Soluble IL-6 receptor induces calcium flux and selectively modulates chemokine expression in human dermal fibroblasts.
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Truncated forms of cytokine receptors have been regarded as modulators of the activity of their cognate ligands. In addition to inhibiting effects of their respective ligands, soluble receptors can also facilitate ligand-mediated signaling. Several studies have demonstrated that exogenous IL-6 in association with the soluble IL-6 receptor alpha (sIL-6Ralpha) can activate cells expressing the gp130 signal transducer lacking the specific, membrane-bound IL-6Ralpha. Since cell cultures of human dermal fibroblasts express high amounts of IL-6, we examined whether the addition of sIL-6Ralpha in association with endogenous IL-6 would be sufficient to stimulate these cells via gp130. As an early rapid signal we analyzed changes in intracellular free calcium concentrations ([Ca2+]i). Addition of sIL-6Ralpha induced an acute and transient increase in cytosolic free calcium concentrations in a dose-dependent fashion. This Ca2+-signal was abolished when cells were pretreated with anti-IL-6 or anti-gp130 antibodies. Using flow cytometric analysis we could demonstrate membrane-associated IL-6 and gp130, but not IL-6Ralpha on fibroblasts. We also analyzed MCP-1 and IL-8 expression as a response involved in the more recently recognized chemoattractant functions of fibroblasts, and found MCP-1 to be up-regulated, but not IL-8. These data suggest that sIL-6Ralpha binds to cell-associated, endogenous IL-6 produced by fibroblasts and this complex then activates the cells via gp130. This pathway of fibroblast activation by sIL-6Ralpha adds another dimension to the role of fibroblasts in the cytokine network. (+info)
Leptin induces oxidative stress in human endothelial cells.
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Human umbilical vein endothelial cells (HUVEC) express functional receptors to leptin, the product of the ob gene. As human obesity is associated with atherosclerosis and hyperleptinemia, we investigated whether leptin, in addition to its angiogenic properties, exerts atherogenic effects through the generation of oxidative stress in endothelial cells. In HUVEC leptin increased the accumulation of reactive oxygen species (ROS), as assessed by the oxidation of 2', 7'- dichlorodihydrofluorescein, in a time- and concentration-dependent manner. In addition, leptin activated the NH2-terminal c-Jun kinase/stress-activated protein kinase pathway as demonstrated by enhanced JNK activity and AP-1 DNA binding. Both effects were sensitive to antioxidant treatment with N-acetylcysteine. NF-kappaB, another redox-sensitive transcription factor, was also activated by leptin stimulation in an oxidant-dependent manner. Finally, activation of both AP-1 and NF-kappaB was associated with an enhanced expression of the monocyte chemoattractant protein-1 in HUVEC. These findings demonstrate that ROS are second messengers involved in leptin-induced signaling in endothelial cells. Thus, chronic oxidative stress in endothelial cells under hyperleptinemia may activate atherogenic processes and contribute to the development of vascular pathology. (+info)
The significance of measuring monocyte tissue factor activity in patients with breast and colorectal cancer.
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Monocytes express tissue factor (mTF) in several conditions including cancer where levels may be valuable in assessing tumour presence and progression. Using a two-stage kinetic chromogenic assay (KCA), mTF levels were measured in controls [normal subjects (n = 60) and patients undergoing hernia repair or cholecystectomy (n = 60)], in patients with benign and malignant disease of the breast (n = 83) and of the large bowel (n = 62). This was performed under fresh (resting) conditions and after incubation for 6 h without (unstimulated) and with (stimulated) Escherichia coli endotoxin. The malignant groups showed higher mTF levels than each of the three controls for resting (P < 0.05 breast, P < 0.05 colorectal) unstimulated (P < 0.05 breast, P < 0.05 colorectal) and stimulated cells (P < 0.001 breast, P < 0.01 colorectal). Similarly, the benign inflammatory groups had higher mTF levels than controls for resting (P < 0.05 colorectal), unstimulated (P < 0.05 colorectal) and stimulated cells (P < 0.01 breast, P < 0.01 colorectal). There was no significant difference between malignant and benign inflammatory groups in each organ. mTF levels showed an increase corresponding to that of histological tumour progression and were higher in non-surviving patients. In conclusion, mTF levels are raised in malignant and inflammatory disease compared to controls and patients with non-inflammatory conditions. Stimulated cells give better discrimination between the groups and may be of value in identifying high risk individuals. mTF levels showed an association with tumour grade or stage and the patients' survival time. (+info)
Inhibition of prostaglandin E2 synthesis in abdominal aortic aneurysms: implications for smooth muscle cell viability, inflammatory processes, and the expansion of abdominal aortic aneurysms.
