Live imaging of platelets and neutrophils during antibody-mediated neurovascular thrombosis.

Immune complexes form in systemic disorders such as rheumatological, autoimmune, and allergic diseases or in response to infections or medications. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) adenoviral vector vaccines have been associated with rare yet serious thrombotic complications in the brain due to the formation of immune complexes that activate platelets. There are currently no data visualizing the interplay of platelets with leukocytes and the brain vasculature endothelium in response to immune complexes. This is in part due to the absence of FcγRIIA in mice, a receptor for immune complexes implicated in these thrombotic incidents. Here, we describe and illustrate events at the cellular level that take place in the brain vasculature in response to systemic administration of surrogate immune complexes. We used Ly6gCre+/-::Rosa26-TdT+/-::CD41-YFP+/- mice expressing the FcγRIIA transgene and fluorescence in neutrophils and platelets. Using real-time videomicroscopy to capture high-velocity events in conjunction with unbiased computer-assisted analyses, we provide images and quantifications of the cellular responses downstream of FcγRIIA stimulation. We observed transient and stable platelet-neutrophil interactions, platelets forming thrombi, and neutrophil adhesion to blood vessel walls. This imaging approach in a quadruple transgenic animal model can be used for the study of the pathogenic roles of immune complexes in disease.

Rapid Onset of Ceftriaxone-Induced Cholelithiasis in an Adult Patient.

Ceftriaxone is a third-generation cephalosporin with broad-spectrum antibacterial activity. Here, we report a case of ceftriaxone-induced cholelithiasis in an adult patient after a short period of administering ceftriaxone. A 57-year-old female was admitted to our hospital for meningitis and treated empirically with ceftriaxone 2 g every 12 hours. Other medications given included vancomycin, ampicillin and acyclovir. Based on culture results and a sensitivity report, ceftriaxone was continued while other medications were discontinued on day three after admission. Her liver function test (LFT) demonstrated an elevation in hepatic transaminases, and alanine and aspartate transaminases peaked on the fifth day (339 and 153 IU/L, respectively). Computed tomography (CT) and ultrasound (US) confirmed the presence of uncomplicated cholelithiasis. Ceftriaxone was discontinued and switched to cefotaxime 2 g every four hours. Hepatic transaminases started declining after ceftriaxone discontinuation and dropped to normal levels on day nine. After the administration of cefotaxime on the 25th day, repeated US imaging revealed the persistence of biliary sludge. The patient was discharged in a good and stable clinical condition with follow-up planned at the outpatient clinic. When the concentration of ceftriaxone in the gallbladder exceeds the solubility of its calcium salt, precipitation occurs, forming a biliary sludge. Using the Naranjo Probability scale, the score was found to be 4, indicating a possible relationship between ceftriaxone and cholelithiasis. Multiple case reports of ceftriaxone-induced cholelithiasis have been documented previously, most of which focused on children or on the prolonged use of ceftriaxone. However, our case report highlights the development of cholelithiasis in adults after administering ceftriaxone for a short time.

Identification of Mitofusin 1 and Complement Component 1q Subcomponent Binding Protein as Mitochondrial Targets in Systemic Lupus Erythematosus.

