Rho is involved in superoxide formation during phagocytosis of opsonized zymosans

Phagocytosis is accompanied by the production of superoxide by the NADPH oxidase complex, for which GTP-bound Rac is essential. We wanted to determine whether Rho is also involved in the production of superoxide during phagocytosis. Inhibition of Rho by Tat-C3 exoenzyme (Tat-C3) blocked superoxide formation and curtailed the phagocytosis of serum- (SOZ), C3bi- (COZ), and IgG-opsonized zymosan (IOZ) particles. Tat-C3 did not affect superoxide formation in response to phorbol myristate acetate (PMA), formyl Met-Leu-Phe (fMLP), or macrophage colony-stimulating factor (M-CSF). Superoxide formation was also reduced in J774 cells transfected with a cDNA expressing dominant-negative form of RhoA (N19RhoA). However, purified prenylated recombinant RhoA did not activate NADPH oxidase in vitro, suggesting that Rho does not interact directly with NADPH oxidase. Tat-C3 inhibited the activity of RhoA, but did not affect that of Rac in vitro or in vivo. It also inhibited the phosphorylation of p47(PHOX), one of the cytosolic components of NADPH oxidase. Taken together, these results suggest that Rho plays an important role in superoxide formation during phagocytosis of SOZ, COZ, and IOZ via phosphorylation of p47(PHOX).     

Ras-related GTPases Rap1 and RhoA collectively induce the phagocytosis of serum-opsonized zymosan particles in macrophages

Phagocytosis occurs primarily through two main processes in macrophages: the Fcγ receptor- and the integrin αMβ2-mediated processes. Complement C3bi-opsonized particles are known to be engulfed through integrin αMβ2-mediated process, which is regulated by RhoA GTPase. C3 toxin fused with Tat-peptide (Tat-C3 toxin), an inhibitor of the Rho GTPases, was shown to markedly inhibit the phagocytosis of serum (C3bi)-opsonized zymosans (SOZs). However, 8CPT-2Me-cAMP, an activator of exchange protein directly activated by cAMP (Epac, Rap1 guanine nucleotide exchange factor), restored the phagocytosis of the SOZs that was previously inhibited by the Tat-C3 toxin. In addition, a constitutively active form of Rap1 GTPase (CA-Rap1) also restored the phagocytosis that was previously reduced by a dominant negative form of RhoA GTPase (DN-RhoA). This suggests that Rap1 can replace the function of RhoA in the phagocytosis. Inversely, CA-RhoA rescued the phagocytosis that was suppressed by DN-Rap1. These findings suggest that both RhoA and Rap1 GTPases collectively regulate the phagocytosis of SOZs. In addition, filamentous actin was reduced by the Tat-C3 toxin, which was again restored by 8CPT-2Me-cAMP. Small interfering profilin suppressed the phagocytosis, suggesting that profilin is essential for the phagocytosis of SOZs. Furthermore, 8CPT-2Me-cAMP increased the co-immunoprecipitation of profilin with Rap1, whereas Tat-C3 toxin decreased that of profilin with RhoA. Co-immunoprecipitations of profilin with actin, Rap1, and RhoA GTPases were augmented in the presence of GTPγS rather than GDP. Therefore, we propose that both Rap1 and RhoA GTPases regulate the formation of filamentous actin through the interaction between actin and profilin, thereby collectively inducing the phagocytosis of SOZs in macrophages.     

