ABSTRACT
Phagocytosis of apoptotic cells (efferocytosis) induces macrophage differentiation towards a regulatory phenotype (IL-10(high)/IL-12p40(low)). CD36 is involved in the recognition of apoptotic cells (AC), and we have shown that the platelet-activating factor receptor (PAFR) is also involved. Here, we investigated the contribution of PAFR and CD36 to efferocytosis and to the establishment of a regulatory macrophage phenotype. Mice bone marrow-derived macrophages were cocultured with apoptotic thymocytes, and the phagocytic index was determined. Blockage of PAFR with antagonists or CD36 with specific antibodies inhibited the phagocytosis of AC (~70-80%). Using immunoprecipitation and confocal microscopy, we showed that efferocytosis increased the CD36 and PAFR colocalisation in the macrophage plasma membrane; PAFR and CD36 coimmunoprecipitated with flotillin-1, a constitutive lipid raft protein, and disruption of these membrane microdomains by methyl-ß-cyclodextrin reduced AC phagocytosis. Efferocytosis induced a pattern of cytokine production, IL-10(high)/IL-12p40(low), that is, characteristic of a regulatory phenotype. LPS potentiated the efferocytosis-induced production of IL-10, and this was prevented by blocking PAFR or CD36. It can be concluded that phagocytosis of apoptotic cells engages CD36 and PAFR, possibly in lipid rafts, and this is required for optimal efferocytosis and the establishment of the macrophage regulatory phenotype.
Subject(s)
Apoptosis , CD36 Antigens/physiology , Macrophages/immunology , Phagocytosis , Platelet Membrane Glycoproteins/physiology , Receptors, G-Protein-Coupled/physiology , Animals , Interleukin-10/physiology , Interleukin-12 Subunit p40/physiology , Male , Mice , Mice, Inbred C57BL , PhenotypeABSTRACT
Cerebral malaria is a severe form of the disease that may result, in part, from an overt inflammatory response during infection by Plasmodium falciparum. The understanding of the pathogenesis of cerebral malaria may aid in the development of better therapeutic strategies for patients. The immune response in cerebral malaria involves elevation of circulating levels of cytokines and chemokines associated with leukocyte accumulation and breakdown of the blood-brain barrier in the central nervous system. Platelet-activating factor (PAF) is a mediator of inflammation shown to orchestrate inflammatory processes, including recruitment of leukocytes and increase of vascular permeability. Using mice lacking the PAF receptor (PAFR(-/-)), we investigated the relevance of this molecule for the outcome and the neuroinflammatory process triggered by P. berghei ANKA, an experimental model of cerebral malaria. In PAFR(-/-) mice, lethality was markedly delayed and brain inflammation was significantly reduced, as demonstrated by histology, accumulation, and activation of CD8(+) T cells, changes in vascular permeability and activation of caspase-3 on endothelial cells and leukocytes. Similarly, treatment with the PAFR antagonist UK-74,505 delayed lethality. Taken together, the results suggest that PAFR signaling is crucial for the development of experimental cerebral malaria. Mechanistically, PAFR activation is crucial for the cascade of events leading to changes in vascular permeability, accumulation, and activation of CD8(+) T cells and apoptosis of leukocytes and endothelial cells.
Subject(s)
Malaria, Cerebral/etiology , Platelet Membrane Glycoproteins/physiology , Receptors, G-Protein-Coupled/physiology , Animals , Brain Chemistry , Caspase 3/metabolism , Chemokines/metabolism , Cytokines/biosynthesis , Cytokines/metabolism , Dihydropyridines/pharmacology , Imidazoles/pharmacology , Leukocytes/physiology , Lymphocyte Activation , Malaria, Cerebral/prevention & control , Mice , Mice, Inbred C57BL , Platelet Aggregation Inhibitors/pharmacology , Platelet Membrane Glycoproteins/antagonists & inhibitors , Platelet Membrane Glycoproteins/deficiency , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/deficiencyABSTRACT
The uptake of oxLDL by CD36 is not regulated by intracellular levels of cholesterol, leading to macrophage differentiation into foam cells which play a major role in atherosclerosis. Furthermore, oxLDL competes with PAF in macrophages for binding to PAF receptors (PAFR). Here we investigated the involvement of PAFR in CD36 expression and uptake of oxLDL by human monocytes/macrophages. Adherent peripheral blood mononuclear cells were treated with PAFR-antagonists (WEB2170, CV3988); inhibitors of ERK1/2 (PD98059), p38 (SB203580), JNK (SP600125) or diluents, before stimulation with oxLDL or PAF. After 24 h, uptake of FITC-oxLDL and expression of CD36 was determined by flow cytometry and phosphorylation of MAP-kinases by Western blot. It was shown that the uptake of oxLDL was reduced by PAFR antagonists. CD36 expression was up-regulated by oxLDL, an effect reversed by PAFR antagonists. The up-regulation of CD36 and oxLDL uptake both required MAP-kinases activation. The oxLDL-induced ERK1/2 and JNK but not p38 phosphorylation was reversed by PAFR-antagonists suggesting that oxLDL signalling involves PAFR dependent and independent pathways. In macrophages from PAFR(-/-) mice, oxLDL was unable to up-regulate CD36 expression and the oxLDL uptake was reduced compared to wild type. These results suggest that oxLDL interacts with PAFR in macrophages to increase CD36 expression and oxLDL uptake. Whereas pharmacological intervention at the level of PAFR would be beneficial in atherosclerosis remains to be determined.
