Morphine stimulates superoxide formation by glomerular mesangial cells

Focal glomerulosclerosis is the predominant glomerular lesion in heroin addicts. We studied whether morphine, a metabolite of heroin, could directly affect the formation of superoxide by glomerular mesangial cells. Mesangial cells preincubated with morphine (10^–8 M) showed a higher (P<0.001) production of superoxide when compared to control cells (control) 401±21 vs. morphine 610±41 nM/mg protein/h). This effect of morphine on mesangial cells was dose dependent. Naloxone, an opiate antagonist, attenuated morphine-induced formation of Superoxide by mesangial cells [control, 317±4; morphine (10^–8 M), 573±9; and naloxone (10^–8 M) + morphine (10^–8 M), 333±6 nM/mg protein/h]. We conclude that morphine enhances formation of superoxide by mesangial cells and this effect of morphine seems to be mediated through opiate receptors. Since superoxide has been demonstrated to cause mesangiolysis, we propose that morphine may be playing a role in the induction of mesangial injury in patients with opiate abuse.This work was supported by National Institute of Health Grant R01-DA-06753.     

Bradykinin stimulates interleukin-8 production by human lung fibroblasts.

Bradykinin (BK) is a potent inflammatory mediator that is generated from kininogens by the actions of plasma and tissue kallikreins. Lung fibroblasts have the potential to participate in the inflammatory responses by releasing proinflammatory cytokines in response to a variety of stimuli. We postulated that human lung fibroblasts might produce interleukin-8 (IL-8) in response to BK stimulation. The present study showed that BK stimulated human lung fibroblasts to produce IL-8 in a dose- and time-dependent manner. Furthermore, Northern blot analysis showed that BK increased IL-8 mRNA expression. The stimulatory effect of BK on IL-8 production was detected at the concentration of 10 nm, and the maximal stimulation was achieved with 100 to 1000 nm. Phorbol 12-myristate 13-acetate pretreatment diminished the ability of BK to stimulate IL-8 production. In addition, GF109203X, a selective protein kinase C inhibitor, blocked BK-induced IL-8 production. These observations suggest that the stimulatory effect of BK on IL-8 production by lung fibroblasts is, at least partially, mediated through protein kinase C. These data suggest that BK may be involved in the inflammatory reaction leading to interstitial lung disorders through stimulating IL-8 production by lung fibroblasts.     

抵抗素刺激软骨细胞趋化因子CCL3及CCL4基因表达及机制

背景：既往研究表明抵抗素可刺激软骨细胞产生大量趋化因子,在炎症性关节病变中具重要作用,但具体作用机制未明。 目的：进一步探讨抵抗素刺激软骨细胞趋化因子CCL3及CCL4基因表达上调的机制。 方法：培养人源性软骨细胞,T/C-28a2细胞及ATDC5细胞,采用qPCR检测抵抗素刺激趋化因子基因的作用,C/EBPβ表达,核因子κB亚型及软骨特异性miRNAs。给予核因子κB抑制剂(IKK-NBD)和C/EBPβ抑制剂(SB303580),对C/EBPβ 及核因子κB的共同调节作用进行检测。在给予抵抗素刺激或无抵抗素刺激时分别进行亚细胞结构定位检测。 结果与结论：①抵抗素可非依赖性上调趋化因子基因表达。②软骨细胞对抵抗素刺激应答具有非严格细胞特异性,通过C/EBPβ抑制剂、核因子κB及一些软骨细胞特异性miRNAs,可对趋化因子基因表达进行联合调控。③一过性核因子κB活性增高可增强C/EBPβ活性,且两个转录因子对趋化因子基因CCL3及CCL4的作用均为非依赖性。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Regulation of CCL2 and CCL3 expression in human brain endothelial cells by cytokines and lipopolysaccharide

Background  

Chemokines are emerging as important mediators of CNS inflammation capable of activating leukocyte integrins and directing the migration of leukocyte subsets to sites of antigenic challenge. In this study we investigated the expression, release and binding of CCL2 (MCP-1) and CCL3 (MIP-1α) in an in vitro model of the human blood-brain barrier.     

