The Interleukin-1 receptor antagonist is a direct target gene of PPARalpha in liver.

BACKGROUND/AIMS: The Peroxisome Proliferator-Activated Receptor (PPAR) alpha belongs to the superfamily of Nuclear Receptors and plays an important role in numerous cellular processes, including lipid metabolism. It is known that PPARalpha also has an anti-inflammatory effect, which is mainly achieved by down-regulating pro-inflammatory genes. The objective of this study was to further characterize the role of PPARalpha in inflammatory gene regulation in liver. RESULTS: According to Affymetrix micro-array analysis, the expression of various inflammatory genes in liver was decreased by treatment of mice with the synthetic PPARalpha agonist Wy14643 in a PPARalpha-dependent manner. In contrast, expression of Interleukin-1 receptor antagonist (IL-1ra), which was acutely stimulated by LPS treatment, was induced by PPARalpha. Up-regulation of IL-1ra by LPS was lower in PPARalpha -/- mice compared to Wt mice. Transactivation and chromatin immunoprecipitation studies identified IL-1ra as a direct positive target gene of PPARalpha with a functional PPRE present in the promoter. Up-regulation of IL-1ra by PPARalpha was conserved in human HepG2 hepatoma cells and the human monocyte/macrophage THP-1 cell line. CONCLUSIONS: In addition to down-regulating expression of pro-inflammatory genes, PPARalpha suppresses the inflammatory response by direct up-regulation of genes with anti-inflammatory properties.

Stress- (and diet-) related regulation of hepatic nuclear receptors and its relevance for ABC-transporter functions.

Nuclear receptors (NRs) play an important role in maintaining cellular homeostasis. With clearly established roles in fatty acid metabolism and inflammation, peroxisome proliferator activated receptors (PPARs) and other nuclear receptors are essential in liver functioning. However, much less is known about the regulation of NRs themselves during inflammatory processes in the liver. Interestingly PPARs and other NRs are negative acute phase proteins because they become rapidly downregulated during the acute phase response. However, PPARs have important roles in modulating inflammatory responses. One of the mechanisms by which dietary or inflammatory stress is relieved involves the hepatic adenosine triphosphate-binding cassette (ABC) transporter proteins, which import and export a wide variety of substrates. These ABC transporters are under close control of several NRs. Because NRs play important roles in fatty acid metabolism and inflammation as well as in the regulation of bile production, they are reviewed here with respect to their role in dietary and stress-related responses of the liver and their impact on the regulation and function of hepatic ABC transporters.

Differential expression of somatostatin receptor subtypes in human peripheral blood mononuclear cell subsets.

BACKGROUND: Somatostatin (SS)-binding sites have been demonstrated in human lymphoid tissues and peripheral blood cells. However, not much is known with respect to the SS receptor subtype (sst) expression pattern and the expression of SS itself in the immune system. OBJECTIVE: The aim of this study was to evaluate the mRNA expression of the five known sst (sst(1-5)) in peripheral blood mononuclear cell (sub)populations. Moreover, the expression of the mRNAs encoding SS and the SS-like peptide cortistatin (CST) in immune cell subsets was studied. METHODS: RT-PCR and quantitative PCR were performed to evaluate sst, SS and CST mRNA expression in cells in the basal or activated state. Fluorescence-activated cell sorter (FACS) analysis using fluorescent SS was performed to visualize sst protein on cell membranes. RESULTS: B- and T-lymphocytes selectively expressed sst(3) mRNA. sst(3) expression in B-lymphocytes was significantly lower compared with T-lymphocytes. Unstimulated, freshly isolated monocytes did not express any sst mRNA. Upon activation, monocytes selectively expressed sst(2) mRNA, whereas T-lymphocyte activation upregulated sst(3) expression. sst(2) mRNA expression on monocytes was confirmed by FACS analysis. B- and T-lymphocytes did not express SS mRNA, while both cell types expressed CST mRNA. CST mRNA expression was downregulated following T-lymphocyte activation. CONCLUSION: We demonstrate for the first time unequivocally that human peripheral blood B- and T-lymphocytes selectively express sst(3), whereas monocytes do not express sst. However, upon activation, monocytes are induced to express sst(2A). No expression of SS mRNA was detected in any cell type, whereas all cell types expressed CST mRNA. The differential expression of sst and CST mRNA in lymphocytes and monocytes suggests a functional significance for the CST-sst interaction in immune cells, but further studies should be performed to evaluate the significance of sst and CST in these cells.

