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Biomedical subjects

O D Rotstein

Publications and source records attributed to O D Rotstein.

At least 19 recordsLinked to original sources
PubMed1999

Cell shrinkage regulates Src kinases and induces tyrosine phosphorylation of cortactin, independent of the osmotic regulation of Na+/H+ exchangers.

The signaling pathways by which cell volume regulates ion transporters, e.g. Na+/H+ exchangers (NHEs), and affects cytoskeletal organization are poorly understood. We have previously shown that shrinkage induces tyrosine phosphorylation in CHO cells, predominantly in an 85-kDa band. To identify volume-sensitive kinases and their substrates, we investigated the effect of hypertonicity on members of the Src kinase family. Hyperosmolarity stimulated Fyn and inhibited Src. Fyn activation was also observed in nystatin-permeabilized cells, where shrinkage cannot induce intracellular alkalinization. In contrast, osmotic inhibition of Src was prevented by permeabilization or by inhibiting NHE-1. PP1, a selective Src family inhibitor, strongly reduced the hypertonicity-induced tyrosine phosphorylation. We identified one of the major targets of the osmotic stress-elicited phosphorylation as cortactin, an 85-kDa actin-binding protein and well known Src family substrate. Cortactin phosphorylation was triggered by shrinkage and not by changes in osmolarity or pHi and was abrogated by PP1. Hyperosmotic cortactin phosphorylation was reduced in Fyn-deficient fibroblasts but remained intact in Src-deficient fibroblasts. To address the potential role of the Src family in the osmotic regulation of NHEs, we used PP1. The drug affected neither the hyperosmotic stimulation of NHE-1 nor the inhibition of NHE-3. Thus, members of the Src family are volume-sensitive enzymes that may participate in the shrinkage-related reorganization of the cytoskeleton but are probably not responsible for the osmotic regulation of NHE.

Animals↗
PubMed1998

Immunomodulatory effects of hypertonic resuscitation on the development of lung inflammation following hemorrhagic shock.

Hypertonic resuscitation fluids are known to be effective in restoring circulating volume in the hypovolemic trauma patient. Previous studies have suggested that hypertonicity might exert effects on immune cells leading to an altered host response. The present studies evaluated the effect of hypertonic resuscitation on the development of lung injury in a hemorrhagic shock model in which antecedent shock primes for increased lung neutrophil sequestration in response to intratracheal LPS. Resuscitation with hypertonic saline significantly reduced albumin leak, bronchoalveolar lavage fluid neutrophil counts, and the degree of histopathologic injury compared with resuscitation with Ringer's lactate. Both in vivo and in vitro data suggest that this beneficial effect may be related to altered adhesion molecule expression by the neutrophil. Specifically, hypertonicity induced shedding of L-selectin and prevented LPS-stimulated expression and activation of CD11b, both of which might contribute to reduced sequestration in the lung. Impaired up-regulation of lung ICAM-1 may have also participated, although ex vivo studies suggest that alterations in neutrophils were sufficient to account for the effect. Lung cytokine-induced neutrophil chemoattractant did not differ between animals resuscitated with hypertonic saline vs Ringer's lactate. Considered together, these studies demonstrate a possible novel approach to inhibiting organ injury in disease processes characterized by neutrophil-mediated damage.

Adjuvants, Immunologic↗
PubMed1998

Murine hepatitis virus strain 3 induces the macrophage prothrombinase fgl-2 through p38 mitogen-activated protein kinase activation.

