CD46/CD3 costimulation induces morphological changes of human T cells and activation of Vav, Rac, and extracellular signal-regulated kinase mitogen-activated protein kinase.

Efficient T cell activation requires at least two signals, one mediated by the engagement of the TCR-CD3 complex and another one mediated by a costimulatory molecule. We recently showed that CD46, a complement regulatory receptor for C3b as well as a receptor for several pathogens, could act as a potent costimulatory molecule for human T cells, highly promoting T cell proliferation. Indeed, we show in this study that CD46/CD3 costimulation induces a synergistic activation of extracellular signal-related kinase mitogen-activated protein kinase. Furthermore, whereas T lymphocytes primarily circulate within the bloodstream, activation may induce their migration toward secondary lymphoid organs or other tissues to encounter APCs or target cells. In this study, we show that CD46/CD3 costimulation also induces drastic morphological changes of primary human T cells, as well as actin relocalization. Moreover, we show that the GTP/GDP exchange factor Vav is phosphorylated upon CD46 stimulation alone, and that CD46/CD3 costimulation induces a synergistic increase of Vav phosphorylation. These results prompted us to investigate whether CD46/CD3 costimulation induced the activation of GTPases from the Rho family. Indeed, we report that the small GTPase Rac is also activated upon CD46/CD3 costimulation, whereas no change of Rho and Cdc42 activity could be detected. Therefore, CD46 costimulation profoundly affects T cell behavior, and these results provide important data concerning the biology of primary human T cells.

Signaling across the platelet adhesion receptor glycoprotein Ib-IX induces alpha IIbbeta 3 activation both in platelets and a transfected Chinese hamster ovary cell system.

In platelets, alpha(IIb)beta(3) exists in a form that cannot bind adhesive proteins in the plasma; although it can interact with immobilized fibrinogen it cannot interact with immobilized von Willebrand factor in the vessel wall. Soluble agonists such as thrombin convert alpha(IIb)beta(3) to a form that recognizes soluble and immobilized ligands. Attempts to reconstitute alpha(IIb)beta(3) activation in a non-hematopoietic, nucleated cell system have been unsuccessful. In the present study, we have developed a transfected Chinese hamster ovary cell model in which alpha(IIb)beta(3) activation is induced by signaling across glycoprotein (GP) Ib-IX by its ligand, von Willebrand factor. GPIb-IX activates not only the transfected alpha(IIb)beta(3) but also endogenous alpha(v)beta(3). Activation of the pathways leading to integrin activation occurred even in cells transfected with GPIb-IX lacking the domain on GPIbalpha that binds 14-3-3 or that which binds actin-binding protein. These studies demonstrate that signals induced by interaction of GPIb-IX with von Willebrand factor lead to alpha(IIb)beta(3) activation and suggest that the signaling pathways by which GPIb-IX induces alpha(IIb)beta(3) activation are different to those used by thrombin. Elucidation of these differences may provide insights into therapeutic ways in which to inhibit integrin activation in selective clinical settings.

Subversion of monocyte functions by coxiella burnetii: impairment of the cross-talk between alphavbeta3 integrin and CR3.

Several intracellular pathogens exploit macrophages as a niche for survival and replication. The success of this strategy requires the subversion or the avoidance of microbicidal functions of macrophages. Coxiella burnetii, the agent of Q fever, is a strictly intracellular bacterium that multiplies in myeloid cells. The survival of C. burnetii may depend on the selective use of macrophage receptors. Virulent C. burnetii organisms were poorly internalized but survived successfully in human monocytes, whereas avirulent variants were efficiently phagocytosed but were also rapidly eliminated. The uptake of avirulent organisms was mediated by leukocyte response integrin (alphavbeta3 integrin) and CR3 (alphaMbeta2 integrin), as demonstrated by using specific Abs and RGD sequence-containing peptides. The phagocytic efficiency of CR3 depends on its activation via alphavbeta3 integrin and integrin-associated protein. Indeed, CR3-mediated phagocytosis of avirulent C. burnetii was abrogated in macrophages from integrin-associated protein-/- mice. In contrast, the internalization of virulent C. burnetii organisms involved the engagement of alphavbeta3 integrin but not that of CR3. The pretreatment of monocytes with virulent C. burnetii organisms prevented the CR3-mediated phagocytosis of zymosan particles and CR3 activation assessed by the expression of the 24 neo-epitope. We conclude that the virulence of C. burnetii is associated with the engagement of alphavbeta3 integrin and the impairment of CR3 activity, which probably results from uncoupling alphavbeta3 integrin from integrin-associated protein. This study describes a strategy not previously reported of phagocytosis modulation by intracellular pathogens.

Role of CR4 in Mycobacterium tuberculosis-human macrophages binding and signal transduction in the absence of serum.