A role for mammalian diaphanous-related formins in complement receptor (CR3)-mediated phagocytosis in macrophages

Macrophages, dendritic cells, and neutrophils use phagocytosis to capture and clear off invading pathogens. The process is triggered by the interaction of ligands on the pathogens' surface with specific phagocytic receptors, including immunoglobulin (FcR) and complement C3bi (CR3) receptors (integrin alpha(M)beta2, Mac1) . Localized actin-filament assembly that acts as the driving force for particle engulfment is controlled by Rho-family small GTPases . RhoA regulates CR3-mediated phagocytosis through a mechanism that is still unclear . Mammalian Diaphanous-related (mDia) formins participate in the generation of a diverse set of actin-remodeling events downstream of RhoA , and mDia1 is recruited around fibronectin-coated beads in a RhoA-dependent manner in fibroblasts . Here, we set out to examine whether mDia proteins are involved in CR3-mediated phagocytosis in macrophages. We show that the RhoA effector mDia1 is recruited early during CR3-mediated phagocytosis and colocalizes with polymerized actin in the phagocytic cup. Interfering with mDia activity inhibits CR3-mediated phagocytosis while having no effect on FcR-mediated phagocytosis. These results indicate a new function for mDia proteins in the regulation of actin polymerization during CR3-mediated phagocytosis.     

Inactivation during phagocytosis of leucine aminopeptidase, an ecto-enzyme of polymorphonuclear neutrophils

Changes in enzyme activities of the plasma membrane makers were examined during phagocytosis using guinea-pig polymorphonuclear neutrophils. Incubation of neutrophils with fresh serum-opsonized zymosan particles showed a significant reduction in leucine aminopeptidase activity, whereas 5′-nucleotidase and alkaline phosphodieterase activities remained unchanged. Inactivation of leucine aminopeptidase activity was also observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or polysterene latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of leucine aminopeptidase activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor(s) which are probably activated by the attachment of an opsonized zymosan particle to a specific membrane receptor, probably the C3b receptor.     

Biochemical analysis and subcellular localization of a neutrophil-specific antigen, PMN-7, involved in the respiratory burst

The adherence of serum-opsonized yeast to neutrophils results in phagocytosis of these particulate stimuli and activation of the respiratory burst. Both events are mediated or modulated in part by the surface receptors for IgG and complement. The link between the binding of complex particulate stimuli to the cell surface, and the triggering of these neutrophil functions, is not completely understood. We have previously described an anti-human neutrophil, murine monoclonal antibody PMN7C3, which specifically inhibits the respiratory burst of neutrophils stimulated with serum-opsonized yeast. In the present study, we show that the antigen recognized by PMN7C3 (PMN7 antigen) is present on a number of neutrophil proteins, including the recently described group of related leukocyte membrane glycoproteins CR3, LFA-1, and p150,95. The PMN-7 antigen differs from other antigens associated with the C3bi receptor complex (MAC 1, MO 1, OKM1, OKM10) in that it is present only on neutrophils among peripheral blood cells. Furthermore, the binding of PMN7C3 to the neutrophil surface inhibits the activation of the respiratory burst by serum opsonized zymosan without affecting phagocytosis of these particulate stimuli. The cross-linking of cell surface PMN7 antigen by multivalent antibody is associated with the capping and internalization of antigen-antibody complexes, and appears to be necessary for the expression of maximum inhibition of opsonized zymosan-triggered respiratory burst activity. PMN7C3 also binds to a group of granule-associated proteins biochemically distinct from CR3, LFA-1, and p150,95. These granule-associated proteins containing PMN7 antigen can be mobilized to the cell surface with secretion. PMN7 antigen-bearing proteins may play a role in modulating the activation of the respiratory burst associated with phagocytosis of serum-opsonize zymosan.     

Action of two phospholipases A2 purified from Bothrops alternatus snake venom on macrophages

The in vitro effects of BaltTX-I, a catalytically inactive Lys49 variant of phospholipase A₂ (PLA₂), and BaltTX-II, an Asp49 catalytically active PLA₂ isolated from Bothrops alternatus snake venom, on thioglycollate-elicited macrophages (TG-macrophages) were investigated. At non-cytotoxic concentrations, the secretory PLA₂ BaltTX-I but not BaltTX-II stimulated complement receptor-mediated phagocytosis. Pharmacological treatment of TG-macrophages with staurosporine, a protein kinase C (PKC) inhibitor, showed that this kinase is involved in the increase of serum-opsonized zymosan phagocytosis induced by BaltTX-I but not BaltTX-II secretory PLA₂, suggesting that PKC may be involved in the stimulatory effect of this toxin in serum-opsonized zymosan phagocytosis. Moreover, BaltTX-I and -II induced superoxide production by TG-macrophages. This superoxide production stimulated by both PLA₂s was abolished after treatment of cells with staurosporine, indicating that PKC is an important signaling pathway for the production of this radical. Our experiments showed that, at non-cytotoxic concentrations, BaltTX-I may upregulate phagocytosis via complement receptors, and that both toxins upregulated the respiratory burst in TG-macrophages.     

