Platelet EVs contain an active proteasome involved in protein processing for antigen presentation via MHC-I molecules.

In addition to their hemostatic role, platelets play a significant role in immunity. Once activated, platelets release extracellular vesicles (EVs) formed by the budding of their cytoplasmic membranes. Because of their heterogeneity, platelet EVs (PEVs) are thought to perform diverse functions. It is unknown, however, whether the proteasome is transferred from platelets to PEVs or whether its function is retained. We hypothesized that functional protein processing and antigen presentation machinery are transferred to PEVs by activated platelets. Using molecular and functional assays, we found that the active 20S proteasome was enriched in PEVs, along with major histocompatibility complex class I (MHC-I) and lymphocyte costimulatory molecules (CD40L and OX40L). Proteasome-containing PEVs were identified in healthy donor blood, but did not increase in platelet concentrates that caused adverse transfusion reactions. They were augmented, however, after immune complex injections in mice. The complete biodistribution of murine PEVs after injection into mice revealed that they principally reached lymphoid organs, such as spleen and lymph nodes, in addition to the bone marrow, and to a lesser extent, liver and lungs. The PEV proteasome processed exogenous ovalbumin (OVA) and loaded its antigenic peptide onto MHC-I molecules, which promoted OVA-specific CD8+ T-lymphocyte proliferation. These results suggest that PEVs contribute to adaptive immunity through cross-presentation of antigens and have privileged access to immune cells through the lymphatic system, a tissue location that is inaccessible to platelets.

Human Inflammatory Neutrophils Express Genes Encoding Peptidase Inhibitors: Production of Elafin Mediated by NF-κB and CCAAT/Enhancer-Binding Protein ß.

The concept of plasticity of neutrophils is highlighted by studies showing their ability to transdifferentiate into APCs. In this regard, transdifferentiated neutrophils were found at inflammatory sites of autoimmune arthritis (AIA). Exposure of neutrophils to inflammatory stimuli prolongs their survival, thereby favoring the acquisition of pathophysiologically relevant phenotypes and functions. By using microarrays, quantitative RT-PCR, and ELISAs, we showed that long-lived (LL) neutrophils obtained after 48 h of culture in the presence of GM-CSF, TNF, and IL-4 differentially expressed genes related to apoptosis, MHC class II, immune response, and inflammation. The expression of anti-inflammatory genes mainly of peptidase inhibitor families is upregulated in LL neutrophils. Among these, the PI3 gene encoding elafin was the most highly expressed. The de novo production of elafin by LL neutrophils depended on a synergism between GM-CSF and TNF via the activation and cooperativity of C/EBPß and NF-κB pathways, respectively. Elafin concentrations were higher in synovial fluids (SF) of patients with AIA than in SF of osteoarthritis. SF neutrophils produced more elafin than blood counterparts. These results are discussed with respect to implications of neutrophils in chronic inflammation and the potential influence of elafin in AIA.

Phospholipase A1 Member A Deficiency Alleviates Mannan-Induced Psoriatic Arthritis in Mice Model.

Synovial fluids from rheumatoid and psoriatic arthritis patients have high levels of PLA1A. The current study was to understand PLA1A functions in the pathophysiology of rheumatic diseases. We generated Pla1a−/− mice to assess their phenotype and the impact of PLA1A deficiency on the development of mannan-induced psoriatic arthritis (MIP). Mice were evaluated routinely for the induced symptoms. On the day of sacrifice, blood samples were collected for hematology analysis and prepared for plasma. Livers were collected. Lymph node immune cells were analyzed using flow cytometry. We performed µCT scans of hind paws from naïve and mannan-induced female mice. Cytokines/chemokines were quantified using Luminex in hind paw tissues and plasma of female mice. Pla1a−/− mice showed a slight increase in circulating and lymph node lymphocytes. CD4+ T cells contributed most to this increase in lymph nodes (p = 0.023). In the MIP model, the lymph node ratios of CD3+ to CD19+ and CD4+ to CD8+ were higher in Pla1a−/− mice. Pla1a−/− mice were less susceptible to MIP (p < 0.001) and showed reduced bone erosions. Pla1a−/− mice also showed reduced IL-17, KC, IP-10, MIP-1ß, LIF, and VEGF in hind paw tissues as compared to WT mice (p < 0.05). These findings indicated that PLA1A deficiency protected from the development of the MIP disease. The data suggested that PLA1A could contribute to MIP through increased activation of lymphocytes, possibly those producing IL-17.