OBJECTIVE: Mitochondria are organelles that exhibit several bacterial features, such as a double-stranded genome with hypomethylated CpG islands, formylated proteins, and cardiolipin-containing membranes. In systemic lupus erythematosus (SLE), mitochondria and their inner components are released into the extracellular space, potentially eliciting a proinflammatory response from the immune system. While cardiolipin and mitochondrial DNA and RNA are confirmed targets of autoantibodies, other antigenic mitochondrial proteins in SLE remain to be identified. The present study was undertaken to characterize the protein repertoire recognized by antimitochondrial antibodies (AMAs) in patients with SLE. METHODS: Using shotgun proteomic profiling, we identified 1,345 proteins, 431 of which were associated with the mitochondrial proteome. Immunoreactivities to several of these candidate proteins were assessed in serum samples from a local cohort (n = 30 healthy donors and 87 patients with SLE) using enzyme-linked immunosorbent assay, and further analyzed for associations with demographic and disease characteristics. RESULTS: We determined that IgG antibodies to the complement component C1q binding protein were significantly elevated in the patients with SLE (P = 0.049) and were also associated with lupus anticoagulant positivity (P = 0.049). Elevated levels of IgG antibodies against mitochondrial protein mitofusin 1 (MFN-1) were promising predictors of SLE diagnosis in our cohort (adjusted odds ratio 2.99 [95% confidence interval 1.39-6.43], P = 0.0044). Moreover, increased levels of anti-MFN-1 were associated with the presence of antiphospholipids (P = 0.011) and anti-double-stranded DNA (P = 0.0005). CONCLUSION: In this study, we characterized the mitochondrial repertoire targeted by AMAs in the setting of SLE. Our results indicate that autoantibodies can recognize secreted and/or surface proteins of mitochondrial origin. Profiling of the AMA repertoire in large prospective cohorts may improve our knowledge of mitochondrial biomarkers and their usefulness for patient stratification.

The interaction of secreted phospholipase A2-IIA with the microbiota alters its lipidome and promotes inflammation.

Secreted phospholipase A2-IIA (sPLA2-IIA) hydrolyzes phospholipids to liberate lysophospholipids and fatty acids. Given its poor activity toward eukaryotic cell membranes, its role in the generation of proinflammatory lipid mediators is unclear. Conversely, sPLA2-IIA efficiently hydrolyzes bacterial membranes. Here, we show that sPLA2-IIA affects the immune system by acting on the intestinal microbial flora. Using mice overexpressing transgene-driven human sPLA2-IIA, we found that the intestinal microbiota was critical for both induction of an immune phenotype and promotion of inflammatory arthritis. The expression of sPLA2-IIA led to alterations of the intestinal microbiota composition, but housing in a more stringent pathogen-free facility revealed that its expression could affect the immune system in the absence of changes to the composition of this flora. In contrast, untargeted lipidomic analysis focusing on bacteria-derived lipid mediators revealed that sPLA2-IIA could profoundly alter the fecal lipidome. The data suggest that a singular protein, sPLA2-IIA, produces systemic effects on the immune system through its activity on the microbiota and its lipidome.

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.

Platelets release mitochondrial antigens in systemic lupus erythematosus.

The accumulation of DNA and nuclear components in blood and their recognition by autoantibodies play a central role in the pathophysiology of systemic lupus erythematosus (SLE). Despite the efforts, the sources of circulating autoantigens in SLE are still unclear. Here, we show that in SLE, platelets release mitochondrial DNA, the majority of which is associated with the extracellular mitochondrial organelle. Mitochondrial release in patients with SLE correlates with platelet degranulation. This process requires the stimulation of platelet FcγRIIA, a receptor for immune complexes. Because mice lack FcγRIIA and murine platelets are completely devoid of receptor capable of binding IgG-containing immune complexes, we used transgenic mice expressing FcγRIIA for our in vivo investigations. FcγRIIA expression in lupus-prone mice led to the recruitment of platelets in kidneys and to the release of mitochondria in vivo. Using a reporter mouse with red fluorescent protein targeted to the mitochondrion, we confirmed platelets as a source of extracellular mitochondria driven by FcγRIIA and its cosignaling by the fibrinogen receptor α2bß3 in vivo. These findings suggest that platelets might be a key source of mitochondrial antigens in SLE and might be a therapeutic target for treating SLE.