The beta2 integrin CR4 is involved in Mycobacterium tuberculosis phagocytosis by human mononuclear phagocytes through the opsonin C3bi. In this study, we demonstrate that M. tuberculosis can bind directly to monocyte-derived macrophages via CR4 in the absence of any opsonins. CR4-transfected CHO cells gave similar results, suggesting recognition by CR4 of bacterial structure. Furthermore, binding of M. tuberculosis transduced a potent signal, resulting in tyrosine phosphorylation of macrophage proteins, which was in part mediated by CR4.

Production of interleukin-10 and transforming growth factor beta by peripheral blood mononuclear cells in Q fever endocarditis.

The pathophysiology of Q fever endocarditis is characterized by the suppression of antigen-specific cell-mediated immune responses. We investigated the production of interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta), known to interfere with the development of protective cell immunity. IL-10 was markedly released by unstimulated peripheral blood mononuclear cells (PBMC) from patients with Q fever endocarditis. This release resulted from the upregulation of IL-10 gene transcription. Similarly, the release of TGF-beta1 and TGF-beta2 was significantly higher in patient PBMC than in control cells, but the expression of their respective mRNA was not enhanced in patient cells. In contrast, lipopolysaccharide-stimulated transcription and release of IL-10 and TGF-beta were similar in patients and controls. The release of IL-10 by PBMC but not that of TGF-beta was correlated with the clinical status of the patients. First, IL-10 production was correlated with specific antibody levels. Second, IL-10 release remained elevated in patients prone to relapse. Taken together, our results suggest that the release of IL-10 and TGF-beta is upregulated in Q fever endocarditis. IL-10 might be considered as a marker of disease relapses and might be instrumental in monitoring the efficiency of the treatment.

Trypanosoma cruzi-induced tyrosine phosphorylation in murine peritoneal macrophages.

Trypanosoma cruzi, the etiological agent of Chagas' disease, binds to and invades macrophages and other cells (fibroblasts, muscle cells) via a complicated set of interactions, but the changes induced by parasite-to-cell interactions are largely unknown. This report investigates the ability of T. cruzi to elicit a tyrosine kinase pathway in immature and mature resident murine peritoneal macrophages (MPM) that differ in their susceptibility to parasite infection. T. cruzi stimulated the phosphorylation of tyrosine residues in several endogenous substrates (proteins of 40-42, 53-56, 66, 75, 80, 90, 95, 100, and 112 kDa), but only in immature MPM. Mature MPM had high levels of spontaneous tyrosine phosphorylation. Upstream tyrosine kinases, such as src-like tyrosine kinases, were not responsible for the differential patterns of tyrosine phosphorylation since they were present in both mature and immature MPM. We suggest that the tyrosine phosphorylation patterns stimulated by T. cruzi reflect most of the biochemical events that occur in parasite host-cell interactions.

Zymosan-triggered association of tyrosine phosphoproteins and lyn kinase with cytoskeleton in human monocytes.

Phagocytosis of pathogens and inert particles such as zymosan by macrophages, and related secretory functions require the combination of several intracellular signals and the reorganization of cytoskeleton. We recently reported that zymosan stimulated the tyrosine phosphorylations of several endogenous substrates in human monocytes. In this work, the relationship between zymosan-stimulated tyrosine phosphoproteins and detergent-insoluble material considered as cytoskeleton was investigated. Triton X-100-insoluble fraction contained two proteins of 53 and 56 kDa that were tyrosine phosphorylated after only 5 min of stimulation with zymosan and remained labeled for 30 min. Because 53- and 56-kDa phosphoproteins migrated, as did some components of the src tyrosine kinase family, namely p53-56lyn, we wondered if 53- and 56-kDa phosphoproteins were related to lyn kinase. First, the amount of immunoreactive p53-56lyn increased in Triton X-100-insoluble fraction as did zymosan-stimulated tyrosine phosphoproteins. This property of p53-56lyn was unique, as no other member of the src family was found in this fraction. Second, when the immunoblots were reprobed with anti-phosphotyrosine mAb, the m.w. of p53-56lyn and tyrosine-phosphorylated proteins were identical in apparent size. Third, p53-56lyn was probably activated after cell stimulation with zymosan, because the phosphorylation levels of a synthetic copolymer of glutamine-tyrosine were increased in Triton X-100-insoluble fraction. In addition, we studied the distribution of lyn kinase and tyrosine phosphoproteins in phagocytozing monocytes. By using immunofluorescence, we showed that lyn kinase was located preferentially in the periphagosomal region in a specific manner, as an src tyrosine kinase such as p59hck, which was not associated with cytoskeleton, was not concentrated around the vacuoles. Moreover, periphagosomal phosphoproteins were also detected and found to be colocalized with polymerized actin. Because zymosan interacts with human monocytes via beta 2 integrins, which are known to be cytoskeleton-associated, we suggest that p53-56lyn provides the molecular link between zymosan receptors and cytoskeleton, and directs the cytoskeletal reorganization in the periphagosomal area.

