Modulation of sphingosine 1-phosphate,a new lipid mediator,on nitric oxide production by vascular smooth muscle cells

The effects of sphingosine 1-phosphate (S1P) on nitric oxide (NO) production induced by interleukin (IL)-1 beta in cultured rat vascular smooth muscle cells (VSMCs) have been investigated. Rat VSMCs abundantly expressed mRNA of Edg-3 and Edg-5 receptor subtypes in both unstimulated and IL-1 beta-stimulated cells. S1P at higher than 0.1 microM inhibited the NO production induced by IL-1 beta in a concentration-dependent manner, in which S1P at 10 microM caused over 90% inhibition. S1P also inhibited inducible NO synthase (iNOS) protein and mRNA expressions induced by IL-1 beta in a concentration-dependent manner, which effects were smaller than that in the NO production. S1P also significantly inhibited GTP cyclohydrolase I (GTPCH) mRNA expression induced by IL-1 beta. PTX pretreatment partially prevented the inhibitory effects of S1P on the NO production and iNOS induction, while completely prevented the inhibitory effects on iNOS and GTPCH mRNA expressions. In PTX-pretreated VSMC, S1P showed a bell-shaped enhancing and inhibitory effect on iNOS protein expression. These results suggest that S1P modulates IL-1 beta induction of NO production by rat VSMCs through multiple mechanisms, partially via Edg-3 and Edg-5 receptor subtypes coupled to PTX-sensitive G proteins.     

Expression of cyclooxygenase-2 and pro-inflammatory cytokines induced by 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) in human mast cells requires NF-kappa B activation

Mast cells are critical for initiating innate immune and inflammatory responses by releasing a number of pro-inflammatory mediators. The potential immunomodulatory properties of hydrogenated aromatic hydrocarbons have been the subject of extensive investigation, as the immune system is a sensitive target for hydrogenated aromatic hydrocarbon toxicity. In this report, the effects of polychlorinated biphenyl (PCB) on the expression of cyclooxygenase-2 and pro-inflammatory cytokines such as interleukin-1beta (IL-1beta), IL-6 and tumor necrosis factor (TNF)-alpha in the human leukemic mast cell line were investigated. TNF-alpha and IL-1beta expressed their respective mRNA in the presence or absence of PCB, while cyclooxygenase-2 (COX-2) and IL-6 mRNA expression were highly induced by PCB after 2 h. Moreover, pre-treatment with the nuclear factor (NF)-kappaB pathway inhibitor, pyrrolidine dithiocarbamate, suppressed COX-2, TNF-alpha and IL-1beta induction and reduced the IL-6 mRNA levels induced by PCB. The NF-kappaB activity was determined by electrophoretic mobility shift analysis (EMSA) using an oligonucleotide containing a consensus NF-kappaB binding sequence. Stimulating the cells with PCB activated NF-kappaB. However, pre-treating them with a NF-kappaB pathway inhibitor, pyrrolidine dithiocarbamate, suppressed PCB-induced NF-kappaB activation. This suggests that PCB induces cycloxoygenase-2 and pro-inflammatory cytokine expression, and that this induction occurs through NF-kappaB.     

Induction of fibroblast growth factor-9 and interleukin-1alpha gene expression by motorcycle exhaust particulate extracts and benzo(a)pyrene in human lung adenocarcinoma cells.

