Analysis of FcgammaRIIA cytoplasmic tail requirements in signaling for serotonin secretion: evidence for an ITAM-dependent, PI3K-dependent pathway.

The human Fc receptor, FcgammaRIIA, is known to mediate phagocytosis and endocytosis, yet the greatest numbers of these receptors are expressed on the surface of non-phagocytic platelets, where they are involved in serotonin secretion. FcgammaRIIA harbours three tyrosine (Y) residues within its cytoplasmic domain. Y1 is upstream of both Y2 and Y3, which are contained within an immunoreceptor tyrosine-based activation motif (ITAM), required for many signaling events. We have demonstrated that the two ITAM tyrosines are required for phagocytic signaling and that mutation of a single ITAM tyrosine decreases but does not abolish phagocytic signaling. Furthermore, we have identified that the YMTL motif is required for endocytosis. These observations suggest that FcgammaRIIA utilizes different sequences for various signaling events. Therefore, we investigated the sequence requirements for another important FcgammaRIIA-mediated signaling event, serotonin secretion, using Rat Basophilic Leukemia (RBL-2H3) cells transfected with wildtype (WT) FcgammaRIIA or mutant FcgammaRIIA. Stimulation of cells expressing WT FcgammaRIIA induced release of serotonin at a level 7-fold greater than that in nonstimulated WT FcgammaRIIA-transfected cells or nontransfected RBL cells. Mutation of either ITAM tyrosine (Y2 or Y3) to phenylalanine was sufficient to abolish serotonin secretion. Further, while inhibition of Syk with piceatannol blocked phagocytosis as expected, it did not inhibit serotonin secretion. Additionally, inhibition of phosphoinositol-3-kinase (PI3K) with wortmannin only had a partial effect on serotonin signaling, despite the fact that the concentrations used completely abolished phagocytic signaling. These data suggest that the requirements for serotonin secretion differ from those for phagocytosis mediated by FcgammaRIIA.

The cytoplasmic domain of FcgammaRIIA (CD32) participates in phagolysosome formation.

Signaling motifs located within the cytoplasmic domain of certain receptors contribute to lysosome fusion. Most studies have described lysosome fusion with respect to endocytic receptors. Phagolysosome fusion has not been extensively studied. To test the hypothesis that the tail of FcgammaRIIA participates in phagolysosomal fusion, a "reverse" genetic complementation system was used. It was previously shown that complement receptor type 3 (CR3) can rescue the phagocytic activity of a mutant FcgammaRIIA lacking its cytoplasmic domain (tail-minus form). This system has allowed us to study Fcgamma receptor-dependent phagocytosis and phagolysosome fusion in the presence and absence of the cytoplasmic domain of FcgammaRIIA. Fluorescent dextran was used to label lysosomes. After target internalization, wild-type FcgammaRIIA-mediated phagolysosome formation was observed as indicated by colocalization of fluorescent dextran and the phagosome. In addition, when studying mutants of FcgammaRIIA containing a full-length cytoplasmic tail with the 2 ITAM tyrosines mutated to phenylalanine, (1) phagocytosis was abolished, (2) CR3 restored phagocytosis, and (3) lysosomal fusion was similar to that observed with the wild-type receptor. In contrast, in the presence of CR3 and the tail-minus form of FcgammaRIIA, internalized particles did not colocalize with dextran. Electron microscopy revealed that the lysosomal enzyme acid phosphatase colocalized with immunoglobulin G-coated targets internalized by wild-type FcgammaRIIA but not by tail-minus FcgammaRIIA and CR3. Thus, the tail of FcgammaRIIA contributes to phagolysosome fusion by a mechanism that does not require a functional ITAM sequence.

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.

Indirect role for COPI in the completion of FCgamma receptor-mediated phagocytosis.