The clinical syndrome of acute liver failure produced by fulminant viral hepatitis can be reproduced in mice by infection with murine hepatitis virus strain 3 (MHV-3). Although it is clear that MHV-3-induced hepatitis depends upon macrophage activation and the expression of a specific prothrombinase, fgl-2, the signaling pathways involved in virally stimulated cell activation are unclear. Since we had previously found that MHV-3 induces the tyrosine phosphorylation of cellular proteins, we investigated the roles of the mitogen-activated protein kinase (MAPK) proteins. In a series of Western blots, immunoprecipitation and in vitro kinase assay studies, we found that both the extracellular signal-related kinase (ERK) and p38 MAPK proteins are tyrosine-phosphorylated and activated following exposure of murine peritoneal exudative macrophages (PEM) to MHV-3. Although p38 phosphorylation and activity are induced soon after MHV-3 exposure, peaking by 1-5 min, ERK phosphorylation and activity increase more gradually, peaking at 20-30 min and gradually fading thereafter. Interestingly, whereas selective p38 inhibition with SB203580 (1-20 microM) abolished the virally stimulated induction of fgl-2 mRNA, protein, and functional activity, selective ERK inhibition with PD98059 (1-50 microM) limited fgl-2 functional activity but had little to no effect on fgl-2 mRNA or protein levels. Moreover, whereas inhibition of ERK had no effect on p38 activity, p38 inhibition consistently increased MHV-3-induced ERK activity. To ensure that these pathways were relevant in vivo, MHV-3 was injected intraperitoneally, and peritoneal exudative macrophages were collected. Again, MHV-3 exposure led to increased p38 and ERK tyrosine phosphorylation. These data argue that MHV-3 induces tightly interconnected ERK and p38 MAPK cascades in the macrophage both in vitro and in vivo. Although the ERK and p38 MAPK proteins have discordant effects at the level of fgl-2 expression, both converge at the level of its activity, suggesting that targeted MAPK inhibition may ultimately be useful in the modulation of viral hepatitis.

Animals↗
PubMed1998

v-SNARE-dependent secretion is required for phagocytosis.

Phagosomes are generally believed to form by gradual apposition of the plasma membrane of leukocytes onto the surface of invading microorganisms. The internalization of the encapsulated particle is therefore predicted to reduce the surface area of the phagocyte. Contrary to this prediction, we observed that phagocytosis is associated with a net increase in cell surface area, suggesting the concomitant occurrence of exocytosis. Selective cleavage of components of the secretory machinery by microinjection or transfection of bacterial neurotoxins induced a pronounced inhibition of phagocytosis. These observations indicate that vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis of endomembranes is essential for optimal completion of particle internalization during phagocytosis.

Animals↗
PubMed1998

Host resistance to intracellular infection: mutation of natural resistance-associated macrophage protein 1 (Nramp1) impairs phagosomal acidification.

The mechanisms underlying the survival of intracellular parasites such as mycobacteria in host macrophages remain poorly understood. In mice, mutations at the Nramp1 gene (for natural resistance-associated macrophage protein), cause susceptibility to mycobacterial infections. Nramp1 encodes an integral membrane protein that is recruited to the phagosome membrane in infected macrophages. In this study, we used microfluorescence ratio imaging of macrophages from wild-type and Nramp1 mutant mice to analyze the effect of loss of Nramp1 function on the properties of phagosomes containing inert particles or live mycobacteria. The pH of phagosomes containing live Mycobacterium bovis was significantly more acidic in Nramp1- expressing macrophages than in mutant cells (pH 5.5 +/- 0.06 versus pH 6.6 +/- 0.05, respectively; P <0.005). The enhanced acidification could not be accounted for by differences in proton consumption during dismutation of superoxide, phagosomal buffering power, counterion conductance, or in the rate of proton "leak", as these were found to be comparable in wild-type and Nramp1-deficient macrophages. Rather, after ingestion of live mycobacteria, Nramp1-expressing cells exhibited increased concanamycin-sensitive H+ pumping across the phagosomal membrane. This was associated with an enhanced ability of phagosomes to fuse with vacuolar-type ATPase-containing late endosomes and/or lysosomes. This effect was restricted to live M. bovis and was not seen in phagosomes containing dead M. bovis or latex beads. These data support the notion that Nramp1 affects intracellular mycobacterial replication by modulating phagosomal pH, suggesting that Nramp1 plays a central role in this process.

Animals↗
PubMed1998

The IL-1 beta-converting enzyme (caspase-1) inhibits apoptosis of inflammatory neutrophils through activation of IL-1 beta.

