Independent regulation of M-CSF and G-CSF gene expression in human monocytes

The macrophage and granulocyte colony-stimulating factors, M-CSF and G- CSF, act in vitro to induce proliferation and differentiation of monocyte and granulocyte progenitor cells, respectively. We show here that both of these CSFs can be produced by stimulated human blood monocytes, but the M-CSF and G-CSF genes are independently regulated. Recombinant human interleukin-3 (IL-3) and GM-CSF primarily induce expression of the M-CSF gene and secretion of M-CSF, whereas bacterial lipopolysaccharide primarily induces expression of the G-CSF gene and secretion of G-CSF. These results suggest that under different conditions of in vitro stimulation the monocyte secretes factors that could lead selectively to either granulocyte or monocyte production.     

ARB affects nicotine-induced gene expression profile in human coronary artery endothelial cells

AIM: To investigate the effects of nicotine and nicotine plus angiotensin II receptor blocker (ARB) on the gene expression profile of human coronary artery endothelial cells (HCAECs). METHODS: The changes in gene expression profiles in HCAECs treated with nicotine and nicotine plus ARB olmesartan were analyzed by DNA microarray. In nicotine-treated HCAECs, 432 genes selected by P < 0.01 were greater than 1.5-fold compared with the untreated cells. Data were analyzed using IPA (Ingenuity^® Systems, www.ingenuity.com). RESULTS: The gene expression levels of tumor necrosis factor-α, collagen type 1, matrix metalloproteinase-10, and disintegrin and metalloprotease domain 8, which are related to “cardiovascular function and disease”, were significantly increased. In canonical pathway analyses using IPA, “atherosclerosis signaling” was strongly affected by nicotine treatment and this effect was reduced by co-incubation with ARB olmesartan. These data indicate that the deleterious cardiovascular consequences of cigarette smoking may, at least in part, be due to the nicotine-induced gene expression profile related to “atherosclerosis signaling”. CONCLUSION: The inhibitory effect of ARB against the nicotine-induced gene expression profile may possibly induce anti-atherosclerotic effects that are independent of those from lowering the blood pressure.     

Methods to profile gene expression

Molecular biology has been influenced tremendously by recent technological advancements in miniaturization and automation. One consequence has been the development of robust and sensitive methods to analyze gene expression. The ability to evaluate systematically the expression of every mammalian gene is now technically feasible. The methods available for gene expression profiling, and their application in cardiovascular research, are the topics of this review.     

Enterovirus-induced gene expression profile is critical for human pancreatic islet destruction

Aims/hypothesis

Virally induced inflammatory responses, beta cell destruction and release of beta cell autoantigens may lead to autoimmune reactions culminating in type 1 diabetes. Therefore, viral capability to induce beta cell death and the nature of virus-induced immune responses are among key determinants of diabetogenic viruses. We hypothesised that enterovirus infection induces a specific gene expression pattern that results in islet destruction and that such a host response pattern is not shared among all enterovirus infections but varies between virus strains.

Methods

The changes in global gene expression and secreted cytokine profiles induced by lytic or benign enterovirus infections were studied in primary human pancreatic islet using DNA microarrays and viral strains either isolated at the clinical onset of type 1 diabetes or capable of causing a diabetes-like condition in mice.

Results

The expression of pro-inflammatory cytokine genes (IL-1-α, IL-1-β and TNF-α) that also mediate cytokine-induced beta cell dysfunction correlated with the lytic potential of a virus. Temporally increasing gene expression levels of double-stranded RNA recognition receptors, antiviral molecules, cytokines and chemokines were detected for all studied virus strains. Lytic coxsackievirus B5 (CBV-5)-DS infection also downregulated genes involved in glycolysis and insulin secretion.

Conclusions/interpretation

The results suggest a distinct, virus-strain-specific, gene expression pattern leading to pancreatic islet destruction and pro-inflammatory effects after enterovirus infection. However, neither viral replication nor cytotoxic cytokine production alone are sufficient to induce necrotic cell death. More likely the combined effect of these and possibly cellular energy depletion lie behind the enterovirus-induced necrosis of islets.     