Genetic variations in the interleukin-12/interleukin-23 receptor (beta1) chain, and implications for IL-12 and IL-23 receptor structure and function.

Cell-mediated immunity (CMI) plays an essential role in human host defense against intracellular bacteria. Type-1 cytokines, particularly gamma interferon (IFN-gamma), interleukin-12 (IL-12), and IL-23, the major cytokines that regulate IFN-gamma production, are essential in CMI. This is illustrated by patients with unusual severe infections caused by poorly pathogenic mycobacteria and Salmonella species, in whom genetic deficiencies have been identified in several key genes in the type-1 cytokine pathway, including IL12RB1, the gene encoding the beta1 chain of the IL-12 and IL-23 receptors. Several mutations in IL12RB1 with deleterious effects on human IL-12R function have been identified, including nonsense and missense mutations. In addition, a number of coding IL12RB1 polymorphisms have been reported. In order to gain more insight into the effect that IL12RB1 mutations and genetic variations can have on IL-12Rbeta1 function, three approaches have been followed. First, we determined the degree of conservation at the variant amino acid positions in IL-12Rbeta1 between different species, using known deleterious mutations, known variations in IL-12Rbeta1, as well as novel coding variations that we have identified at position S74R and R156H. Second, we analyzed the potential impact of these amino acid variations on the three-dimensional structure of the IL-12Rbeta1 protein. Third, we analyzed the putative functions of different IL-12Rbeta1 domains, partly based on their homology with gp130, and analyzed the possible effects of the above amino acid variations on the function of these domains. Based on these analyses, we propose an integrated model of IL-12Rbeta1 structure and function. This significantly enhances our molecular understanding of the human IL-12 and IL-23 systems.

Severe Mycobacterium bovis BCG infections in a large series of novel IL-12 receptor beta1 deficient patients and evidence for the existence of partial IL-12 receptor beta1 deficiency.

Cell mediated immunity plays a critical role in human host defence against intracellular bacteria. In patients with unusual, severe infections caused by poorly pathogenic species of mycobacteria and salmonellae, genetic deficiencies have been identified in key genes in the type-1 cytokine pathway, especially in IFNGR1 and IL12RB1. Here, we analyzed 11 patients originating from Turkey and suffering from unusual Mycobacterium bovis Bacille Calmette-Guerin infections following vaccination, and found that most patients (n=8) are deficient in IL-12Rbeta1 expression and function. No defects were found in patients' IFN-gammaR or IL-18R. In addition, a first patient suffering from partial IL-12Rbeta1 deficiency is described. This patient presented with an intermediate cellular and immunological phenotype: a consistent, low response to IL-12 was found, which could be further augmented by IL-18. Despite a lack of cell surface IL-12Rbeta1 expression, normal levels of intracellular IL-12Rbeta1 protein were detectable, which was not seen in the other, completely IL-12Rbeta1 deficient patients examined. Moreover, this patient had a relatively mild clinical phenotype and was the only individual with a single homozygous amino acid substitution in IL-12Rbeta1 (C198R). Collectively, our findings indicate that idiopathic, unusually severe infections due to M. bovis BCG can be caused by complete as well as partial IL-12Rbeta1 deficiency.

Quantitative and functional expression of somatostatin receptor subtypes in human thymocytes.