FcγRIIA expression accelerates nephritis and increases platelet activation in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease characterized by deposits of immune complexes (ICs) in organs and tissues. The expression of FcγRIIA by human platelets, which is their unique receptor for immunoglobulin G antibodies, positions them to ideally respond to circulating ICs. Whereas chronic platelet activation and thrombosis are well-recognized features of human SLE, the exact mechanisms underlying platelet activation in SLE remain unknown. Here, we evaluated the involvement of FcγRIIA in the course of SLE and platelet activation. In patients with SLE, levels of ICs are associated with platelet activation. Because FcγRIIA is absent in mice, and murine platelets do not respond to ICs in any existing mouse model of SLE, we introduced the FcγRIIA (FCGR2A) transgene into the NZB/NZWF1 mouse model of SLE. In mice, FcγRIIA expression by bone marrow cells severely aggravated lupus nephritis and accelerated death. Lupus onset initiated major changes to the platelet transcriptome, both in FcγRIIA-expressing and nonexpressing mice, but enrichment for type I interferon response gene changes was specifically observed in the FcγRIIA mice. Moreover, circulating platelets were degranulated and were found to interact with neutrophils in FcγRIIA-expressing lupus mice. FcγRIIA expression in lupus mice also led to thrombosis in lungs and kidneys. The model recapitulates hallmarks of human SLE and can be used to identify contributions of different cellular lineages in the manifestations of SLE. The study further reveals a role for FcγRIIA in nephritis and in platelet activation in SLE.

Platelets Disseminate Extracellular Vesicles in Lymph in Rheumatoid Arthritis.

OBJECTIVE: The lymphatic system is a circulatory system that unidirectionally drains the interstitial tissue fluid back to blood circulation. Although lymph is utilized by leukocytes for immune surveillance, it remains inaccessible to platelets and erythrocytes. Activated cells release submicron extracellular vesicles (EV) that transport molecules from the donor cell. In rheumatoid arthritis, EV accumulate in the joint where they can interact with numerous cellular lineages. However, whether EV can exit the inflamed tissue to recirculate is unknown. Here, we investigated whether vascular leakage that occurs during inflammation could favor EV access to the lymphatic system. Approach and Results: Using an in vivo model of autoimmune inflammatory arthritis, we show that there is an influx of platelet EV, but not EV from erythrocytes or leukocytes, in joint-draining lymph. In contrast to blood platelet EV, lymph platelet EV lacked mitochondrial organelles and failed to promote coagulation. Platelet EV influx in lymph was consistent with joint vascular leakage and implicated the fibrinogen receptor α2bß3 and platelet-derived serotonin. CONCLUSIONS: These findings show that platelets can disseminate their EV in fluid that is inaccessible to platelets and beyond the joint in this disease.

Respective contribution of cytosolic phospholipase A2α and secreted phospholipase A2 IIA to inflammation and eicosanoid production in arthritis.

Phospholipase A2s (PLA2) play a key role in generation of eicosanoids. Cytosolic PLA2α (cPLA2α) is constitutively expressed in most cells, whereas IIA secreted PLA2 (sPLA2-IIA) is induced during inflammation and is present at high levels in the synovial fluid of rheumatoid arthritis patients. In mice, both cPLA2α and sPLA2-IIA have been implicated in autoimmune arthritis; however, the respective contribution of these two enzymes to the pathogenesis and production of eicosanoids is unknown. We evaluated the respective role of cPLA2α and sPLA2-IIA with regard to arthritis and eicosanoid profile in an in vivo model of arthritis. While arthritis was most severe in mice expressing both enzymes, it was abolished when both cPLA2α and sPLA2-IIA were lacking. cPLA2α played a dominant role in the severity of arthritis, although sPLA2-IIA sufficed to significantly contribute to the disease. Several eicosanoids were modulated during the course of arthritis and numerous species involved sPLA2-IIA expression. This study confirms the critical role of PLA2s in arthritis and unveils the distinct contribution of cPLA2α and sPLA2-IIA to the eicosanoid profile in arthritis.

Anti-mitochondrial autoantibodies in systemic lupus erythematosus and their association with disease manifestations.

