Suppressed expression of surface Ia on macrophages by lipopolysaccharide: evidence for regulation at the level of accumulation of mRNA

The surface expression of class II major histocompatibility molecules (immune associated or Ia antigens) is an acquired property of macrophages, essential to their ability to interact effectively with T lymphocytes. Surface expression of Ia is induced by stimulants such as interferon-gamma and is suppressed by agents such as lipopolysaccharide (LPS). Recent studies on several cultured cell lines indicate that interferon-gamma can heighten cellular levels of mRNA encoding Ia, and the level of such mRNA may represent an important regulatory focus for controlling expression of surface Ia. Murine peritoneal macrophages were treated with interferon-gamma and/or LPS and expression of Ia mRNA determined by Northern blot analysis with a probe specific for the murine beta-chain of I-A. mRNA specific for I-A beta was not detectable in explanted macrophages obtained from sites of sterile inflammation but was induced by treatment of purified recombinant interferon-gamma. This effect was dose dependent and was optimal by 24 hr after stimulation. Ia-specific mRNA preceded the surface expression of Ia as monitored by a radioimmunoassay using a monoclonal antibody specific for I-A beta. When a physiologic dose of LPS was added concomitantly with the interferon-gamma, the time course of induction if Ia-specific mRNA was not altered, but the amount of such mRNA detected was suppressed 40 to 80%. This effect was dependent on the dose of LPS, and the levels of mRNA correlated closely with subsequent surface expression of Ia. The ability of LPS to suppress both mRNA and cell surface Ia expression required that the suppressive agent be added within 12 hr of the inducing stimulus. This is the time frame during which accumulation of mRNA occurs. Thus the data demonstrates that accumulation of specific mRNA is a major regulatory focus governing expression of Ia both by interferon-gamma and LPS.     

Fatty acids differentially modify the expression of urokinase type plasminogen activator receptor in monocytes

The urokinase plasminogen activator system with its receptor uPAR contributes to the migratory potential of macrophages, a key event in atherosclerosis. We here investigated whether free fatty acids (FFA) modify the expression for uPAR in the PMA-differentiated human monocyte/macrophage-like cell line U937. Two hundred micromolar palmitate induced a threefold increase of the uPAR mRNA expression. Although the mono- and polyunsaturated fatty acids oleate and linoleate also stimulated uPAR expression, oleate had a significantly lower effect than palmitate. The observed effects were time and dose dependent. Inhibition of PKC-and ERK-pathways resulted in a strong down-regulation of basal uPAR expression whereas the FFA induced up-regulation remained unchanged. In contrast, FFA induced uPAR up-regulation was abolished by the specific inhibition of p38 MAPK. In conclusion we demonstrate that uPAR expression in human monocytes/macrophages is differentially stimulated by FFA. These effects are partially mediated by the p38 MAP-kinase signaling pathway.     

The effect of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on granulocyte macrophage-colony stimulating factor (GM-CSF) production by neuronal precursor cells

AIMS: To determine whether granulocyte macrophage-colony stimulating factor (GM-CSF) production by neuronal precursor (NT2) cells can be regulated by IL-1beta and TNF-alpha. BACKGROUND: We have previously demonstrated GM-CSF expression by neurons of the developing human brain, as well as by NT2 cells. IL-1beta and TNF-alpha upregulate GM-CSF production in glial cells, but GM-CSF regulation in neurons is as yet undefined. We hypothesized that IL-1beta and TNF-alpha would increase GM-CSF mRNA and protein production in NT2 cells. METHODS: The effect of IL-1beta and TNF-alpha on GM-CSF production was assessed by dose response (0 to 2,000 U/ml), and time course (0 to 48 hours incubation) experiments. GM-CSF mRNA and protein production were assessed by quantitative RT-PCR and by ELISA. The effect of these cytokines on cell turnover was determined by BrdU incorporation. RESULTS: IL-1beta increased GM-CSF mRNA and protein expression by NT2 cells. This effect was time and dose dependent, and the effective dose ranging from (20-200 U/ml). TNF-alpha increased GM-CSF mRNA expression to a lesser extent than did IL-1beta (maximal stimulation at 200 U/ml), and a minimal increase in net protein accumulation was noted. Neither cytokine increased NT2 cell turnover. CONCLUSIONS: IL-1beta and TNF-alpha both increase GM-CSF mRNA expression by NT2 cells, but only IL-1beta increases net GM-CSF protein accumulation.     