Subject(s)
CD36 Antigens/metabolism , Lipoproteins, LDL/metabolism , Macrophages/metabolism , Platelet Membrane Glycoproteins/physiology , Receptors, G-Protein-Coupled/physiology , Animals , Cells, Cultured , Fluorescein-5-isothiocyanate/chemistry , Fluorescent Dyes/chemistry , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Macrophages/immunology , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , Platelet Membrane Glycoproteins/genetics , Platelet Membrane Glycoproteins/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Up-Regulation , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Experimental autoimmune encephalomyelitis (EAE) is a condition induced in some susceptible species to the study of multiple sclerosis (MS). The platelet activating factor (PAF) is an important mediator of immune responses and seems to be involved in MS. However, the participation of PAF in EAE and MS remains controversial. Thus, in this study, we aimed to evaluate the role of PAF receptor in the pathogenesis of EAE. EAE was induced using an emulsion containing MOG(35-55). EAE-induced PAF receptor knock out (PAFR(-/-)) mice presented milder disease when compared to C57BL/6 wild type (WT) animals. PAFR(-/-) animals had lower inflammatory infiltrates in central nervous system (CNS) tissue when compared to WT mice. However, intravital microscopy in cerebral microvasculature revealed similar levels of rolling and adhering leukocytes in both WT and PAFR(-/-) mice. Interleukine (IL)-17 and chemokines C-C motif legends (CCL)2 and CCL5 were significantly lower in PAFR(-/-) mice when compared to WT mice. Brain infiltrating cluster of differentiation (CD)4(+) leukocytes and IL-17(+) leukocytes was diminished in PAFR(-/-) when compared to WT mice. Taken together, our results suggest that PAF receptor is important in the induction and development of EAE, although it has no influence in rolling and adhesion steps of cell recruitment. The absence of PAF receptor results in milder disease by altering the type of inflammatory mediators and cells that are present in CNS tissue.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/pathology , Inflammation Mediators/physiology , Leukocytes/metabolism , Leukocytes/pathology , Platelet Membrane Glycoproteins/deficiency , Receptors, G-Protein-Coupled/deficiency , Animals , Cell Adhesion/immunology , Cell Differentiation/physiology , Central Nervous System/metabolism , Central Nervous System/pathology , Encephalomyelitis, Autoimmune, Experimental/genetics , Female , Inflammation Mediators/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Platelet Membrane Glycoproteins/genetics , Platelet Membrane Glycoproteins/physiology , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/physiologyABSTRACT
Nuclear migration is regulated by the LIS1 protein, which is the regulatory subunit of platelet activating factor (PAF) acetyl-hydrolase, an enzyme complex that inactivates the lipid mediator PAF. Among other functions, PAF modulates cell proliferation, but its effects upon mechanisms of the cell cycle are unknown. Here we show that PAF inhibited interkinetic nuclear migration (IKNM) in retinal proliferating progenitors. The lipid did not, however, affect the velocity of nuclear migration in cells that escaped IKNM blockade. The effect depended on the PAF receptor, Erk and p38 pathways and Chk1. PAF induced no cell death, nor a reduction in nucleotide incorporation, which rules out an intra-S checkpoint. Notwithstanding, the expected increase in cyclin B1 content during G2-phase was prevented in the proliferating cells. We conclude that PAF blocks interkinetic nuclear migration in retinal progenitor cells through an unusual arrest of the cell cycle at the transition from S to G2 phases. These data suggest the operation, in the developing retina, of a checkpoint that monitors the transition from S to G2 phases of the cell cycle.
Subject(s)
Cell Nucleus/physiology , G2 Phase , Platelet Activating Factor/physiology , Platelet Membrane Glycoproteins/physiology , Receptors, G-Protein-Coupled/physiology , S Phase , Animals , Biological Transport , Cell Proliferation , Checkpoint Kinase 1 , Extracellular Signal-Regulated MAP Kinases , Protein Kinases , Rats , Retina/cytology , Stem Cells , p38 Mitogen-Activated Protein KinasesABSTRACT
Influenza A virus causes annual epidemics which affect millions of people worldwide. A recent Influenza pandemic brought new awareness over the health impact of the disease. It is thought that a severe inflammatory response against the virus contributes to disease severity and death. Therefore, modulating the effects of inflammatory mediators may represent a new therapy against Influenza infection. Platelet activating factor (PAF) receptor (PAFR) deficient mice were used to evaluate the role of the gene in a model of experimental infection with Influenza A/WSN/33 H1N1 or a reassortant Influenza A H3N1 subtype. The following parameters were evaluated: lethality, cell recruitment to the airways, lung pathology, viral titers and cytokine levels in lungs. The PAFR antagonist PCA4248 was also used after the onset of flu symptoms. Absence or antagonism of PAFR caused significant protection against flu-associated lethality and lung injury. Protection was correlated with decreased neutrophil recruitment, lung edema, vascular permeability and injury. There was no increase of viral load and greater recruitment of NK1.1(+) cells. Antibody responses were similar in WT and PAFR-deficient mice and animals were protected from re-infection. Influenza infection induces the enzyme that synthesizes PAF, lyso-PAF acetyltransferase, an effect linked to activation of TLR7/8. Therefore, it is suggested that PAFR is a disease-associated gene and plays an important role in driving neutrophil influx and lung damage after infection of mice with two subtypes of Influenza A. Further studies should investigate whether targeting PAFR may be useful to reduce lung pathology associated with Influenza A virus infection in humans.