Phospholipase A1 Member A Activates Fibroblast-like Synoviocytes through the Autotaxin-Lysophosphatidic Acid Receptor Axis.

Lysophosphatidylserine (lysoPS) is known to regulate immune cell functions. Phospholipase A1 member A (PLA1A) can generate this bioactive lipid through hydrolysis of sn-1 fatty acids on phosphatidylserine (PS). PLA1A has been associated with cancer metastasis, asthma, as well as acute coronary syndrome. However, the functions of PLA1A in the development of systemic autoimmune rheumatic diseases remain elusive. To investigate the possible implication of PLA1A during rheumatic diseases, we monitored PLA1A in synovial fluids from patients with rheumatoid arthritis and plasma of early-diagnosed arthritis (EA) patients and clinically stable systemic lupus erythematosus (SLE) patients. We used human primary fibroblast-like synoviocytes (FLSs) to evaluate the PLA1A-induced biological responses. Our results highlighted that the plasma concentrations of PLA1A in EA and SLE patients were elevated compared to healthy donors. High concentrations of PLA1A were also detected in synovial fluids from rheumatoid arthritis patients compared to those from osteoarthritis (OA) and gout patients. The origin of PLA1A in FLSs and the arthritic joints remained unknown, as healthy human primary FLSs does not express the PLA1A transcript. Besides, the addition of recombinant PLA1A stimulated cultured human primary FLSs to secrete IL-8. Preincubation with heparin, autotaxin (ATX) inhibitor HA130 or lysophosphatidic acid (LPA) receptor antagonist Ki16425 reduced PLA1A-induced-secretion of IL-8. Our data suggested that FLS-associated PLA1A cleaves membrane-exposed PS into lysoPS, which is subsequently converted to LPA by ATX. Since primary FLSs do not express any lysoPS receptors, the data suggested PLA1A-mediated pro-inflammatory responses through the ATX-LPA receptor signaling axis.

Assessment of Arf6 Deletion in PLB-985 Differentiated in Neutrophil-Like Cells and in Mouse Neutrophils: Impact on Adhesion and Migration.

Chemoattractant sensing, adhesiveness, and migration are critical events underlying the recruitment of neutrophils (PMNs) to sites of inflammation or infection. Defects in leukocyte adhesion or migration result in immunodeficiency disorders characterized by recurrent infections. In this study, we evaluated the role of Arf6 on PMN adhesion in vitro and on migration to inflammatory sites using PMN-Arf6 conditional knockout (cKO) mice. In PMN-like PLB-985 silenced for Arf6 fMLP-mediated adhesion to the ß2 integrin ligands, ICAM-1 and fibrinogen or the ß1/ß2 integrin ligand fibronectin was significantly reduced. Furthermore, overexpression of wild-type Arf6 promoted basal and fMLP-induced adhesion to immobilized integrin ligands, while overexpression of the dominant-negative Arf6 has the opposite effects. Using the Elane-Cre deleting mouse strains, we report that the level of Arf6 deletion in inflammatory PMNs isolated from the dorsal air pouches was stronger when compared to naïve cells isolated from the bone marrow. In PMN-Arf6 cKO mice, the recruitment of PMNs into the dorsal air pouch injected with LPS or the chemoattractant fMLP was significantly diminished. Impaired cell migration correlated with reduced cell surface expression of CD11a and CD11b in Arf6 cKO PMNs. Our results highlight that Arf6 regulates the activity and possibly the recycling of PMN integrins, and this compromises PMN migration to inflammatory sites.

