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International audience; Pathogen recognition and engulfment by phagocytic cells of the blood cell lineage constitute the first line of defense against invading pathogens. This cellular immune response is conserved throughout evolution and depends strictly on cytoskeletal changes regulated by the RhoGTPases family. Many pathogens have developed toxins modifying RhoGTPases activity to their own benefit. In particular, the Exoenzyme S (ExoS) toxin of the Gram-negative bacteria Pseudomonas aeruginosa is directly injected into the host cell cytoplasm and contains a GAP domain (ExoSGAP) targeting RhoGTPases. Searching for the contribution of each RhoGTPases, Rho1, Rac1, Rac2, Mtl (Mig2-like) and Cdc42 to fly resistance to P. aeruginosa infections, we found that Rac2 is required to resist to P. aeruginosa and to other Gram-negative or Gram-positive bacteria. The Rac2 immune-deficient phenotype is attributable to defective engulfment of pathogens since Rac2-mutant macrophages exhibited strong reduction in the phagocytosis level of both Gram-negative and Gram-positive bacterial particles whereas systemic immune signaling pathways, including Toll, Immune deficiency and Jun kinases, were not affected. Co-expression of Rac2 and ExoSGAP rescued the increased sensitivity to P. aeruginosa observed in ExoSGAP-expressing flies suggesting that Rac2 is the main host factor whose function is inhibited by the GAP domain of the ExoS toxin.

International audience; The NADPH-oxidase complex of phagocytic cells plays a key role in the defense against invading pathogens, through the release of superoxide anion, precursor of other reactive oxygen species (ROS). NADPH-oxidase deficiency is called Chronic Granulomatous Disease (CGD), in which patients suffer from recurrent infections and from the formation of granulomas in various organs. Research on NADPH-oxidase has much benefited from the discovery of cell-free systems, i.e. reconstitution assays from broken resting (unstimulated) phagocytes, in which activation of the oxidase is elicited in vitro. Cell-free systems were developed in parallel to studies of molecular defects of patients with CGD, both approaches leading to the identification of the major proteins implicated in enzyme activation. Variations around the cell-free system allowed molecular dissection of the mechanism of NADPH-oxidase activation and provided insights into its relationship to phagocytosis.

The 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10 of Mycobacterium tuberculosis are secreted by the ESX-1 system into the host cell and thereby contribute to pathogenicity. Although different studies performed at the organismal and cellular levels have helped to explain ESX-1-associated phenomena, not much is known about how ESAT-6 and CFP-10 contribute to pathogenesis at the molecular level. In this study we describe the interaction of both proteins with lipid bilayers, using biologically relevant liposomal preparations containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol, and cholesterol. Using floatation gradient centrifugation, we demonstrate that ESAT-6 showed strong association with liposomes, and in particular with preparations containing DMPC and cholesterol, whereas the interaction of CFP-10 with membranes appeared to be weaker and less specific. Most importantly, binding to the biomembranes no longer occurred when the proteins were present as a 1:1 ESAT-6·CFP-10 complex. However, lowering of the pH resulted in dissociation of the protein complex and subsequent protein-liposome interaction. Finally, cryoelectron microscopy revealed that ESAT-6 destabilized and lysed liposomes, whereas CFP-10 did not. In conclusion, we propose that one of the main features of ESAT-6 in the infection process of M. tuberculosis is the interaction with biomembranes that occurs after dissociation from its putative chaperone CFP-10 under acidic conditions typically encountered in the phagosome.

International audience; Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. Patients with CGD suffer from recurrent bacterial and fungal infections because of the absence of superoxide anions (O2- degrees ) generatingsystem. The NADPH oxidase complex is composed of a membranous cytochrome b558, cytosolic proteins p67phox, p47phox, p40phox and two small GTPases Rac2 and Rap1A. Cytochrome b558 consists of two sub-units gp91phox and p22phox. The most common form of CGD is due to mutations in CYBB gene encoding gp91phox. In some rare cases, the mutated gp91phox is normally expressed but is devoided of oxidase activity. These variants called X+ CGD, have provided interesting informations about oxidase activation mechanisms. However modelization of such variants is necessary to obtain enough biological material for studies at the molecular level. A cellular model (knock-out PLB-985 cells) has been developed for expressing recombinant mutated gp91phox for functional analysis of the oxidase complex. Recent works demonstrated that this cell line genetically deficient in gp91phox is a powerful tool for functional analysis of the NADPH oxidase complex activation.