Motorcycle exhaust particulates (MEP) contain carcinogenic polycyclic aromatic hydrocarbons including benzo(a)pyrene. This study has determined the ability of MEP to alter the expression of select genes from drug metabolism, cytokine, oncogene, tumor suppressor, and estrogen signaling families of human lung adenocarcinoma CL5 cells. cDNA microarray analyses and confirmation studies were performed using CL5 cells treated with 100 microg/ml MEP extract for 6 h. The results showed that MEP increased the mRNA levels of metabolic enzymes CYP1A1 and CYP1B1, proinflammatory cytokines interleukin (IL)-1alpha, IL-6, and IL-11, fibroblast growth factor (FGF)-6 and FGF-9, vascular endothelial growth factor (VEGF)-D, oncogene fra-1, and tumor suppressor p21. In contrast, MEP decreased tumor suppressor Rb mRNA in CL5 lung epithelial cells. Treatment with 10 microM benzo(a)pyrene for 6 h altered gene expression profiles, in a manner similar to those by MEP. Induction of IL-1alpha, IL-6, IL-11, and FGF-9 mRNA by MEP and benzo(a)pyrene was concentration and time dependent. Cotreatment with 2 mM N-acetylcysteine blocked the MEP- and benzo(a)pyrene-mediated induction. Treatment with MEP or benzo(a)pyrene increased IL-6 and IL-11 releases to CL5 cell medium. Incubation of human lung fibroblast WI-38 with MEP- or benzo(a)pyrene-induced CL5 conditioned medium for 4 days stimulated cell growth of the fibroblasts. Inhalation exposure of rats to 1:10 diluted motorcycle exhaust 2 h daily for 4 weeks increased CYP1A1, FGF-9, and IL-1alpha mRNA in lung. This present study shows that MEP and benzo(a)pyrene can induce metabolic enzyme, inflammatory cytokine, and growth factor gene expression in CL5 cells and stimulate lung epithelium-fibroblast interaction.     

Cytotoxic activity and cytokine gene induction of Asp-hemolysin to vascular endothelial cells

We examined the effects of Asp-hemolysin from Aspergillus fumigatus Fresenius-Muramatsu strain on the viability and cytokine gene expression of human umbilical vein endothelial cells (HUVEC). The cell viability of HUVEC was reduced to 50% by 100 micrograms/ml of Asp-hemolysin. However, lower concentration of Asp-hemolysin (< 30 micrograms/ml) had no effect on the cell viability. The mRNA expression of such cytokines as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, IL-6, IL-8 and granulocyte-macrophage colony stimulating factor (GM-CSF) were also observed in HUVEC cultured with 30 micrograms/ml of Asp-hemolysin.     

Responses of exposure to cigarette smoke at three dosage levels on soleus muscle fibers in Wistar-Kyoto and spontaneously hypertensive rats

Overproduction of nitric oxide (NO) from inducible nitric oxide synthase (iNOS) is importantly involved in the pathogenesis of endotoxemia and atherosclerosis. Calcium antagonists are commonly used as cardiovascular drugs and have a beneficial effect on prolonging survival in various models of endotoxin shock. The present study was to investigate the effect of a calcium antagonist amlodipine on nitrite, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) formation and iNOS induction both in lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-treated rat aortic smooth muscle cells (RASMC) and in a rat model of endotoxemia. Incubation with amlodipine (0.1 - 10 microM) for 24 h resulted in a significant and dose-dependent attenuation in medium nitrite, TNF-alpha and IL-1beta formation as well as iNOS protein expression in LPS/IFN-gamma-treated RASMC. In addition, amlodipine inhibited leucigenin-induced superoxide formation in RASMC. In the rat endotoxic model, the serum nitrite/nitrate, TNF-alpha and IL-1beta levels as well as iNOS protein expression of lungs were also suppressed by administration of amlodipine (50 microg/kg, i.v.). These results suggest that amlodipine may exert vascular beneficial effects by suppressing pro-inflammatory cytokines and free radical generation as well as iNOS induction in smooth muscle cells during activation of inflammatory mechanism.     