Subject(s)
Apoptosis , Influenza A Virus, H1N1 Subtype/pathogenicity , Lung Injury/metabolism , Lung Injury/virology , Orthomyxoviridae Infections/prevention & control , Platelet Membrane Glycoproteins/physiology , Receptors, G-Protein-Coupled/physiology , Animals , Blotting, Western , Chickens , Dihydropyridines/pharmacology , Disease Models, Animal , Inflammation Mediators/metabolism , Influenza A Virus, H1N1 Subtype/genetics , Lung Injury/prevention & control , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Orthomyxoviridae Infections/metabolism , Orthomyxoviridae Infections/virology , Platelet Activating Factor/genetics , Platelet Activating Factor/metabolism , Platelet Membrane Glycoproteins/antagonists & inhibitors , RNA, Messenger/genetics , Receptors, G-Protein-Coupled/antagonists & inhibitors , Reverse Transcriptase Polymerase Chain Reaction , Survival Rate , Toll-Like Receptors/genetics , Toll-Like Receptors/metabolism , Viral LoadABSTRACT
Severe dengue infection in humans causes a disease characterized by thrombocytopenia, increased levels of cytokines, increased vascular permeability, hemorrhage, and shock. Treatment is supportive. Activation of platelet-activating factor (PAF) receptor (PAFR) on endothelial cells and leukocytes induces increase in vascular permeability, hypotension, and production of cytokines. We hypothesized that activation of PAFR could account for the major systemic manifestations of dengue infection. Inoculation of adult mice with an adapted strain of Dengue virus caused a systemic disease, with several features of the infection in humans. In PAFR(-/-) mice, there was decreased thrombocytopenia, hemoconcentration, decreased systemic levels of cytokines, and delay of lethality, when compared with WT infected mice. Treatment with UK-74,505, an orally active PAFR antagonist, prevented the above-mentioned manifestations, as well as hypotension and increased vascular permeability, and decreased lethality, even when started 5 days after virus inoculation. Similar results were obtained with a distinct PAFR antagonist, PCA-4246. Despite decreased disease manifestation, viral loads were similar (PAFR(-/-)) or lower (PAFR antagonist) than in WT mice. Thus, activation of PAFR plays a major role in the pathogenesis of experimental dengue infection, and its blockade prevents more severe disease manifestation after infection with no increase in systemic viral titers, suggesting that there is no interference in the ability of the murine host to deal with the infection. PAFR antagonists are disease-modifying agents in experimental dengue infection.
Subject(s)
Dengue Virus/metabolism , Dengue/metabolism , Dengue/virology , Platelet Membrane Glycoproteins/physiology , Receptors, G-Protein-Coupled/physiology , Aedes , Animals , Brain/metabolism , Brain/virology , Cell Line , Cytokines/metabolism , Dihydropyridines/pharmacology , Disease Models, Animal , Humans , Imidazoles/pharmacology , Mice , Mice, Inbred BALB C , Platelet Membrane Glycoproteins/antagonists & inhibitors , Receptors, G-Protein-Coupled/antagonists & inhibitors , Viral LoadABSTRACT
Tumor growth is associated with angiogenesis and inflammation and the endogenous lipid, platelet activating factor (PAF), is a pro-inflammatory and pro-angiogenic mediator. We therefore measured tumor growth, angiogenesis and inflammation in normal (WT) mice and those lacking the receptor for PAF, through gene deletion (PAFR-KO). Growth of solid tumors derived from colon 26 cells was not altered but that from Ehrlich cells was markedly (5-fold) increased in the PAFR-KO mice, relative to the WT strain. Angiogenesis, as tumor content of VEGF or hemoglobin, was increased in both tumors from the mutant strain. Inflammation, as neutrophil and macrophage accumulation and chemokine (CXCL2 and CCL2) content of tumors, was decreased or unchanged in the tumors implying an overall decrease in the inflammatory response in the PAFR-KO strain. We also assessed growth of the Ehrlich tumor in its ascites form, after i.p. injection. Here growth (ascites volume) was inhibited by about 30%, but neutrophil and macrophage numbers were increased in the ascites fluid from the PAFR-KO mice. Angiogenesis in the peritoneal wall, which is not invaded by the tumor cells, was increased but leukocyte infiltration decreased in the mutant strain. Our results show, unexpectedly, that tumor-induced angiogenesis was increased in mice lacking response to PAF, from which we infer that in normal (WT) mice, PAF is anti-angiogenic. Further, although growth was still associated with angiogenesis in PAFR-KO mice, growth was not correlated with inflammation (leukocyte accumulation).