Gene expression profile in human leukocytes

Leukocytes are classified as myelocytic or lymphocytic, and each class of leukocytes consists of several types of cells that have different phenotypes and different roles. To define the gene expression in these cells, we have performed serial analysis of gene expression (SAGE) using human leukocytes and have provided the gene database for these cells not only at the resting stage but also at the activated stage. A total of 709,990 tags from 17 libraries were analyzed for the manifestation of gene expression profiles in various types of human leukocytes. Types of leukocytes analyzed were as follows: peripheral blood monocytes, colony-stimulating factor-induced macrophages, monocyte-derived immature dendritic cells, mature/activated dendritic cells, granulocytes, natural killer (NK) cells, resting B cells, activated B cells, naive T cells, CCR4(-) memory T cells (resting T(H)1 cells), CCR4(+) memory T cells (resting T(H)2 cells), activated T(H)1 cells, and activated T(H)2 cells. Among 38,961 distinct tags that appeared more than once in the combined total libraries, 27,323 tags were found to represent unique genes in certain type(s) of leukocytes. Using probability (P) and hierarchical clustering analysis, we identified the genes selectively expressed in each type of leukocytes. Identification of the genes specifically expressed in different types of leukocytes provides not only a novel molecular signature to define different subsets of resting and activated cells but also contributes to further understanding of the biologic function of leukocytes in the host defense system.     

Downregulation of c-fms gene expression in human monocytes treated with phorbol esters and colony-stimulating factor 1

The colony-stimulating factor-1 (CSF-1) regulates survival, growth, and differentiation of monocytes by binding to a single class of high-affinity receptors. The CSF-1 receptor is identical to the product of the c-fms protooncogene. The present studies monitored the effects of TPA and CSF-1 on c-fms gene expression in human monocytes. The results demonstrate that TPA downmodulates the constitutive expression of c-fms mRNA to low but detectable levels. Treatment of human monocytes with TPA was similarly associated with decreases in levels of the 138- and 125-Kd c-fms-encoded proteins. However, the kinetics of c-fms protein downmodulation indicated independent effects of TPA on c-fms expression at the RNA and protein levels. Furthermore, c-fms protein levels subsequently recovered despite persistently low levels of c-fms mRNA. Although previous studies demonstrated that c-fms protein is down-regulated in the presence of CSF-1, the present results indicate that CSF-1 also downregulates levels of c-fms mRNA. Moreover, the results indicate that CSF-1 increases protein kinase C activity in the membrane fraction. Together, these findings suggest that c-fms gene expression is differentially regulated at both the RNA and protein levels after activation of protein kinase C in human monocytes treated with TPA and CSF-1.     

Expression of ornithine decarboxylase gene in elderly human monocytes

The proliferative capacity of the immune system is impaired in elderly subjects and the expression of various genes involved in cell cycle progression is reduced in PHA stimulated lymphocytes during the aging process. Macrophages play a fundamental role in the immune system response. It has recently been demonstrated that the process of macrophage activation is accompanied by a rapid, transient rise of ornithine decarboxylase (ODC) mRNA levels. In fact, the ODC gene seems to be involved in macrophage activation and differentiation. The authors demonstrated that the steady-state levels of ODC mRNA and the correlated superoxide anion production are lower in the monocytes of elderly subjects with respect to those in young subjects used as control. These results confirmed the impaired immune function of the elderly.     

酪丝缬肽对人肝癌细胞SMMC-7721血管生成相关基因表达谱的影响

目的:观察小分子三肽化合物酪丝缬hk(tyroscryatide,YSV)对人肝癌细胞系SMMC-7721的抑制作用,并分析YSV对肿瘤细胞血管生成相关基因表达的影响.方法:建立人肝癌细胞SMMC-7721的体外培养体系,采用四甲基偶氮唑蓝(MTT)比色法检测YSV对体外培养的SMMC-7721的增殖抑制作用,应用功能分类基因芯片分析人肝癌细胞SMMC-7721经药物作用后血管生成相关基因表达的差异变化,同时应用RT-PCR方法验证相关基因的表达.结果:YSV能显著抑制人肝癌SMMC-7721的生长,10mg/L YSV作用72h对SMMC-7721的增殖抑制率为42.34%,与对照组比较有统计学意义(P＜0.05).在113个血管生成相关基因中,YSV组相比对照组,基因下调0.667倍以上的有7个.RT-PCR在基因水平上证实YSV能显著抑制血管内皮生长因子(VEGF)和白介素8(IL-8)的表达.结论:YSV在体外能够显著抑制人肝癌细胞SMMC-7721的生长,并能在基因水平上下调肿瘤细胞血管生成相关因子的表达.     

Interleukin-4 inhibits indoleamine 2,3-dioxygenase expression in human monocytes

Indoleamine 2,3-dioxygenase (IDO), a flavin-dependent enzyme that catalyzes the conversion of tryptophan to kynurenine, is induced in peripheral blood mononuclear cells by interferon-gamma (IFN gamma). Interleukin-4 (IL-4) is a cytokine that modulates the functional properties of monocytes/macrophages, and we investigated the effects of IL-4 on IDO. We showed that IL-4 inhibited the induction of IDO mRNA and IDO activity by IFN gamma in human monocytes. The inhibitory effect was evident with as little as 2 U/mL of IL-4. These results provide the first evidence that a cytokine can provide a negative signal for IDO expression and that IL-4 can influence the catabolism of tryptophan.     