A transgenic mouse model for the in vivo bioluminescence imaging of the expression of the lysophosphatidic acid receptor 3: relevance for inflammation and uterine physiology research.

Lysophosphatidic acid (LPA) is a lipid-derived signaling molecule that plays key roles in diverse biological processes including inflammation and uterine remodeling. Although the function of LPA and its receptors has been extensively studied using knock-out mice, the temporal-spatial expression of LPA receptors is less well-characterized. To gain further insight into the dynamic regulation of LPA receptor 3 (Lpar3) expression in vivo by bioluminescence imaging, we generated and characterized mice transgenic for a putative Lpar3 promoter fragment. A non-coding region of the Lpar3 gene immediately upstream of the start site was subcloned adjacent to the luciferase gene. Promoter activity was determined by in vitro luciferase assays, in vivo bioluminescent imaging or by semi-quantitative real-time PCR. The air-pouch model was used to investigate Lpar3 promoter activity in the context of inflammation. The putative Lpar3 promoter fragment behaved similarly to the endogenous promoter in vitro and in vivo. In male mice, elevated levels of Lpar3-induced luciferase activity were observed in the testis. In female mice, the basal level of luciferase activity in the uterus significantly increased during pseudopregnancy. Moreover, luciferase activity was upregulated by TNF-α in the air-pouch model. We report the identification of a functional Lpar3 promoter fragment and the generation of a transgenic mouse model to investigate the regulation of Lpar3 promoter activity non-invasively in vivo by bioluminescence imaging. This mouse model is a valuable tool for reproductive biology and inflammation research as well as other biological processes in which this receptor is involved.

LPA Promotes T Cell Recruitment through Synthesis of CXCL13.

Lysophosphatidic acid (LPA) is a bioactive phospholipid playing an important role in various inflammatory diseases by inducing expression and secretion of many inflammatory cytokines/chemokines. Here we report in a murine air pouch model of inflammation that LPA induced CXCL13 secretion in a time-dependent manner and with exacerbation of the response when LPA was administered after a pretreatment with TNF-α, a key inflammatory cytokine. LPA mediates recruitment of leukocytes, including that of CD3(+) cells into unprimed and TNF-α-primed air pouches. CXCL13 neutralization using a blocking antibody injected into air pouches prior to administration of LPA into TNF-α-primed air pouches decreased CD3(+) cell influx. Our data highlight that LPA-mediated CXCL13 secretion plays a role in T cell recruitment and participates in regulation of the inflammatory response.

Chemical Hypoxia Brings to Light Altered Autocrine Sphingosine-1-Phosphate Signalling in Rheumatoid Arthritis Synovial Fibroblasts.

Emerging evidence suggests a role for sphingosine-1-phosphate (S1P) in various aspects of rheumatoid arthritis (RA) pathogenesis. In this study we compared the effect of chemical hypoxia induced by cobalt chloride (CoCl2) on the expression of S1P metabolic enzymes and cytokine/chemokine secretion in normal fibroblast-like synoviocytes (FLS) and RAFLS. RAFLS incubated with CoCl2, but not S1P, produced less IL-8 and MCP-1 than normal FLS. Furthermore, incubation with the S1P2 and S1P3 receptor antagonists, JTE-013 and CAY10444, reduced CoCl2-mediated chemokine production in normal FLS but not in RAFLS. RAFLS showed lower levels of intracellular S1P and enhanced mRNA expression of S1P phosphatase 1 (SGPP1) and S1P lyase (SPL), the enzymes that are involved in intracellular S1P degradation, when compared to normal FLS. Incubation with CoCl2 decreased SGPP1 mRNA and protein and SPL mRNA as well. Inhibition of SPL enhanced CoCl2-mediated cytokine/chemokine release and restored autocrine activation of S1P2 and S1P3 receptors in RAFLS. The results suggest that the sphingolipid pathway regulating the intracellular levels of S1P is dysregulated in RAFLS and has a significant impact on cell autocrine activation by S1P. Altered sphingolipid metabolism in FLS from patients with advanced RA raises the issue of synovial cell burnout due to chronic inflammation.

