CD 36和胞外信号调节激酶通路在脂多糖诱导炎症因子中的作用研究

本研究以鼠源巨噬细胞RAW264．7为模型,研究CD36和胞外信号调节激酶（ERK ）通路对脂多糖（LPS）诱导巨噬细胞分泌炎症因子的影响。首先用100 ng／ml LPS刺激正常及小干扰RNA （siRNA ）技术沉默CD36表达的巨噬细胞16 h ,检测巨噬细胞的ERK活性及分泌炎症因子如肿瘤坏死因子α（TNF‐α）、白细胞介素6（IL‐6）和IL‐10的水平;继而以20 nmol／L ERK抑制剂处理细胞,再用LPS刺激,检测以上各项指标的变化,进一步明确ERK通路与LPS诱导巨噬细胞分泌炎症因子的相关性。结果显示,经LPS刺激,巨噬细胞的ERK活性显著增强,分泌的促炎因子 TNF‐α和 IL‐6显著增高,抑炎因子 IL‐10水平无明显变化;与CD36正常表达的巨噬细胞相比,CD36表达下降的巨噬细胞ERK活性及促炎因子TNF‐α、IL‐6水平显著下降,抑炎因子IL‐10显著增多。与未处理组相比,ERK抑制剂预处理的巨噬细胞中LPS诱导的ERK活性显著降低,促炎因子 TNF‐α和 IL‐6水平降低,抑炎因子 IL‐10水平升高。结果提示,LPS能通过其受体———CD36,激活巨噬细胞内ERK活性,进而促进巨噬细胞促炎因子的分泌。     

IL-4 regulation of a protein kinase C independent pathway for the generation of alpha CD3-induced activated killer cells.

alpha CD3 induced the generation of activated killer cells from resting T cells. Pretreatment of the splenic responders with PMA, a phorbol ester, depleted protein kinase C and induced unresponsiveness to the generation of alpha CD3-induced activated killer (CD3-AK) cells. Addition of exogenous IL-4 (1 U/ml) restored the cytotoxic response, with the maximal effect achieved with 30 to 100 U/ml. The phenotypes of CD3-AK cells maintained in IL-2 or in IL-4, with or without PMA, were the same: Thy1+ and CD8+. These results were reproduced with purified T cells and purified CD8+ cells, indicating that both the effectors and precursors were CD8+ cells and IL-4 had a selective effect to upregulate the CD8+ cells. Similar results were obtained by using SSP (staurosporine), another PKC inhibitor. At 2 days prior to testing, switching the lymphokine added to 2-week PMA- and IL-2-maintained CD3-AK cells reversed their cytolytic activity: switching from IL-2 to IL-4 restored cytolytic activity, and switching from IL-4 to IL-2 reduced cytolytic activity. The cytolytic activity of these CD3-AK cells correlated with their ability to produce BLT-esterase. In the absence of PMA, CD3-AK cells cultured in either IL-2 or IL-4 were cytolytic and contained high levels of BLT-esterase. In contrast, in the presence of PMA, only the IL-4-maintained CD3-AK cells were cytolytic and produced significant amounts of BLT-esterase. The effect of IL-4 was abrogated by the alpha IL-4 antibody 11B11, which reduced the cytolytic activity of CD3-AK and the ability to produce BLT-esterase. The requirement of IL-2 was less stringent and its major role appeared to be maintaining the cell growth. These findings indicate that IL-4 may participate in the regulation of a PKC-independent pathway for the generation of CD3-AK cells by regulating the production of cytolytic granules.     

Arachidonate metabolism and the signaling pathway of induction of apoptosis by oxidized LDL/oxysterol

Owing at least in part to oxysterol components that can induce apoptosis, oxidized LDL (oxLDL) is cytotoxic to mammalian cells with receptors that can internalize it. Vascular cells possess such receptors, and it appears that the apoptotic response of vascular cells to the oxysterols borne by oxLDL is an important part of the atherogenic effects of oxLDL. Thus, an analysis of the signaling pathway of apoptotic induction by oxysterols is of value in understanding the development of atherosclerotic plaque. In a prior study, we demonstrated an induction of calcium ion flux into cells treated with 25-hydroxycholesterol (25-OHC) and showed that this response is essential for 25-OHC-induced apoptosis. One possible signal transduction pathway initiated by calcium ion fluxes is the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we demonstrate that activation of cPLA2 does occur in both macrophages and fibroblasts treated with 25-OHC or oxLDL. Activation is evidenced by 25-OHC-induced relocalization of cPLA2 to the nuclear envelope and arachidonic acid release. Loss of cPLA2 activity, either through genetic knockout in mice, or by treatment with a cPLA2 inhibitor, results in an attenuation of arachidonic acid release as well as of the apoptotic response to oxLDL in peritoneal macrophages or to 25-OHC in cultured fibroblast and macrophage cell lines.     