IL-6 and Crohn's disease

Interleukin-6 (IL-6) is a pleiotropic cytokine with central roles in immune regulation, inflammation, hematopoiesis, and oncogenesis. Its biological activities are shared by IL-6-family of cytokines such as leukemia inhibitory factor and oncostatin M. When IL-6 binds to IL-6R, the IL-6/IL-6R complex then associates with gp130, the common signal transducer of cytokines related to IL-6. IL-6R does not have to be expressed on the cell surface for IL-6 signaling because soluble form of IL-6R (sIL-6R) can bind to IL-6 and function through gp130. Increased levels of IL-6 and sIL-6R have been demonstrated in both serum and intestinal tissues of the patients with active Crohn's disease. In animal model studies, anti-IL-6R monoclonal antibody (mAb) successfully prevented intestinal inflammation and systemic wasting disease by suppressing adhesion molecule expression by vascular endothelium. It also reduced colonic expression of tumor necrosis factor alpha, IL-1beta, and interferon gamma mRNA without affecting the production of transforming growth factor beta, IL-10, and IL-4. Moreover, the treatment displayed therapeutic efficacy against established colitis through the induction of lamina propria T-cell apoptosis. These results strongly suggest that specific targeting of IL-6/sIL-6R pathway will be a promising new approach for the treatment of Crohn's disease, and the clinical trial of humanized anti-IL-6R mAb has been carried out.     

Inhibition of p38 and ERK MAP kinases blocks endotoxin-induced nitric oxide production and differentially modulates cytokine expression.

Mitogen-activated protein kinases (MAPKs) are thought to have a critical role in lipopolysaccharide (LPS)-induced immune responses but the molecular mechanisms underlying the mediation of these signaling are not clear. The roles of p38 and extracellular signal-regulated kinase (ERK) in the regulation of nitric oxide (NO) and proinflammatory cytokine expression in J774A.1 macrophages in response to LPS were examined. Specific inhibitors for p38 and ERK, SB203580 and PD98059, respectively, were used. LPS (30ng/ml) activated inducible nitric oxide synthase (iNOS), subsequent NO production, and gene expression for tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, and IL-12. Treatment of cultures with SB203580 increased LPS-induced reactive oxygen species (ROS) production, whereas both SB203580 and PD98059 decreased LPS-induced NO production. Concomitant decreases in the expression of iNOS mRNA and protein were detected. SB203580 and PD98059 decreased LPS-induced gene expression of IL-1beta and IL-6. SB203580 increased LPS-induced expression of TNF-alpha and IL-12; PD98059 had no effect on these cytokines. Results indicated that both p38 and ERK pathways are involved in LPS-stimulated NO synthesis and the expression of IL-1beta and IL-6. p38 signaling pathway is involved in LPS-induced ROS, TNF-alpha and IL-12 production.     

WY-14 643, a selective PPAR{alpha} agonist, induces proinflammatory and proangiogenic responses in human ocular cells

Peroxisome proliferator-activated receptor α (PPARα) agonism in ocular inflammation has not been thoroughly investigated. The objective of this investigation was to determine the effect of WY-14 643, a selective PPARα agonist, on inflammatory cytokine release in human ocular cells. Stimulation of primary human corneal epithelial cells, keratocytes, and retinal endothelial cells with 1 to 10 ng/mL interleukin 1β (IL-1β) resulted in a significant increase in numerous inflammatory cytokines, including IL-6, IL-8, and tumor necrosis factor α (TNF-α); and dexamethasone was able to significantly inhibit these effects. However, WY-14 643 did not effectively block IL-1β-induced cytokine release in ocular cells; rather, significant increases in IL-1β-induced inflammatory cytokines were observed in these cells but not in aortic smooth muscle cells. WY-14 643 also significantly upregulated vascular endothelial growth factor (VEGF) expression in corneal epithelial cells and keratocytes. These studies demonstrate for the first time that PPARα agonism may be proinflammatory and proangiogenic in a variety of ocular cells and suggest that therapeutic applications of such agents in ophthalmology may be limited.     