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BACKGROUND: There is no treatment proven to limit the growth of abdominal aortic aneurysms, in which the histological hallmarks include inflammation and medial atrophy, with apoptosis of smooth muscle cells and destruction of elastin. METHODS AND RESULTS: Aneurysm biopsies were used for explant cultures, the preparation of smooth muscle cell cultures, and isolation of macrophages. Tissue macrophages stained strongly for cyclooxygenase 2. Prostaglandin E2 (PGE2) concentrations in aneurysm tissue homogenates, conditioned medium from explants, and isolated macrophages were 49+/-22 ng/g, 319+/-38 ng/mL, and 22+/-21 ng/mL, respectively. PGE2 inhibited DNA synthesis and proliferation in normal aortic smooth muscle cells (IC50, 23.2+/-3.8 and 23.6+/-4.5 ng/mL, respectively). In smooth muscle cells derived from aneurysmal aorta, PGE2 also caused cell death, with generation of oligonucleosomes. Conditioned medium from the mixed smooth muscle and monocyte cultures derived from explants also had potent growth-inhibitory effects, and fractionation of this medium showed that the growth-inhibitory molecule(s) coeluted with PGE2. In explants, indomethacin 10 micromol/L or mefenamic acid 10 micromol/L abolished PGE2 secretion and significantly reduced IL-1beta and IL-6 secretion. In a separate case-control study, the expansion of abdominal aortic aneurysms was compared in 15 patients taking nonsteroidal anti-inflammatory drugs and 63 control subjects; median growth rates were 1.5 and 3.2 mm/y, respectively, P=0.001. CONCLUSIONS: The adverse effects of PGE2 on aortic smooth muscle cell viability and cytokine secretion in vitro and the apparent effect of anti-inflammatory drugs to lower aneurysm growth rates suggest that selective inhibition of PGE2 synthesis could be an effective treatment to curtail aneurysm expansion. (+info)
Interferon-gamma (IFN-gamma)-dependent protection and synthesis of chemoattractants for mononuclear leucocytes caused by IL-12 in the lungs of mice infected with Cryptococcus neoformans.
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We have recently demonstrated that IL-12 induced cellular inflammatory responses consisting mainly of accumulation of mononuclear leucocytes in the lungs of mice infected with Cryptococcus neoformans and protected mice against fulminant infection. We examined the involvement of endogenously synthesized IFN-gamma in such a response by investigating the effects of a neutralizing monoclonal antibody against this cytokine. The latter treatment completely abrogated the positive effects of IL-12 on survival of infected mice and prevented IL-12-induced elimination of microbials from the lungs. Histopathological examination showed that accumulation of mononuclear leucocytes in the infected lungs caused by IL-12 was clearly inhibited by anti-IFN-gamma MoAb. We also examined the local production of mononuclear cell-attracting chemokines such as monocyte chemotactic protein-1 (MCP-1), regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta and IFN-gamma-inducible protein 10 (IP-10) in the lungs using a reverse transcriptase-polymerase chain reaction (RT-PCR) method. We found that these chemokines were not synthesized in the infected lungs, while IL-12 treatment markedly induced their production. Interestingly, neutralizing anti-IFN-gamma MoAb strongly suppressed IL-12-induced production of these chemokines. Similar results were obtained with MCP-1 and MIP-1alpha when their synthesis was measured at the protein level using respective ELISA kits. Our results indicate that IFN-gamma plays a central role in the protective effects of IL-12 by inducing mononuclear leucocyte-attracting chemokines and cellular inflammatory responses. (+info)
Augmented production of chemokines (monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta) in patients with systemic sclerosis: MCP-1 and MIP-1alpha may be involved in the development of pulmonary fibrosis.
