The GPR40 Agonist GW9508 Enhances Neutrophil Function to Aid Bacterial Clearance During E. coli Infections.

G-protein-coupled receptor 40 (GPR40) is known to play a role in the regulation of fatty acids, insulin secretion, and inflammation. However, the function of this receptor in human neutrophils, one of the first leukocytes to arrive at the site of infection, remains to be fully elucidated. In the present study, we demonstrate that GPR40 is upregulated on activated human neutrophils and investigated the functional effects upon treatment with a selective agonist; GW9508. Interestingly, GPR40 expression was up-regulated after neutrophil stimulation with platelet-activating factor (10 nM) or leukotriene B4 (LTB4, 10 nM) suggesting potential regulatory roles for this receptor during inflammation. Indeed, GW9508 (1 and 10 µM) increased neutrophil chemotaxis in response to the chemokine IL-8 (30 ng/ml) and enhanced phagocytosis of Escherichia coli by approximately 50% when tested at 0.1 and 1 µM. These results were translated in vivo whereby administration of GW9508 (10 mg/kg, i.p.) during E. coli infections resulted in elevated peritoneal leukocyte infiltration with a higher phagocytic capacity. Importantly, GW9508 administration also modulated the lipid mediator profile, with increased levels of the pro-resolving mediators resolvin D3 and lipoxins. In conclusion, GPR40 is expressed by activated neutrophils and plays an important host protective role to aid clearance of bacterial infections.

Comparative analysis of Annexin A1-formyl peptide receptor 2/ALX expression in human leukocyte subsets.

Recent studies have associated the dysregulated expression of Annexin-A1/Formyl peptide receptor 2 (FPR2/ALX) system with the development of autoimmune diseases. In this study we systematically scanned human leukocyte subsets for the presence of this pathway aiming to provide a roadmap that will help investigators to explore possible links between the development of immune related disorders and the expression of this system. Our results show that neutrophils, monocytes and NK cells express higher levels of both AnxA1 and FPR2/ALX compared to T or B cells. Further analysis of specific T cell subsets revealed higher levels in activated CD25(+) and memory CD45RO CD4 T cells compared to resting CD25(-) or naïve CD45RA CD4 T cells. Together the results expand our knowledge of the AnxA1-FPR2/ALX system in immune cells and provide new avenues for investigation into the functions of this signalling pathway in systems other than that classically described for neutrophils.

Rheumatoid peripheral blood phagocytes are primed for activation but have impaired Fc-mediated generation of reactive oxygen species.

Significant levels of circulating immune complexes (ICs) containing rheumatoid factors and immunoglobulin G in peripheral blood are a characteristic feature of rheumatoid arthritis (RA). ICs interact through Fc gamma receptors (Fc gammaR) to activate phagocytes in numerous inflammatory processes. The high concentration of neutrophils in synovial fluid during active phases of the disease, together with their destructive capacity, pose important questions as to their role in the pathogenesis of RA. Functional defects in RA or control peripheral blood neutrophil Fc gammaRs were examined with a specific Fc gammaR-mediated reactive oxygen species (ROS) assay. Heterologous cross-linking of Fc gammaRIIa and Fc gammaRIIIb on neutrophils resulted in a significantly decreased production of ROS by RA cells compared with controls matched for age and sex. However, expression and homologous ligation of receptors did not differ between these groups. These data suggest that neutrophil priming does occur before emigration into the joint and that blood neutrophils from patients with RA have a functional impairment in cooperative Fc gammaR-mediated ROS generation. This may account for the increased susceptibility to bacterial infection that arises in patients with severe disease.

Ligand-specific glucocorticoid receptor activation in human platelets.

Few studies have addressed the effects of classical anti-inflammatory glucocorticoids on platelet function. Here, we report for the first time that human platelets contain the glucocorticoid receptor (GR) as identified by a combination of biochemical and functional techniques. Ligand-binding studies revealed the presence of a high- and low-affinity binding site for [3H]-dexamethasone in platelets. The 2 GR ligands prednisolone and dexamethasone competed for [3H]-dexamethasone binding, as did the mineralocorticoid aldosterone. However, while prednisolone (1-10 microM) reduced adenosine diphosphate (ADP, 4 microM) and thromboxane A2 receptor agonist U46619 induced platelet aggregation (up to 75%), dexamethasone had no effect. The inhibition produced by prednisolone was reversed by preincubation with the GR antagonist mifepristone (10 microM; RU486), suggesting the functional importance of the ligand-receptor complex. In addition, prednisolone caused a marked (approximately 50%) reduction in thromboxane B2 levels, whereas dexamethasone was without effect. The apparently anomalous binding data were clarified by the fact that washed platelets (1) contained mineralocorticoid receptor and that (2) it was associated with GR. Taken together, our data suggest that platelet GR forms a heterodimeric complex with the mineralocorticoid receptor that is susceptible to differential activation by specific receptor ligands.

The molecular complexity of glucocorticoid actions in inflammation - a four-ring circus.

Glucocorticoids have been the mainstay of anti-inflammatory therapy for the past 50 years despite the significant risks associated with their long-term use. Recent research into the molecular mechanisms of action of these compounds can be aggregated into two major themes: first, those that focus on the involvement of the glucocorticoid receptor in regulating the transcription of pro-inflammatory genes; and second, studies that seek to identify alternative pathways of glucocorticoid action either via the induction of anti-inflammatory mediators or through rapid non-genomic mechanisms. Understanding the temporal-spatial patterns of these multiple mechanisms on relevant cell types will be key to explaining the profound effects that these four-ring steroids exert on the inflammatory process.

Regulation and localization of endogenous human tristetraprolin.

Tumor necrosis factor (TNF) has been implicated in the development and pathogenicity of infectious diseases and autoimmune disorders, such as septic shock and arthritis. The zinc-finger protein tristetraprolin (TTP) has been identified as a major regulator of TNF biosynthesis. To define its intracellular location and examine its regulation of TNF, a quantitive intracellular staining assay specific for TTP was developed. We establish for the first time that in peripheral blood leukocytes, expression of endogenous TTP is confined to the cytoplasm. Baseline expression of TTP was higher in monocytes than in lymphocytes or neutrophils. After in vitro incubation with lipopolysaccharide (LPS), leukocyte TTP levels increased rapidly, peaking after approximately 2 hours. Monocytes showed the greatest response to LPS stimulation and lymphocytes the least. TTP levels were also studied in leukocytes isolated from healthy volunteers infused with a bolus dose of LPS. TTP expression and initial upregulation in response to LPS infusion were consistent with the in vitro data. Neutrophil TTP levels responded first, reaching an initial peak within 1 hour, monocyte levels peaked next at 2 hours, followed by lymphocytes at 4 hours. This response paralleled plasma TNF levels, which peaked 2 hours after infusion and were no longer detectable after 12 hours. A second rise in intracellular TTP levels, which did not parallel plasma TNF levels, was observed in all leukocyte populations, starting 12 hours after infusion. These data establish the cytoplasmic location of TTP, supporting a major role for this protein in regulating TNF production, and suggest that TTP levels are not regulated solely by TNF.