Cytomegalovirus infects human lymphocytes and monocytes: virus expression is restricted to immediate-early gene products.

In this investigation, we studied the ability of human cytomegalovirus to infect peripheral blood mononuclear cells. With monoclonal antibody technology, we demonstrated that cytomegalovirus could infect human lymphocytes of T- and B-cell lineage, natural killer cells, and monocytes. Furthermore, virus expression was limited to the synthesis of immediate-early cytomegalovirus polypeptides. These peripheral blood mononuclear cells did not produce infectious virus, nor were mature virions visualized by electron microscopy. This abortive infection of mononuclear cells was most convincingly shown with stocks of cytomegalovirus that had been recently isolated from infected patients and passaged minimally in fibroblasts. This argues for an increased lymphotropic effect of some isolates of cytomegalovirus, compared to strains of virus that are extensively adapted to growth in fibroblasts. Furthermore, immunocompetent cells that were shown to be abortively infected with cytomegalovirus lost selected differentiated functions.     

Interleukin-2-induced tumor necrosis factor-alpha (TNF-alpha) gene expression in human alveolar macrophages and blood monocytes

Recent investigations have demonstrated interleukin-2 receptor (IL-2R) expression on both human alveolar macrophages (AM phi) and blood monocytes (PBM), but the function of these receptors has not been fully elucidated. In this study, we demonstrate that human AM phi, as well as PBM, can be induced to express biologically active TNF-alpha after challenge with interleukin-2 (IL-2). Furthermore, we examined the expression of TNF-alpha at the mRNA level via Northern blot and in situ hybridization analysis. Normal AM phi, obtained by bronchoalveolar lavage, and PBM were stimulated with either IL-2 (2,000 U/ml) or lipopolysaccharide (LPS) (10 micrograms/ml) for 18 h. Specificity was demonstrated by neutralizing TNF-alpha activity with a polyclonal rabbit anti-human TNF-alpha antibody. PBM TNF-alpha biologic activity from 11 subjects challenged with either IL-2 or LPS was 19 +/- 6 and 85 +/- 15 U/ml/10(6) cells, respectively, which represented 5-fold and 21-fold increases over control values. AM phi TNF-alpha biologic activity from nine subjects was 110 +/- 28 (IL-2-mediated) and 304 +/- 69 (LPS-mediated) U/ml/10(6) cells, which represented 2- and 6-fold increases over controls. AM phi exhibited statistically greater (p less than 0.05) TNF production in response to both IL-2 and LPS as compared to PBM. IL-2 challenge resulted in an induction of TNF-alpha mRNA accumulation, as demonstrated by Northern blot and in situ hybridization analyses. TNF-alpha mRNA was quantitated by laser densitometry for Northern blots or by counting the number of silver grains/mononuclear phagocytic cell in the in situ hybridization analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

PPARalpha activators inhibit tissue factor expression and activity in human monocytes

BACKGROUND: Tissue factor (TF), expressed on the surface of monocytes and macrophages in human atherosclerotic lesions, acts as the major procoagulant initiating thrombus formation in acute coronary syndromes. Peroxisome proliferator-activated receptor-alpha (PPARalpha), a nuclear receptor family member, regulates gene expression in response to certain fatty acids and fibric acid derivatives. Given that some of these substances reduce TF activity in patients, we tested whether PPARalpha activators limit TF responses in human monocytic cells. METHODS AND RESULTS: Pretreatment of freshly isolated human monocytes or monocyte-derived macrophages with PPARalpha activators WY14643 and eicosatetraynoic acid (ETYA) led to reduced lipopolysaccharide (LPS)-induced TF activity in a concentration-dependent manner (maximal reduction to 43+/-8% with 250 micromol/L WY14643 [P:<0.05, n=5] and to 42+/-12% with 30 micromol/L ETYA [P:>0.05, n=3]). Two different PPARgamma activators (15-deoxy(_Delta12,14)-prostaglandin J(2) and BRL49653) lacked similar effects. WY14643 also decreased tumor necrosis factor-alpha protein expression in supernatants of LPS-stimulated human monocytes. Pretreatment of monocytes with WY14643 inhibited LPS-induced TF protein and mRNA expression without altering mRNA half-life. Transient transfection assays of a human TF promoter construct in THP-1 cells revealed WY14643 inhibition of LPS-induced promoter activity, which appeared to be mediated through the inhibition of nuclear factor-kappaB but not to be due to reduced nuclear factor-kappaB binding. CONCLUSIONS: PPARalpha activators can reduce TF expression and activity in human monocytes/macrophages and thus potentially reduce the thrombogenicity of atherosclerotic lesions. These data provide new insight into how PPARalpha-activating fibric acid derivatives and certain fatty acids might influence atherothrombosis in patients with vascular disease.