IL-17 suppresses TNF-alpha-induced CCL27 production through induction of COX-2 in human keratinocytes

BACKGROUND: The chemokine CCL27 attracts skin-homing T cells. CCL27 production by keratinocytes is dependent on nuclear factor kappaB (NF-kappaB) activity and enhanced in lesions of patients with atopic dermatitis, psoriasis vulgaris, or allergic contact dermatitis. IL-17 is released from activated memory T cells and modulates skin inflammation. OBJECTIVE: We examined the in vitro effects of IL-17 on TNF-alpha-induced CCL27 production in human keratinocytes. METHODS: Keratinocytes were incubated with TNF-alpha, IL-17, or both. CCL27 secretion and mRNA levels were analyzed by means of ELISA and RT-PCR, respectively. COX-2 promoter and NF-kappaB activities were analyzed by using luciferase assays. COX-2 protein levels were analyzed by means of Western blotting. RESULTS: IL-17 suppressed TNF-alpha-induced CCL27 secretion and mRNA expression and NF-kappaB activity in keratinocytes. The COX-2 inhibitor NS398 counteracted the effects of IL-17, and prostaglandin E(2) prevented counteraction by NS398. IL-17 alone or synergistically with TNF-alpha increased prostaglandin E(2) release from keratinocytes, and the increase was suppressed by NS398. IL-17 alone or synergistically with TNF-alpha increased COX-2 mRNA and protein levels, promoter activity, and mRNA stability. The stimulatory effects of IL-17 on COX-2 expression were suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) kinase. IL-17 alone or synergistically with TNF-alpha induced dual phosphorylation of p38 MAPK and ERK. CONCLUSION: IL-17 might suppress TNF-alpha-induced CCL27 production by inhibiting NF-kappaB through induction of COX-2. The induction of COX-2 might be mediated by activation of p38 MAPK and ERK. T cell-derived IL-17 might alleviate T-cell skin infiltration through inhibition of CCL27 production.     

Interleukin-1 stimulates human uterine prostaglandin production through induction of cyclooxygenase-2 expression

PROBLEM: Uterine infection occurs in as much as 20% of preterm labor and results in increased decidual cytokines. The objective of this study was to examine the effect of interleukin-1 (IL-1) and the cyclooxygenase-2 (COX-2) inhibitor, NS-398, on myometrial prostaglandin (PG) production and COX-2 expression. METHOD OF STUDY: Human uterine myocytes were stimulated with IL-1 (0-50 ng/mL) over 24 hr. PGE2, PGF2alpha, and 6-keto F1alpha were measured by enzyme-linked immunosorbent assay. Both COX-1 and COX-2 proteins and mRNA were measured by western and northern blot, respectively. RESULTS: IL-1 increased PG production beginning at 6 hr, COX-2 protein increased beginning at 4 hr and continued to increase at 24 hr. COX-2 mRNA increased at 2 hr and peaked at 4 hr. NS-398 blocked PG production but had no effect on COX-2 protein or mRNA. CONCLUSIONS: IL-1 increases PG production by myometrium by increased COX-2 expression. NS-398 completely blocks IL-1-induced PG production. With intrauterine infection, IL-1 may induce labor through the autocrine production of uterotonic PGs.     

CCL2 modulates cytokine production in cultured mouse astrocytes

Background  

The chemokine CCL2 (also known as monocyte chemoattractant protein-1, or MCP-1) is upregulated in patients and rodent models of traumatic brain injury (TBI), contributing to post-traumatic neuroinflammation and degeneration by directing the infiltration of blood-derived macrophages into the injured brain. Our laboratory has previously reported that Ccl2 -/- mice show reduced macrophage accumulation and tissue damage, corresponding to improved motor recovery, following experimental TBI. Surprisingly, Ccl2 -deficient mice also exhibited delayed but exacerbated secretion of key proinflammatory cytokines in the injured cortex. Thus we sought to further characterise CCL2's potential ability to modulate immunoactivation of astrocytes in vitro.     