We recently demonstrated the expression of somatostatin (SS) and SS receptor (SSR) subtype 1 (sst1), sst2A, and sst3 in normal human thymic tissue and of sst1 and sst2A on isolated thymic epithelial cells (TEC). We also found an inhibitory effect of SS and octreotide on TEC proliferation. In the present study, we further investigated the presence and function of SSR in freshly purified human thymocytes at various stages of development. Thymocytes represent a heterogeneous population of lymphoid cells displaying different levels of maturation and characterized by specific cell surface markers. In this study, we first demonstrated specific high-affinity 125I-Tyr(11)-labeled SS-14 binding on thymocyte membrane homogenates. Subsequently, by RT-PCR, sst2A and sst3 mRNA expression was detected in the whole thymocyte population. After separation of thymocytes into subpopulations, we found by quantitative RT-PCR that sst2A and sst3 are differentially expressed in intermediate/mature and immature thymocytes. The expression of sst3 mRNA was higher in the intermediate/mature CD3+ fraction compared with the immature CD2+CD3- one, whereas sst2A mRNA was less abundant in the intermediate/mature CD3+ thymocytes. In 7-day-cultured thymocytes, SSR subtype mRNA expression was lost. SS-14 significantly inhibited [3H]thymidine incorporation in all thymocyte cultures, indicating the presence of functional receptors. Conversely, octreotide significantly inhibited [3H]thymidine incorporation only in the cultures of immature CD2+CD3- thymocytes. Subtype sst3 is expressed mainly on the intermediate/mature thymocyte fraction, and most of these cells generally die by apoptosis. Because SS-14, but not octreotide, induced a significant increase in the percentage of apoptotic thymocytes, it might be that sst3 is involved in this process. Moreover, sst3 has recently been demonstrated on peripheral human T lymphocytes, which derive directly from mature thymocytes, and SS analogs may induce apoptosis in these cells. Interestingly, CD14+ thymic cells, which are cells belonging to the monocyte-macrophage lineage, selectively expressed sst2A mRNA. Finally, SSR expression in human thymocytes seems to follow a developmental pathway. The heterogeneous expression of SSR within the human thymus on specific cell subsets and the endogenous production of SS as well as SS-like peptides emphasize their role in the bidirectional interactions between the main cell components of the thymus involved in intrathymic T cell maturation.

Genetics, cytokines and human infectious disease: lessons from weakly pathogenic mycobacteria and salmonellae.

Host genetic factors are important in determining the outcome of infections caused by intracellular pathogens, including mycobacteria and salmonellae, but until now have been poorly characterized. Recently, some individuals with severe infections due to otherwise weakly pathogenic mycobacteria (non-tuberculous mycobacteria or Mycobacterium bovis bacille Calmette-Guérin) or Salmonella species have been shown to be unable to produce or respond to interferon-gamma. This inability results from mutations in any of five genes encoding essential proteins of the type 1 cytokine cascade: interleukin-12p40, interleukin-12R beta 1, interferon-gamma R1, interferon-gamma R2 or STAT1. Ten syndromes have thus far been identified. Recent insights in genetically controlled host defense and susceptibility to mycobacterial disease are discussed.

Somatostatin is a selective chemoattractant for primitive (CD34(+)) hematopoietic progenitor cells.

OBJECTIVE: Somatostatin (SST) is a regulatory peptide with a wide variety of activities in different tissues. SST activates G(alpha i)-protein-coupled receptors of a family comprising five members (SSTR1-5). Despite the broad use of SST and its analogs in clinical practice, the spectrum of activities of SST is incompletely defined. Here, we examined the role of SST and its receptors in hematopoiesis. MATERIALS AND METHODS: SSTR expression on human and mouse hematopoietic cells was analyzed by flow cytometry and reverse transcriptase polymerase chain reaction. The effects of SST on cell migration were measured in transwell assays. Using selective inhibitors, signaling mechanisms involved in SSTR2-mediated migration were studied in 32D cell transfectants expressing SSTR2. RESULTS: Human hematopoietic cells exclusively expressed SSTR2, whereas mouse bone marrow cells expressed SSTR2 and SSTR4. SSTR levels were high on primitive (CD34(+), Lin(-)) but low or absent on more mature (CD34(-), Lin(+)) cell types. Both SST and its analog octreotide acted as chemoattractants for primitive hematopoietic cells. Despite the presence of SSTR4, bone marrow cells from SSTR2-deficient mice failed to migrate toward SST gradients, suggesting that SSTR2 and SSTR4 are functionally different in this respect. SST activated phosphatidylinositol 3-kinase and the MAP kinases Erk1/2 and p38 in 32D[SSTR2] cells. While chemical inhibitors of these kinases had some effect, SST-induced migration was most strongly affected by blocking G(alpha i) activity or by elevating intracellular cAMP levels. CONCLUSIONS: Somatostatin acts as a selective chemoattractant for immature hematopoietic cells via activation of multiple intracellular pathways.