow cytometry combined with the appropriate fluorescent markers allowed for quantification of the phagolysosomal oxidative burst after Fc receptor-mediated phagocytosis of immune complexes, neutrophil phagocytosis of opsonized Escherichia coli, and the intracellular concentration of reduced glutathione as a measure of oxidative stress. The phagolysosomal oxidative burst after Fc receptor-mediated phagocytosis was significantly lower in neutrophils from septic dogs (mean fluorescence intensity +/- standard deviation; 118 +/- 13 and 165 +/- 27 for septic and control dogs, respectively, p = 0.001), although the phagocytosis of opsonized E. coli was significantly increased (155 +/- 74 and 77 +/- 44 for septic and control dogs, respectively, p = 0.004). Intracellular reduced glutathione was not significantly different in neutrophils from septic and healthy control dogs. An important component of neutrophil function is decreased in septic dogs. The diminished oxidative burst after Fc receptor-mediated phagocytosis in neutrophils from septic dogs could hinder the ability of the innate immune system to clear bacterial infections or it might help protect these patients from the systemic consequences of infection.

Surfactant protein A binds to IgG and enhances phagocytosis of IgG-opsonized erythrocytes

Surfactant protein (SP)-A and SP-D, immunoglobulins, and complement all modulate inflammation within the lung by regulating pathogen clearance. For example, SP-A binds to and opsonizes a variety of bacteria and viruses, thereby enhancing their phagocytosis by innate immune cells such as alveolar macrophages. Immunoglobulins, which bind to antigen and facilitate Fc receptor-mediated phagocytosis, can also activate complement, a family of soluble proteins with multiple host defense functions. Previous studies showed that SP-A and complement protein C1q interact. Since complement protein C1q binds to IgG and IgM immune complexes, the hypothesis tested in this study was that SP-A, which is structurally homologous to C1q, also binds to IgG and affects its functions. SP-A binds to the Fc, rather than the Fab, region of IgG. Binding is calcium dependent but not inhibited by saccharides known to bind to SP-A's carbohydrate recognition domain. The binding of SP-A does not inhibit the formation of immune complexes or the binding of IgG to C1q. In contrast, SP-A enhances the uptake of IgG-coated erythrocytes, suggesting that SP-A might be influencing Fc receptor-mediated uptake. In summary, this study shows a novel interaction between SP-A and IgG and a functional consequence of the binding.

Chemical compounds that target thiol‐disulfide groups on mononuclear phagocytes inhibit immune mediated phagocytosis of red blood cells

Patients having immune cytopenias produce antibodies that target hematopoietic cells resulting in their phagocytosis and intracellular destruction. Early reports suggested that phagocytosis could be inhibited by interfering with membrane thiol (SH) groups on phagocytes. Thus, whether chemical compounds that interact with SH or disulfide (SS) groups on mononuclear phagocytes can inhibit phagocytosis of antibody-coated cells was examined. A monocyte monolayer assay (MMA), which examines the in vitro monocyte-macrophage (Mphi) interaction with anti-Rh(D)-coated red cells (RBCs), was used to study the ability of different SH and SS chemicals to inhibit the Fc receptor-mediated phagocytosis of sensitized RBCs. The compounds examined included thimerosal, dithiothreitol (DTT), pentane-1-thiol, and two recently described SH and two SS chemicals that have been synthesized. All compounds were found to be able to inhibit phagocytosis to varying degrees correlating to the structure of the molecule. In general, those compounds that interact with free SH groups to inhibit phagocytosis were found better than SH-containing compounds that interact with SSs. Thimerosal and p-nitrophenyl methyl disulfide were the most effective compounds inhibiting phagocytosis. Both chemicals showed greater than 50 percent inhibition at concentrations as low as 10(-9) mol per L. DTT was the least effective compound tested. Only thimerosal showed significant toxicity, as determined by decreased cell viability and increased apoptosis, but only at concentrations of 10(-8) mol per L. The effect of chemical treatment was on attachment rather than on phagocytosis itself. Fcgamma receptor-independent endocytosis was not affected by the chemical treatment. These studies indicate that pharmacologic strategies that target SH groups on mononuclear phagocytes may have future efficacy for the treatment of immune cytopenias.