To combat infection, the fruit fly Drosophila melanogaster relies on multiple innate defense reactions, many of which are shared with higher organisms. These reactions include the use of physical barriers together with local and systemic immune responses. First, epithelia, such as those beneath the cuticle, in the alimentary tract, and in tracheae, act both as a physical barrier and local defense against pathogens by producing antimicrobial peptides and reactive oxygen species. Second, specialized hemocytes participate in phagocytosis and encapsulation of foreign intruders in the hemolymph. Finally, the fat body, a functional equivalent of the mammalian liver, produces humoral response molecules including antimicrobial peptides. Here we review our current knowledge of the molecular mechanisms underlying Drosophila defense reactions together with strategies evolved by pathogens to evade them.

ATP-binding cassette transporter 1 and Transglutaminase 2 act on the same genetic pathway in the apoptotic cell clearance

International audience; As first defensive line, monocytes are a pivotal cell population of innate immunity. Monocyte activation can be relevant to a range of immune conditions and responses. Here we present new insights into the activation of monocytes by a series of phosphonic acid-terminated, phosphorus-containing dendrimers. Various dendritic or subdendritic structures were synthesized and tested, revealing the basic structural requirements for monocyte activation. We showed that multivalent character and phosphonic acid capping of dendrimers are crucial for monocyte targeting and activation. Confocal videomicroscopy showed that a fluorescein-tagged dendrimer binds to isolated monocytes and gets internalized within a few seconds. We also found that dendrimers follow the phagolysosomial route during internalization by monocytes. Finally, we performed fluorescence resonance energy transfer (FRET) experiments between a specifically designed fluorescent dendrimer and phycoerythrin-coupled antibodies. We showed that the typical innate Toll-like receptor (TLR)-2 is clearly involved, but not alone, in the sensing of dendrimers by monocytes. In conclusion, phosphorus-containing dendrimers appear as precisely tunable nanobiotools able to target and activate human innate immunity and thus prove to be good candidates to develop new drugs for immunotherapies.

Chronic exposure to inorganic arsenic, a widely distributed environmental contaminant, can lead to toxic effects, including immunosuppression. Owing to the established roles of human macrophages in immune defense, we determined, in the present study, whether inorganic arsenic can affect these major immune cells. Our results demonstrate that noncytotoxic concentrations of arsenic trioxide (As2O3), an inorganic trivalent form, markedly impair differentiated features of human blood monocyte-derived macrophages. First, treatment of macrophages with 1 μM As2O3 induced a rapid cell rounding and a subsequent loss of adhesion. These morphologic alterations were associated with a marked reorganization of actin cytoskeleton, which includes retraction of peripheral actin extensions and formation of a cortical actin ring. In addition, As2O3 reduced expression of various macrophagic surface markers, enhanced that of the monocytic marker CD14, and altered both endocytosis and phagocytosis; unexpectedly, exposure of macrophages to the metalloid also strongly potentiated expression of TNFα and IL-8 induced by LPS. Finally, like monocytes, As2O3-treated macrophages can be differentiated into dendritic-like cells. Impairment of macrophage function by As2O3 mainly resulted from activation of a RhoA/Rho-associated kinase pathway; indeed, pretreatment of macrophages with the Rho-associated kinase inhibitor Y-27632 prevented metalloid effects on cytoskeleton and phagocytosis. Moreover, As2O3 was found to increase level of the active GTP-bound form of RhoA and that of phosphorylated-Moesin, a major cytoskeleton adaptor protein involved in RhoA regulation. Taken together, our results demonstrated that human macrophages constitute sensitive targets of inorganic arsenic, which may contribute to immunotoxicity of this environmental contaminant.

Comment in :- Microbiology. Malaria's stealth shuttle. [Science. 2006]- Fooling the liver: malaria incognito. [Hepatology. 2007]; International audience; The merozoite stage of the malaria parasite that infects erythrocytes and causes the symptoms of the disease is initially formed inside host hepatocytes. However, the mechanism by which hepatic merozoites reach blood vessels (sinusoids) in the liver and escape the host immune system before invading erythrocytes remains unknown. Here, we show that parasites induce the death and the detachment of their host hepatocytes, followed by the budding of parasite-filled vesicles (merosomes) into the sinusoid lumen. Parasites simultaneously inhibit the exposure of phosphatidylserine on the outer leaflet of host plasma membranes, which act as "eat me" signals to phagocytes. Thus, the hepatocyte-derived merosomes appear to ensure both the migration of parasites into the bloodstream and their protection from host immunity.