Metabolic regulation of neutrophil functions in homeostasis and diseases.

Neutrophils are the most abundant leukocytes in humans and play a role in the innate immune response by being the first cells attracted to the site of infection. While early studies presented neutrophils as almost exclusively glycolytic cells, recent advances show that these cells use several metabolic pathways other than glycolysis, such as the pentose phosphate pathway, oxidative phosphorylation, fatty acid oxidation, and glutaminolysis, which they modulate to perform their functions. Metabolism shifts from fatty acid oxidation-mediated mitochondrial respiration in immature neutrophils to glycolysis in mature neutrophils. Tissue environments largely influence neutrophil metabolism according to nutrient sources, inflammatory mediators, and oxygen availability. Inhibition of metabolic pathways in neutrophils results in impairment of certain effector functions, such as NETosis, chemotaxis, degranulation, and reactive oxygen species generation. Alteration of these neutrophil functions is implicated in certain human diseases, such as antiphospholipid syndrome, coronavirus disease 2019, and bronchiectasis. Metabolic regulators such as AMPK, HIF-1α, mTOR, and Arf6 are linked to neutrophil metabolism and function and could potentially be targeted for the treatment of diseases associated with neutrophil dysfunction. This review details the effects of alterations in neutrophil metabolism on the effector functions of these cells.

Cytohesin-1 regulates the Arf6-phospholipase D signaling axis in human neutrophils: impact on superoxide anion production and secretion.

Polymorphonuclear neutrophil (PMN) stimulation with fMLP stimulates small G proteins such as ADP-ribosylation factors (Arfs) Arf1 and Arf6, leading to phospholipase D (PLD) activation and functions such as degranulation and the oxidative burst. However, the molecular links between fMLF receptors and PLD remain unclear. PMNs express cytohesin-1, an Arf-guanine exchange factor that activates Arfs, and its expression is strongly induced during the acquisition of the neutrophilic phenotype by neutrophil-like cells. The role of cytohesin-1 in the activation of the fMLF-Arf-PLD signaling axis, and the accomplishment of superoxide anion production, and degranulation was investigated in PMNs using the selective inhibitor of cytohesin, Sec 7 inhibitor H3 (secinH3). Cytohesin-1 inhibition with secinH3 leads to Arf6 but not Arf1 inhibition, demonstrating the specificity for Arf6, and fMLF-mediated activation of PLD and of the oxidative burst as well. We observed a decrease in fMLF-mediated protein secretion and expression of cell surface markers corresponding to primary (CD63/myeloperoxidase), secondary (CD66/lactoferrin), and tertiary (matrix metalloproteinase-9) granules in PMNs incubated with secinH3. Similarly, silencing cytohesin-1 or Arf6 in PLB-985 cells negatively affected fMLF-induced activation of PLD, superoxide production, and expression of granule markers on the cell surface. In contrast, stable overexpression of cytohesin-1 in PLB-985 cells enhanced fMLF-induced activation of Arf6, PLD, and NADPH oxidase. The results of this study provide evidence for an involvement of cytohesin-1 in the regulation of the functional responses of human PMNs and link these events, in part at least, to the activation of Arf6.

Functional Role of AGAP2/PIKE-A in Fcγ Receptor-Mediated Phagocytosis.