Recent evidence suggests that extension of pseudopods during phagocytosis requires localized insertion of endomembrane vesicles. The nature of these vesicles and the processes mediating their release and insertion are unknown. COPI plays an essential role in the budding and traffic of membrane vesicles in intracellular compartments. We therefore assessed whether COPI is also involved in phagosome formation. We used ldlF cells, a mutant line derived from Chinese hamster ovary cells that express a temperature-sensitive form of epsilonCOP. To confer phagocytic ability to ldlF cells, they were stably transfected with Fc receptors type IIA (FcgammaRIIA). In the presence of functional COPI, FcgammaRIIA-transfected ldlF cells effectively internalized opsonized particles. In contrast, phagocytosis was virtually eliminated after incubation at the restrictive temperature. Similar results were obtained impairing COPI function in macrophages using brefeldin A. Notably, loss of COPI function preceded complete inhibition of phagocytosis, suggesting that COPI is indirectly required for phagocytosis. Despite their inability to internalize particles, COPI-deficient cells nevertheless expressed normal levels of FcgammaRIIA, and signal transduction appeared unimpeded. The opsonized particles adhered normally to COPI-deficient cells and were often found on actin-rich pedestals, but they were not internalized due to the inability of the cells to extend pseudopods. The failure to extend pseudopods was attributed to the inability of COPI-deficient cells to mobilize endomembrane vesicles, including a VAMP3-containing compartment, in response to the phagocytic stimulus.

Fc gamma receptors differ in their structural requirements for interaction with the tyrosine kinase Syk in the initial steps of signaling for phagocytosis.

Receptors for the constant region of IgG, Fc gamma receptors, are expressed on the surface of hematopoietic cells, where they mediate signaling events, such as phagocytosis, essential for host defense. Fc gamma receptors also play a role in the pathophysiology of autoimmune diseases. We have demonstrated that members of each of the three classes of human Fc gamma receptors, Fc gamma RI, Fc gamma RII, and Fc gamma RIII, mediate phagocytosis, but that important differences exist in their requirements for phagocytic signaling. For example, the Fc gamma receptors Fc gamma RI and Fc gamma RIIIA induce signaling largely by association with a gamma subunit containing a conserved cytoplasmic motif (ITAM) whose tyrosines are phosphorylated following receptor stimulation. Fc gamma RIIA contains a similar motif in its own cytoplasmic domain and does not require the gamma chain for phagocytic signaling. The tyrosine kinase Syk associates with the cytoplasmic domain of both the Fc gamma receptor gamma chain and Fc gamma RIIA and is required for phagocytosis by both Fc gamma receptor systems. To elucidate the differences in phagocytic signaling by the gamma chain and Fc gamma RIIA, we investigated the requirements for Fc gamma receptor/Syk co-immunoprecipitation, tyrosine phosphorylation, and phagocytosis. Both Fc gamma RIIA and the human gamma chain contain a tyrosine seven amino acids upstream of the ITAM motif. We observed that the upstream tyrosine plays a role in Fc gamma RIIA phagocytic signaling but is not involved in phagocytic signaling by the human gamma chain. Our data also indicate that the two ITAM tyrosines of the human gamma chain and Fc gamma RIIA do not contribute equally to Fc gamma receptor association with Syk kinase and phagocytic signaling. The data indicate that the carboxy-terminal tyrosine of the receptor cytoplasmic domain is especially important both for the interaction with Syk kinase and for phagocytosis. Elucidating such differences in gamma chain and Fc gamma RIIA signaling may be valuable in designing strategies for therapeutic intervention in hematopoietic and immunological disorders.

Requirement for N-ethylmaleimide-sensitive factor activity at different stages of bacterial invasion and phagocytosis.

Bacterial invasion, like the process of phagocytosis, involves extensive and localized protrusion of the host cell plasma membrane. To examine the molecular mechanisms of the membrane remodeling that accompanies bacterial invasion, soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic was studied in cultured cells during infection by Salmonella typhimurium. A green fluorescent protein-tagged chimera of VAMP3, a SNARE characteristic of recycling endosomes, was found to accumulate at sites of Salmonella invasion. To analyze the possible role of SNARE-mediated membrane traffic in bacterial infection, invasion was measured in cells expressing a dominant-negative form of N-ethylmaleimide-sensitive factor (NSF), an essential regulator of membrane fusion. Inhibition of NSF activity did not affect cellular invasion by S. typhimurium nor the associated membrane remodeling. By contrast, Fcgamma receptor-mediated phagocytosis was greatly reduced in the presence of the mutant NSF. Most important, dominant-negative NSF significantly impaired the fusion of Salmonella-containing vacuoles with endomembranes. These observations indicate that the membrane protrusions elicited by Salmonella invasion, unlike those involved in phagocytosis, occur via an NSF-independent mechanism, whereas maturation of Salmonella-containing vacuoles is NSF-dependent.