International audience; Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy.

International audience; We previously showed that the phenotypes of adipocyte progenitors and macrophages were close. Using functional analyses and microarray technology, we first tested whether this intriguing relationship was specific to adipocyte progenitors or could be shared with other progenitors. Measurements of phagocytic activity and gene profiling analysis of different progenitor cells revealed that the latter hypothesis should be retained. These results encouraged us to pursue and to confirm our analysis with a gold-standard stem cell population, embryonic stem cells or ESC. The transcriptomic profiles of ESC and macrophages were clustered together, unlike differentiated ESC. In addition, undifferentiated ESC displayed higher phagocytic activity than other progenitors, and they could phagocytoze apoptotic bodies. These data suggest that progenitors and stem cells share some characteristics of macrophages. This opens new perspectives on understanding stem cell phenotype and functionalities such as a putative role of stem cells in tissue remodeling by discarding dead cells but also their immunomodulation or fusion properties.

International audience; The amoeba Dictyostelium discoideum shares many traits with mammalian macrophages, in particular the ability to phagocytose and kill bacteria. In response, pathogenic bacteria use conserved mechanisms to fight amoebae and mammalian phagocytes. Here we developed an assay using Dictyostelium to monitor phagocyte-bacteria interactions. Genetic analysis revealed that the virulence of Klebsiella pneumoniae measured by this test is very similar to that observed in a mouse pneumonia model. Using this assay, two new host resistance genes (PHG1 and KIL1) were identified and shown to be involved in intracellular killing of K. pneumoniae by phagocytes. Phg1 is a member of the 9TM family of proteins, and Kil1 is a sulphotransferase. The loss of PHG1 resulted in Dictyostelium susceptibility to a small subset of bacterial species including K. pneumoniae. Remarkably, Drosophila mutants deficient for PHG1 also exhibited a specific susceptibility to K. pneumoniae infections. Systematic analysis of several additional Dictyostelium mutants created a two-dimensional virulence array, where the complex interactions between host and bacteria are visualized.

International audience; Phospholipases play an important role as virulence factors in human pathogens. Candida albicans, the major fungal pathogen of humans, encodes phospholipases of type A, B, C and D. Type B Plb2 and type D Pld1 phospholipases have been shown to contribute to virulence in this organism. We analyzed, in C. albicans, PLC2 and PLC3, two highly conserved genes coding for phosphatidylinositol-dependent phospholipases C with homology to the known virulence factor PlcA in the human pathogen Listeria monocytogenes. We show that expression of PLC2 and PLC3 is upregulated under different filament-inducing conditions and in the constitutive filamentous mutant tup1Δ. In order to analyze PLC2 and PLC3 function in C. albicans, we constructed strains that carry PLC2 or PLC3 under a constitutive promoter and strains that lack all four PLC2/3 alleles. These strains were not affected in their ability to produce filaments under non-inducing conditions, nor was filamentation modified under inducing conditions, suggesting that PLC2/3 are not critical determinants of the yeast-to-hypha switch. In a cell culture model for macrophage interaction, phagocytosis of C. albicans and subsequent killing were not influenced by PLC2/3. These results demonstrate that C. albicans PLC2 and PLC3 are dispensable for virulence; moreover, they underline the sharp contrast with the function of plcA in L. monocytogenes.

International audience; We show here that transgenic Drosophila can be used to decipher the effect of a bacterial toxin on innate immunity and demonstrate the contribution of blood cells in fly resistance to bacterial infection. ExoS is a Pseudomonas aeruginosa exotoxin directly translocated into the host cell cytoplasm through the type III secretion system found in many Gram-negative bacteria. It contains a N-terminal GTPase activating (GAP) domain that prevents cytoskeleton reorganization by Rho family of GTPases in cell culture. Directed expression of the ExoS GAP domain (ExoSGAP) during fly eye morphogenesis inhibited Rac1-, Cdc42- and Rho-dependent signalling, demonstrating for the first time its activity on RhoGTPases in a whole organism. We further showed that fly resistance to P. aeruginosa infections was altered when ExoSGAP was expressed either ubiquitously or in haemocytes, but not when expressed into the fat body, the major source of NF-(kappa)B-dependent anti-microbial peptide synthesis. Fly sensitivity to infection was also observed with Gram-positive Staphylococcus aureus strain and was associated to a reduced phagocytosis capacity of ExoSGAP-expressing haemocytes. Our results highlight the major contribution of cellular immunity during the first hours after Drosophila infection by P. aeruginosa, an opportunist pathogen affecting patients with pathologies associated to a reduced leukocyte number.