Subject(s)
Inflammation/pathology , Neoplasms/pathology , Neovascularization, Pathologic/pathology , Platelet Membrane Glycoproteins/physiology , Receptors, G-Protein-Coupled/physiology , Acetylglucosaminidase/metabolism , Animals , Ascites/metabolism , Carcinoma, Ehrlich Tumor/pathology , Chemokine CCL2/biosynthesis , Chemokines, CXC/biosynthesis , Inflammation/genetics , Mice , Mice, Inbred BALB C , Mice, Knockout , Neoplasm Transplantation , Neoplasms/blood supply , Neoplasms/genetics , Neovascularization, Pathologic/genetics , Peritoneal Cavity , Peroxidase/metabolism , Platelet Activating Factor/pharmacology , Platelet Activating Factor/physiology , Platelet Membrane Glycoproteins/genetics , Receptors, G-Protein-Coupled/genetics , Regional Blood Flow/genetics , Regional Blood Flow/physiology , Tumor Necrosis Factor-alpha/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Activation of cellular receptors by diverse stimuli induces dramatic changes in shape and function to respond to the new circumstances of the cell. This modified behavior depends on the reorganization of the peripheral actin meshwork. An outstanding example of these processes can be found in platelets, from which much of the information available on cytoskeletal function has been obtained. Among the many actin-crosslinking proteins like spectrin, fimbrin or alpha actinin, filamin a (FLNa) emerges as the one with the highest potential in initiating the polimerization of actin filaments (F-actin) during the formation of tridimensional actin gels. FLNa also links actin filaments to the cytosolic domain of many membrane glycoproteins in platelets through its C-terminal region. In addition to participating in cell shape changes, FLNa is a scaffoldding protein that recruits numerous proteins involved in a completely different set of functions, including signal transduction, gene transcription regulation, and receptor translocation; however, the physiological role of FLNa in these processes has remained elusive. The purpose of the present communication is to briefly describe the characteristics of the macromolecules able to interact with FLNa and to discuss a possible role of FLNa during the transduction of signals from those molecular elements in platelets.
Subject(s)
Animals , Humans , Blood Platelets/physiology , Contractile Proteins/physiology , Cytoskeletal Proteins/physiology , Microfilament Proteins/physiology , Platelet Activation , Platelet Membrane Glycoproteins/physiology , Receptors, Cell Surface/physiology , Signal Transduction , Actins/physiology , Contractile Proteins , Contractile Proteins , Cytoskeletal Proteins , Cytoskeletal Proteins , Drosophila , Integrins/physiology , Microfilament Proteins , Microfilament Proteins , Phosphorylation , Platelet Activation/physiology , Platelet Membrane Glycoproteins , Receptors, Cell Surface , Signal Transduction/physiologyABSTRACT
There is evidence that apoptotic cells and oxidized low density lipoprotein (oxLDL) particles have common ligands on their surface consisting of oxidized phospholipids which bind to scavenger receptors in macrophages leading to phagocytosis. Some effects of oxLDL binding to its receptor(s) were shown to be inhibited by Platelet Activating Factor (PAF)-receptor antagonists. Thus, we investigated the effect of PAF-receptor antagonists on the phagocytosis of apoptotic, necrotic and viable thymocytes by murine peritoneal macrophages. It was found that phagocytosis of altered cells is significantly increased compared to viable cells, a phenomenon reversed by pre-treatment of macrophages with PAF-receptor antagonists (WEB2170 and CV3988), PAF or oxLDL. Phagocytosis of altered cells induced negligible expression of cyclooxygenase-2 (COX-2) but strongly potentiated the LPS-induced expression of this enzyme. This phenomenon was restricted to altered cells and was reversed by pre-treatment of macrophages with PAF-receptor antagonists. These findings indicate that apoptotic and necrotic cells share common ligands with PAF and oxLDL and suggest the involvement of PAF-like receptors in the enhanced clearance of these cells.