IL-1 beta-converting enzyme (ICE), also known as caspase-1, subserves two dichotomous biologic roles. It processes newly synthesized pro-IL-1 beta to yield the active cytokine and, as the human homologue of the Caenorhabditis elegans gene product, ced-3, it also induces cellular apoptosis through the cleavage of key intracellular structural and regulatory proteins and through the catalytic activation of other caspase family members. We show here that two different proinflammatory stimuli, LPS and granulocyte-macrophage-CSF, up-regulate the expression of both ICE and IL-1 beta in human polymorphonuclear neutrophils, and that the ICE-dependent cleavage of pro-IL-1 beta results in delayed expression of the constitutive cell death program. The apoptotic delay can be blocked by inhibiting tyrosine kinases or NF-kappa B activation and by inhibiting protein synthesis. Since an antisense oligonucleotide for IL-1 beta, a blocking Ab to IL-1 beta, and preincubation with the IL-1R antagonist all prevent the delay in apoptosis, we conclude that IL-1 beta acts in an autocrine manner to inhibit granulocyte programmed cell death. We conclude that caspase-1 (ICE) subserves both pro- and antiapoptotic roles; the latter role is evident during inflammation as an inhibition of spontaneous neutrophil apoptosis through the processing of IL-1 beta. The ICE-dependent activation of IL-1 beta may represent a common autocrine pathway for the divergent stimuli that inhibit the constitutive expression of neutrophil programmed cell death during inflammation.

Apoptosis↗
PubMed1998

Hemorrhagic shock primes for increased expression of cytokine-induced neutrophil chemoattractant in the lung: role in pulmonary inflammation following lipopolysaccharide.

Recent studies have suggested that hemorrhagic shock followed by resuscitation renders patients more susceptible to lung injury by priming for an exaggerated response to a second stimulus, the so-called "two-hit" hypothesis. We investigated the role of C-X-C chemokines in mediating the augmented lung inflammation in response to LPS following resuscitated shock. In a rodent model, animals exposed to antecedent shock exhibited enhanced lung neutrophil sequestration and transpulmonary albumin flux in response to intratracheal LPS. This effect correlated with an exaggerated expression of cytokine-induced neutrophil chemoattractant (CINC) protein and mRNA, but not macrophage-inflammatory protein 2. Strategies designed to inhibit CINC, both anti-CINC Ab and supplementation with the antioxidant N-acetyl-cysteine, prevented the enhanced neutrophil sequestration, suggesting that CINC played a central role in the enhanced leukocyte accumulation following shock plus LPS treatment. Shock alone increased lung nuclear factor-kappaB expression and augmented the response to LPS. Prevention of this effect by N-acetylcysteine supplementation of the resuscitation fluid implicates a role for oxidant stress in the priming for lung inflammation following shock. Finally, alveolar macrophages recovered from shock-resuscitated animals released more CINC protein in vitro in response to LPS than macrophages from sham animals. Considered together, these findings show that augmented release of CINC, in part from primed alveolar macrophages, contributes significantly to the enhanced lung leukosequestration and transpulmonary albumin flux in response to LPS following resuscitated shock.

Acetylcysteine↗
PubMed1998

Thiol-mediated regulation of ICAM-1 expression in endotoxin-induced acute lung injury.

The intracellular redox state regulates several aspects of cell function, suggesting that strategies directed toward altering the cellular redox state may modulate cell activation in inflammatory states. As the most abundant intracellular thiol, glutathione plays a critical role as an intracellular redox buffer. Using diethylmaleate (DEM) as a glutathione-depleting agent, we evaluated the effects of GSH depletion in a rodent model of polymorphonuclear neutrophil (PMN)-dependent acute lung injury. Rats received 500 microg of LPS by intratracheal challenge, inducing a 5.5-fold increase in lung permeability and sixfold increase in lung PMN content. Pretreatment with DEM prevented the LPS-induced increase in lung PMN influx and lung permeability. Northern analysis and immunohistochemical studies suggest that this effect may be mediated by preventing up-regulation of lung ICAM-1 mRNA and protein expression. This effect is specific to ICAM-1, because lung cytokine-induced neutrophil chemoattractant and TNF-alpha mRNA levels are unaffected. This finding is not unique to the lung, because a similar effect on PMN influx was recapitulated in a rodent model of chemical peritonitis. Further, in vitro studies demonstrated that pretreatment of HUVEC monolayers with DEM prevented both ICAM-1 up-regulation and PMN transendothelial migration. These data indicate the presence of a thiol-sensitive mechanism for modulating ICAM-1 gene expression and suggest a potential novel therapeutic strategy for diseases characterized by PMN-mediated tissue injury.

Animals↗
PubMed1998

Bioavailability of oral ciprofloxacin in early postsurgical patients.