Insulin suppresses collagenase stimulatory effect of stratifin in dermal fibroblasts

A delicate balance between synthesis and degradation of extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is an essential feature of tissue remodeling. We have recently demonstrated that keratinocyte releasable stratifin, also known as 14-3-3 sigma protein, plays a critical role in modulating collagenase (MMP-1) mRNA expression in human dermal fibroblasts. In this study, we further characterized the collagenase stimulatory effect of stratifin in dermal fibroblasts and evaluated its effect in the presence and absence of insulin. Our data indicate that stratifin increases the expression of collagenase mRNA more than 20-fold in dermal fibroblasts, grown in either Dulbecco's modified Eagle's medium (DMEM) plus 2% or 10% fetal bovine serum (FBS). Collagenase stimulatory effect of stratifin was completely blocked, when fibroblasts were cultured in test medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% DMEM. The collagenase suppressive effect of test medium was directly proportional to the volume of KSFM used. As this medium contained insulin, we then evaluated the collagenase stimulatory effect of stratifin in dermal fibroblasts in the presence and absence of insulin. The results revealed that stratifin significantly increased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio, while insulin significantly decreased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio. The insulin inhibitory effect on collagenase mRNA expression was time and dose dependent. The maximal inhibitory effect of insulin was seen at 36 h post treatment. In conclusion, stratifin stimulates the expression of collagenase mRNA expression in dermal fibroblasts and this effect is suppressed by insulin treatment.     

Comparative study of cathinone and amphetamine on brown adipose thermogenesis

The effect of cathinone and amphetamine on brown adipose tissue thermogenesis and its modification with propranolol and timolol has been studied in rats. Both cathinone and amphetamine produced significant dose dependent increases in intracapsular brown adipose tissue (IBAT) and rectal temperatures. Amphetamine was found to be three times more potent as compared to cathinone, on a dose basis. Pretreatment of animals with propranolol and timolol individually inhibited cathinone and amphetamine induced hyperthermia. These findings suggest the involvement of beta adrenergic receptors in cathinone and amphetamine induced thermogenesis.     

Activation of gelatinases by fibrin is PA/plasmin system-dependent in human glomerular endothelial cells

Evidence suggests that fibrin deposit is related to severity of glomerulonephropathy. Fibrin is considered to play an active role beyond a haemostatic plug or temporary matrix in response to injury. We have reported that fibrin induced specific morphological changes and up-regulated intercellular adhesion molecule-1 expression of glomerular endothelial cells (GECs). Changes of gelatinases activity have been implicated playing a prominent role in glomerular diseases involving matrix turnover. This study examined whether overlying fibrin influences the expression of gelatinase A and B in cultured human GECs and mechanism underlying the activation. No gelatinase activity was detectable in supernatant of cultured GECs; however, physiological concentration of fibrin (0.5–2.0 mg/ml) induced a dramatic expression of activated MMP-2 and MMP-9 at both mRNA and protein level in a dose and time dependent manner. Increased mRNA level of membrane-type 1 matrix metalloproteinases (MT1-MMPs) was also found. Interestingly, we observed that fibrin also induced the expression of tissue type plasminogen activator (tPA), urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 by casein zymographic and reverse zymographic analysis. Fibrin plate assay revealed the net activity was PA predominant. Serine protease inhibitor aprotinin blocked the conversion of pro-gelatinase A and B to their active forms. The results demonstrate that overlying fibrin increased the secretion of gelatinase A and B from GECs. PA/plasmin proteolytic pathways contributed to the activation of gelatinases.     