Thrombosis and shock induced by activating antiplatelet antibodies in human Fc gamma RIIA transgenic mice: the interplay among antibody, spleen, and Fc receptor.

Transgenic mouse lines were created that express Fc gamma RIIA on platelets and macrophages at human physiologic levels, and they were used to explore the consequences in vivo of activating antiplatelet antibodies. Anti-CD9 antibody activated platelets of Fc gamma RIIA transgenic (tg) mice and, following injection in vivo, caused more rapid severe thrombocytopenia than nonactivating antiplatelet antibody. Anti-CD9 injected into Fc gamma RIIA tg crossed with FcR gamma-chain knockout (gamma-KO) mice caused thrombosis and shock in all mice, and death in 16 of 18 mice. The shock depended on platelet Fc receptor density and antibody dose. On histologic examination, the lung vasculature of anti-CD9-treated Fc gamma RIIA tg x gamma-KO mice contained extensive platelet-fibrin thrombi. Thrombosis and shock in Fc gamma RIIA tg mice in the context of the FcR gamma-chain knockout suggested the importance of the interplay of intravascular platelet activation and splenic clearance. Reduction of splenic clearance surgically (splenectomy) or functionally (monoclonal antibody treatment) also facilitated anti-CD9-mediated shock in Fc gamma RIIA tg mice. The spleen, which clears nonactivating antibody-coated platelets leading to thrombocytopenia, appears to play a protective role in the thrombosis and shock observed with activating antiplatelet antibody. The data indicate that antibodies, which activate platelets in an Fc gamma RIIA-dependent manner, can lead to thrombosis, shock, and death. Furthermore, antibody titer, platelet Fc receptor density, and splenic clearance are likely important determinants of the outcome. (Blood. 2000;96:4254-4260)

Focal exocytosis of VAMP3-containing vesicles at sites of phagosome formation.

Phagocytosis involves the receptor-mediated extension of plasmalemmal protrusions, called pseudopods, which fuse at their tip to engulf a particle. Actin polymerizes under the nascent phagosome and may propel the protrusion of pseudopods. Alternatively, membrane extension could result from the localized insertion of intracellular membranes into the plasmalemma next to the particle. Here we show focal accumulation of VAMP3-containing vesicles, likely derived from recycling endosomes, in the vicinity of the nascent phagosome. Using green fluorescent protein (GFP) as both a fluorescent indicator and an exofacial epitope tag, we show that polarized fusion of VAMP3 vesicles precedes phagosome sealing. It is therefore likely that targeted delivery of endomembranes contributes to the elongation of pseudopods. In addition to mediating pseudopod formation, receptor-triggered focal secretion of endosomes may contribute to polarized membrane extension in processes such as lamellipodial elongation or chemotaxis.

Role of COPI in phagosome maturation.

Phagosomes mature by sequentially fusing with endosomes and lysosomes. Vesicle budding is presumed to occur concomitantly, mediating the retrieval of plasmalemmal components and the regulation of phagosomal size. We analyzed whether fission of vesicles from phagosomes requires COPI, a multimeric complex known to be involved in budding from the Golgi and endosomes. The role of COPI was studied using ldlF cells, that harbor a temperature-sensitive mutation in epsilon-COP, a subunit of the coatomer complex. These cells were made phagocytic toward IgG-opsonized particles by heterologous expression of human FcgammaRIIA receptors. Following incubation at the restrictive temperature, epsilon-COP was degraded in these cells and their Golgi complex dispersed. Nevertheless, phagocytosis persisted for hours in cells devoid of epsilon-COP. Retrieval of transferrin receptors from phagosomes became inefficient in the absence of epsilon-COP, while clearance of the FcgammaRIIA receptors was unaffected. This indicates that fission of vesicles from the phagosomal membrane involves at least two mechanisms, one of which requires intact COPI. Traffic of fluid-phase markers and aggregated IgG-receptor complexes along the endocytic pathway was abnormal in epsilon-COP-deficient cells. In contrast, phagosome fusion with endosomes and lysosomes was unimpaired. Moreover, the resulting phagolysosomes were highly acidic. Similar results were obtained in RAW264.7 macrophages treated with brefeldin A, which precludes COPI assembly by interfering with the activation of adenosine ribosylation factor. These data indicate that neither phagosome formation nor maturation are absolutely dependent on COPI. Our findings imply that phagosomal maturation differs from endosomal progression, which appears to be more dependent on COPI-mediated formation of carrier vesicles.