Mitochondria are organelles that govern energy supply and control cell death. Mitochondria also express bacterial features, such as the presence of inner membrane cardiolipin and a circular genome rich in hypomethylated CpG motifs. While mitochondrial extrusion by damaged organs or activated cells is thought to trigger innate immunity, it is unclear whether extracellular mitochondria also stimulate an adaptive immune response. We describe the development of novel assays to detect autoantibodies specific to two distinct components of the mitochondrion: the mitochondrial outer membrane and mitochondrial DNA. Antibodies to these two mitochondrial constituents were increased in both human and murine systemic lupus erythematosus (SLE), compared to controls, and were present at higher levels than in patients with antiphospholipid syndrome or primary biliary cirrhosis. In both bi- and multi-variate regression models, antibodies to mitochondrial DNA, but not whole mitochondria, were associated with increased anti-dsDNA antibodies and lupus nephritis. This study describes new and optimized methods for the assessment of anti-mitochondrial antibodies, and demonstrates their presence in both human and murine SLE. These findings suggest that different mitochondrial components are immunogenic in SLE, and support the concept that extracellular mitochondria may provide an important source of circulating autoantigens in SLE.

Platelets release pathogenic serotonin and return to circulation after immune complex-mediated sequestration.

There is a growing appreciation for the contribution of platelets to immunity; however, our knowledge mostly relies on platelet functions associated with vascular injury and the prevention of bleeding. Circulating immune complexes (ICs) contribute to both chronic and acute inflammation in a multitude of clinical conditions. Herein, we scrutinized platelet responses to systemic ICs in the absence of tissue and endothelial wall injury. Platelet activation by circulating ICs through a mechanism requiring expression of platelet Fcγ receptor IIA resulted in the induction of systemic shock. IC-driven shock was dependent on release of serotonin from platelet-dense granules secondary to platelet outside-in signaling by αIIbß3 and its ligand fibrinogen. While activated platelets sequestered in the lungs and leaky vasculature of the blood-brain barrier, platelets also sequestered in the absence of shock in mice lacking peripheral serotonin. Unexpectedly, platelets returned to the blood circulation with emptied granules and were thereby ineffective at promoting subsequent systemic shock, although they still underwent sequestration. We propose that in response to circulating ICs, platelets are a crucial mediator of the inflammatory response highly relevant to sepsis, viremia, and anaphylaxis. In addition, platelets recirculate after degranulation and sequestration, demonstrating that in adaptive immunity implicating antibody responses, activated platelets are longer lived than anticipated and may explain platelet count fluctuations in IC-driven diseases.

Microparticle and mitochondrial release during extended storage of different types of platelet concentrates.

On activation, platelets release vesicles called microparticles (MPs). MPs are heterogeneous with regard to the presence or absence of mitochondria. We quantified MPs in platelet concentrates (PCs) taking their mitochondrial content into account. Platelet-rich plasma (PRP), buffy coat (BC) and apheresis (AP) PCs were tested through 7 days of storage. A combination of flow cytometry and spanning-tree progression analysis of density-normalized events (SPADE) was used to determine MP and mitochondrial release during storage. All the PC biochemical parameters complied with transfusion standards at all times. Platelet activation markers increased during storage and were higher for PRP than other types of PCs. Concentrations of MPs and extracellular mitochondria interpreted by SPADE algorithm were significantly higher in PRP than other in PCs and were stable throughout storage. The mode of preparation, rather than storage duration, impacts the release of MPs and mitochondria in PCs.

The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection.

Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.

Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA.

Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

Detection and quantification of microparticles from different cellular lineages using flow cytometry. Evaluation of the impact of secreted phospholipase A2 on microparticle assessment.

Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.

Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation.

Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside the cells in blood in various pathologies. Platelets are abundant in blood where they promote hemostasis. Although lacking a nucleus, platelets contain functional mitochondria. On activation, platelets produce extracellular vesicles known as microparticles. We hypothesized that activated platelets could also release their mitochondria. We show that activated platelets release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. Extracellular mitochondria are found in platelet concentrates used for transfusion and are present at higher levels in those that induced acute reactions (febrile nonhemolytic reactions, skin manifestations, and cardiovascular events) in transfused patients. We establish that the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), a phospholipase otherwise specific for bacteria, likely reflecting the ancestral proteobacteria origin of mitochondria. The hydrolysis of the mitochondrial membrane by sPLA2-IIA yields inflammatory mediators (ie, lysophospholipids, fatty acids, and mtDNA) that promote leukocyte activation. Two-photon microscopy in live transfused animals revealed that extracellular mitochondria interact with neutrophils in vivo, triggering neutrophil adhesion to the endothelial wall. Our findings identify extracellular mitochondria, produced by platelets, at the midpoint of a potent mechanism leading to inflammatory responses.

Influenza virus H1N1 activates platelets through FcγRIIA signaling and thrombin generation.

Platelets play crucial functions in hemostasis and the prevention of bleeding. During H1N1 influenza A virus infection, platelets display activation markers. The platelet activation triggers during H1N1 infection remain elusive. We observed that H1N1 induces surface receptor activation, lipid mediator synthesis, and release of microparticles from platelets. These activation processes require the presence of serum/plasma, pointing to the contribution of soluble factor(s). Considering that immune complexes in the H1N1 pandemic were reported to play a pathogenic role, we assessed their contribution in H1N1-induced platelet activation. In influenza-immunized subjects, we observed that the virus scaffolds with immunoglobulin G (IgG) to form immune complexes that promote platelet activation. Mechanistically, this activation occurs through stimulation of low-affinity type 2 receptor for Fc portion of IgG (FcγRIIA), a receptor for immune complexes, independently of thrombin. Using a combination of in vitro and in vivo approaches, we found that the antibodies from H3N2-immunized mice activate transgenic mouse platelets that express FcγRIIA when put in the presence of H1N1, suggesting that cross-reacting influenza antibodies suffice. Alternatively, H1N1 can activate platelets via thrombin formation, independently of complement and FcγRIIA. These observations identify both the adaptive immune response and the innate response against pathogens as 2 intertwined processes that activate platelets during influenza infections.

Thyroid hormone status regulates the expression of secretory phospholipases.

Thyroid hormone (T3) stimulates various metabolic pathways and the hepatic actions of T3 are mediated primarily through the thyroid hormone receptor beta (TRß). Hypothyroidism has been linked with low grade inflammation, elevated risk of hepatic steatosis and atherosclerosis. Secretory phospholipases (sPLA2) are associated with inflammation, hyperlipidemia and atherosclerosis. Due to potential linkage between thyroid hormone and sPLA2, we investigated the effect of thyroid hormone status on the regulation of secretory phospholipases in mice, rats and human liver. T3 suppressed the expression of the sPLA2 group IIa (PLA2g2a) gene in the liver of BALB/c mice and C57BL/6 transgenic mice expressing the human PLA2g2a. PLA2g2a was elevated with hypothyroidism and high fat diets which may contribute to the low grade inflammation associated with hypothyroidism and diet induced obesity. We also examined the effects of the TRß agonist eprotirome on hepatic gene regulation. We observed that eprotirome inhibited the expression of selected sPLA2 genes and furthermore the cytokine mediated induction PLA2g2a was suppressed. In addition, eprotirome induced genes involved in fatty acid oxidation and cholesterol clearance while inhibiting lipogenic genes. Our results indicate that in vivo thyroid hormone status regulates the abundance of sPLA2 and the inhibition of PLA2g2a by T3 is conserved across species. By regulating sPLA2 genes, T3 may impact processes associated with atherosclerosis and inflammation and TRß agonists may ameliorate inflammation and hyperlipidemia.