Complement receptor 3 (CD11b/CD18) mediates type I and type II phagocytosis during nonopsonic and opsonic phagocytosis,respectively

Two types of opsonic phagocytosis have been defined depending on the receptor engaged: FcgammaRs mediate type I phagocytosis of IgG-coated particles; complement receptor 3 (CR3) mediates type II phagocytosis of complement-coated particles. In addition to opsonic phagocytosis, CR3 also mediates nonopsonic phagocytosis of zymosan (Z) and Mycobacterium kansasii through engagement of distinct sites. Using Chinese hamster ovary cells stably expressing human CR3, we studied CR3-mediated ingestion of nonopsonized particles, Z or M. kansasii, compared with opsonized zymosan (OZ). We show that 1) while OZ sinks into cells, Z is engulfed by pseudopodia as visualized by electron microscopy; 2) in contrast to OZ, nonopsonic phagocytosis of Z and M. kansasii depends on Rac and Cdc42 but not on Rho activity; and 3) CR3-mediated phagocytosis of Z depends on the kinase activity of the Src family tyrosine kinase Hck, while OZ internalization does not. Therefore, CR3 mediates type I phagocytosis under nonopsonic conditions and type II under opsonic conditions. This is the first evidence that a single receptor can mediate both types of phagocytosis depending on the ligand used.     

Two distinct cytoplasmic regions of the beta2 integrin chain regulate RhoA function during phagocytosis

AlphaMbeta2 integrins mediate phagocytosis of opsonized particles in a process controlled by RhoA, Rho kinase, myosin II, Arp2/3, and actin polymerization. AlphaMbeta2, Rho, Arp2/3, and F-actin accumulate underneath bound particles; however, the mechanism regulating Rho function during alphaMbeta2-mediated phagocytosis is poorly understood. We report that the binding of C3bi-opsonized sheep red blood cells (RBCs) to alphaMbeta2 increases Rho-GTP, but not Rac-GTP, levels. Deletion of the cytoplasmic domain of beta2, but not of alphaM, abolished Rho recruitment and activation, as well as phagocytic uptake. Interestingly, a 16-amino acid (aa) region in the membrane-proximal half of the beta2 cytoplasmic domain was necessary for activating Rho. Three COOH-terminal residues (aa 758-760) were essential for beta2-induced accumulation of Rho at complement receptor 3 (CR3) phagosomes. Activation of Rho was necessary, but not sufficient, for its stable recruitment underneath bound particles or for uptake. However, recruitment of active Rho was sufficient for phagocytosis. Our data shed light on the mechanism of outside-in signaling, from ligated integrins to the activation of Rho GTPase signaling.     

Nonopsonic phagocytosis of Mycobacterium kansasii by human neutrophils depends on cholesterol and is mediated by CR3 associated with glycosylphosphatidylinositol-anchored proteins