Subject(s)
Apoptosis , Cyclooxygenase 2/biosynthesis , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/physiology , Necrosis , Phagocytosis/drug effects , Platelet Membrane Glycoproteins/antagonists & inhibitors , Receptors, G-Protein-Coupled/antagonists & inhibitors , T-Lymphocytes/pathology , Animals , Azepines/pharmacology , Enzyme Induction , Lipoproteins, LDL/pharmacology , Macrophages, Peritoneal/drug effects , Male , Mice , Mice, Inbred BALB C , Phospholipid Ethers/pharmacology , Platelet Membrane Glycoproteins/physiology , Receptors, G-Protein-Coupled/physiology , Triazoles/pharmacologyABSTRACT
Sepsis is a systemic inflammatory response that results from the inability of the immune system to limit bacterial spread during an ongoing infection. Recently, we have documented an impaired neutrophil migration toward the infectious focus in severe sepsis. This impairment seems to be mediated by circulating cytokines, chemokines, and NO. Platelet-activating factor (PAF) plays an important role in the orchestration of different inflammatory reactions, including the release of cytokines, chemokines, and free radicals. Using a PAFR antagonist, PCA-4248, and PAFR-deficient mice, we investigated whether signaling via PAFR was relevant for the failure of neutrophils to migrate to the site of infection after lethal sepsis caused by cecum ligation and puncture in mice. In PAFR-deficient mice or mice pretreated with PCA-4248 (5 mg/kg) and subjected to lethal sepsis, neutrophil migration failure was prevented, and bacterial clearance was more efficient. There was also reduced systemic inflammation (low serum cytokine levels), lower nitrate levels in plasma, and higher survival rate. Altogether, the results firmly establish a role for PAFR in mediating the early impairment of neutrophil migration toward the infectious focus. Blockade of PAFR may prevent the establishment of severe sepsis.
Subject(s)
Neutrophil Infiltration/immunology , Platelet Membrane Glycoproteins/physiology , Receptors, G-Protein-Coupled/physiology , Sepsis/immunology , Sepsis/microbiology , Signal Transduction/immunology , Animals , Cecum , Cell Adhesion/genetics , Cell Adhesion/immunology , Cell Movement/genetics , Cell Movement/immunology , Chemokines/biosynthesis , Immunity, Innate , Ligation , Mice , Mice, Inbred BALB C , Mice, Knockout , Neutrophil Infiltration/genetics , Platelet Activating Factor/metabolism , Platelet Membrane Glycoproteins/antagonists & inhibitors , Platelet Membrane Glycoproteins/deficiency , Platelet Membrane Glycoproteins/genetics , Punctures , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/deficiency , Receptors, G-Protein-Coupled/genetics , Sepsis/metabolism , Sepsis/pathology , Signal Transduction/geneticsABSTRACT
Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.
Subject(s)
Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Crotalid Venoms/chemistry , Crotalid Venoms/pharmacology , Lectins, C-Type/chemistry , Platelet Aggregation/drug effects , Amino Acid Sequence , Animals , Anti-Infective Agents/isolation & purification , Crotalid Venoms/isolation & purification , Crotalus/physiology , Integrins/physiology , Lectins, C-Type/isolation & purification , Molecular Sequence Data , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIb-IX Complex/metabolism , Platelet Membrane Glycoproteins/chemistry , Platelet Membrane Glycoproteins/physiology , Receptors, Collagen/drug effectsABSTRACT
Activation of cellular receptors by diverse stimuli induces dramatic changes in shape and function to respond to the new circumstances of the cell. This modified behavior depends on the reorganization of the peripheral actin meshwork. An outstanding example of these processes can be found in platelets, from which much of the information available on cytoskeletal function has been obtained. Among the many actin-crosslinking proteins like spectrin, fimbrin or alpha actinin, filamin a (FLNa) emerges as the one with the highest potential in initiating the polimerization of actin filaments (F-actin) during the formation of tridimensional actin gels. FLNa also links actin filaments to the cytosolic domain of many membrane glycoproteins in platelets through its C-terminal region. In addition to participating in cell shape changes, FLNa is a scaffoldding protein that recruits numerous proteins involved in a completely different set of functions, including signal transduction, gene transcription regulation, and receptor translocation; however, the physiological role of FLNa in these processes has remained elusive. The purpose of the present communication is to briefly describe the characteristics of the macromolecules able to interact with FLNa and to discuss a possible role of FLNa during the transduction of signals from those molecular elements in platelets.
Subject(s)
Blood Platelets/physiology , Contractile Proteins/physiology , Cytoskeletal Proteins/physiology , Microfilament Proteins/physiology , Platelet Activation , Platelet Membrane Glycoproteins/physiology , Receptors, Cell Surface/physiology , Signal Transduction , Actins/physiology , Animals , Contractile Proteins/chemistry , Contractile Proteins/metabolism , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Drosophila , Filamins , Humans , Integrins/physiology , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Phosphorylation , Platelet Activation/physiology , Platelet Membrane Glycoproteins/genetics , Receptors, Cell Surface/genetics , Signal Transduction/physiologyABSTRACT
We analyzed herein whether members of the tetraspanin superfamily are involved in human immature dendritic cell (DC) functions such as foreign antigen internalization, phagocytosis, and cell migration. We show that CD63, CD9, CD81, CD82, and CD151 are present in immature DCs. Whereas CD9 and CD81 are mostly expressed at the cell surface, CD63 and CD82 are also located in intracellular organelles. Complexes of monoclonal antibody (Mab) FC-5.01-CD63 or Fab-5.01-CD63 were rapidly translocated "outside-in" and followed the endocytic pathway through early endosomes and lysosomes, reaching major histocompatibility complex (MHC) class II-enriched compartments (MIICs) in less than one hour. Internalization of CD63 was also observed during Saccharomyces cerevisiae phagocytosis. Moreover, an association of CD63 with the beta-glycan receptor dectin-1 was observed. Mabs against CD9, CD63, CD81, and CD82 enhanced by 50% the migration induced by the chemokines macrophage inflammatory protein-5 (MIP-5) and MIP-1alpha. Concomitantly, Mabs against CD63 and CD82 diminished the surface expression of CD29, CD11b, CD18, and alpha5 integrins. By immunoprecipitation experiments we found that CD63 associated with integrins CD11b and CD18. These results suggest that CD9, CD63, CD81, and CD82 could play a role in modulating the interactions between immature DCs and their environment, slowing their migratory ability. However, only CD63 would intervene in the internalization of complex antigens.