In phagocytes, cytoskeletal and membrane remodeling is finely regulated at the phagocytic cup. Various smaFll G proteins, including those of the Arf family, control these dynamic processes. Human neutrophils express AGAP2, an Arf GTPase activating protein (ArfGAP) that regulates endosomal trafficking and focal adhesion remodeling. We first examined the impact of AGAP2 on phagocytosis in CHO cells stably expressing the FcγRIIA receptor (CHO-IIA). In unstimulated CHO-IIA cells, AGAP2 only partially co-localized with cytoskeletal elements and intracellular compartments. In CHO-IIA cells, AGAP2 transiently accumulated at actin-rich phagocytic cups and increased Fcγ receptor-mediated phagocytosis. Enhanced phagocytosis was not dependent on the N-terminal GTP-binding protein-like (GLD) domain of AGAP2. AGAP2 deleted of its GTPase-activating protein (GAP) domain was not recruited to phagocytic cups and did not enhance the engulfment of IgG-opsonized beads. However, the GAP-deficient [R618K]AGAP2 transiently localized at the phagocytic cups and enhanced phagocytosis. In PLB-985 cells differentiated towards a neutrophil-like phenotype, silencing of AGAP2 reduced phagocytosis of opsonized zymosan. In human neutrophils, opsonized zymosan or monosodium urate crystals induced AGAP2 phosphorylation. The data indicate that particulate agonists induce AGAP2 phosphorylation in neutrophils. This study highlights the role of AGAP2 and its GAP domain but not GAP activity in FcγR-dependent uptake of opsonized particles.

Red blood cell-derived phosphatidylserine positive extracellular vesicles are associated with past thrombotic events in patients with systemic erythematous lupus.

BACKGROUND: Extracellular vesicles (EVs) released by blood cells have proinflammation and procoagulant action. Patients with systemic lupus erythematosus (SLE) present high vascular inflammation and are prone to develop cardiovascular diseases. Therefore, we postulated that the EV populations found in blood, including platelet EVs (PEVs) and red blood cell EVs (REVs), are associated with SLE disease activity and SLE-associated cardiovascular accidents. METHOD: We assessed autotaxin (ATX) plasma levels by ELISA, the platelet activation markers PAC1 and CD62P, ATX bound to platelets and the amounts of plasma PEVs and REVs by flow cytometry in a cohort of 102 patients with SLE, including 29 incident cases of SLE and 30 controls. Correlation analyses explored the associations with the clinical parameters. RESULT: Platelet activation markers were increased in patients with SLE compared with healthy control, with the marker CD62P associated with the SLE disease activity index (SLEDAI). The incident cases show additional associations between platelet markers (CD62P/ATX and PAC1/CD62P) and the SLEDAI. Compared with controls, patients with SLE presented higher levels of PEVs, phosphatidylserine positive (PS+) PEVs, REVs and PS+ REVs, but there is no association with disease activity. When stratified according to the plasma level of PS+ REVs, the group of patients with SLE with a high level of PS+ REVs presented a higher number of past thrombosis events and higher ATX levels. CONCLUSION: Incident and prevalent forms of SLE cases present similar levels of platelet activation markers, with CD62P correlating with disease activity. Though EVs are not associated with disease activity, the incidence of past thrombotic events is higher in patients with a high level of PS+ REVs.

TNF-alpha promotes LPA1- and LPA3-mediated recruitment of leukocytes in vivo through CXCR2 ligand chemokines.

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid present in low concentrations in serum and biological fluids but in high concentrations at sites of inflammation. LPA evokes a variety of cellular responses via binding to and activation of its specific G protein-coupled receptors (GPCR), namely LPA(1-6). Even though LPA is a chemoattractant for inflammatory cells in vitro, such a role for LPA in vivo remains largely unexplored. In the present study, we used the murine air pouch model to study LPA-mediated leukocyte recruitment in vivo using selective LPA receptor agonist/antagonist and LPA(3)-deficient mice. We report that 1) LPA injection into the air pouch induced leukocyte recruitment and that both LPA(1) and LPA(3) were involved in this process; 2) LPA stimulated the release of the pro-inflammatory chemokines keratinocyte-derived chemokine (KC) and interferon-inducible protein-10 (IP-10) in the air pouch; 3) tumor necrosis factor-α (TNF-α) injected into the air pouch prior to LPA strongly potentiated LPA-mediated secretion of cytokines/chemokines, including KC, IL-6, and IP-10, which preceded the enhanced leukocyte influx; and 4) blocking CXCR2 significantly reduced leukocyte infiltration. We suggest that LPA, via LPA(1) and LPA(3) receptors, may play a significant role in inducing and/or sustaining the massive infiltration of leukocytes during inflammation.