Morphological polarization of human polymorphonuclear leucocytes in response to three different chemoattractants: an effector response independent of calcium rise and tyrosine kinases.

Chemoattractants such as interleukin-8, C5a and N-formylmethionyl-leucyl-phenylalanine induce a cytosolic calcium rise involved in triggering the secretory functions of human polymorphonuclear leucocytes. We studied the possible role of calcium rise in membrane ruffling, actin polymerization, filamentous actin distribution, and morphological polarization, which are all events contributing to chemotaxis. Membrane ruffling was assessed by right-angle light-scatter changes, the cellular content of polymerized actin by fluorescence of bodipy phallacidin, the intracellular distribution of filamentous actin by fluorescence microscopy and image digitization, and morphological polarization by scanning electron microscopy. Pretreatment of polymorphonuclear leucocytes with 50 microM BAPTA/AM, an intracellular calcium chelator, lowered the basal level in cell calcium and inhibited the transient calcium rise stimulated by 2 nM interleukin-8, 2 nM C5a, and 10 nM N-formylmethionyl-leucyl-phenylalanine. However, BAPTA pretreatment of polymorphonuclear leucocytes did not modify membrane ruffling, actin polymerization, filamentous actin distribution, and morphological polarization stimulated by chemoattractants. Downstream effectors may be protein tyrosine kinases. However, the tyrosine kinase inhibitor tyrphostin did not affect the cytoskeletal characteristics elicited by chemoattractants. Taken together, our results suggest that the transductional pathway leading to cytoskeleton organization and morphological polarization of polymorphonuclear leucocytes is different from that leading to secretion.

F-actin content and spatial distribution in resting and chemoattractant-stimulated human polymorphonuclear leucocytes. Which role for intracellular free calcium?

Intracellular free calcium concentration ([Ca2+]i) plays a pivotal role for many responses in polymorphonuclear leucocytes (PMNs) stimulated by chemoattractants such as N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). The importance of [Ca2+]i in the morphological polarization was investigated by using calcium-manipulated PMNs. We loaded human PMNs with BAPTA/AM to buffer or chelate [Ca2+]i in the presence or the absence of extracellular calcium by using fluo-3/AM as calcium indicator. The shape changes of PMNs were determined by microscopic examination, and membrane ruffling by right-angle light-scatter changes. Actin polymerization and F-actin distribution were recorded by staining PMNs with bodipy-phallacidin and quantified by quantitative fluorescence microscopy. We found that calcium-free incubation of PMNs loaded or not with 50 microM BAPTA/AM did not modify morphological polarization, membrane ruffling, actin assembly and F-actin distribution of PMNs stimulated with fMet-Leu-Phe, suggesting that these responses were probably functionally linked. It should be noted that incubation of PMNs in calcium-free conditions resulted in a radial distribution of F-actin and a moderate polymerization of actin, but not in morphological polarization of PMNs. Moreover, both calcium-sensitive and calcium-insensitive mechanisms of actin polymerization were additive, and inhibitable by 5 micrograms/ml cytochalasin B.

Role of calcium in the shape control of human granulocytes.

The possible role of cytoplasmic calcium in the shape control of human blood granulocytes was explored with two complementary sets of experiments. First, cells were stimulated with N-formyl methionyl leucyl phenylalanine (FMLP) with or without depletion of free intracellular calcium (by incubation with BAPTA/AM in calcium-deprived medium). Control cells displayed a rapid and transient rise of free intracellular calcium (peaked after a few seconds, returned to the basal level after 2-3 minutes), extensive morphological polarization (more than 90% polarized cells after 15 minutes), and reorganization of actin filaments (as assessed by image analysis on cells labeled with a fluorescent phallacidin derivative). Calcium-depleted cells displayed no calcium rise, but both morphological polarization and actin reorganization were indistinguishable from those of controls. In a second set of experiments, individual granulocytes were labeled with fluorescent calcium probes and aspirated in a micropipette on the stage of a confocal laser microscope. About half of the cells displayed a transient calcium rise, which was concomitant with the formation of a protrusion in most cases. However, extensive protrusions were shown by the cells that did not exhibit calcium flashes. Furthermore, if cells were treated with ionomycin, in calcium-containing or calcium-deprived medium, then aspirated, they showed protrusions comparable to those of controls. Finally, when cells were treated with ionomycin during micropipette aspiration, a calcium rise was followed by a moderate increase of the rate of protrusion growth. It is concluded that free calcium alone cannot play a major role in the control of cell shape.