Morphine induces CD4+ T cell IL-4 expression through an adenylyl cyclase mechanism independent of the protein kinase A pathway

Impaired host defense mechanisms after major operative procedures and trauma are recognized as important factors in the development of infectious complication. Trauma is associated with impaired cellular immunity and CD4+ T cell Th2 differentiation. We have previously implicated morphine treatment as a possible mechanism for Th2 differentiation after injury. In this investigation we first establish that morphine treatment in vivo results in Th2 differentiation and that this effect is mediated through a naltrexone-sensitive opioid receptor. We investigated the intracellular mechanism by which morphine controls CD4+ T cell differentiation and demonstrate that morphine treatment in vitro 1) increases anti CD3/CD28 Ab-induced CD4+ T cell IL-4 protein synthesis, IL-4 mRNA, and GATA-3 mRNA accumulation through a pertussis toxin-sensitive receptor; 2) results in a dose-dependent increase in anti-CD3/CD28 Ab-induced CD4+ T cell cytoplasmic cAMP concentration; and 3) increases the forskolin-stimulated cytoplasmic cAMP level through a pertussis toxin-sensitive receptor. We also demonstrate that chronic morphine treatment increases anti-CD3/CD28 Ab-induced IL-4 promoter activity and IL-4 immunoprotein expression through a p38 MAPK-dependent, but protein kinase A- and Erk1/Erk2-independent, mechanism.     

Phorbol ester-induced apoptosis of C4-2 cells requires both a unique and a redundant protein kinase C signaling pathway

Phorbol 12-myristate 13-acetate (PMA) potently induces apoptosis of LNCaP human prostate cancer cells. Here, we show that C4-2 cells, androgen-hypersensitive derivatives of LNCaP cells, also are sensitive to PMA-induced apoptosis. Previous reports have implicated activation of protein kinase C (PKC) isozymes alpha and delta in PMA-induced LNCaP apoptosis using overexpression, pharmacological inhibitors, and dominant-negative constructs, but have left unresolved if other isozymes are involved, if there are separate requirements for individual PKC isozymes, or if there is redundancy. We have resolved these questions in C4-2 cells using stable expression of short hairpin RNAs to knock down expression of specific PKC isozymes individually and in pairs. Partial knockdown of PKCdelta inhibited PMA-induced C4-2 cell death almost completely, whereas near-complete knockdown of PKCalpha had no effect. Knockdown of PKCepsilon alone had no effect, but simultaneous knockdown of both PKCalpha and PKCepsilon in C4-2 cells that continued to express normal levels of PKCdelta inhibited PMA-induced apoptosis. Thus, our data indicate that there is an absolute requirement for PKCdelta in PMA-induced C4-2 apoptosis but that the functions of PKCalpha and PKCepsilon in apoptosis induction are redundant, such that either one (but not both) is required. Investigation of PMA-induced events required for LNCaP and C4-2 apoptosis revealed that p38 activation is dependent on PKCdelta, whereas induction of retinoblastoma protein hypophosphorylation requires both PKC signaling pathways and is downstream of p38 activation in the PKCdelta pathway.     

Oxidised LDL up-regulate CD36 expression by the Nrf2 pathway in 3T3-L1 preadipocytes

The effect of oxLDL on CD36 expression has been assessed in preadipocytes induced to differentiate. Novel evidence is provided that oxLDL induce a peroxisome proliferator-activated receptor gamma-independent CD36 overexpression, by up-regulating nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2). The nuclear translocation of Nrf2 appeared to depend on PKC pathway activation. In adipocytes, the CD36 up-regulation may indicate a compensation mechanism to meet the demand of excess oxLDL and oxidised lipids in blood, reducing the risk of atherogenesis. Besides strengthening the hypothesis that oxLDL can contribute to the onset of insulin-resistance, data herein presented highlight the significance of oxLDL-induced CD36 overexpression within the cellular defence response.     

Regulation of CD93 cell surface expression by protein kinase C isoenzymes

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.     

Contribution of protein kinase A and protein kinase C pathways in ultraviolet B-induced IL-8 expression by human keratinocytes

We have previously demonstrated that treatment of the human keratinocyte cell line NCTC 2544 with a UVB dose equivalent to 1h exposure (100 mJ/cm2) results in a significant increase of IL-8 production. In this study, we use specific inhibitors to investigate the role of both PKA- and PKC-mediated pathways in the regulation of UVB-induced IL-8 expression in NCTC 2544 cell line. We show here that the treatment of irradiated human keratinocytes with PKA inhibitors [H89 and PKA inhibitor (PKAi)] induced a significant decrease of IL-8 production at both mRNA and protein levels. However, the regulation of IL-8 production seems to be mediated via a cAMP-independent PKA pathway, since drugs known to enhance cAMP concentrations [PGE2, cholera toxin and dibutyryl cAMP] decrease IL-8 production in irradiated cells by down-regulating NF-kappa B activation in response to UVB radiation. Using PMA (a potent pharmacological activator of PKC) and calphostin C (a specific PKC inhibitor), we demonstrated an up-regulation of IL-8 in NCTC 2544 cells and a down-regulation of the cytokine in UVB-irradiated cells, respectively. We also observed that in our experimental conditions, staurosporine, an inhibitor of both PKC and PMA-stimulated cellular responses, does not involve PKC inhibition in irradiated cells and significantly decreased NF-kappa B activity in response to UVB radiation. Finally, we concluded that a cAMP-independent PKA activation and a PKC-associated pathway are probably involved in the regulation of UVB-induced IL-8 synthesis in human keratinocytes.     