Aerosolized Syk antisense suppresses Syk expression, mediator release from macrophages, and pulmonary inflammation.

Syk protein tyrosine kinase (PTK) is involved in signaling in leukocytes. In macrophages, Fcgamma-receptor cross-linking induces Syk PTK phosphorylation and activation, resulting in Syk-dependent events required for phagocytosis and mediator release. We hypothesized that Syk antisense oligodeoxynucleotides (ASO) delivered by aerosol to rat lungs in vivo would depress Syk PTK expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation. RT-PCR and RT-in situ PCR demonstrated that aerosolized Syk ASO administration reduced Syk mRNA expression from alveolar macrophages compared with cells isolated from sham-treated rats. Western blot analysis confirmed that Syk PTK expression was reduced after Syk ASO treatment. Compared with sham-treated rats (scrambled oligodeoxynucleotide), Syk ASO treatment suppressed Fcgamma-receptor-mediated nitric oxide (86.0 +/- 8.3%) and TNF (73.1 +/- 3.1%) production by alveolar macrophages stimulated with IgG-anti-IgG complexes. In contrast, Fcgamma-receptor-induced IL-1beta release was unaffected by Syk ASO treatment. Additionally, Syk ASO suppressed Ag-induced pulmonary inflammation, suggesting that Syk ASO may prove useful as an anti-inflammatory therapy in disorders such as asthma.

Target site search and effective inhibition of leukaemic cell growth by a covalently closed multiple anti-sense oligonucleotide to c-myb.

Systematic secondary structure simulation of a target mRNA sequence is shown to be effective for locating a good anti-sense target site. Multiple selected anti-sense sequences were placed in a single molecule. The anti-sense oligonucleotide (oligo) was covalently closed to avoid exonuclease activities and was designated CMAS (covalently closed multiple anti-sense)-oligo. CMAS-oligo was found to be stable, largely preserving its structural integrity after 24 h of incubation in the presence of either exonuclease III or serum. When human c-myb mRNA was targeted by the c-myb CMAS-oligo, expression of the gene was completely abolished. Further, tumour cell growth was inhibited by 82+/-3% as determined by an MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and by 90+/-1% by [(3)H]thymidine incorporation. When a leukaemic cell line K562 was treated with CMAS-oligo, colony formation on soft agarose was also decreased by 93%. In contrast, treatment with a scrambled control oligo did not significantly inhibit leukaemic cell growth. These results suggest that a rational target site search is possible for an anti-sense oligo and that CMAS-oligo can be employed as an effective anti-sense agent with enhanced stability.

Potent growth inhibition of leukemic cells by novel ribbon-type antisense oligonucleotides to c-myb1.

We studied the effects of antisense oligonucleotides (AS oligos) with a novel structure. The AS oligos were covalently closed to avoid exonuclease activities by enzymatic ligation of two identical molecules. The AS oligos of a ribbon type (RiAS oligos) consist of two loops containing multiple antisense sequences and a stem connecting the two loops. Three antisense sequences targeting different binding sites were placed in a loop that was designed to form a minimal secondary structure by itself. RiAS oligos were found to be stable because they largely preserved their structural integrity after 24 h incubation in the presence of either exonuclease III or serums. When a human promyelocytic cell line, HL-60, was treated with RiAS oligos to c-myb, c-myb expression was effectively ablated. Cell growth was inhibited by >90% determined by both the 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and [(3)H]thymidine incorporation. Further, when the leukemic cell line K562 was treated with c-myb RiAS oligos, colony formation on soft agarose was reduced by 92 +/- 2%. These results suggest that RiAS oligos may be employed for developing molecular antisense drugs as well as for the functional study of a gene.

Activation of macrophage CD8: pharmacological studies of TNF and IL-1 beta production.

