A cDNA microarray assessment of gene expression in the liver of rainbow trout (Oncorhynchus mykiss) in response to a handling and confinement stressor.

A purpose-designed microarray platform (Stressgenes, Phase 1) was utilised to investigate the changes in gene expression within the liver of rainbow trout during exposure to a prolonged period of confinement. Tissue and blood samples were collected from trout at intervals up to 648 h after transfer to a standardised confinement stressor, together with matched samples from undisturbed control fish. Plasma ACTH, cortisol, glucose and lactate were analysed to confirm that the neuroendocrine response to confinement was consistent with previous findings and to provide a phenotypic context to assist interpretation of gene expression data. Liver samples for suppression subtractive hybridisation (SSH) library construction were selected from within the experimental groups comprising "early" stress (2-48 h) and "late" stress (96-504 h). In order to reduce redundancy within the four SSH libraries and yield a higher number of unique clones an additional subtraction was carried out. After printing of the arrays a series of 55 hybridisations were executed to cover 6 time points. At 2 h, 6 h, 24 h, 168 h and 504 h 5 individual confined fish and 5 individual control fish were used with control fish only at 0 h. A preliminary list of 314 clones considered differentially regulated over the complete time course was generated by a combination of data analysis approaches and the most significant gene expression changes were found to occur during the 24 h to 168 h time period with a general approach to control levels by 504 h. Few changes in expression were apparent over the first 6 h. The list of genes whose expression was significantly altered comprised predominantly genes belonging to the biological process category (response to stimulus) and one cellular component category (extracellular region) and were dominated by so-called acute phase proteins. Analysis of the gene expression profile in liver tissue during confinement revealed a number of significant clusters. The major patterns comprised genes that were up-regulated at 24 h and beyond, the primary examples being haptoglobin, beta-fibrinogen and EST10729. Two representative genes from each of the six k-means clusters were validated by qPCR. Correlations between microarray and qPCR expression patterns were significant for most of the genes tested. qPCR analysis revealed that haptoglobin expression was up-regulated approximately 8-fold at 24 h and over 13-fold by 168 h.

Regulation of expression of granulocyte-macrophage colony-stimulating factor in human bronchial epithelial cells: roles of protein kinase C and mitogen-activated protein kinases.

GM-CSF has a major role in the immune and inflammatory milieu of the airway. Airway epithelial cells (AEC) are among the first targets of environmental stimuli and local cytokines, in response to which they can produce GM-CSF. The regulation of GM-CSF is only minimally understood in AEC. We hypothesized that GM-CSF expression in AEC would result from activation of protein kinase C (PKC) and subsequent activation of the extracellular signal-regulated kinase (MAPKerk1/2) pathway, so we investigated signal transduction pathways in human primary culture bronchial epithelial cells (HBECs). TNF-alpha, IL-1beta, and PMA induced the release of GM-CSF in HBECs. The robust response to PMA was not detected in SV40 adenovirus-transformed normal human bronchial epithelial cells (BEAS-2B). PMA and TNF-alpha stimulation of GM-CSF required activation of PKC (inhibition by staurosporine and bisindolylmaleimide I). GM-CSF expression was up-regulated by a nonphorbol PKC activator, but not by an inactive PMA analogue. PMA-induced GM-CSF production in HBECs did not require a Ca2+ ionophore and was not inhibited by cyclosporin A. Activation of MAPKerk1/2 via PKC was associated with and was required for GM-CSF production induced by PMA and TNF-alpha. The data demonstrate regulation of GM-CSF in HBECs by PKC pathways converging on the MAPKerk1/2 pathway and further define cell-specific regulation critical for local airway responses.

Early inhibition of mycobacterial growth by human alveolar macrophages is not due to nitric oxide.

Phagocytic cells provide the first line of defense against mycobacteria. We examined the relative mycobacteriostatic contributions of normal human alveolar macrophages (HAM), peripheral blood monocytes (PBM), and polymorphonuclear leukocytes (PMN) in the early time period after infection with mycobacteria (48 h). Cells were infected with Mycobacterium bovis (BCG) or M. tuberculosis H37Ra and their ability to inhibit growth was determined by mycobacterial incorporation of [3H]uracil. HAM inhibited the growth of both mycobacteria (44.2 +/- 7.9 and 37.6 +/- 10.5% inhibition, respectively). Two populations of HAM donors were subsequently defined: inhibitors and noninhibitors. The ability to inhibit growth of H37Ra correlated with that of BCG. In contrast to HAM, PBM and PMN did not inhibit mycobacterial growth. Because nitric oxide (NO) has been proposed to mediate growth inhibition in murine models, we examined whether NO was responsible for the early growth inhibition of mycobacteria by HAM. As expected, in murine peritoneal macrophages (MPM) IFN-gamma (2,500 U/ml) enhanced growth inhibition of BCG; the effect was abolished by the nitric oxide synthase (NOS) inhibitor NMMA. In contrast, IFN-gamma failed to enhance growth inhibition by HAM or PBM and NMMA had no effect. MPM expressed inducible nitric oxide synthase (NOS2) mRNA in response to LPS and IFN-gamma and produced NO. Neither NOS2 mRNA nor NO could be detected in HAM stimulated with LPS and IFN-gamma or mycobacteria. These data demonstrate that HAM, but not PBM or PMN, have NO-independent mycobacteriostatic activity in the early time period after infection with mycobacteria.