Cooperative role of endogenous leucotrienes and platelet-activating factor in ischaemia-reperfusion-mediated tissue injury.

Insufficient oxygen delivery to organs leads to tissue dysfunction and cell death. Reperfusion, although vital to organ survival, initiates an inflammatory response that may both aggravate local tissue injury and elicit remote organ damage. Polymorphonuclear neutrophil (PMN) trafficking to remote organs following ischaemia/reperfusion (I/R) is associated with the release of lipid mediators, including leucotriene (LT) B4 , cysteinyl-LTs (CysLTs) and platelet-activating factor (PAF). Yet, their potentially cooperative role in regulating I/R-mediated inflammation has not been thoroughly assessed. The present study aimed to determine the cooperative role of lipid mediators in regulating PMN migration, tissue oedema and injury using selective receptor antagonists in selected models of I/R and dermal inflammation. Our results show that rabbits, pre-treated orally with BIIL 284 and/or WEB 2086 and MK-0571, were protected from remote tissue injury following I/R or dermal inflammation in an additive or synergistic manner when the animals were pre-treated with two drugs concomitantly. The functional selectivity of the antagonists towards their respective agonists was assessed in vitro, showing that neither BIIL 284 nor WEB 2086 prevented the inflammatory response to IL-8, C5a and zymosan-activated plasma stimulation. However, these agonists elicited LTB4 biosynthesis in isolated rabbit PMNs. Similarly, a cardioprotective effect of PAF and LTB4 receptor antagonists was shown following myocardial I/R in mice. Taken together, these results underscore the intricate involvement of LTB4 and PAF in each other's responses and provide further evidence that targeting both LTs and PAF receptors provides a much stronger anti-inflammatory effect, regulating PMN migration and oedema formation.

Extra domain A of fibronectin primes leukotriene biosynthesis and stimulates neutrophil migration through activation of Toll-like receptor 4.

OBJECTIVE: There is increasing evidence of a role for Toll-like receptors (TLRs) in inflammatory arthritis. The extra domain A (ED-A)-containing isoform of fibronectin is generated under pathologic conditions such as rheumatoid arthritis, and ED-A has been identified as an endogenous TLR-4 ligand. Leukotriene B4 (LTB4) and polymorphonuclear neutrophils (PMNs) play a critical role in murine models of inflammatory arthritis. The aim of this study was therefore to investigate the putative effects of ED-A on leukotriene biosynthesis and PMN migration through TLR signaling. METHODS: The effect of recombinant human ED-A (rhED-A) on leukotriene biosynthesis was evaluated in isolated human blood PMNs and monocytes by high-performance liquid chromatography. The capacity of rhED-A to stimulate PMN migration was evaluated using a transendothelial/matrix migration assay in vitro and the mouse air-pouch model in vivo. RESULTS: Recombinant human ED-A efficiently primed the biosynthesis of LTB4 in PMN and monocyte suspensions. This priming effect was dependent on TLR-4 activation, since the TLR-4-signaling inhibitor CLI-095 completely blocked the effect of rhED-A but not that of other TLR ligands (R-848, Pam2 CSK4) or cytokines. Moreover, rhED-A stimulated transendothelial migration of PMNs in vitro, which was inhibited by 50-60% with the LTB4 receptor 1 (BLT1) antagonist CP105,696 or the cytosolic phospholipase A2 α inhibitor pyrrophenone. In vivo, rhED-A induced a significant PMN recruitment into the air pouch of C3H/HeOuJ mice (expressing functional TLR-4), but not in C3H/HeJ mice (expressing nonsignaling TLR-4). CONCLUSION: These results demonstrate the ability of rhED-A to promote LTB4 biosynthesis and PMN migration through TLR-4 activation, thus providing new insights on TLR-dependent mechanisms of regulation of LTB4 biosynthesis and PMN infiltration in inflammatory joint diseases.