Impact of animal strain on gene expression in a rat model of acute cardiac rejection

<p>Abstract</p> <p>Background</p> <p>The expression levels of many genes show wide natural variation among strains or populations. This study investigated the potential for animal strain-related genotypic differences to confound gene expression profiles in acute cellular rejection (ACR). Using a rat heart transplant model and 2 different rat strains (Dark Agouti, and Brown Norway), microarrays were performed on native hearts, transplanted hearts, and peripheral blood mononuclear cells (PBMC).</p> <p>Results</p> <p>In heart tissue, strain alone affected the expression of only 33 probesets while rejection affected the expression of 1368 probesets (FDR 10% and FC ≥ 3). Only 13 genes were affected by both strain and rejection, which was < 1% (13/1368) of all probesets differentially expressed in ACR. However, for PBMC, strain alone affected 265 probesets (FDR 10% and FC ≥ 3) and the addition of ACR had little further effect. Pathway analysis of these differentially expressed strain effect genes connected them with immune response, cell motility and cell death, functional themes that overlap with those related to ACR. After accounting for animal strain, additional analysis identified 30 PBMC candidate genes potentially associated with ACR.</p> <p>Conclusion</p> <p>In ACR, genetic background has a large impact on the transcriptome of immune cells, but not heart tissue. Gene expression studies of ACR should avoid study designs that require cross strain comparisons between leukocytes.</p

Concentration-Dependent Noncysteinyl Leukotriene Type 1 Receptor-Mediated Inhibitory Activity of Leukotriene Receptor Antagonists

BACKGROUND: The use of leukotriene antagonists (LTRAs) for asthma therapy has been associated with a significant degree of inter-patient variability in response to treatment. Some of that variability may be attributable to non-cysteinyl leukotriene type 1 receptor (CysLT(1)) mediated inhibitory mechanisms that have been demonstrated for this group of drugs. OBJECTIVE: We have used a model of CysLT(1) signaling in human monocytes to characterize CysLT(1)-dependent and CysLT(1)-independent anti-inflammatory activity of two chemically different, clinically relevant, LTRAs (montelukast and zafirlukast). RESULTS: Using receptor desensitization experiments in monocytes and CysLT(1) transfected HEK293 cells, and IL-10 and CysLT(1) siRNA induced downregulation of CysLT(1) expression, we showed that reported CysLT(1) agonists, LTD(4) and uridine diphosphate (UDP), signal through calcium mobilization, acting on separate receptors and that both pathways were inhibited by montelukast and zafirlukast. However, 3 logs higher concentrations of LTRAs were required for inhibition of UDP induced signaling. In monocytes, UDP, but not LTD(4), induced IL-8 production that was significantly inhibited by both drugs at micromolar concentrations. Both LTRAs, at low micromolar concentrations, also inhibited calcium ionophore induced leukotriene (LTB(4) and LTC(4)) production, indicating 5-lipoxygenase inhibitory activities. CONCLUSION: We report here that montelukast and zafirlukast, acting in a concentration dependent manner, can inhibit non-CysLT(1) mediated, proinflammatory reactions, suggesting activities potentially relevant for inter-patient variability in response to treatment. Higher doses of currently known LTRAs or new compounds derived from this class of drugs may represent a new strategy for finding more efficient therapy for bronchial asthma

IFN-γ Induces Cysteinyl Leukotriene Receptor 2 Expression and Enhances the Responsiveness of Human Endothelial Cells to Cysteinyl Leukotrienes

Cysteinyl leukotrienes (cysLTs) are important mediators of cell trafficking and innate immune responses, involved in the pathogenesis of inflammatory processes, i.e., atherosclerosis, pulmonary fibrosis, and bronchial asthma. The aim of this study was to examine the regulation of cysLT signaling by IFN-gamma in human primary endothelial cells. IFN-gamma increased cysLT receptor 2 (CysLTR2) mRNA expression and CysLTR2-specific calcium signaling in endothelial cells. IFN-gamma signaled through Jak/STAT1, as both AG490, a Jak2 inhibitor, and expression of a STAT1 dominant-negative construct, significantly inhibited CysLTR2 mRNA expression in response to IFN-gamma. To determine mechanisms of IFN-gamma-induced CysLTR2 expression, the human CysLTR2 gene structure was characterized. The CysLTR2 gene has a TATA-less promoter, with multiple transcription start sites. It consists of six variably spliced exons. Eight different CysLTR2 transcripts were identified in endothelial and monocytic cells. Gene reporter assay showed potent basal promoter activity of a putative CysLTR2 promoter region. However, there were no significant changes in gene reporter and mRNA t(1/2) assays in response to IFN-gamma, suggesting transcriptional control of CysLTR2 mRNA up-regulation by IFN-gamma response motifs localized outside of the cloned CysLTR2 promoter region. Stimulation of endothelial cells by cysLTs induced mRNA and protein expression of early growth response genes 1, 2, and 3 and cycloxygenase-2. This response was mediated by CysLTR2 coupled to G(q/11), activation of phospholipase C, and inositol-1,4,5-triphosphate, and was enhanced further 2- to 5-fold by IFN-gamma stimulation. Thus, IFN-gamma induces CysLTR2 expression and enhances cysLT-induced inflammatory responses.</p