Effect of Inositol-phosphatase on Fc Receptor-mediated Phagocytosis of Macrophages

Fc receptor-mediated phagocytosis is a complex process involving the activation of kinases and phosphatases. FcgammaRIIB has been known to transduces inhibitory signals through an immunoreceptor tyrosine-based inhibitory motif (ITIM) in cytoplasmic domains. In this study, we examined the involvement of inositol-phosphatase in the Fc receptor-mediated phagocytosis. Methods: J774 cells were infected using vaccinia viral vector containing SH2 domain-containing inositol-phosphatase (SHIP) cDNA and stimulated with the sensitized sheep red blood cells. Results: Stimulation of J774 cells induced the tyrosine phosphorylation of SHIP which was maximal at 5 minutes. Phosphatidylinositol-3 (PI-3) kinase inhibitor (wortmannin) inhibits J774 cell phagocytosis of sensitized sheep red blood cells in a dose-dependent manner. Heterologious expression of SHIP in J774 cells inhibits phagocytosis of sensitized sheep red blood cells in a dose-dependency manner, but catalytically dead mutants of SHIP has no effect on phagocytosis. Conclusion: These results strongly suggest that the active signals mediated by PI-3 kinase are opposed by inhibitory signals through SHIP in the regulation of Fc receptor-mediated phagocytosis.

Actin-based molecular machineries and phosphoinositide signals in Fc receptor-mediated phagocytosis

Actin-based molecular machineries and phosphoinositide signals in Fc receptor-mediated phagocytosis

Therapeutic Action of Helicobacter pylori (H. pylori) Eradication in Patients with Chronic ITP - Lessons from Eradication Therapy on H. pylori-Negative Patients.

Helicobacter pylori (H. pylori), a gram-negative bacterium, is suspected to be involved in the pathogenesis of idiopathic thrombocytopenic purpura (ITP). Recent studies from Italy and Japan showed that more than a half of H. pylori-positive patients with ITP achieved a partial or complete platelet recovery after eradication of H. pylori. To examine therapeutic action of H. pylori eradication therapy, we performed a prospective study in which ITP patients were treated with a standard eradication regimen (a combination of lansoprazole, amoxicillin and clarithromycin for one week) irrespective of the presence or absence of H. pylori infection. Thirty-seven adult patients (mean 56.4 years of age) with chronic ITP and a platelet count below 50 x 109/L were enrolled. Platelet counts and circulating anti-GPIIb/IIIa antibody-producing B cell frequencies were monitored at 0, 1, 12, and 24 weeks after initiation of the eradication therapy. H. pylori infection was found in 26 (70%) patients by means of a urea breath test, while the remaining 11 patients were negative for all of a urea breath test, a stool antigen test, and a serum antibody test. Although H. pylori-positive patients tended to be older than H. pylori-negative patients (P = 0.06), other characteristics, such as disease duration, previous treatment regimens, platelet count and anti-GPIIb/IIIa antibody-producing B cells, were not different between these 2 groups. Twenty-five (96%) H. pylori-positive patients were successfully eradicated. At 24 weeks, a significant response (platelet count > 100 x 109/L) was observed in 16 (64%) of 25 eradicated patients, but not in a H. pylori-positive patient who failed in the eradication. In addition, a platelet count did not change at all in 11 H. pylori-negative patients, indicating that platelet recovery results from eradication of H. pylori itself, but not from immunomodulatory effects of the drugs used or eradication of microorganisms other than H. pylori. Anti-GPIIb/IIIa antibody-producing B cells were significantly reduced at 12 and 24 weeks in H. pylori-positive responders (P < 0.0001) as well as, to a lesser extent, in H. pylori-positive non-responders (P = 0.02), but not in H. pylori-negative patients (P = 1.0). In the majority of responders, a platelet count was already increased at one week when anti-GPIIb/IIIa antibody-producing B cells were not decreased. To further evaluate therapeutic action of H. pylori eradication, changes of anti-GPIIb/IIIa antibody-producing B cell frequency at one week after initiation of varuous therapies were additionally examined in ITP patients who responded to intravenous immunoglobulin (n = 6), corticosteroids (n = 7), or splenectomy (n = 7). The B cell frequency was significantly decreased after treatment with corticosteroids and splenectomy (both for P < 0.0001), whereas a stable B cell frequency observed at one week after H. pylori eradication was compatible with intravenous immunoglobulin that primarily inhibits Fc receptor-mediated platelet phagocytosis. In summary, this prospective study confirms effectiveness of H. pylori eradication for chronic ITP and a direct role of H. pylori infection in the pathogenesis of ITP. The platelet recovery after H. pylori eradication is likely to be mediated through complex processes; inhibition of Fc receptor-mediated phagocytosis followed by suppression of anti-platelet autoantibody production.