Subject(s)
Antigens, CD/metabolism , Antigens, CD/physiology , Dendritic Cells/metabolism , Endocytosis , Platelet Membrane Glycoproteins/metabolism , Platelet Membrane Glycoproteins/physiology , Antigens, CD/analysis , Cell Movement , Dendritic Cells/physiology , Endosomes/metabolism , Histocompatibility Antigens Class I/metabolism , Humans , Kangai-1 Protein , Lysosomes/metabolism , Membrane Glycoproteins/physiology , Phagocytosis , Protein Transport , Proto-Oncogene Proteins/physiology , Tetraspanin 28 , Tetraspanin 29 , Tetraspanin 30ABSTRACT
We describe the parasitological kinetics and histopathological and immunological alterations in platelet-activating factor receptor-deficient (PAFR(-/-)) and wild-type mice after a single Strongyloides venezuelensis infection (subcutaneous inoculation of 500 L3 larvae). There was no difference in the numbers of worms that reached and became established in the small intestines of PAFR(-/-) and wild-type mice. However, at 12 days after infection, significantly more worms were recovered from PAFR(-/-) mice. Although PAFR(-/-) infected mice showed a delay in elimination of adult worms, worms established in the small intestine of these mice produced a significantly lower number of eggs due to a reduction in worm fecundity. There were also significant reductions in the number of circulating and tissue eosinophils and tumor necrosis factor levels in the small intestines of PAFR(-/-) mice infected for 7 days compared to the number and level in wild-type mice. Histological analysis confirmed the reduced inflammatory process and revealed that the PAFR(-/-) mice had a smaller number of goblet cells. The concentrations of the type 2 cytokines interleukin-4 (IL-4), IL-5, and IL-10 were lower in small intestine homogenates and in supernatants of antigen-stimulated lymphocytes from spleens or mesenteric lymph nodes of PAFR(-/-) mice than in the corresponding preparations from wild-type mice. Thus, in S. venezuelensis-infected PAFR(-/-) mice, decreased intestinal inflammation is associated with enhanced worm survival but decreased fecundity. We suggest that although a Th2-predominant inflammatory response decreases worm survival, the worm may use factors produced during this response to facilitate egg output and reproduction. PAFR-mediated responses appear to modulate these host-derived signals that are important for worm fecundity.
Subject(s)
Platelet Membrane Glycoproteins/physiology , Receptors, G-Protein-Coupled/physiology , Strongyloides/isolation & purification , Strongyloidiasis/immunology , Animals , Cytokines/biosynthesis , Female , Intestine, Small/parasitology , Intestine, Small/pathology , Leukocyte Count , Mice , Mice, Inbred C57BL , Parasite Egg Count , Platelet Membrane Glycoproteins/deficiency , Receptors, G-Protein-Coupled/deficiency , Strongyloidiasis/parasitology , Strongyloidiasis/pathology , Th2 Cells/immunologyABSTRACT
The generation of an inflammatory response driven by Trypanosoma cruzi or its subproducts appears to be essential for tissue injury and disease pathogenesis. However, this inflammatory response is also relevant in the control of T. cruzi replication. The lipid mediator platelet-activating factor (PAF) has been implicated in a number of pathological conditions characterized by tissue inflammation. In the present study, we aimed at evaluating the role of PAF during T. cruzi infection by using mice that were genetically deficient in the PAF receptor. We observed that infected hearts of PAFR(-/-) mice had an increased number of parasite nests, associated with a more intense inflammatory infiltrate. This was associated with greater parasitemia and lethality. When wild-type and PAFR(-/-) mice were compared, there were no marked changes in the kinetics of the expression of MCP-1, RANTES, IFN-gamma and TNF-alpha in heart tissue of infected animals. Moreover, serum concentrations of TNF-alpha, nitrate and parasite-specific IgM were similar in both groups of mice. In vitro, macrophages from PAFR(-/-) animals did not phagocytose trypomastigote forms when activated with PAF, leukotriene B(4) or MCP-1 and produced less nitric oxide when infected and activated with IFN-gamma. These results are consistent with the hypothesis that endogenous synthesis of PAF and activation of PAF receptors control T. cruzi replication in mice in great part via facilitation of the uptake of the parasite and consequent activation of macrophages.