Interaction between target cell CD47 and the inhibitory macrophage receptor signal regulatory protein α (SIRPα) counteracts macrophage phagocytosis of CD47-expressing host cells. As platelets also express CD47, we asked whether inhibitory CD47/SIRPα signaling regulates normal platelet turnover and clearance of platelets in immune thrombocytopenic purpura (ITP). CD47-/- mice had a mild spontaneous thrombocytopenia, which was not due to a decreased platelet half-life as a result of increased expression of P-selectin, CD61, or phosphatidylserine. In contrast, CD47-/- platelets were rapidly cleared when transfused into CD47+/+ recipients, whereas CD47+/- platelets had a nearly normal half-life in CD47+/+ mice under nonautoimmune conditions. CD47-/- mice were more sensitive to ITP, as compared with CD47+/+ mice. In vitro, macrophage phagocytosis of immunoglobulin G (IgG)–opsonized CD47-/- platelets was significantly higher than that for equally opsonized CD47+/+ platelets. However, when SIRPα was blocked, phagocytosis of CD47+/+ platelets increased to the level of CD47-/- platelets. Phagocytosis of opsonized CD47+/- platelets was higher than that for CD47+/+ platelets, but lower than that for CD47-/- platelets, suggesting a gene-dose effect of CD47 in this system. In conclusion, we suggest that inhibitory CD47/SIRPα signaling is involved in regulating platelet phagocytosis in ITP, and that targeting SIRPα may be a new means of reducing platelet clearance in ITP.

International audience; BACKGROUND AND OBJECTIVES: The residual tumor cells remaining after completion of standard chemotherapy and radiation treatment in B lymphoma patients, may be eradicated by active immunotherapy that stimulates tumor-specific T lymphocytes. Irradiated autologous lymphoma cells expressing tumor-associated antigens (TAA) may serve as a potential tumor vaccine, provided that they are effectively targeted to the antigen-presenting cells (APC). We propose exploiting the natural anti-Gal antibody in order to target vaccinating tumor cells to APC. Anti-Gal constitutes 1% of IgG in human serum and interacts specifically with the alpha-gal epitope (Galalpha1-3Galphalbeta1-4GlcNAc-R). DESIGN AND METHODS: Alpha-gal epitopes were synthesized in vitro on the membrane of primary lymphoma cells by using the recombinant glycosylation enzyme alpha1,3galactosyltransferase (alpha1,3GT). Processed tumor cells were opsonized by purified anti-Gal antibodies and studied for uptake (phagocytosis) by APC including monocyte-derived macrophages and dendritic cells. Cross-presentation of tumor antigens after phagocytosis of processed MHC-I negative lymphoma cells was measured by activation of a tumor-specific CD8+ T-cell line. RESULTS: We demonstrate synthesis of alpha-gal epitopes on freshly isolated B lymphoma cells of various types following the use of the recombinant enzyme alpha1,3GT. The subsequent binding of anti-Gal to the de novo synthesized alphagal epitopes opsonizes these tumor cells for effective uptake by macrophages and dendritic cells, through phagocytosis mediated by FcgammaR1 (CD64). Moreover, anti-Gal-mediated phagocytosis resulted in cross-presentation of TAA by dendritic cells. INTERPRETATION AND CONCLUSIONS: This study suggests that immunization with irradiated autologous lymphoma cells processed to express alpha-gal epitopes will result in anti-Gal-mediated, in vivo targeting of the autologous tumor vaccine to APC.