The coordination of signaling during Fc receptor-mediated phagocytosis.

Phagocytosis by macrophages can be initiated by Fcgamma receptors (FcR) in membranes that bind to Fc regions of immunoglobulin G (IgG). Activated FcR transduce signals to cytoplasm, which regulate the internalization of IgG-coated particles into plasma membrane-derived vacuoles, phagosomes. Particles internalized by phagocytosis are much larger than FcR, which prompts questions of if and how the receptors are coordinated with each other. FcR-mediated signal transduction entails recruitment of proteins from cytoplasm to the receptor, largely via protein phosphorylation. These FcR signaling complexes then activate proteins that regulate actin, myosin, membrane fusion, and the production of reactive oxygen intermediates. Recent fluorescence microscopic studies of phagocytosis in macrophages indicate that signaling by FcR occurs as a sequence of distinct stages, evident in the spatial and temporal patterns of phosphoinositides, protein kinase C, and Rho-family GTPase activation on forming phagosomes. The coordination of these stages may be regulated by lipids or lipid-anchored proteins, which diffuse away from FcR complexes. Lateral diffusion of FcR-derived signals could integrate FcR-dependent responses over large areas of membrane in the forming phagosome.

Statin inhibition of Fc receptor-mediated phagocytosis by macrophages is modulated by cell activation and cholesterol.

An inflammatory response to altered lipoproteins that accumulate in the arterial wall is a major component of the pathogenesis of atherosclerosis. Statins reduce plasma levels of low-density lipoprotein (LDL) and are effective treatments for atherosclerosis. It is hypothesized that they also modulate inflammation. The aim of this study was to examine whether lovastatin inhibits macrophage inflammatory processes and clarify its mechanism of action. We examined the effects of statins on phagocytosis of antibody-coated red blood cells by cultured human monocytes and mouse peritoneal macrophages. Lovastatin, simvastatin, and zaragozic acid, a squalene synthase inhibitor, blocked Fc receptor-mediated phagocytosis by cultured human monocytes and mouse peritoneal macrophages. The inhibitory effect of lovastatin on Fc receptor-mediated phagocytosis was prevented completely by addition of mevalonate, farnesyl pyrophosphate, LDL, or cholesterol to the culture medium. The inhibitory effect of zaragozic acid was reversed by addition of LDL, but not by the addition of geranylgeranyl pyrophosphate, to the medium. In addition, the effect of lovastatin on phagocytosis is a function of cell activation because treatment of cells with tumor necrosis factor-alpha or lipopolysaccharide prevented inhibition of phagocytosis by lovastatin. The inhibition of Fc receptor-mediated phagocytosis of lovastatin is related to its effect on cholesterol biosynthesis rather than its effect on the formation of isoprenoids.

Kv1.3 potassium channels in human alveolar macrophages.

Human alveolar macrophages were obtained from macroscopically normal lung tissue obtained at surgical resections, isolated by adherence, and identified by morphology. Whole cell recordings were made from cells 1-3 h in culture, using electrodes containing potassium chloride. From a holding potential of -100 mV, depolarizing pulses to -40 mV or greater activated an outward current. Tail current reversals showed that this current was potassium selective. Margatoxin completely blocked the current; the concentration giving half-maximal block was 160 pM. In current clamp recordings, the resting membrane potential was -34 mV; margatoxin depolarized cells to close to 0 mV. A pure macrophage population was isolated by fluorescence-activated cell sorting, using the phagocytosis of BODIPY-labeled zymosan particles. Reverse transcription-polymerase chain reaction showed that, of 13 voltage-gated K+ (Kv) potassium channels sought, only Kv1.3 mRNA was present. Margatoxin (1 nM) did not affect the percentage of cells showing phagocytosis sorted from the total population. Under these experimental conditions Kv1.3 sets the resting potential of the cells, but it is not required for Fc receptor-mediated phagocytosis.