Previously, we demonstrated that rat macrophages express CD8 and that Ab to CD8 stimulates NO production. We confirm that CD8 is expressed by rat macrophages and extend understanding of its functional significance. Activation of CD8 alpha (OX8 Ab) on alveolar macrophages stimulated mRNA expression for TNF and IL-1 beta and promoted TNF and IL-1 beta secretion. Similarly, OX8 Ab (CD8 alpha) stimulated NR8383 cells to secrete TNF, IL-1 beta, and NO. Activation of CD8 beta (Ab 341) on alveolar macrophages increased mRNA expression for TNF and IL-1 beta and stimulated secretion of TNF, but not IL-1 beta. Interestingly, anti-CD8 Abs did not stimulate IFN-gamma or PGE2 production, or phagocytosis by macrophages. OX8 (CD8 alpha)-induced TNF and IL-1 beta production by macrophages was blocked by inhibitors of protein tyrosine kinase(s), PP1, and genistein, but not by phosphatidylinositol-3 kinase inhibitor, wortmannin. Moreover, OX8 stimulated protein tyrosine kinase activity in NR8383 cells. Further analysis of kinase dependence using antisense to Syk kinase demonstrated that TNF, but not IL-1 beta, stimulation by CD8 alpha is Syk dependent. By contrast, protein kinase C inhibitor Ro 31-8220 had no effect on OX8-induced TNF production, whereas OX8-induced IL-1 beta production was blocked by Ro 31-8220. Thus, there are distinct signaling mechanisms involved in CD8 alpha (OX8)-induced TNF and IL-1 beta production. In summary, macrophages express CD8 molecules that, when activated, stimulate TNF and IL-1 beta expression, probably through mechanisms that include activation of Src and Syk kinases and protein kinase C. These findings identify a previously unknown pathway of macrophage activation likely to be involved in host defense and inflammation.

Serum lipids and arterial plaque load are altered independently with high-dose progesterone in hypercholesterolemic male rabbits.

Antiatherogenic effects of sex steroids in premenopausal women are not well defined. Therefore, we employed an established rabbit model for atherosclerosis to study the effects of exogenous estrogen and a progesterone analogue (P) on serum lipids and aortic plaque load. Serum cholesterol (C) and triglyceride (T) levels and atherosclerotic plaque loads were compared in 5 groups of male New Zealand White rabbits fed a 12-week, C-rich diet: 1 control group (CG) and 4 groups treated with estriol (E), haloperidol (H), low-dose 17-hydroxyprogesterone (LDP), or high-dose 17-hydroxyprogesterone (HDP). Serum P was measured in the LDP and HDP groups. Serial histologic sections (15 each of 27 ascending aortas) were studied by light microscopy and computerized morphometric analysis. Plaque load is defined as the ratio of intimal area to medial area (I/M). Exogenous E (p<0.001), H (P = 0. 02), LDP and HDP (P<0.001, each) were found to be significantly associated with less aortic plaque load than controls. In a multivariate analysis, after controlling for the differences in serum C and T levels, HDP (p = 0.014) was found to be associated with less aortic plaque load than controls, and this association approached statistical significance in the E (p = 0.052) and H (p = 0.069) groups. These data suggest that the mechanism(s) involved with the antiatherogenic effect of HDP in this animal model is, or are, independent of an alteration in serum lipids.

Enhancement of fcgamma receptor-mediated phagocytosis by transforming mutants of Cbl.

Phagocytosis mediated by FcgammaR plays an important role in host defense. The molecular events involved in this process have not been completely defined. The adapter protein Cbl has been implicated in FcgammaR signaling, but the function of Cbl in phagocytosis is unknown. Here we show that overexpression of the transforming mutants of Cbl, Cbl-70Z, and v-Cbl, but not wild-type (wt) Cbl, enhance phagocytosis mediated by FcgammaR in COS cells. Cbl-70Z, but not Cbl-wt, also enhanced FcgammaR-mediated phagocytosis in P388D1 murine macrophage cells. Cbl-70Z did not affect tyrosine phosphorylation or in vitro kinase activity of Syk, indicating that Syk may not be the direct target of Cbl-70Z in the enhancement of phagocytosis. A point mutation (G306E) in the phosphotyrosine domain of Cbl-70Z, as well as a C-terminal 67-aa deletion, partially abolished the enhancing effect on FcgammaR-mediated phagocytosis. A double mutant of Cbl-70Z containing both the G306E mutation and the C-terminal deletion completely lacked the ability to enhance phagocytosis. Thus, both the phosphotyrosine binding domain and the carboxyl-terminal tail were required for optimal enhancement of phagocytosis by Cbl-70Z. Functional phosphatidylinositol 3-kinase was required for Cbl-70Z to enhance phagocytosis, since wortmannin, a phosphatidylinositol 3-kinase inhibitor, inhibited FcgammaR-mediated phagocytosis in the presence of Cbl-70Z. These studies demonstrate that mutants of Cbl can modulate the phagocytic pathway mediated by FcgammaR and imply a functional involvement of c-Cbl in Fcgamma receptor-mediated phagocytosis.