Leptin对猪原代脂肪细胞脂解及其关键脂酶mRNA表达的影响

Leptin是由脂肪组织分泌的内源因子,在调节机体能量平衡过程中起重要作用.Leptin促进脂解的研究由来已久,但其作用机理尚不完善.本研究旨在通过系统检测关键脂酶mRNA的表达变化来探讨Leptin促进脂解的分子机理.运用形态学观察,油红O染色和RT-PCR鉴定培养的猪原代脂肪细胞;甘油测定试剂盒和游离脂肪酸(FFA)测定试剂盒分别检测甘油和FFA的释放;半定量RT-PCR检测关键脂酶mRNA的表达.结果显示:100 nmol/L的Leptin可显著上调ATGL、TGH-2、HSL、MGL和LPL mRNA的表达(P<0.01),但同时下调Perilipin mRNA的表达(P<0.01);Leptin呈浓度依赖性促进甘油的释放(P<0.01),但对FFA的释放影响不显著(P>0.05).以上结果提示,Leptin可能主要通过上调ATGL、MGL、LPL和下调Perilipin的表达促进猪原代脂肪细胞的脂解;同时推测,FFA释放的相对稳定可能是由Leptin通过上调UCPs的表达而增加FFA的消耗引起的.     

Transcriptional induction of urokinase in cultured human kidney carcinoma cells by tetradecanoyl-phorbol-acetate

A cell line derived from a human kidney carcinoma produces in vitro the urinary type of plasminogen activator (urokinase). The synthesis of plasminogen activator is enhanced by 12-O-tetradecanoyl-phorbol-13-acetate(TPA); the increase can be followed both in the cell lysate and in the culture medium. The effect requires RNA and protein synthesis as well as the continuous presence of the inducer. Immunofluorescence and immunoprecipitation experiments with monospecific antiurokinase IgG show that kidney carcinoma cells synthesize a 50,000-dalton urokinase and TPA enhances the synthesis of the same molecular species. Hybridization of the total cellular RNA to a human urokinase cDNA probe shows that TPA increases the urokinase mRNA level.     

Evidence that triiodothyronine decreases rat serum leptin concentration by down-regulation of leptin gene expression in white adipose tissue

Conflicting results have been reported regarding the effect of triiodothyronine (T(3)) on serum leptin and adipose tissue leptin gene expression in human and animals. The aim of the present study was to evaluate the effect of administration of increasing doses of T(3) on serum leptin concentration and on leptin mRNA abundance in white adipose tissue of rats. The results presented in this paper indicate that administration of single different doses of T(3) to euthyroid rats resulted dose dependent increases of serum total T(3) concentrations which are associated with a decrease in white adipose tissue leptin mRNA level. The leptin mRNA level in white adipose tissue was negatively correlated with serum total T(3) concentration (r=-0.8, p<0.001). Like white adipose tissue leptin mRNA level, serum leptin concentration decreased after T(3) administration, and was also negatively correlated with the serum T(3) concentration (r=-0.8, p<0.001). In contrast, administration of T(3) to the same rats led to a significant increase in white adipose tissue expression of the malic enzyme gene (malic enzyme activity and malic enzyme mRNA level), a known target gene for T(3). The results indicate that T(3) exerts a selective inhibitory effect on white adipose tissue leptin gene expression in vivo. A conclusion is that T(3) decreases rat serum leptin concentration by down-regulation of leptin gene expression in white adipose tissue.     