Receptors involved in the phagocytosis of microorganisms under nonopsonic conditions have been little studied in neutrophils. Complement receptor type 3 (CR3) is a pattern recognition receptor able to internalize zymosan and C3bi-coated particles. We report that Abs directed against CR3 strongly inhibited nonopsonic phagocytosis of Mycobacterium kansasii in human neutrophils. In these cells CR3 has been found associated with several GPI-anchored proteins localized in cholesterol-rich microdomains (rafts) of the plasma membrane. Cholesterol sequestration by nystatin, filipin, or beta-cyclodextrin as well as treatment of neutrophils with phosphatidylinositol phospholipase C to remove GPI-anchored proteins from the cell surface markedly inhibited phagocytosis of M. kansasii, without affecting phagocytosis of zymosan or serum-opsonized M. kansasii. Abs directed against several GPI-anchored proteins inhibited phagocytosis of M. kansasii, but not of zymosan. N:-acetyl-D-glucosamine, which is known to disrupt interactions between CR3 and GPI proteins, also strongly diminished phagocytosis of these mycobacteria. In conclusion, phagocytosis of M. kansasii involved CR3, GPI-anchored receptors, and cholesterol. In contrast, phagocytosis of zymosan or opsonized particles involved CR3, but not cholesterol or GPI proteins. We propose that CR3, when associated with a GPI protein, relocates in cholesterol-rich domains where M. kansasii are internalized. When CR3 is not associated with a GPI protein, it remains outside of these domains and mediates phagocytosis of zymosan and opsonized particles, but not of M. kansasii.     

Complement-mediated phagocytosis--the role of Syk

Phagocytosis is a central event in the innate immune responses that are triggered by the association between ligands on the surface of pathogens and receptors on the membrane of phagocytes. Particularly, complement-mediated phagocytosis is accomplished by specific recognition of bound complement components by the corresponding complement receptors on the phagocytes. The protein-tyrosine kinase, Syk, plays a central role in Fcgamma receptor-mediated phagocytosis in the adaptive immune system. From recent studies using a macrophage-like differentiated cell line and serum-treated zymosan, it was found that Syk also plays an essential role in complement-mediated phagocytosis in innate immunity. Serum-treated zymosan particles promptly attached to the cells and were subsequently engulfed via complement receptor3. During this process, Syk became tyrosine-phosphorylated and accumulated around the nascent phagosomes. The transfer of Syk-siRNA or dominant-negative Syk (DN-Syk) into macrophages resulted in impaired engulfment of pathogen. Collectively, Syk is required for the engulfment of pathogen in complement-mediated phagocytosis.     

RhoB affects macrophage adhesion, integrin expression and migration

Rho GTPases regulate multiple cellular responses, including cell motility and cell cycle progression. The Rho isoform RhoB represses transformation and affects endosomal trafficking, but its effects on cell adhesion and migration have not been investigated in detail. Here we show that RhoB-null macrophages are more rounded than wild-type macrophages on fibronectin and uncoated glass, and have reduced adhesion to ICAM-1 and glass but not fibronectin. This correlated with lower cell surface expression of beta2 and beta3 integrins but not beta1 integrin. RhoB-null cells migrated faster than Wt cells on fibronectin, consistent with their smaller spread area, but slower than Wt cells on glass, reflecting their reduced adhesion. C3 transferase, which inhibits RhoA, RhoB and RhoC, induced cell spreading but this effect was reduced in RhoB-null cells. However, RhoB is not required for assembly of podosomes, which are integrin-based adhesion sites, whereas C3 transferase induced a decrease in podosomes and defects in tail retraction. Since macrophages do not express RhoC, these effects of C3 transferase are due to inhibition of RhoA rather than RhoB. Our results suggest that RhoB affects cell shape and migration by regulating surface integrin levels.     

Phagocytic and macropinocytic activity in MARCKS-deficient macrophages and fibroblasts