Phosphatidylserine-specific phospholipase A1: A friend or the devil in disguise.

Various human tissues and cells express phospholipase A1 member A (PLA1A), including the liver, lung, prostate gland, and immune cells. The enzyme belongs to the pancreatic lipase family. PLA1A specifically hydrolyzes sn-1 fatty acid of phosphatidylserine (PS) or 1-acyl-lysophosphatidylserine (1-acyl-lysoPS). PS externalized by activated cells or apoptotic cells or extracellular vesicles is a potential source of substrate for the production of unsaturated lysoPS species by PLA1A. Maturation and functions of many immune cells, such as T cells, dendritic cells, macrophages, and mast cells, can be regulated by PLA1A and lysoPS. Several lysoPS receptors, including GPR34, GPR174 and P2Y10, have been identified. High serum levels and high PLA1A expression are associated with autoimmune disorders such as Graves' disease and systemic lupus erythematosus. Increased expression of PLA1A is associated with metastatic melanomas. PLA1A may contribute to cardiometabolic disorders through mediating cholesterol transportation and producing lysoPS. Furthermore, PLA1A is necessary for hepatitis C virus assembly and can play a role in the antivirus innate immune response. This review summarizes recent findings on PLA1A expression, lysoPS and lysoPS receptors in autoimmune disorders, cancers, cardiometabolic disorders, antivirus immune responses, as well as regulations of immune cells.

Interplay between LPA2 and LPA3 in LPA-mediated phosphatidylserine cell surface exposure and extracellular vesicles release by erythrocytes.

Evidence is growing for the role of red blood cells (RBCs) in vascular homeostasis, including thrombogenic events and inflammation. Lysophosphatidic acid (LPA) is known to induce phosphatidylserine (PS) exposure and the release of RBC Extracellular Vesicles (REVs). Using high sensitivity flow cytometry, we examined the effects and the mechanisms by which the LPA species commonly found in human plasma could activate RBCs. We report that LPA 16:0, 18:0 and 18:1, but not LPA 20:4, induced PS exposure and the release of small PS- and large PS+ REVs through LPA3 receptor signalling in RBCs. The release of large PS+ REVs required higher concentrations of LPA. RBCs were not activated by LPA 20:4. Interestingly, blockade of LPA2 enhanced LPA-mediated PS- REV release in RBCs. Furthermore, LPA receptor agonists and antagonists highlighted that LPA 20:4 inhibited LPA3-dependent PS exposure and, through the LPA2 receptor, inhibited PS- REV production. Activation of RBCs with LPA 18:1 in normal plasma stimulated the release of PS- and PS+ REVs. REVs released in response to LPA were similar to those found in the plasma of systemic lupus erythematosus patients. Our results suggest that LPA species exhibit different biological activities in RBCs through targeting LPA2 and/or LPA3 receptors.

Arf6 regulates energy metabolism in neutrophils.

The small GTPase Arf6 regulates many cellular processes, including cytoskeletal remodeling, receptor endocytosis, and pathogen phagocytosis. Arf6 silencing in neutrophil (PMN)-like cells is well-known to inhibit chemotactic peptide-mediated activation of phospholipase D, the oxidative burst, and ß2 integrin-dependent adhesion. In conditional knockout (cKO) mice, the migration to inflammatory sites of Arf6-deficient PMNs was diminished and associated with reduced cell surface expression of ß2 integrins. In this study we assessed the impact of Arf6 depletion on the functions and gene expression profile of PMNs isolated from the mouse air pouch. Numerous genes involved in response to oxygen levels, erythrocyte and myeloid differentiation, macrophage chemotaxis, response to chemicals, apoptosis, RNA destabilization, endosome organization, and vesicle transport were differentially expressed in PMNs cKO for Arf6. Lpar6 and Lacc-1 were the most up-regulated and down-regulated genes, respectively. The deletion of Arf6 also decreased Lacc-1 protein level in PMNs, and silencing of Arf6 in THP-1 monocytic cells delayed LPS-mediated Lacc-1 expression. We report that fMLP or zymosan-induced glycolysis and oxygen consumption rate were both decreased in air pouch PMNs but not in bone marrow PMNs of Arf6 cKO mice. Reduced oxygen consumption correlated with a decrease in superoxide and ROS production. Deletion of Arf6 in PMNs also reduced phagocytosis and interfered with apoptosis. The data suggest that Arf6 regulates energy metabolism, which may contribute to impaired phagocytosis, ROS production, and apoptosis in PMN-Arf6 cKO. This study provides new information on the functions and the inflammatory pathways influenced by Arf6 in PMNs.