IL-2 and IL-6 synergize to augment the pore-forming protein gene expression and cytotoxic potential of human peripheral blood T cells

Our previous studies have demonstrated that high dose IL-2 (1000 U/ml) alone can induce human peripheral blood T cell pore-forming protein (PFP) mRNA expression and cytotoxic potential. We now report that the levels of IL-2 needed to induce these effects in T cells can be significantly reduced in the presence of IL-6. IL-6 and suboptimal doses of IL-2 (10 U/ml) were found to costimulate PFP mRNA expression and cytotoxic potential in resting human peripheral blood T cells, whereas IL-6 or low dose IL-2 alone had no effect. The induction of T cell PFP mRNA by IL-2/IL-6 was extremely rapid and increases in both PFP mRNA expression and cytotoxic potential were IL-6 dose dependent. The costimulatory effect of IL-6 did not appear to involve the IL-2/IL-2R pathway in as much as IL-6 did not induce IL-2 production or detectably increase IL-2R surface expression in T cells. These findings, in addition to the rapid induction of PFP mRNA by IL-2/IL-6, suggested that IL-6 can directly and independently provide an additional signal to augment the differentiation of CTL. In contrast to the results observed in T cells, IL-6 and IL-2 could enhance CD3- large granular lymphocyte (LGL) NK activity, but IL-6 either alone or in combination with IL-2 had no effect on constitutive PFP mRNA expression in resting LGL. These data further confirm that different mechanisms may be responsible for lymphokine activation of CTL and LGL in human peripheral blood. In particular it appears that IL-6 acts as a costimulatory signal with IL-2 in generating CTL and that IL-6 functions in part by acting in synergy with IL-2 to induce PFP, a major lytic protein involved in lymphocyte cytotoxicity.     

Oxygen-mediated regulation of tumor cell invasiveness. Involvement of a nitric oxide signaling pathway

Tumor hypoxia is associated with a poor prognosis for patients with various cancers, often resulting in an increase in metastasis. Moreover, exposure to hypoxia increases the ability of breast carcinoma cells to invade the extracellular matrix, an important aspect of metastasis. Here, we demonstrate that the hypoxic up-regulation of invasiveness is linked to reduced nitric oxide signaling. Incubation of human breast carcinoma cells in 0.5% versus 20% oxygen increased their in vitro invasiveness and their expression of the urokinase receptor, an invasion-associated molecule. These effects of hypoxia were inhibited by nitric oxide-mimetic drugs; and in a manner similar to hypoxia, pharmacological inhibition of nitric oxide synthesis increased urokinase receptor expression. The nitric oxide signaling pathway involves activation of soluble guanylyl cyclase (sGC) and the subsequent activation of protein kinase G (PKG). Culture of tumor cells under hypoxic conditions (0.5% versus 20% oxygen) resulted in lower cGMP levels, an effect that could be prevented by incubation with glyceryl trinitrate. Inhibition of sGC activity with a selective blocker or with the heme biosynthesis inhibitor desferrioxamine increased urokinase receptor expression. These compounds also prevented the glyceryl trinitrate-mediated suppression of urokinase receptor expression in cells incubated under hypoxic conditions. In contrast, direct activation of PKG using 8-bromo-cGMP prevented the hypoxia- and desferrioxamine-induced increases in urokinase receptor expression as well as the hypoxia-mediated enhanced invasiveness. Further involvement of PKG in the regulation of invasion-associated phenotypes was established using a selective PKG inhibitor, which alone increased urokinase receptor expression. These findings reveal that an important mechanism by which hypoxia increases tumor cell invasiveness (and possibly metastasis) requires inhibition of the nitric oxide signaling pathway involving sGC and PKG activation.     

Cell culture of bivalves: tool for the study of the effects of environmental stressors.

Spontaneous beating cells can be isolated from the heart of the oyster (Crassostrea gigas) and cultured for more than two months. They form adherent contractile networks in culture conditions. They show muscarinic and beta-adrenergic reactivity thus showing that they are functional cardio-myocytes: Acetylcholine induced a dose dependent decrease in spontaneous beating rate via an increase in potassium conductance, this effect being blocked by atropine. Epinephrine induced a dramatic increase in calcium conductance which was blocked by high concentrations of propranolol but not by sotalol and reversed by verapamil. Tributyltin and cadmium induced a dose and time dependent decrease mainly in inward ionic conductances, leading to a decrease or even a total suppression of the beating rate. Present study indicates that this model could be used as a sensitive test to study the effects of some marine pollutants at the cellular level in molluscs.