Macrophages express high levels of the myristoylated,alanine-rich, C kinase substrate (MARCKS), an actin cross-linkingprotein. To investigate a possible role of MARCKS in macrophagefunction, fetal liver-derived macrophages were generated from wild-type and MARCKS knockout mouse embryos. No differences between the wild-typeand MARCKS-deficient macrophages with respect to morphology (Wright'sstain) or actin distribution (staining with rhodamine-phalloidin, underbasal conditions or after treatment with phorbol esters, lipopolysaccharide, or both) were observed. We then evaluated phagocytosis mediated by different receptors: Fc receptors tested withIgG-coated sheep red blood cells, complement C3b receptors tested withC3b-coated yeast, mannose receptors tested with unopsonized zymosan,and nonspecific phagocytosis tested with latex beads. We also studiedfluid phase endocytosis in macrophages and mouse embryo fibroblasts byusing FITC-dextran to quantitate this process. In most cases, therewere no differences between the cells derived from wild-type andMARCKS-deficient mice. However, a minor but significant andreproducible difference in rates of zymosan phagocytosis at 45-60min was observed, with lower rates of phagocytosis in theMARCKS-deficient cells. Our data indicate that MARCKS deficiency maylead to slightly decreased rates of zymosan phagocytosis.

     

HUVEC take up opsonized zymosan particles and secrete cytokines IL-6 and IL-8 in vitro

Uptake of zymosan A particles by human umbilical vein endothelial cells (HUVEC) and its effect on cellular cytokine and oxygen radical production was examined. HUVEC took up more serum-opsonized than -unopsonized zymosan as demonstrated by flow cytometry with fluorescence-labeled particles. The former uptake was inhibited in the presence of anti-C3c antibodies and thus complement-mediated. It probably occurred via CR1 (CD35), although participation of other receptors cannot be ruled out. Scanning electron microscopy indicated that HUVEC with fully internalized zymosan particles were damaged. Prolonged incubation of both serum-opsonized and -unopsonized zymosan particles with HUVEC induced increased secretion of the proinflammatory cytokines IL-6 and IL-8 to the cell culture supernatants, but had no effect on production of oxygen radicals. The results confirm previous reports that EC can internalize yeast and other pathogens and points to complement as a mechanism of uptake, but illustrates that the cells may be damaged in the process. Moreover, EC may participate in the anti-infection defense effort by secreting proinflammatory and chemotactic cytokines in response to the contact with pathogens.     

Rac2-deficient murine macrophages have selective defects in superoxide production and phagocytosis of opsonized particles

The Rho family GTPase Rac is a crucial participant in numerous cellular functions and acts as a molecular switch for signal transduction. Mice deficient in hemopoietic-specific Rac2 exhibited agonist-specific defects in neutrophil functions including chemoattractant-stimulated filamentous actin polymerization and chemotaxis, and superoxide production elicited by phorbol ester, fMLP, or IgG-coated particles, despite expression of the highly homologous Rac1 isoform. In this study, functional responses of Rac2-null murine macrophages were characterized to examine whether Rac2 also has nonredundant functions in this phagocytic lineage. In contrast to murine neutrophils, in which Rac1 and Rac2 are present in similar amounts, Rac1 was approximately 4-fold more abundant than Rac2 in both bone marrow-derived and peritoneal exudate macrophages, and macrophage Rac1 levels were unchanged by the absence of Rac2. Accumulation of exudate macrophages during peritoneal inflammation was reduced in rac2(-/-) mice. FcgammaR-mediated phagocytosis of IgG-coated SRBC was also significantly decreased in Rac2-null macrophages, as was NADPH oxidase activity in response to phorbol ester or FcgammaR stimulation. However, phagocytosis and oxidant production stimulated by serum-opsonized zymosan was normal in rac2(-/-) macrophages. Macrophage morphology was also similar in wild-type and Rac2-null cells, as was actin polymerization induced by FcgammaR-mediated phagocytosis or M-CSF. Hence, Rac2-null macrophages have selective defects paralleling many of the observed functional defects in Rac2-null neutrophils. These results provide genetic evidence that although Rac2 is a relatively minor isoform in murine macrophages, it plays a nonoverlapping role with Rac1 to regulate host defense functions in this phagocyte lineage.     