Induction of angiogenic chemokine CCL2 by human herpesvirus 8 chemokine receptor

Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma (KS), an endothelial cell lesion believed to be initiated and driven primarily by cytokine dysregulation. Among the viral proteins suspected as contributing to viral pathogenesis is the lytically expressed viral G protein-coupled receptor (vGPCR), which can induce various cellular cytokines. CC ligand-2 (CCL2/MCP-1) is a vGPCR-regulated angiogenic chemokine found at elevated levels in KS lesions and induced by HHV-8 infection of endothelial cells. Here we show that vGPCR induces CCL2 in endothelial cells via activation of C/EBPβ and that vGPCR and C/EBPβ are critical components of CCL2 induction by HHV-8 infection of endothelial cultures. To our knowledge, this is the first report of vGPCR-mediated cytokine induction, and its characterization, in the context of virus infection. Our results identify a mechanism by which vGPCR can contribute, in a host cell shutoff-independent manner, to viral pathogenesis.     

Interleukin-16 stimulates the expression and production of pro-inflammatory cytokines by human monocytes

Interleukin-16 (IL-16) acts as a chemoattractant for CD4+ cells, as a modulator of T-cell activation, and plays a key role in asthma. This report describes the cytokine-inducing effects of IL-16 on total peripheral blood mononuclear cells (PBMC) and PBMC subpopulations. While CD4+ T lymphocytes did not secrete cytokines in response to rhIL-16, CD14+ CD4+ monocytes and maturing macrophages secrete IL-1beta, IL-6, IL-15 and tumour necrosis factor-alpha (TNF-alpha) upon rhIL-16 stimulation. The mRNA species for these four cytokines were detected as early as 4 hr post-stimulation, with protein being secreted by 24 hr. Secretion of IL-1beta and IL-6 by total PBMC was dose dependent, with maximal secretion being observed using 50 ng/ml rhIL-16. However, for IL-15 or TNF-alpha maximal secretion by total PBMC occurred with all concentrations between 5 ng/ml to 500 ng/ml rhIL-16. Purified monocytes/macrophages secreted maximal concentrations of all four cytokines in the presence of 500 ng/ml rhIL-16, except for monocytes where maximal secretion of IL-15 was, interestingly, observed with only 50 ng/ml rhIL-16. The use of higher concentrations of rhIL-16 (1000 ng/ml) inhibited secretion of all four cytokines. While these IL-16-induced cytokines are likely to be involved in the immune system's response to antigen, the data suggest that IL-16 may play a key role in initiating and/or sustaining an inflammatory response.     

前列腺素D2受体调节PGD2诱导的人气道黏液高分泌

目的研究前列腺素D2(PGD2)通过D类前列腺素受体2(DP2)对人气道上皮细胞黏液分泌的效应及其作用机制。方法用PGD2刺激人气道上皮16HBE细胞,以DP2拮抗剂AZD6430、DP1拮抗剂BWA868C及三磷酸肌醇蛋白激酶(PI3K)抑制剂LY294002为干预因素,将细胞分为对照组、PGD2刺激组、PGD2刺激+AZD6430组、PGD2刺激+BWA868C组、PGD2刺激+LY294002组。ELISA检测细胞中肿瘤坏死因子(TNF)-α、白介素(IL)-4和IL-13的蛋白含量,激光共聚焦技术检测细胞内黏蛋白(MUC)5AC含量;Western blot检测DP2、DP1、PI3K和磷酸化AKT(p-AKT)表达水平;实时荧光定量PCR检测TNF-α、IL-4、IL-13和MUC5AC mRNA表达水平。结果 PGD2刺激后MUC5AC、TNF-α、IL-4、IL-13、DP2、PI3K和p-AKT表达显著高于对照组(P0.05);AZD6430拮抗组中MUC5AC、TNF-α、IL-4、IL-13、PI3K及p-AKT表达较PGD2刺激组明显减弱(P0.05);LY294002组TNF-α、IL-4、IL-13和MUC5AC表达较PGD2组亦显著减弱(P0.05)。结论 PGD2激活人气道上皮细胞的DP2受体,活化PI3K/AKT引起黏液高分泌。     

Prostaglandin E2 inhibits production of the inflammatory chemokines CCL3 and CCL4 in dendritic cells