The macrophage mannose receptor (MR) appears to play an important role in the binding and phagocytosis of several human pathogens, but its phagocytic property and signaling pathways have been poorly defined. The general strategy to explore such topics is to express the protein of interest in nonphagocytic cells, but in the case of MR, there are few reports using the full-length MR cDNA. When we searched to clone de novo the human MR (hMR) cDNA, problems were encountered, and full-length hMR cDNA was only obtained after devising a complex cloning strategy. Chinese hamster ovary cells, which have a fully functional phagocytic machinery when expressing professional phagocytic receptors, were stably transfected, and cell clones expressing hMR at quantitatively comparable levels than human macrophages or J774E cells were obtained. They exhibited a functional hMR-mediated endocytic capacity of a soluble ligand but failed to ingest classical particulate ligands of MR such as zymosan, Mycobacterium kansasii, or trimannoside bovine serum albumin-coated latex beads. Transient expression of hMR in two human cell lines did not provide a phagocytic capacity either. In conclusion, we show that MR is not a professional phagocytic receptor, as it does not possess the ability to promote particle ingestion in nonphagocytic cells on its own. We propose that MR is a binding receptor, which requires a partner to trigger phagocytosis in some specialized cells such as macrophages. Our new expression vector could represent a useful tool to study the receptor and its partnership further.

In epithelial cells, endocytic activity is mostly dedicated to nutrient and macromolecule uptake. To invade these cells, Chlamydiaceae, like other pathogens, have evolved strategies that utilise the existing endocytic machineries and signalling pathways, but little is known about the host cell molecules involved. In this report, we show that within five minutes of infection of HeLa cells by Chlamydia caviae GPIC strain several events take place in the immediate vicinity of invasive bacteria: GM1-containing microdomains cluster, tyrosine-phosphorylated proteins accumulate, and intense actin polymerization occurs. We show that actin polymerization is controlled by the small GTPases Cdc42 and Rac, which become activated upon infection. Expression of dominant negative forms of these GTPases inhibits C. caviae entry and leads to abnormal actin polymerization. In contrast, the small GTPase Rho does not seem essential for bacterial entry. Finally, phosphatidylinositol 3-kinase activity is also required for internalization of C. caviae, probably downstream of the other molecular events reported here. We present the first scheme of the events occurring at the sites of invasion of epithelial cells by a member of the Chlamydiaceae family.

International audience; Bordetella pertussis, the causative agent of whooping cough, secretes among other virulence factors an adenylate cyclase (AC) toxin that is able to enter into eukaryotic cells where it is activated upon binding to endogenous calmodulin (CaM) and synthesizes supraphysiological cAMP levels. In vivo, the AC toxin, through its specific interaction with the CD11b/CD18 integrin, primarily targets phagocytic cells such as neutrophils and macrophages. Because neutrophil priming and activation result in the production of reactive oxygen species that may cause intracellular oxidation, we have examined the biological consequences of the oxidation of CaM methionines upon its interaction with AC. We show here that the interaction of CaM with AC is dependent on the reduced state of methionines, because oxidation of all methionine residues of CaM dramatically decreases its affinity for AC. Peptide methionine sulfoxide reductases A (MsrA) and B (MsrB) were able to partially reduce the oxidized CaM, and these partially "repaired" forms could interact with AC nearly as efficiently as the native protein. We further showed that the CaM.AC complex is resistant to oxidation with tert-butylhydroperoxide, and we identified methionine residues 109, 124, and 145 as critical for binding to AC. The resistance of the AC.CaM complex to oxidation and the ability of AC to be efficiently activated by partially oxidized CaM molecules should allow the toxin to exert its cytotoxic effects on activated neutrophils and contribute to the host colonization.

Hydroxyapatite (HA) is widely used to coat the metal parts of prosthetic implants in order to improve their biocompatibility and as a bone defect filling material. HA has been demonstrated to produce particles at the prosthetic interface that lead to an activation of phagocytic cells that induce a cascade reaction leading to bone resorption and aseptic loosening. Monocytes/macrophages are commonly observed in the interface tissue, and are among the first cells to colonize the inflammatory site where they play a key role in the immune response. IL-18 is a pro-inflammatory cytokine. Monocytes/macrophages were described as IL-18 producing cells. IL-18 works antagonistically to IL-6, which activates osteoclastogenesis. In the present study, we investigated the ability of HA particles to induce the production of active IL-18 by human monocytes according to particle characteristics (size, sintering temperature and shape). Our study demonstrates, for the first time, that HA particles are capable of stimulating the production of the proinflammatory cytokine IL-18 in human monocytes according to their particle characteristics. The expression and the production of IL-18 was modified by the parameter studied. The difference observed between the expression and the production could be explain by the production of ICE. The needle shaped particles induced the larger production of IL-18.