OBJECTIVE: To evaluate the absorption of oral ciprofloxacin within 24 hours of laparotomy for major elective surgery or peritonitis. DESIGN: In this prospective trial, patients were given a 750-mg oral close the morning after major elective surgery (n=15) or surgery for peritonitis (n=7). Healthy volunteers served as controls (n=9). Serial urine and blood samples were drawn during the subsequent 12 hours, and pharmacokinetic measures were determined by standard high-performance liquid chromatography assay procedures. SETTING: Multicenter, university-affiliated hospitals. MAIN OUTCOME MEASURES: Drug absorption as determined by area under the concentration time curve, maximum concentration, and time to maximum concentration. RESULTS: Oral bioavailability was reduced in elective surgery and peritonitis patients compared with controls. Among the 15 elective surgery patients, 27% (4/15) showed no absorption. The remaining 73% (11/15) had an area under the curve comparable with that of controls (8.3+/-1.6 (mg/[L x h]). Among all patients, those who showed drug absorption vs those who showed no absorption did not differ with respect to malignant neoplasm, case type, age, or biochemistry. However, patients showing no absorption were significantly heavier than patients showing absorption (patients showing absorption, 15%+/-3% over ideal body weight vs patients showing no absorption, 29%+/-6% over ideal body weight; P<.05). When elective surgery patients were stratified by presence or absence of obesity (25% above ideal body weight), mean area under the curve in nonobese patients was 9.80+/-2.37 vs 0.91+/-0.56 (mg/(L x h) in obese patients (P<.05). CONCLUSIONS: Oral bioavailability was reduced for peritonitis surgery patients on the first day postoperatively, and for obese elective surgery patients. To achieve adequate serum levels requires continuation of intravenous antibiotics in patients with peritonitis, and adjustment of oral dosage in obese patients in the early period after elective surgery.

Administration, Oral↗
PubMed1998

MAP-kinase dependent induction of monocytic procoagulant activity by beta2-integrins.

beta2-Integrin adhesion molecules play crucial roles in monocyte transmigration and adherence to the inflamed extracellular matrix. While integrin engagement contributes to inflammatory cell activation, little is known about the precise signaling pathways that are important to integrin-dependent monocyte activation. We examined the role of tyrosine phosphorylation and extracellular-signal regulated kinase (ERK) activity in beta2-integrin signaling in monocytes. Cross-linking of the LFA-1 (CD11a/CD18) and MAC-1 (CD11b/CD18) integrins on the surface of THP-1 monocytic cells induced the accumulation of tyrosine phosphoproteins. As part of this signal both ERK-1 and ERK-2 are tyrosine phosphorylated. In vitro kinase assays documented an increase in ERK-2 activity following both LFA-1 and MAC-1 cross-linking. beta2-Integrin cross-linking also led to a marked increase in 4-h procoagulant activity (PCA) in THP-1 cells and purified human monocytes. Inhibition of tyrosine phosphorylation by genistein (10 microg/ml), or selective ERK inhibition with PD98059 (10 microM), was able to block the integrin-dependent induction of PCA in both THP-1 cells and human monocytes. Thus, beta2 integrin signaling in monocytic cells can flow through the tyrosine phosphorylation and activation of the ERK mitogen activated protein kinases, which is essential for the subsequent expression of tissue factor. These results suggest that the ERK proteins likely function to integrate various adhesion-dependent signals during the process of monocyte transmigration.

Blood Coagulation Factors↗
PubMed1998

The glutathione depleting agent diethylmaleate prolongs renal allograft survival.