Targeting the autotaxin - Lysophosphatidic acid receptor axis in cardiovascular diseases.

Lysophosphatidic acid (LPA) is a well-characterized bioactive lipid mediator, which is involved in development, physiology, and pathological processes of the cardiovascular system. LPA can be produced both inside cells and in biological fluids. The majority of extracellularLPAis produced locally by the secreted lysophospholipase D, autotaxin (ATX), through its binding to various ß integrins or heparin sulfate on cell surface and hydrolyzing various lysophospholipids. LPA initiates cellular signalling pathways upon binding to and activation of its G protein-coupled receptors (LPA1-6). LPA has potent effects on various blood cells and vascular cells involved in the development of cardiovascular diseases such as atherosclerosis and aortic valve sclerosis. LPA signalling drives cell migration and proliferation, cytokine production, thrombosis, fibrosis, as well as angiogenesis. For instance, LPA promotes activation and aggregation of platelets through LPA5, increases expression of adhesion molecules in endothelial cells, and enhances expression of tissue factor in vascular smooth muscle cells. Furthermore, LPA induces differentiation of monocytes into macrophages and stimulates oxidized low-density lipoproteins (oxLDLs) uptake by macrophages to form foam cells during formation of atherosclerotic lesions through LPA1-3. This review summarizes recent findings of the roles played by ATX, LPA and LPA receptors (LPARs) in atherosclerosis and calcific aortic valve disease. Targeting the ATX-LPAR axis may have potential applications for treatment of patients suffering from various cardiovascular diseases.

Impairment of chemical hypoxia-induced sphingosine kinase-1 expression and activation in rheumatoid arthritis synovial fibroblasts: A signature of exhaustion?

Sphingosine kinase 1 (SphK1) and 2 (SphK2) have been shown contribute to synovial inflammation in animal models of arthritis. However, low levels of intracellular sphingosine-1 phosphate (S1P) were reported in fibroblast-like synoviocytes (FLS) from patients in the end stage of rheumatoid arthritis (RA) compared to normal FLS. Moreover, the S1P receptor-mediated chemokine synthesis was altered in RAFLS in response to chemical hypoxia. Since the mechanisms responsible for low levels of intracellular S1P in RAFLS are not fully identified, we evaluated the contribution of SphKs to the S1P-induced synthesis of chemokines under conditions of chemical hypoxia. Our results show that a chemical hypoxia mimetic cobalt chloride (CoCl2) increased SphK1 expression and activation in normal FLS but not in RAFLS. Using selective inhibitors of SphKs and gene silencing approaches, we provide evidence that both SphK1 and SphK2 are involved in hypoxia-induced chemokine production in normal FLS. In contrast, only SphK2 mediates hypoxia-induced chemokine production in RAFLS. Moreover, CoCl2 increased S1P2 and S1P3 receptor mRNA levels in normal FLS but not in RAFLS. The data suggest that altered expression and/or activation of SphK1 combined with reduced induction of S1P receptor expression by CoCl2 impaired the CoCl2-mediated autocrine S1P receptor signaling loop and chemokine production in RAFLS.

Regulation of lysophosphatidic acid receptor expression and function in human synoviocytes: implications for rheumatoid arthritis?