Interaction of two phagocytic host defense systems: Fcγ receptors and complement receptor 3

Phagocytosis of foreign pathogens by cells of the immune system is a vitally important function of innate immunity. The phagocytic response is initiated when ligands on the surface of invading microorganisms come in contact with receptors on the surface of phagocytic cells such as neutrophils, monocytes/macrophages, and dendritic cells. The complement receptor CR3 (CD11b/CD18, Mac-1) mediates the phagocytosis of complement protein (C3bi)-coated particles. Fcγ receptors (FcγRs) bind IgG-opsonized particles and provide a mechanism for immune clearance and phagocytosis of IgG-coated particles. We have observed that stimulation of FcγRs modulates CR3-mediated phagocytosis and that FcγRIIA and FcγRI exert opposite (stimulatory and inhibitory) effects. We have also determined that an intact FcγR immunoreceptor tyrosine-based activation motif is required for these effects, and we have investigated the involvement of downstream effectors. The ability to up-regulate or down-regulate CR3 signaling has important implications for therapeutics in disorders involving the host defense system.     

Detection of a neoantigen on human C3bi and C3d by monoclonal antibody

A neoantigen was detected on human C3bi and C3d by using the monoclonal antibody (MoAb) 130. The antibody bound to EC3bi and EC3d cells but not to EC3b. Although highly purified C3bi or C3d strongly inhibited the binding of the antibody to EC3d, highly purified C3c had no such effect. Native C3, C3b, or C3(H2O) inhibited this binding only weakly. The neoantigen was also detected in serum after activation with zymosan or heat-aggregated IgG, and it was found bound to the aggregated IgG and zymosan particles. Plasma samples from patients with immunologic disorders were tested for this neoantigen, and 25 out of 43 samples tested were found to have levels of neoantigen corresponding to 2 to 11.5% complement activation, whereas 13 out of 14 normal donor plasmas contained amounts of neoantigen indicating much less than 1% complement activation.     

Decreased Fc and C3 receptor function in macrophage populations which are refractory to migration inhibitory factor,C3 activators,and immune complex

Guinea pig macrophage populations previously established to be either responsive or refractory to activation by migration inhibitory factor (MIF) and Lotus fucolectin in the macrophage migration inhibition (MMI) assay were further characterized for their MMI response to diverse effectors as correlated with their Fc and C3b receptor function. MIF-refractory populations were found to be uniformly unresponsive to the complement activators: bacterial lipopolysaccharide, cobra venom factor, zymosan, and immune complex. MIF-responsive macrophages were responsive to the same activators. Fc-mediated binding and phagocytosis of IgG-coated sheep erythrocytes (EA) were markedly depressed in freshly harvested refractory macrophages as compared to responsive cells. Fc phagocytosis by refractory populations increased rapidly during 24–28 hr in vitro culture to levels equal to that of responsive cells which corresponded with an increase in their MMI response to MIF. Refractory macrophages also had decreased C3b receptor function as shown by reduced binding and phagocytosis of EAC or serum-coated zymosan and displayed a greater loss in C3b binding capacity than responsive cells during 48 hr in vitro culture. Trypsinization of responsive macrophages rendered them refractory in their MMI response to the various activators and selectively reversed C3b-dependent binding without effect on Fc binding. The plasmin esterase inhibitors, ?-amino-n-caproic acid, tranexamic acid, and l-lysine, previously established to reverse the MMI response to MIF, FBP, and C3 activators were found to inhibit both Fc- and C3-dependent phagocytosis. These results indicate that macrophage populations which are refractory to migration inhibition by MIF and C3 activators also have reduced Fc- and C3b-mediated phagocytic functions as compared to more mature responsive populations.     

Arachidonic acid-mediated and serum-opsonized-zymosan-mediated inhibition of complement production by macrophages. Lack of requirement for endogenous arachidonic acid metabolites

The ability of exogenous prostaglandins to inhibit complement production (CP) by monocytes and macrophages (Mφ) suggests that endogenous arachidonic acid metabolites produced by these cells may also regulate their rate of CP. We assessed the regulatory influence of endogenous metabolites on CP by Mφ utilizing exogenous arachidonic acid and serum-opsonized zymosan as stimulators of production of cyclooxygenase and lipoxygenase metabolites. The results of this study show that (i) the inhibition of CP caused by both agents is independent of arachidonic acid metabolites, suggesting that endogenously produced metabolites do not influence CP, and (ii) arachidonic acid and serum-opsonized zymosan inhibit production by independent mechanisms.     