INTRODUCTION: Intercellular adhesion molecule 1 (ICAM-1) plays an important role in mediating allograft rejection through its role in cellular trafficking and as an important costimulatory signal mediating T cell activation. We have previously reported that systemic administration of the glutathione (GSH) depleting agent diethylmaleate (DEM) prevents upregulation of ICAM-1 in various inflammatory models, suggesting that this agent may offer benefit in preventing allograft rejection. Thus we evaluated the effects of DEM in a murine model of renal transplantation. METHODS: Kidneys from C57BL/6 mice were transplanted into MHC incompatible C3H mice. Donors were treated with DEM 1 h prior to sacrifice, whereas recipients received DEM 1 h following transplantation. Animals were followed until the time of death. In separate studies, renal ICAM-1 mRNA expression was evaluated by polymerase chain reaction and the CD4(+) T cell cytokine profile evaluated in a mixed lymphocyte reaction using C3H responder splenocytes and C57BL/6 stimulator cells. RESULTS: Pretreatment with DEM increased survival from 18.9 +/- 3.6 to 30.6 +/- 10 days (P < 0.05). This increase in survival was associated with a reduction in renal ICAM-1 mRNA expression. Mixed lymphocyte cultures derived from animals pretreated with DEM demonstrated a reduction in the Th1 cytokines IFN-gamma and IL-2 and an increase in the Th2 cytokines IL-4 and IL-10. CONCLUSION: Administration of DEM with consequent systemic GSH depletion significantly reduces allograft ICAM-1 expression and prolongs graft survival. Although speculative, a shift from a Th1 to a Th2 cytokine response raises the possibility that tolerance induction plays a role in prolonged allograft survival.

Animals↗
PubMed1998

Tertiary peritonitis: clinical features of a complex nosocomial infection.

The objective of this study was to define risk factors for and the clinical course of recurrent or tertiary peritonitis. Intensive supportive care of patients with life-threatening intraabdominal infections has led to the emergence of a new clinical syndrome, tertiary peritonitis, defined as the persistence or recurrence of intraabdominal infection following apparently adequate therapy of primary or secondary peritonitis. We undertook a retrospective study of 59 patients admitted with intraabdominal infection to a surgical intensive care unit (ICU). Tertiary peritonitis developed in 74% (44/59) of patients. Despite comparable premorbid health status, source of peritonitis, and admission APACHE II scores, patients with tertiary peritonitis had a significantly longer ICU stay (21.8 +/- 14.9 vs. 8.5 +/- 7.9 days), more advanced organ dysfunction reflected in higher organ dysfunction scores (13.3 +/- 5.1 vs. 7.7 +/- 3.3), and higher ICU mortality (64% vs. 33%) than patients with uncomplicated secondary peritonitis. The most common infecting organisms in patients with tertiary peritonitis were Enterococcus, Candida, Staphylococcus epidermidis, and Enterobacter. Infectious foci were rarely amenable to percutaneous drainage and were found to be poorly localized at laparotomy. Recurrent, or tertiary, peritonitis is a common complication of intraabdominal infection in patients admitted to an ICU. It differs from uncomplicated secondary peritonitis in its microbial flora and lack of response to appropriate surgical and antibiotic therapy. Like nosocomial pneumonia in the critically ill patient, the syndrome appears to be more a reflection than a cause of adverse outcome.

Adult↗
PubMed1998

Role of the coagulation system in the local and systemic inflammatory response.

Activation of the coagulation cascade during the inflammatory response is an essential component of the host response to infection. Coagulation represents a double-edged sword. Necessary for hemostasis and the acute containment of an infective focus, it also amplifies the inflammatory response, decreases bacterial clearance, and in the critically ill patient contributes to end-organ damage and death. Evidence for these concepts is found in the fact that critically ill and septic patients consistently demonstrate marked abnormalities in their biochemical indices of coagulation. Moreover, there is excellent experimental evidence to suggest that replacement or inhibition of individual coagulation factors, either consumed or up-regulated during the inflammatory response, can attenuate both local and systemic injury and protect animals from otherwise fatal outcomes. Based on these facts, novel therapeutic approaches to the treatment of the systemic response to infection have been developed and tested in intensive care settings. Although promising results have been obtained, much work remains to be done in defining exactly which coagulation factors (or combination of factors) should be inhibited or replaced during treatment of the septic, critically ill patient. The experimental and clinical evidence linking the coagulation cascade to local and systemic inflammation is reviewed, and the status of the clinical trials performed to date is overviewed.

Animals↗
PubMed1998

Inhibition of amiloride-sensitive sodium-channel activity in distal lung epithelial cells by nitric oxide.

Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current (Isc) without affecting total Isc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the Isc. The effect was reversed by the addition of N-monomethyl-L-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.

Amiloride↗
PubMed1998

Host response to laparoscopic surgery: mechanisms and clinical correlates.