International audience; Invasive bacteria actively induce their own uptake by phagocytosis in normally nonphagocytic cells and then either establish a protected niche within which they survive and replicate, or disseminate from cell to cell by means of an actin-based motility process. The mechanisms underlying bacterial entry, phagosome maturation, and dissemination reveal common strategies as well as unique tactics evolved by individual species to establish infection.

International audience; The aim of the present study was to investigate possible mechanisms that could be involved in the deactivation of the assembled, catalytically active NADPH oxidase of phagocytic cells and thereby lead to termination of O(2)(.-) production. Our major findings are the following: (1) Addition of GDP to the active oxidase is able to reduce O(2)(.-) production both in the fully purified and in a semi-recombinant cell-free activation system. (2) p67(phox) inhibits GTP hydrolysis on Rac whereas p47(phox) has no effect on Rac GTPase activity. (3) Soluble regulatory proteins (GTPase activating protein, guanine nucleotide dissociation inhibitor, and the Rac-binding domain of the target protein p21-activated kinase) inhibit activation of the NADPH oxidase but have no effect on electron transfer via the assembled enzyme complex. (4) Membrane-associated GTPase activating proteins (GAPs) have access also to the assembled, catalytically active oxidase. Taken together, we propose that the GTP-bound active form of Rac is required for sustained enzyme activity and that membrane-localized GAPs have a role in the deactivation of NADPH oxidase.

Complement receptor type 3 (CR3) was initially described as an opsonic receptor. Subsequently, CR3-mediated lectin-sugar recognition mechanisms have been shown to play a major role in the nonopsonic phagocytosis of several pathogens, among themMycobacterium tuberculosis. Little is known about the binding and signal transduction mechanisms operating during nonopsonic ingestion through CR3 of different microorganisms. In the present study, we used CHO cells stably transfected with CR3 to show that CR3 was able to mediate internalization of zymosan and pathogenic mycobacteria (Mycobacterium kansasiiandMycobacterium avium) but not that of nonpathogenic species (Mycobacterium smegmatisandMycobacterium phlei). A combination of mannan and β-glucan inhibited the phagocytosis of zymosan but had no effect onM. kansasiiingestion. Among six monoclonal antibodies (MAbs) directed against the CD11b subunit of CR3 that decreased zymosan ingestion, only three inhibitedM. kansasiiphagocytosis. In particular, MAbs known to block the CR3 lectin site affected only internalization of zymosan. Using U937 macrophages, we observed that zymosan ingestion through CR3 induced superoxide production measured by cytochromecreduction and by translocation of the NADPH oxidase cytosolic component p47phox to the phagosomal membrane, whereas phagocytosis of viable or heat-killedM. kansasiidid not. Furthermore, lack of superoxide anion production during phagocytosis ofM. kansasiiwas not due to inhibition of NADPH oxidase per se or superoxide anion scavenging. Together, our results indicate that (i) nonopsonic phagocytosis of zymosan andM. kansasiiby CR3 implicates different molecular mechanisms involving multiple and distinct epitopes of CD11b and (ii) CR3 may transduce different cellular responses depending on the sites mediating nonopsonic phagocytosis.

International audience; On adhesion to host cells the flagellate Trichomonas vaginalis switches to an amoeboid form rich in actin microfilaments. We have undertaken the identification of actin-associated proteins that regulate actin dynamics. A monoclonal antibody 4C12 raised against a cytoskeletal fraction of T. vaginalis labeled a protein doublet at circa 50 kDa. These two bands were recognized by the antibody against Dictyostelium discoideum coronin. During cell extraction and actin polymerization, T. vaginalis coronin cosedimented with F-actin. By two-dimensional gel electrophoresis, the protein doublet was separated into two sets of isoforms covering two Ip zones around 6 and 7. By screening a T. vaginalis library with 4C12, two clones Cor 1 and Cor 2 were isolated. This gene duplicity is a particularity among unicellular organisms examined. The complete sequence of the gene Cor 1 encodes a 435-residue protein with a calculated molecular mass of 48 kDa and Ip of 5.58. The incomplete sequence Cor 2 was very similar but with a more basic calculated Ip than Cor 1 on the same region. T. vaginalis coronin had 50% similarity with the coronin family, possessing the five WD-repeats and a leucine zipper in its C-terminal part. Double immunofluorescence labeling showed that coronin mainly colocalized with actin at the periphery of the adherent amoeboid cells. However, coronin labeling displayed patches within a reticular array. Immunogold electron microscopy confirmed the coronin labeling in the actin-rich microfilamentous fringe beneath the plasma membrane, with accumulation in phagocytic zones and pseudopodial extensions. In T. vaginalis, one of the first emerging lineage of eukaryotes, coronin seems to play an important role in actin dynamics and may be a downstream target of a signaling mechanism for the cytoskeleton reorganization.