Subject(s)
Chagas Cardiomyopathy/immunology , Platelet Activating Factor/physiology , Platelet Membrane Glycoproteins/physiology , Receptors, Cell Surface/physiology , Receptors, G-Protein-Coupled , Trypanosoma cruzi/growth & development , Trypanosoma cruzi/immunology , Animals , Antibodies, Protozoan/blood , Cells, Cultured , Chagas Cardiomyopathy/metabolism , Chagas Cardiomyopathy/parasitology , Chagas Cardiomyopathy/pathology , Chemokine CCL2/biosynthesis , Chemokine CCL2/pharmacology , Chemokine CCL5/biosynthesis , Female , Heart/parasitology , Immunoglobulin M/blood , Interferon-gamma/biosynthesis , Leukotriene B4/pharmacology , Macrophages/metabolism , Macrophages/parasitology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardium/metabolism , Myocardium/pathology , Nitric Oxide/blood , Parasitemia , Platelet Activating Factor/pharmacology , Platelet Membrane Glycoproteins/genetics , Receptors, Cell Surface/genetics , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
1 The reperfusion of ischemic tissues may be associated with local and systemic inflammation that prevents the full benefit of blood flow restoration. The present study aimed to confirm a role for platelet-activating factor receptor(s) (PAFR) during ischemia and reperfusion injury by using genetically modified mice deficient in the PAFR (PAFR(-/-) mice) and to evaluate comparatively the effectiveness of pharmacological treatment using the PAFR antagonist UK-74,505 (modipafant). 2 The reperfusion of the ischemic superior mesenteric artery (SMA) induced marked local (intestine) and remote (lungs) tissue injury, as assessed by the increase in vascular permeability, neutrophil influx and intestinal hemorrhage and in the production of TNF-alpha. There was also a systemic inflammatory response, as shown by the increase in serum TNF-alpha concentrations and marked reperfusion-associated lethality. 3 After reperfusion of the ischemic SMA, PAFR(-/-) mice had little tissue or systemic inflammation and lethality was delayed, but not prevented, in these mice. Interestingly, the reperfusion-associated increases in tissue concentrations of IL-10 were significantly greater in PAFR(-/-) than wild-type mice. 4 Pretreatment with PAFR antagonist UK-74,505 (1 mg kg(-1)) markedly prevented tissue injury, as assessed by the increase in vascular permeability, neutrophil accumulation, hemorrhage and TNF-alpha concentrations in the intestine and lungs. In contrast, UK-74,505 failed to affect reperfusion-associated lethality and increases in serum TNF-alpha when used at 1 mg kg(-1). 5 Reperfusion-associated lethality and increase in serum TNF-alpha were only affected when a supra-maximal dose of the antagonist was used (10 mg kg(-1)). At this dose, UK-74,505 also induced a marked enhancement of reperfusion-associated increases in tissue concentrations of IL-10. However, at the same dose, UK-74,505 failed to prevent reperfusion-associated lethality in PAFR(-/-) mice any further. 6 The present studies using genetically modified animals and a receptor antagonist firmly establish a role of PAFR activation for the local, remote and systemic inflammatory injury and lethality which follows reperfusion of the ischemic SMA in mice. Moreover, it is suggested that high doses of PAFR antagonists need to be used if the real efficacy of these compounds is to be tested clinically.
Subject(s)
Intestines/blood supply , Mesenteric Artery, Superior/physiopathology , Mice, Inbred C57BL/genetics , Platelet Activating Factor/therapeutic use , Platelet Membrane Glycoproteins/antagonists & inhibitors , Platelet Membrane Glycoproteins/physiology , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/physiology , Reperfusion Injury/physiopathology , Animals , Dihydropyridines/administration & dosage , Dihydropyridines/pharmacology , Dihydropyridines/therapeutic use , Disease Models, Animal , Imidazoles/administration & dosage , Imidazoles/pharmacology , Imidazoles/therapeutic use , Intestinal Diseases/complications , Intestinal Diseases/drug therapy , Intestinal Diseases/physiopathology , Lung Diseases/complications , Lung Diseases/drug therapy , Lung Diseases/physiopathology , Male , Mesenteric Artery, Superior/drug effects , Mice , Platelet Activating Factor/pharmacology , Platelet Membrane Glycoproteins/genetics , Receptors, G-Protein-Coupled/genetics , Reperfusion Injury/drug therapyABSTRACT
Eosinophils are important inflammatory cells in allergic diseases. In the present study, we have investigated the effects of CCL22 on the recruitment of eosinophils in vivo and in vitro. CCL22 induced a dose- and time-dependent recruitment of eosinophils into the pleural cavity of mice, and this was dependent on the release of platelet-activating factor (PAF) and subsequent generation of CCL11. However, in an allergic pleurisy model, an anti-CCL22 polyclonal antibody given during sensitization or before challenge had no significant effect on eosinophil recruitment. CCL22 did not induce eosinophil chemotaxis in vitro but was able to induce eosinophil degranulation in vitro and in vivo. In conclusion, we show that although exogenously added CCL22 may induce eosinophil migration in vivo via release of PAF and CCL11 (eotaxin), endogenous production of CCL22 does not drive eosinophil migration during allergic inflammation. However, CCL22 may be an important activator of eosinophils once these cells have migrated into tissue.