Minimal access surgery has revolutionized the treatment of a variety of surgical diseases, partly because it is associated with less patient morbidity than nonlaparoscopic surgical procedures. Emerging evidence suggests that alteration in the host response after laparoscopic procedures has significantly contributed to the improved postoperative course. Laparoscopy modulates both afferent stimuli (including tissue trauma, pain and wound size) and efferent responses (via neuroendocrine, metabolic, immunologic and cardiorespiratory systems). These effects lead to a decrease in postoperative pain, fever and disability. Laparoscopy mediates these effects through reduced wound size, the activities of endotoxin and immunomodulatory actions of the insufflated gas, resulting in impaired macrophage activity. Although clearly beneficial in reducing postoperative morbidity after elective surgery, this immunosuppression could increase the risk of complications during procedures for infection or neoplasia.

Humans↗
PubMed1997

Regulation of phagosomal acidification. Differential targeting of Na+/H+ exchangers, Na+/K+-ATPases, and vacuolar-type H+-atpases.

Vacuolar-type (V) ATPases are thought to be the main determinant of phagosomal acidification. In phagosomes containing mycobacteria, which ostensibly impair the delivery of V-ATPases to the phagosomal membrane, the pH would be expected to be near neutral. This prediction was tested by microfluorescence ratio imaging using macrophages from mice susceptible to mycobacterial infection. Although less acidic than their counterparts containing dead bacteria, phagosomes containing live Mycobacteria bovis were nearly 1 pH unit more acidic than the cytosol, suggesting the existence of alternate H+ transport mechanisms. We therefore investigated whether Na+/H+ exchange (NHE) contributes to phagosomal acidification. Immunoblotting, reverse transcriptase-polymerase chain reaction, and pharmacological studies indicated that NHE1 is the predominant isoform of the exchanger in macrophages. Fractionation revealed that NHE1 is incorporated into the phagosomal membrane, and measurements of pH indicated that it is functional in this location. Nevertheless, acidification of the lumen of phagosomes containing either latex beads or live M. bovis was insensitive to (3-methylsulfonyl-4-piperidinobenzoyl)-guanidine methanesulfonate, a potent inhibitor of NHE1. This may have been due to the absence of an appropriate lumen to cytosol Na+ gradient, because the phagosomal membrane was found to be devoid of Na+/K+ pumps. Unexpectedly, the acidification of M. bovis phagosomes was fully reversed by specific inhibitors of the vacuolar H+-ATPase, suggesting that ATPases are present only transiently or in reduced quantities in the phagosomal membrane. Alternatively, acid equivalents accumulated in endosomes by V-ATPases may be delivered to the mycobacterial phagosome by carrier vesicles devoid of ATPases.

Animals↗
PubMed1997

Rho is required for the initiation of calcium signaling and phagocytosis by Fcgamma receptors in macrophages.

Phagocytosis of bacteria by macrophages and neutrophils is an essential component of host defense against infection. The mechanism whereby the interaction of opsonized particles with Fcgamma receptors triggers the engulfment of opsonized particles remains incompletely understood, although activation of tyrosine kinases has been recognized as an early step. Recent studies in other systems have demonstrated that tyrosine kinases can in turn signal the activation of small GTPases of the ras superfamily. We therefore investigated the possible role of Rho in Fc receptor-mediated phagocytosis. To this end we microinjected J774 macrophages with C3 exotoxin from Clostridium botulinum, which ADP-ribosylates and inactivates Rho. C3 exotoxin induced the retraction of filopodia, the disappearance of focal complexes, and a global decrease in the F-actin content of J774 cells. In addition, these cells exhibited increased spreading and the formation of vacuolar structures. Importantly, inactivation of Rho resulted in the complete abrogation of phagocytosis. Inhibition of Fcgamma receptor-mediated phagocytosis by C3 exotoxin was confirmed in COS cells, which become phagocytic upon transfection of the FcgammaRIIA receptor. Rho was found to be essential for the accumulation of phosphotyrosine and of F-actin around phagocytic cups and for Fcgamma receptor-mediated Ca2+ signaling. The clustering of receptors in response to opsonin, an essential step in Fcgamma-induced signaling, was the earliest event shown to be inhibited by C3 exotoxin. The effect of the toxin was specific, since clustering and internalization of transferrin receptors were unaffected by microinjection of C3. These data identify a role for small GTPases in Fcgamma receptor-mediated phagocytosis by leukocytes.

Actins↗