Signal transduction during Fc receptor-mediated phagocytosis

Phagocytosis is the process whereby cells engulf large particles, usually over 0.5 micro m in diameter. Phagocytosis is triggered by the interaction of opsonins that cover the particle to be internalized with specific receptors on the surface of the phagocyte. The best-studied phagocytic receptors include the Fc receptors (FcR) that bind to the Fc portion of immunoglobulins. Cross-linking of FcR on the phagocyte initiates a variety of signals, which lead through the reorganization of the actin cytoskeleton, and membrane remodeling, to the formation of the phagosome. From recent data, it is becoming clear that FcR-mediated phagocytosis occurs as a series of steps that are regulated in a nonlinear manner and that signaling for phagocytosis does not terminate when the phagosome is formed. Several lipid molecules localize around the nascent phagosome and function as initiators of important signaling pathways for the late stages of phagolysosome formation. In addition, the use of particular signaling molecules may change for different receptors and may also vary depending on the activation or differentiation state of the cell. This review focuses on this new information and presents a model of our present understanding of the signal transduction events that regulate phagocytosis mediated by FcR.

Phosphatidylinositol 3-kinase and extracellular signal-regulated kinase are recruited for Fc receptor-mediated phagocytosis during monocyte-to-macrophage differentiation

The molecular mechanism involved in Fc receptor-mediated phagocytosis in the different cell types of the immune system is still poorly defined. We investigated the role of phosphatidylinositol 3-kinase (PI 3-K) and extracellular signal-regulated kinase (ERK) in phagocytosis by monocytes and by monocyte-differentiated macrophages. Peripheral blood monocytes and monocytic cells (THP-1 cell line) were able to ingest IgG-coated erythrocytes in the absence of additional stimulus. Phagocytosis by these cells was not blocked by wortmannin and LY294002, specific inhibitors of PI 3-K, or by PD98059, a specific MEK/ERK inhibitor. However, upon differentiation of THP-1 monocytes to macrophages, through treatment with retinoic acid and interferon-gamma (IFN-gamma), wortmannin and PD98059 blocked Fc receptor-mediated phagocytosis efficiently. Inhibition of phagocytosis by PD98059 was observed after 24 h of IFN-gamma treatment, whereas wortmannin could inhibit phagocytosis only after 48 h of IFN-gamma treatment. Additionally, phagocytosis of IgG-coated erythrocytes by neutrophils, a more efficient phagocyte, was inhibited by wortmannin and PD98059. Neutrophils and monocyte-differentiated macrophages presented significantly more efficient phagocytosis than monocytes upon PMA stimulation. Taken together, these results indicate that poorly phagocytic leukocytes, such as monocytes, do not require PI 3-K and ERK for phagocytosis. Upon differentiation into macrophages, however, ERK first and PI 3-K second are recruited for regulation of phagocytosis. In addition, our data support the idea that professional phagocytes require ERK and PI 3-K for efficient phagocytosis.

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.

Fc receptor-mediated phagocytosis makes a significant contribution to clearance of influenza virus infections.

Fc receptors for IgG expressed on macrophages and NK cells are important mediators of opsonophagocytosis and Ab-dependent cell-mediated cytotoxicity. Phagocyte-mediated opsonophagocytosis is pivotal for protection against bacteria, but its importance in recovery from infection with intracellular pathogens is unclear. We have now investigated the role of opsonophagocytosis in protection against lethal influenza virus infection by using FcR gamma(-/-) mice. Absence of the FcR gamma-chain did not affect the expression of IFN-gamma and IL-10 in the lungs and spleens after intranasal immunization with an influenza subunit vaccine. Titers of serum and respiratory Abs of the IgM, IgG1, IgG2a, and IgA isotypes in FcR gamma(-/-) mice were similar to levels seen in FcR gamma(+/+) mice. Nevertheless, FcR gamma(-/-) mice were highly susceptible to influenza infection, even in the presence of anti-influenza Abs from immune FcR gamma(+/+) mice. NK cells were not necessary for the observed Ab-mediated viral clearance, but macrophages were found to be capable of actively ingesting opsonized virus particles. We conclude that Fc receptor-mediated phagocytosis plays a pivotal role in clearance of respiratory virus infections.