Infection byShigella flexneriis characterized by infiltration of neutrophils in the intestinal mucosa and by a strong inflammatory reaction. Although neutrophils are constitutively programmed to die by apoptosis, we show that isolated human neutrophils undergo necrosis 2 h after infection with virulentS. flexneristrain M90T but not with the virulence plasmid-cured strain BS176. This was demonstrated by the release of azurophil granule proteins concomitant with the release of lactate dehydrogenase (LDH), disruption of the plasma membrane, and absence of DNA fragmentation. Mutants with themxiD1gene, coding for an essential component of the secretion type III machinery, or the genes coding for IpaB or IpaC invasins deleted were not cytotoxic. Neutrophil necrosis occurred independently of the bacterial ability to leave phagosomes, and it involved actin polymerization, as the addition of cytochalasin D after phagocytosis ofShigellainhibited the release of LDH. In conclusion,Shigellakills neutrophils by necrosis, a process characterized by the release of tissue-injurious granular proteins. This probably contributes to disruption of the epithelial barrier, leading to the dysentery observed in shigellosis and allowingShigellato enter its host cells.

The mannose receptor (MR) is involved in the phagocytosis of pathogenic microorganisms. Here we investigated its role in the bactericidal functions of human monocyte-derived macrophages (MDMs), using (i) trimannoside-bovine serum albumin (BSA)-coated latex beads and zymosan as particulate ligands of the MR, and (ii) mannan and mannose-BSA as soluble ligands. We show that phagocytosis of mannosylated latex beads did not elicit the production of O 2 − . Zymosan, which is composed of α-mannan and β-glucan, was internalized by the MR and a β-glucan receptor, but the production of O 2 − was triggered only by phagocytosis through the β-glucan receptor. Activation and translocation of Hck, a Src family tyrosine kinase located on lysosomes, has previously been used as a marker of fusion between lysosomes and phagosomes in human neutrophils. In MDMs, Hck was activated and recruited to phagosomes containing zymosan later than LAMP-1 and CD63. Phagosomes containing mannosylated latex beads fused with LAMP-1 and CD63 vesicles but not with the Hck compartment, and the kinase was not activated. We also demonstrate that the MR was unable to distinguish between nonpathogenic and pathogenic mycobacteria, as they were internalized at similar rates by this receptor, indicating that this route of entry cannot be considered as a differential determinant of the intracellular fate of mycobacteria. In conclusion, MR-dependent phagocytosis is coupled neither to the activation of NADPH oxidase nor to the maturation of phagosomes until fusion with the Hck compartment and therefore constitutes a safe portal of entry for microorganisms.

International audience; Dendritic cells, but not macrophages, efficiently phagocytose apoptotic cells and cross-present viral, tumor, and self-antigens to CD8(+) T cells. This in vitro pathway corresponds to the in vivo phenomena of cross-priming and cross-tolerance. Here, we demonstrate that phagocytosis of apoptotic cells is restricted to the immature stage of dendritic cell (DC) development, and that this process is accompanied by the expression of a unique profile of receptors, in particular the alphavbeta5 integrin and CD36. Upon maturation, these receptors and, in turn, the phagocytic capacity of DCs, are downmodulated. Macrophages engulf apoptotic cells more efficiently than DCs, and although they express many receptors that mediate this uptake, they lack the alphavbeta5 integrin. Furthermore, in contrast to DCs, macrophages fail to cross-present antigenic material contained within the engulfed apoptotic cells. Thus, DCs use unique pathways for the phagocytosis, processing, and presentation of antigen derived from apoptotic cells on class I major histocompatibility complex. We suggest that the alphavbeta5 integrin plays a critical role in the trafficking of exogenous antigen by immature DCs in this cross-priming pathway.