Intrinsic AHR in IL-5 transgenic mice is dependent on CD4(+) cells and CD49d-mediated signaling

Overexpression of interleukin (IL)-5 by the airway epithelium in mice using the rat CC10 promoter (NJ.1726 line) leads to several histopathologies characteristic of human asthma, including airway hyperreactivity (AHR). We investigated the contribution of B and T cells, as well as CD4 expression, to the development of AHR in IL-5 transgenic mice. NJ.1726 mice on a T cell or CD4 knockout background, but not on a B cell knockout background, lost intrinsic AHR. These effects occurred without decreases in IL-5 or eosinophils. We further investigated the contribution of alpha(4)-integrin signaling to the development of AHR in IL-5 transgenic mice through the administration of anti-CD49d (alpha(4)-integrin) antibody (PS/2). Administration of PS/2 resulted in immediate (16-h) inhibition of AHR. The inhibition of AHR was not associated with a decrease in airway eosinophils. These studies demonstrate that, despite the presence of increased levels of IL-5 and eosinophils in the lungs of NJ.1726 mice, CD4(+) cells and alpha(4)-integrin signaling are necessary for the intrinsic AHR that develops in IL-5 transgenic mice.     

Commitment to the CD4 lineage mediated by extracellular signal-related kinase mitogen-activated protein kinase and lck signaling

The development of T cells results in a concordance between the specificity of the TCR for MHC class I and class II molecules and the expression of CD8 and CD4 coreceptors. Based on analogy to simple metazoan models of organ development and lineage commitment, we sought to determine whether extracellular signal-related kinase (Erk) mitogen-activated protein (MAP) kinase pathway signaling acts as an inductive signal for the CD4 lineage. Here, we show that, by altering the intracellular signaling involving the Erk/MAP kinase pathway, T cells with specificity for MHC class I can be diverted to express CD4, and, conversely, T cells with specificity for MHC class II can be diverted to express CD8. Furthermore, we find that activation of the src-family tyrosine kinase, p56lck is an upstream mediator of lineage commitment. These results suggest a simple mechanism for lineage commitment in T cell development.     

Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages

 Macrophages are key players in many aspects of human physiology and disease. It has been hypothesized that in a given microenvironment monocytes differentiate into specific subpopulations with distinct functions. In order to study the role of macrophage heterogeneity in atherogenesis, we established a novel isolation and culture technique for human monocyte-derived macrophages. The present technique does not select for monocyte subpopulations prior to the onset of differentiation. Monocytes were cultured for 2 weeks in the presence of autologous lymphocytes before being plated quantitatively. They differentiated into mature macrophages in terms of morphology, lipid composition, and biological activity. Based on phagocytic activity as well as on the expression of CD14, CD36, and the low-density lipoprotein (LDL) receptor, we have identified macrophage subpopulations that may play distinct roles in atherogenesis. While virtually all adherence-purified monocytes expressed CD14, CD36, and the LDL-R, we characterized three subpopulations of macrophages based on the expression of these antigens: CD36⁺CD14^–LDL-R^– (58±12%), CD36⁺CD14⁺LDL-R⁺(18±5%), the remaining cells being CD36^–CD14^–LDL-R^–. The first two subsets decreased in size during further differentiation (51±12% and 8±3%, respectively). Our culture technique may also serve as a good model for studying the implications of macrophage heterogeneity in diseases other than atherosclerosis. Accepted: 13 February 1998     

Gastrin''s trophic effect in the colon: Identification of a signaling pathway mediated by protein kinase C

In previous studies we have reported that gastrin exerts a trophic effect on rat colonic epithelial cells in vitro. The effect of gastrin appeared to be mediated through a protein kinase C mechanism. In this study, we have characterized the role of protein kinase C in the gastrin-induced stimulation. Gastrin, in a time- and dose-dependent manner, increased protein kinase C translocation from the cytosol to the membrane, an index of enzyme activation. Maximum translocation occurred in 1 to 2 min following exposure to gastrin (10⁻⁸ M), before declining back to baseline level within 5 min. Gastrin did not change total protein kinase C activity in the colonic cells. Staurosporine, an inhibitor of protein kinase C, totally abolished the basal as well as the gastrin-stimulated activity of protein kinase C. The tumor promoter phorbol 12-myristate 13-acetate also stimulated colonic epithelial protein kinase C. However, prolonged treatment of cells with phorbol inhibited their subsequent response to gastrin stimulation. The response to gastrin was also prevented by the gastrin receptor antagonist proglumide. These observations suggest that protein kinase C mediates the stimulatory effect of gastrin on colonic epithelial cells, possibly through a receptor mechanism.