A critical role of Fc receptor-mediated antibody-dependent phagocytosis in the host resistance to blood-stage Plasmodium berghei XAT infection.

Plasmodium berghei XAT is an irradiation-induced attenuated variant derived from the lethal strain P. berghei NK65, and its blood-stage parasites are spontaneously cleared in immune competent mice. In the present study, we studied the mechanism of host resistance to blood-stage malaria infection using P. berghei XAT. Infection enhanced Ab-dependent phagocytosis of PRBC by splenic macrophages in wild-type C57BL/6 mice. In contrast, FcR gamma-chain knockout (FcRgamma(-/-)) mice, which lack the ability to mediate Ab-dependent phagocytosis and Ab-dependent cell-mediated cytotoxicity through FcgammaRI, FcgammaRII, and FcgammaRIII, could not induce Ab-dependent phagocytic activity. These FcRgamma(-/-) mice showed increased susceptibility to the P. berghei XAT infection, with eventually fatal results, although they produced comparable amounts of IFN-gamma by spleen cells and anti-XAT Abs in serum. In addition, passive transfer of anti-XAT IgG obtained from wild-type mice that had recovered from infection into FcRgamma(-/-) mice could not suppress the increase in parasitemia, and almost all of these mice died after marked parasitemia. In contrast, passive transfer of anti-XAT IgG into control wild-type mice inhibited the increase in parasitemia. IFN-gamma(-/-) mice, which were highly susceptible to the P. berghei XAT infection, failed to induce Ab-dependent phagocytic activity and also showed reduced production of serum anti-XAT IgG2a isotype compared with control wild-type mice. These results suggest that FcR-mediated Ab-dependent phagocytosis, which is located downstream of IFN-gamma production, is important as an effector mechanism to eliminate PRBC in blood-stage P. berghei XAT infection.

Phagocytosis and clearance of apoptotic cells is mediated by MER.

Apoptosis is fundamental to the development and maintenance of animal tissues and the immune system. Rapid clearance of apoptotic cells by macrophages is important to inhibit inflammation and autoimmune responses against intracellular antigens. Here we report a new function for Mer, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family. mer(kd) mice with a cytoplasmic truncation of Mer had macrophages deficient in the clearance of apoptotic thymocytes. This was corrected in chimaeric mice reconstituted with bone marrow from wild-type animals. Primary macrophages isolated from mer(kd) mice showed that the phagocytic deficiency was restricted to apoptotic cells and was independent of Fc receptor-mediated phagocytosis or ingestion of other particles. The inability to clear apoptotic cells adequately may be linked to an increased number of nuclear autoantibodies in mer(kd) mice. Thus, the Mer receptor tyrosine kinase seems to be critical for the engulfment and efficient clearance of apoptotic cells. This has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus.

Of umbrellas, jazz and slow-steppin'.

Of umbrellas, jazz and slow-steppin'.

Polyclonal immunoglobulins (IVIg) modulate nitric oxide production and microglial functions in vitro via Fc receptors