When differentiated into mature macrophages by the combination of all-trans retinoic acid and 1,25-dihydroxyvitamin D3, the human promonocytic cell lines U937 and THP-1 expressed inducible nitric oxide synthase (iNOS) transcripts. During their differentiation, the cells acquired the capacity to produce not only superoxide anion (O2.-) but also nitric oxide (.NO) in response to IgG (or IgE)-opsonized zymosan. The inhibitors of the iNOS pathway, aminoguanidine and NG-monomethyl-L-arginine (L-NMMA), suppressed the production of .NO and enhanced the steady-state concentration of O2.- determined. Conversely, superoxide dismutase (SOD) scavenged the O2.- released and increased the .NO-derived nitrite concentration detected. These data suggested a possible interaction between O2.- and .NO. In differentiated U937 (or THP-1) cells, IgG or IgE-opsonized zymosan induced a strong time-dependent luminol-dependent chemiluminescence (LDCL), which was abrogated by SOD and partially inhibited by aminoguanidine or L-NMMA. Since the iNOS inhibitors did not directly scavenge O2.-, LDCL determination in the presence or absence of SOD and/or iNOS inhibitors demonstrated a concomitant production of O2.- and .NO. These radicals induced the formation of a .NO-derived product(s), probably peroxynitrite (ONOO-), which was required to elicit maximal LDCL. Finally, LDCL measurement provided a convenient tool to characterize iNOS triggering and demonstrated an interaction between NADPH oxidase and iNOS products in human macrophagic cells phagocytizing opsonized-zymosan. These findings show that in activated macrophages, iNOS activity can be involved in LDCL and support the debated hypothesis of iNOS participation to the microbicidal activity of human macrophages.

International audience; Intracellular pathogens can be artificially divided into two groups, those which are present only in phagocytes and those which also enter and replicate in nonprofessional phagocytic cells. The process of entry into nonphagocytic cells is often referred to as “parasite-directed endocytosis”or “induced phagocytosis”, since it involves the participation of both the pathogen and the host eukaryotic cell. In those cases where it has been carefully studied, interaction between a bacterial ligand and the host cell receptor results in stimulation of host signaling pathways and the subsequent cytoskeletal rearrangements necessary for uptake (BLISKA et al. 1993). Phagocytosis by professional phagocytes is generally different from “induced phagocytosis”in at least two aspects: (a) Internalization is mainly a “hostdirected” event resulting from deposition on the bacterium of antibodies or other compounds recognized by phagocyte receptors, (b) Professional phagocytes can produce potent microbicidal radicals or compounds that intracellular bacteria must circumvent, alter, or destroy in order to survive.

The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process.

The effects of the trivalent arsenical phenylarsine oxide (PAO) on the activity of NADPH oxidase in human neutrophils were studied. PAO caused a rapid dose-dependent inhibition of superoxide generation which was maximal at a concentration of 1 microM, irrespective of the stimulating agent. This inhibitory effect was not due to impaired transduction of activation signals since neither degranulation nor phagocytosis were modified. When cytosolic and membrane fractions from resting neutrophils were combined to reconstitute the NADPH oxidase, O2-. generation was inhibited by PAO while translocation of the NADPH oxidase components to the plasma membrane fraction was not affected. The inhibition was completely and specifically reversed by 2,3-dimercaptopropanol, not by dithiothreitol or beta-mercaptoethanol, indicating that PAO binds covalently to spatially vicinal thiol groups. PAO inhibited the plasma membrane's capacity to initiate O2-. generation while it apparently did not affect the cytosol. When PAO was added subsequently to NADPH oxidase activation, no inhibition was observed, indicating that PAO cannot reach its target once the oxidase is functionally assembled. In conclusion, PAO is the first complete and reversible inhibitor of NADPH oxidase which could provide the basis for new therapeutical approaches in inflammatory diseases.

International audience; An enzyme-linked immunosorbent assay (ELISA) has been developed using polyclonal antibodies raised against two cytosolic proteins of 47 kDa (p47) and 67 kDa (p67) which behave as activation factors for the superoxide-generating NADPH oxidase of neutrophils at the onset of phagocytosis. These two proteins become associated with the NADPH oxidase complex during activation. This immunological technique has been used to follow the purification steps of p47 and p67. It allows the detection of very small amounts of cytosolic factors in a crude neutrophil extract. It is straightforward and much more sensitive (about 1000 times more) than the classical assay based on the use of a cell-free system of oxidase activation and production of superoxide anion. The percentages of p47 and p67 assessed by ELISA with respect to total cytosolic protein were estimated to amount to 0.13 and 0.20%, respectively. The described method has potential applications for the titration of the cytosolic factors of oxidase activation in autosomal recessive forms of chronic granulomatous disease.