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To determine the role of chemokines in the pathogenesis of systemic sclerosis (SSc), we examined serum levels, spontaneous production by peripheral blood mononuclear cells (PBMC), and histological distribution in the affected skin, of MCP-1, MIP-1alpha and MIP-1beta in SSc patients. Serum levels of these chemokines were examined by ELISA in 58 patients with SSc and 20 normal controls. The levels of these chemokines in culture supernatants from PBMC were also measured by ELISA. Serum levels and spontaneous production levels by PBMC of MCP-1, MIP-1alpha, and MIP-1beta were significantly elevated in patients with SSc compared with normal controls. Elevated serum levels of MCP-1 and MIP-1alpha significantly correlated with the presence of pulmonary fibrosis. MCP-1 expression in the skin of SSc was immunohistochemically examined using anti-MCP-1 MoAb. MCP-1 was strongly expressed in the epidermis, inflammatory mononuclear cells, and vascular endothelial cells in the sclerotic skin of SSc patients, but not expressed in any control skin. Furthermore, the MCP-1 expression in inflammatory mononuclear cells and endothelial cells significantly correlated with earlier onset of SSc. Thus, MCP-1, MIP-1alpha and MIP-1beta may be involved in the disease process, possibly by augmenting leucocyte migration into the affected tissues in SSc. Furthermore, MCP-1 and MIP-1alpha may play an important role in the development of pulmonary fibrosis in SSc. (+info)
Induction of CC chemokines in human peripheral blood mononuclear cells by staphylococcal exotoxins and its prevention by pentoxifylline.
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We investigated the inflammatory processes that might be associated with the arthrogenic activity of Staphylococcus aureus, the principal causative agent of bacterial arthritis. Human peripheral blood mononuclear cells (PBMC) were stimulated with the staphylococcal toxic shock syndrome toxin-1 (TSST-1) or enterotoxin B (SEB) and the production of chemokines was examined. Both TSST-1 and SEB induced high levels (ng/mL) of MIP-1alpha, MIP-1beta, and MCP-1. The induction of these chemokines occurred mostly by direct stimulation of PBMC with staphylococcal exotoxins (SE), without requiring the intervention of IL-1 and TNF-alpha. The production of SE-induced chemokines was blocked partially by anti-DR and anti-CD2 antibodies. Cell separation revealed monocytes as the cell source of these chemokines. However, addition of purified T cells amplified the levels of chemokine produced, suggesting that cognate interaction of SE bound on antigen-presenting cells with T cells also contributes to chemokine production. The activation and recruitment of leukocytes by these chemokines may contribute to the pathophysiology of septic arthritis caused by staphylococci in humans through tissue injury and the recruitment of T lymphocytes, perhaps also initiating autoimmune responses. Pentoxifylline, an anti-inflammatory agent, completely inhibited the production of these chemokines. (+info)
Regulation of monocyte chemotactic protein-1 expression in human endometrial stromal cells by integrin-dependent cell adhesion.
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Shed menstrual endometrium is viable and has the ability to implant and grow in women, who eventually develop endometriosis. Many of the cell-to-cell or cell-to-extracellular matrix (ECM) connections are mediated by integrins. Monocyte chemotactic protein (MCP)-1, a potent chemotactic factor produced in many cell types, is elevated in the peritoneal fluid of women with endometriosis. In this study, we investigated whether endometrial stromal cell (ESC) adhesion itself induces the expression of MCP-1 and whether this process is integrin mediated. ESC were plated on Petri dishes and 24-well plates coated with fibronectin, laminin, collagen IV, poly-L-lysine, or mouse anti-human integrin beta(1) and beta(2) monoclonal antibodies. Adherence of ESC to various ECM substrates, except for poly-L-lysine, a non-integrin-dependent adhesion matrix, induced the expression of MCP-1 at both mRNA and protein levels. Engagement of beta(1)-containing integrins was associated with ESC adhesion and resulted in up-regulation of MCP-1 gene expression and protein secretion. Disruption of the actin cytoskeleton by treating ESC with cytochalasin D completely blocked the increase of MCP-1 induced in response to integrin activation. These findings indicate a novel mechanism of MCP-1 regulation. Cell adhesion to ECM is an important event that leads to stimulation of MCP-1 expression, and this process is mediated by integrins. (+info)