Subject(s)
Chemokines, CC/pharmacology , Chemotaxis/drug effects , Eosinophils/cytology , Pleurisy/pathology , Receptors, G-Protein-Coupled , Animals , Antibodies/pharmacology , Cell Degranulation/drug effects , Chemokine CCL11 , Chemokine CCL22 , Chemokines, CC/administration & dosage , Chemokines, CC/immunology , Chemokines, CC/physiology , Chemotactic Factors, Eosinophil/immunology , Chemotactic Factors, Eosinophil/physiology , Hypersensitivity/pathology , Leukotriene B4/physiology , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Platelet Membrane Glycoproteins/genetics , Platelet Membrane Glycoproteins/physiology , Pleurisy/immunology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/physiologyABSTRACT
The lipid mediator PAF plays an important role in the phagocytosis of particles, including bacteria, and consequent production of pro-inflammatory cytokines, such as TNF-alpha and IL-8. Using a PAF receptor antagonist (UK-74,505) and PAF receptor knock-out mice, we have investigated the relevance of PAF for the inflammatory changes and lethality after pulmonary infection with the gram-negative bacteria Klebsiella pneumoniae in mice. At an inoculum of 3 x 10(6) bacteria, there was marked pulmonary (bronchoalveolar lavage and lung) neutrophilia that started early (2.5 h after infection) and peaked at 48 h. All animals were dead by day 4 of infection. The chemokine KC and the pro-inflammatory cytokine TNF-alpha increased rapidly and persisted for 48 h in the lungs. Pretreatment with UK-74,505 (30 mg kg(-1) per day, p.o.) had no significant effects on the number of infiltrating neutrophils in BAL fluid or lung tissue, as assessed by histology and measuring myeloperoxidase, or on the concentrations of KC. In contrast, concentrations of TNF-alpha and the number of bacteria inside neutrophils were significantly diminished. In order to support a role for the PAF during K. pneumoniae infection, experiments were also carried out in PAFR-deficient mice. In the latter animals, lethality occurred earlier than in wild-type controls. This was associated with greater number of bacteria in lung tissue and diminished percentage of neutrophils containing bacteria in their cytoplasm. Our results suggest that PAF, acting on its receptor, plays a protective role during infection with K. pneumoniae in mice.
Subject(s)
Klebsiella Infections/immunology , Klebsiella pneumoniae/immunology , Platelet Membrane Glycoproteins/physiology , Receptors, Cell Surface/physiology , Receptors, G-Protein-Coupled , Animals , Dihydropyridines/pharmacology , Female , Gram-Negative Bacteria/immunology , Gram-Negative Bacteria/metabolism , Imidazoles/pharmacology , Klebsiella Infections/metabolism , Klebsiella pneumoniae/metabolism , Lung/drug effects , Lung/immunology , Lung/metabolism , Lung/microbiology , Mice , Mice, Inbred BALB C , Platelet Membrane Glycoproteins/antagonists & inhibitors , Receptors, Cell Surface/antagonists & inhibitors , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Platelets are blood cells that participate in the human primary hemostatic mechanism. Platelets need to be activated to perform all their functions and this activation can be initially produced after an endothelial injury that exposes subendothelial structures to the blood flow. Platelet adhesiveness can also occur by the intervention of high shear forces of the blood stream leading to the attachment of platelets to the subendothelium, forming a monolayer with platelet shape changes and pseudopod emission, that covers all the exposed subendothelial surface. Adhesiveness results from the participation of three components: Glycoprotein Ib, von Willebrand factor and collagen. Fibronectin and vitronectin can also participate in this process. Aggregation (platelet-platelet interaction) has as the final purpose to produce a platelet thrombi that constitutes the primary hemostatic plug. Aggregation involves a sequence of biochemical reactions that are initiated by the contact of an agonist agent (ADP, collagen, thrombin) with receptors on the platelet membrane, this is followed by the fibrinogen binding to Glycoprotein IIb/IIIa complex serving as a link with other surrounding platelets, the aggregation process is then amplified and becomes irreversible with the secretion of the platelet intragranular substances through the release reaction. These series of reactions have their own mechanisms of control that regulate or modulate platelet activation as the Ca2+/cAMP relationship, the balance between PGI2 and TxA2 and the enzymatic system of kinases and phosphatases. The relationship between platelets and other blood cells such as erythrocytes and leukocytes probably modulate the platelet aggregatory response during a vascular injury promoting mechanisms of thrombogenesis and wound healing. On the other hand, the multiple compounds released by the endothelial cell that inhibit platelet function, specially prostacyclin (PGI2) and recently the endothelial derived relaxing factor (EDRF) or nitric oxide have focused new understanding on the physiologic role of platelets in the control of hemostatic and thrombotic processes.