Fibronectin-enhanced phagocytosis of an alternative pathway activator by human culture-derived macrophages is mediated by the C4b/C3b complement receptor (CR1)

We examined the ability of human monocytes and culture-derived macrophages under serum-free conditions to phagocytose desialated sheep erythrocytes (E), an activator of the alternative pathway of human complement. Freshly derived monocytes ingested desialated erythrocytes, but the degree of phagocytosis varied among individual donors. However, exposing the phagocyte to intact plasma fibronectin (Fn) had no effect on monocyte phagocytosis. Macrophages derived from monocytes in culture were far more efficient at ingesting desialated E, and the extent of phagocytosis was proportional to the degree of desialation. Although exposure of macrophages to substrate-bound Fn or fluid-phase Fn enhanced the phagocytosis of desialated E, pretreatment of desialated E with Fn did not enhance phagocytosis, demonstrating that Fn acted through an interaction with the macrophages. Fn-enhanced phagocytosis of desialated E was inhibited by treating macrophages with a monoclonal antibody to the C4b/C3b receptor (CR1), but not with a monoclonal antibody to the receptor for C3bi (CR3). Addition of cobra venom factor (CVF) to the macrophages also inhibited Fn-enhanced phagocytosis of desialated E. Phagocytosis of IgG-sensitized E, either in the absence or in the presence of Fn, was not significantly affected by anti-CR1 or CVF, demonstrating that these reagents did not lead to a general inhibition of phagocytosis. These experiments suggest that macrophages may deposit enough C3b onto desialated E to cause CR1-mediated phagocytosis in the presence of Fn. The ability of macrophages to opsonize and ingest foreign particles that activate complement may be critically important in areas of inflammation where concentrations of serum-derived specific opsonins may be inadequate.     

Transglutaminase-dependent RhoA activation and depletion by serotonin in vascular smooth muscle cells

The small G protein RhoA plays a major role in several vascular processes and cardiovascular disorders. Here we analyze the mechanisms of RhoA regulation by serotonin (5-HT) in arterial smooth muscle. 5-HT (0.1-10 microM) induced activation of RhoA followed by RhoA depletion at 24-72 h. Inhibition of 5-HT1 receptors reduced the early phase of RhoA activation but had no effect on 5-HT-induced delayed RhoA activation and depletion, which were suppressed by the 5-HT transporter inhibitor fluoxetine and the transglutaminase inhibitor monodansylcadaverin and in type 2 transglutaminase-deficient smooth muscle cells. Coimmunoprecipitations demonstrated that 5-HT associated with RhoA both in vitro and in vivo. This association was calcium-dependent and inhibited by fluoxetine and monodansylcadaverin. 5-HT promotes the association of RhoA with the E3 ubiquitin ligase Smurf1, and 5-HT-induced RhoA depletion was inhibited by the proteasome inhibitor MG132 and the RhoA inhibitor Tat-C3. Simvastatin, the Rho kinase inhibitor Y-27632, small interfering RNA-mediated RhoA gene silencing, and long-term 5-HT stimulation induced Akt activation. In contrast, inhibition of 5-HT-mediated RhoA degradation by MG132 prevented 5-HT-induced Akt activation. Long-term 5-HT stimulation also led to the inhibition of the RhoA/Rho kinase component of arterial contraction. Our data provide evidence that 5-HT, internalized through the 5-HT transporter, is transamidated to RhoA by transglutaminase. Transamidation of RhoA leads to RhoA activation and enhanced proteasomal degradation, which in turn is responsible for Akt activation and contraction inhibition. The observation of transamidation of 5-HT to RhoA in pulmonary artery of hypoxic rats suggests that this process could participate in pulmonary artery remodeling and hypertension.