Lysophosphatidic acid (LPA), via interaction with its G-protein coupled receptors, is involved in various pathological conditions. Extracellular LPA is mainly produced by the enzyme autotaxin (ATX). Using fibroblast-like synoviocytes (FLS) isolated from synovial tissues of patients with rheumatoid arthritis (RA), we studied the expression profile of LPA receptors, LPA-induced cell migration, and interleukin (IL)-8 and IL-6 production. We report that FLS express LPA receptors LPA(1-3). Moreover, exogenously applied LPA induces FLS migration and secretion of IL-8/IL-6, whereas the LPA(3) agonist l-sn-1-O-oleoyl-2-methyl-glyceryl-3-phosphothionate (2S-OMPT) stimulates cytokine synthesis but not cell motility. The LPA-induced FLS motility and cytokine production are suppressed by LPA(1/3) receptor antagonists diacylglycerol pyrophosphate and (S)-phosphoric acid mono-(2-octadec-9-enoylamino-3-[4-(pyridine-2-ylmethoxy)-phenyl]-propyl) ester (VPC32183). Signal transduction through p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and Rho kinase is involved in LPA-mediated cytokine secretion, whereas LPA-induced cell motility requires p38 MAPK and Rho kinase but not p42/44 MAPK. Treatment of FLS with tumor necrosis factor-alpha (TNF-alpha) increases LPA(3) mRNA expression and correlates with enhanced LPA- or OMPT-induced cytokine production. LPA-mediated superproduction of cytokines by TNF-alpha-primed FLS is abolished by LPA(1/3) receptor antagonists. We also report the presence of ATX in synovial fluid of patients with RA. LPA(1/3) receptor antagonists and ATX inhibitors reduce the synovial fluid-induced cell motility. Together the data suggest that LPA(1) and LPA(3) may contribute to the pathogenesis of RA through the modulation of FLS migration and cytokine production. The above results provide novel insights into the relevance of LPA receptors in FLS biology and as potential therapeutic targets for the treatment of RA.

Dissociation between the translocation and the activation of Akt in fMLP-stimulated human neutrophils--effect of prostaglandin E2.

PGE(2) and other cAMP-elevating agents are known to down-regulate most functions stimulated by fMLP in human polymorphonuclear neutrophils. We reported previously that the inhibitory potential of PGE(2) resides in its capacity to suppress fMLP-stimulated PI-3Kgamma activation via the PGE(2) receptor EP(2) and hence, to decrease phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P(3)] formation. Akt activity is stimulated by fMLP through phosphorylation on threonine 308 (Thr308) and serine 473 (Ser473) by 3-phosphoinositide-dependent kinase 1 (PDK1) and MAPK-AP kinase (APK)-APK-2 (MAPKAPK-2), respectively, in a PI-3K-dependent manner. Despite the suppression of fMLP-induced PI-3Kgamma activation observed in the presence of PGE(2), we show that Akt is fully phosphorylated on Thr308 and Ser473. However, fMLP-induced Akt translocation is decreased markedly in this context. PGE(2) does not affect the phosphorylation of MAPKAPK-2 but decreases the translocation of PDK1 induced by fMLP. Other cAMP-elevating agents such as adenosine (Ado) similarly block the fMLP-induced PI-3Kgamma activation process but do not inhibit Akt phosphorylation. However, Akt activity stimulated by fMLP is down-regulated slightly by agonists that elevate cAMP levels. Whereas protein kinase A is not involved in the maintenance of Akt phosphorylation, it is required for the inhibition of Akt translocation by PGE(2). Moreover, inhibition of fMLP-stimulated PI-3Kdelta activity by the selective inhibitor IC87114 only partially affects the late phase of Akt phosphorylation in the presence of PGE(2). Taken together, these results suggest that cAMP-elevating agents, such as PGE(2) or Ado, are able to induce an alternative mechanism of Akt activation by fMLP in which the translocation of Akt to PI(3,4,5)P(3)-enriched membranes is not required prior to its phosphorylation.