Enhanced liver uptake of opsonized red blood cells after in vivo transfer of FcgammaRIIA cDNA to the liver.

Fcgamma receptors convey to phagocytic cells the ability to recognize, bind, and internalize IgG-coated cells and microorganisms. The present study demonstrates the use of adenovirus (Ad)-mediated gene transfer of human Fcgamma receptor IIA cDNA to convert normally nonphagocytic cells (hepatocytes) into functional equivalents of phagocytic cells. Ad vector in vitro transfer and expression of FcgammaRIIA cDNA in primary rat hepatocytes was confirmed by flow cytometry anti-FcgammaRIIA immunodetection, and the function of the receptor was demonstrated by enhanced binding and phagocytosis of (51)Cr-labeled IgG-opsonized erythrocytes. After in vivo gene transfer to rats, expression of FcgammaRIIA cDNA in hepatocytes was confirmed by Northern analysis and immunohistochemistry. Rats infected with the Ad vector carrying the FcgammaRIIA cDNA demonstrated enhanced clearance of opsonized erythrocytes, but not nonopsonized erythrocytes, from the circulation with increased sequestration within the liver. Together, these data demonstrate that Ad-mediated FcgammaRIIA gene transfer can convert normally IgG-nonphagocytic cells into phagocytic cells capable of recognizing, binding, and ingesting an opsonized particulate antigen, suggesting that gene transfer strategies might be used to transiently augment host defense by enhancing the clearance of immune complexes.

The cytoplasmic domain of human FcgammaRIa alters the functional properties of the FcgammaRI.gamma-chain receptor complex.

The gamma/zeta-chain family of proteins mediate cell activation for multiple immunoglobulin receptors. However, the recognition that these receptors may have distinct biologic functions suggests that additional signaling elements may contribute to functional diversity. We hypothesized that the cytoplasmic domain (CY) of the ligand binding alpha-chain alters the biological properties of the receptor complex. Using macrophage FcgammaRIa as a model system, we created stable transfectants expressing a full-length or a CY deletion mutant of human FcgammaRIa. Both receptors functionally associate with the endogenous murine gamma-chain. However, we have established that the CY of FcgammaRIa directly contributes to the functional properties of the receptor complex. Deletion of the FcgammaRIa CY leads to slower kinetics of receptor-specific phagocytosis and endocytosis as well as lower total phagocytosis despite identical levels of receptor expression. Deletion of the CY also converts the phenotype of calcium independent FcgammaRIa-specific phagocytosis to a calcium-dependent phenotype. Finally, deletion of the CY abrogates FcgammaRIa-specific secretion of interleukin-6 but does not affect production of interleukin-1beta. These results demonstrate a functional role for the CY of FcgammaRIa and provide a general model for understanding how multiple receptors that utilize the gamma-chain can generate diversity in function.

Expression of the protein kinase C substrate pleckstrin in macrophages: association with phagosomal membranes.

Despite evidence suggesting that protein kinase C (PKC) isoforms are important in phagocytosis by Fcgamma receptors, the mechanisms by which the substrates of these kinases act are largely unknown. We have investigated the role of one PKC substrate, pleckstrin, in cells of the monocyte/macrophage lineage. Pleckstrin expression in mouse macrophages was induced severalfold in response to bacterial LPS and IFN-gamma. In unstimulated cells, the protein was largely confined to the cytosol. Upon ingestion of IgG-opsonized zymosan particles (OPZ), however, pleckstrin accumulated on the phagosomal membrane. This association was transient, being maximal after 15 min and declining thereafter. Similar kinetics of association was also seen for both filamentous actin and the delta isoform of PKC. Ingestion of OPZ was found to induce phosphorylation of pleckstrin. To examine whether phosphorylation was required for phagosomal association, pleckstrin was expressed in CHO-IIA cells that stably express the FcgammaRIIA receptor and are competent for phagocytosis of OPZ. In these cells, both wild-type pleckstrin and mutants in which the phosphoacceptor sites had been mutated to either alanine (nonphosphorylatable) or glutamine (pseudophosphorylated) were found to accumulate on OPZ phagosomes. Thus, association of pleckstrin with phagosomes is independent of its phosphorylation. Our findings suggest that pleckstrin may serve as an intracellular adaptor/targeting protein in response to particulate stimuli. By targeting interacting ligands to the phagosomal compartment, pleckstrin may serve to regulate phagocytosis and/or early steps during maturation of the phagosome.