Mitogenic effects of cytokines on smooth muscle are critically dependent on protein kinase A and are unmasked by steroids and cyclooxygenase inhibitors

Excessive smooth muscle growth occurs within the context of inflammation associated with certain vascular and airway diseases. The inflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha) have been shown previously to inhibit mitogen-stimulated smooth muscle growth through a mechanism presumed to be dependent on the induction of cyclooxygenase-2, prostaglandins, and activation of the cAMP-dependent protein kinase (PKA). Using both molecular and pharmacological strategies, we demonstrate that the mitogenic effects of IL-1beta and TNF-alpha on cultured human airway smooth muscle (ASM) cells are tightly regulated by PKA activity. Suppression of induced PKA activity by either corticosteroids or cyclooxygenase inhibitors converts the cytokines from inhibitors to enhancers of mitogen-stimulated ASM growth, and biological variability in the capacity to activate PKA influences the modulatory effect of cytokines. Promitogenic effects of IL-1beta are associated with delayed increases in p42/p44 and phosphoinositide-3 kinase activities, suggesting a role for induced autocrine factors. These findings suggest a mechanism by which mainstream therapies such as corticosteroids or cyclooxygenase inhibitors could fail to address or exacerbate the pathogenic smooth muscle growth that occurs in obstructive airway and cardiovascular diseases.     

Induction of eotaxin expression and release from human airway smooth muscle cells by IL-1beta and TNFalpha: effects of IL-10 and corticosteroids.

Eotaxin is a novel C-C chemokine with selective chemoattractant activity for eosinophils. We determined whether eotaxin could be produced by human airway smooth muscle (HASM) cells in culture and examined its regulation by interleukin-10 (IL-10) and the corticosteroid, dexamethasone. Stimulation of the cells with interleukin-1beta (IL-1beta) or tumour necrosis factor (TNFalpha) each at 10 ng ml(-1) induced the release of eotaxin protein with maximal accumulation by 24 h. Interferon-gamma (IFNgamma) alone at 10 ng ml(-1) had no effect and there was no synergy between these cytokines on the release of eotaxin. Reverse phase high performance liquid chromatographic (HPLC) analysis of supernatents from cells treated with TNFalpha (10 ng ml(-1) for 96 h showed immunoreactivity to eotaxin which eluted with the expected retention time of 34.5-35 min. Both IL-1beta and TNFalpha-induced release of eotaxin was not inhibited by dexamethasone (1 microM), however IL-10 (10 ng ml(-1)) had a significant inhibitory effect. Dexamethasone and IL-10 did not inhibit the induction of eotaxin mRNA induced by IL-1beta or TNFalpha. Thus, human airway smooth muscle cells can release eotaxin and could be an important source of chemokine production during airway inflammatory events.     

Anti-interleukin-6 therapy for Crohn's disease

Proinflammatory cytokines have been demonstrated to play a crucial role in the pathogenesis and physiopathology of various chronic inflammatory conditions including Crohn's disease (CD). Among these cytokines, interleukin-6 (IL-6) must be especially important because increased serum concentrations of acute phase proteins, reduced level of serum albumin, and remarkable thrombocytosis are all well-explained by the increased level of IL-6. Moreover, IL-6 is capable of stimulating even IL-6 receptor (IL-6R) negative cells such as vascular endothelial cells when complexed to soluble form of IL-6R (sIL-6R), and serum level of IL-6 as well as sIL-6R has been demonstrated to increase during inflammation. To investigate the therapeutic potential of IL-6 signaling blockade for CD, anti-IL-6R monoclonal antibody (mAb) was introduced to various murine models of colitis. Anti-IL-6R mAb successfully prevented wasting disease and the development of macroscopic and histological lesions. It suppressed the accumulation of ICAM-1 positive and Mac-1 positive cells in the lamina propria (LP) and the expression of ICAM-1 and VCAM-1 by vascular endothelial cells. Expansion of colonic and splenic CD4(+) T cells was reduced as well as the colonic expression of tumor necrosis factor alpha (TNF-alpha), IL-1beta, and interferon gamma (IFN-gamma) mRNA without affecting the production of transforming growth factor beta (TGF-beta), IL-10, and IL-4 mRNA. The treatment also suppressed established colitis by inducing LP T cell apoptosis. These results strongly suggest that specific targeting of IL-6/sIL-6R pathway will be a promising new approach for the treatment of CD, and the clinical trial of humanized anti-IL-6R mAb is now under way.