Dendritic cells bridge innate and adaptive immunity and participate in both responses. Upon capture of pathogens, dendritic cells release inflammatory cytokines and chemokines, attracting other immune cells to the infection site. Anti-inflammatory cytokines, glucocorticoids, anti-inflammatory neuropeptides, and lipid mediators such as prostaglandin E2 (PGE2) limit and control the inflammatory response. In this study we report that exogenous PGE2 inhibits CCL3 (MIP-1alpha) and CCL4 (MIP-1beta) expression and release from dendritic cells stimulated with either lipopolysaccharide (LPS), a TLR4 ligand, or peptidoglycan, a TLR2 ligand. The inhibition is dose-dependent and occurs at both the mRNA and protein levels. The inhibitory effect is mediated through EP2 and EP4 receptors and requires the presence of PGE2 at the time of LPS stimulation. Intraperitoneal administration of PGE2 together with LPS results in a reduction in the levels of CCL3 and CCL4 released in the peritoneal fluid, a reduction in the number of dendritic cells accumulating in the peritoneal cavity, and a reduction in CCL3 amount per cell in the peritoneal cell population. These results suggest that one of the mechanisms by which endogenous PGE2 acts as an anti-inflammatory agent, is the inhibition of inflammatory chemokine release from activated dendritic cells, preventing the excess accumulation of activated immune cells.     

Chlamydia trachomatis growth stimulates interleukin 8 production by human monocytic U-937 cells.

Growth of Chlamydia trachomatis serotypes L2 and L3 in a human monocytic cell line, U-937, increased the rate of interleukin 8 (IL-8) release 100-fold. Heat-killed chlamydiae induced a 10-fold-lower level of production of IL-8. IL-8 may play an important role in the inflammatory reaction to chlamydial infection.     

Distinct regulation of C3a-induced MCP-1/CCL2 and RANTES/CCL5 production in human mast cells by extracellular signal regulated kinase and PI3 kinase

Complement component C3a causes a robust degranulation in human mast cells. Whether C3a also stimulates chemokine production in human mast cells and what signaling pathway it activates is not known. In the present study, we demonstrate that CD34+ cell-derived primary mast cells and a human mast cell line LAD 2 express surface C3a receptors at similar levels. Furthermore, C3a caused approximately 50% internalization of cell surface C3a receptors in both cell types. We therefore used LAD 2 cells as a model to study C3a-induced biological responses and signaling in human mast cells. We found that C3a stimulated substantial degranulation and induced chemokine monocyte chemoattractant protein 1 (MCP-1/CCL2) and regulated upon activation, normal T-cell expressed and secreted (RANTES/CCL5) production in LAD 2 mast cells. C3a caused a rapid and sustained extracellular-signal-regulated kinase (ERK) phosphorylation and Akt phosphorylation in LAD 2 mast cells. Furthermore, U0126 and LY294002, which respectively inhibit MEK-induced ERK phosphorylation and PI3 kinase-mediated Akt phosphorylation had distinct effects on C3a-induced responses. Thus, U0126, which blocked C3a-induced RANTES/CCL5 production by 50.6+/-2.3%, inhibited MCP-1/CCL2 generation by 85.2+/-0.6%. In contrast, LY294002 had no effect on C3a-induced RANTES/CCL5 production but blocked MCP-1/CCL2 generation by 83.7+/-1.5%. These data demonstrate that C3a activates divergent signaling pathways to induce chemokine production in human mast cells.     

f-Met-Leu-Phe stimulates nitric oxide production in chick embryo neurons: the role of NF-kB

N-formyl-methionyl-leucyl-phenylalanine (fMLP) is a major chemotactic factor produced by Escherichia coli and other Gram-negative bacteria. In avian models the fMLP effects and the possible expression of FPRs have been poorly investigated. This report demonstrates that fMLP stimulation in vitro is able to elicit significant cellular responses from 10-day chick embryo nerve cells. Cell treatment with 10(-7) M fMLP at 37 degrees C induces a dramatic increase of nitric oxide (NO) production, after 24, 48, and 72 h, respectively. After 72 h of treatment with 10(-7) M fMLP the maximum nuclear translocation of the NF-kB complex protein p65 is visible, corresponding to the greatest NO production. In this context, 72 h of fMLP stimulation lead to a marked expression of the antiapoptotic protein Bcl-2, involved in cell survival. This suggests that activation of the NF-kB complex plays a protective role in chick neuronal cells treated with fMLP, confirmed by the significant neuronal cells degeneration observed after NF-kB inhibition with the specific inhibitor, TPCK. Overall, these data suggest a possible protective mechanism displayed by neurons against toxic molecules, like NO, released after cell exposure to bacterial products.