Controlled trials in multiple sclerosis (MS) and case reports in acute demyelinating encephalomyelitis (ADEM) have shown that intravenous immunoglobulins (IVIg) are of therapeutic benefit in central nervous system (CNS) inflammatory diseases. Studies in experimental autoimmune encephalomyelitis (EAE) have suggested these effects are mediated by modulation of the cytokine network and T cell responses. However, there are no data on the influence of IVIg on the local immune reaction in the CNS, the site of inflammation in EAE. We have therefore studied the effect of IVIg on cultured rat microglia, the main immune cell in the CNS. IVIg increased nitric oxide (NO) production in a dose-dependent manner in microglia stimulated with IFNγ. The increase was only marginal in LPS-treated cells, and no effect was seen in untreated microglia or after stimulation with TNFα or PMA. This enhancement of NO production depended on the Fc portion of IVIg and could be abrogated by the pharmacological inhibition of Syk and phosphatidylinositol 3-kinase, two enzymes involved in the signalling cascade of Fc receptors. TNFα secretion was dose-dependently stimulated by IVIg in both untreated microglia and after stimulation with LPS or IFNγ. Again, this effect was mediated through the Fc portion. Finally, we showed that Fc receptor-mediated phagocytosis was inhibited by IVIg, presumably by blockade of the Fc receptor. These different effects may protect oligodendrocytes from antibody mediated phagocytosis and on the other hand could terminate the immune reaction by induction of apoptosis in infiltrating T cells via NO and TNFα. We propose that IVIg, in addition to known effects on the peripheral immune system, may also modulate the local immune reaction in CNS inflammatory disease.

Neutralizing antibodies to adenylate cyclase toxin promote phagocytosis of Bordetella pertussis by human neutrophils.

A previous study showed that opsonization with human immune serum could either promote or antagonize phagocytosis of Bordetella pertussis by human neutrophils depending on whether the bacteria expressed adenylate cyclase toxin. Opsonization of the wild-type strain inhibited phagocytosis relative to unopsonized controls. In contrast, mutants lacking adenylate cyclase toxin were efficiently phagocytosed when opsonized with human immune serum. In this study, we examined opsonization in the presence or absence of monoclonal antibodies to adenylate cyclase toxin. Addition of neutralizing monoclonal antibodies to adenylate cyclase toxin converted a serum that previously inhibited both attachment and phagocytosis of the wild-type strain to one that increased both attachment and phagocytosis compared to the no-serum control. Monoclonal antibodies that recognize the adenylate cyclase toxin but fail to neutralize activity were without effect. These results suggest that adenylate cyclase toxin inhibits both Fc receptor-mediated attachment and phagocytosis of B. pertussis by neutrophils.

Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages.

Phagolysosome biogenesis is essential for the killing and degradation of intracellular pathogens. It involves the fusion of phagosomes with various endocytic organelles, a process known to be regulated in part by Rab proteins. We generated RAW 264.7 macrophages expressing an active mutant of Rab5 (Rab5(Q79L)) to determine the role of Rab5 in phagocytosis and phagolysosome biogenesis. Our results indicate that Rab5 stimulates phagocytosis of latex beads but not Fc or C3 receptor-mediated phagocytosis. Rab5 also acts to restrict the complete fusion of phagosomes with endosomes, a phenomenon allowing exchange of solutes from the two compartments without complete intermixing of their membrane (kiss and run). In Rab5(Q79L)-expressing macrophages, uncontrolled fusion events occurred, leading to the appearance of giant phagosomes. These phagosomes could initiate their maturation and acquire LAMP1, but failed to generate the microbicidal conditions needed to kill intracellular parasites. These results identify Rab5 as a key molecule regulating phagosome-endosome fusion and as an essential component in the innate ability of macrophages to restrict the growth of intracellular parasites.

Membrane recruitment of Rac1 triggers phagocytosis.

Rac1 is a &Rgr;-family GTP-binding protein that controls lamellipodia formation and membrane ruffling in fibroblasts. Recently, Rac1 and Cdc42, another member of the &Rgr;-family, have been shown to regulate Fc receptor-mediated phagocytosis in macrophages by controlling different steps of membrane and actin dynamics leading to particle engulfment. Here, we investigated the function of Rac1 using a membrane recruitment system that mimics phagocytosis. Recruitment of an activated Rac1 protein to the cytoplasmic domain of an engineered membrane receptor by using rapamycin as a bridge induces ingestion of latex beads bound to the receptor. Rac1-mediated bead uptake depends on actin polymerisation since actin filaments accumulate at the bead/membrane binding sites and internalisation is inhibited by cytochalasin D. Internalisation is also abolished upon substitution of Phe37 to Leu in the Rac1 effector region. Our results indicate that by promoting actin polymerisation at particle attachment sites, Rac1 by acting through specific downstream effectors induces plasma membrane remodeling that allows particle internalisation in a membrane-enclosed phagosome.