Phagosomal maturation, acidification, and inhibition of bacterial growth in nonphagocytic cells transfected with FcgammaRIIA receptors.

Phagocytosis and killing of microbial pathogens by professional phagocytes is an essential component of the innate immune response. Recently, heterologous transfection of individual receptors into nonmyeloid cells has been used successfully to elucidate the early steps that signal phagosome formation. It is unclear, however, whether the vacuoles formed by such transfected cells are bona fide phagosomes, capable of fusion with endomembranes, of luminal acidification, and of controlling the growth of microorganisms. The aim of the current study was to determine whether COS-1 and Chinese hamster ovary cells, rendered phagocytic by expression of human FcgammaRIIA receptors, express the cellular machinery required to support phagosomal maturation. Immunolocalization studies demonstrated that early endosomes, as well as late endosomes and/or lysosomes, fuse sequentially with phagosomes in the transfectants. Microfluorescence ratio imaging of particles labeled with pH-sensitive dyes revealed that maturation of the phagosome was accompanied by luminal acidification. The drop in pH, which attained levels comparable to those reported in professional phagocytes, was prevented by inhibitors of vacuolar-type H(+)-ATPases. Optimal phagosomal acidification required elevation of cytosolic [Ca(2+)], suggesting that it results from fusion of endomembranes bearing proton pumps. Moreover, the transfected cells effectively internalized live bacteria. Opsonization was essential for bacterial internalization, implying that it occurred by FcgammaRIIA-mediated phagocytosis, as opposed to invasion. Uptake into phagolysosomes was associated with inhibition of bacterial growth, due at least in part to the low intraphagosomal pH. These studies indicate that the biochemical events that follow receptor-mediated particle internalization in cells transfected with FcgammaRIIA receptors closely resemble the process of phagosomal maturation in neutrophils and macrophages. FcgammaRIIA-transfected cells can, therefore, be used as a model for the study of additional aspects of phagocyte biology.

Augmentation of pulmonary host defense against Pseudomonas by FcgammaRIIA cDNA transfer to the respiratory epithelium.

Fcgamma receptors on the surface of phagocytic cells bind the Fc region of IgG and mediate binding, phagocytosis, and destruction of particulate antigens opsonized by the antigen-specific IgG molecule. The present study evaluates the feasibility of converting lung epithelial cells into phagocytic cells using adenovirus (Ad) vector-mediated gene transfer of FcgammaRIIA cDNA to induce expression of the human FcgammaRIIA receptor. Binding and phagocytosis of opsonized sheep red blood cells (SRBCs) by the A549 human lung epithelial cell line after Ad-mediated FcgammaRIIA gene transfer was demonstrated using light and fluorescence microscopy and phagocytic assays with (51)Cr-labeled SRBCs. When A549 cells were infected with an Ad vector expressing a FcgammaRIIA mutant in which 2 of 3 cytoplasmic tyrosines have been replaced with phenylalanine, only binding, but not phagocytosis, of opsonized SRBCs was observed. In vivo expression of FcgammaRIIA in the lung after intratracheal administration of the AdFcgammaRIIA enhanced clearance of opsonized Pseudomonas aeruginosa from the lung in normal rats and in mice deficient in Fcgamma receptor expression. Similar results were observed with a chimeric FcgammaRIIA construct containing the extracellular domain of FcgammaRIIIA. Together, these data demonstrate that Ad-mediated FcgammaRIIA receptor cDNA expression can mediate the binding and phagocytosis of opsonized particulate antigens by normally nonphagocytic cells, suggesting that gene-transfer strategies might be used to utilize nonphagocytic cells to clear bacteria or other opsonized particulate antigens from the respiratory tract.