Regulation of gelatinase B production in corneal cells is independent of autocrine IL-1alpha.
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PURPOSE: The matrix metalloproteinase gelatinase B is synthesized by cells at the leading edge of the corneal epithelium migrating to heal a wound. Recent data from the authors' laboratory suggest that excessive synthesis contributes to repair defects. The goal of the study reported here was to investigate mechanisms controlling gelatinase B production by corneal epithelial cells. METHODS: Freshly isolated cultures of corneal epithelial cells and early passage stromal fibroblasts from rabbit were used for these studies. RESULTS: In a previous study, it was found that the cytokine interleukin (IL)-1alpha is released into the culture medium of corneal epithelial cells more efficiently when they are plated at low density with limited cell-cell contact than when plated at high density. In this study, we show that production of gelatinase B by these cells is similarly affected by cell plating density. However, it is further demonstrated that these two events are not dependent on one another but occur in parallel: IL-1alpha does not regulate gelatinase B production (synthesis), nor was there evidence that any other secreted autocrine cytokine acts as mediator. Instead, our data suggest that gelatinase B production is downregulated directly by high cell density and indicate a connection to the level of protein kinase C activity. Nevertheless, the anticancer agent suramin, which blocks collagenase synthesis by interfering with autocrine cytokine-receptor interactions, still inhibits synthesis of gelatinase B. CONCLUSIONS: Unlike collagenase synthesis by corneal stromal fibroblasts, production (synthesis) of gelatinase B does not appear to be controlled by secreted autocrine cytokines but can still be inhibited by suramin. Suramin may make an effective therapeutic agent for controlling pathologic overproduction of gelatinase B in corneal ulcers. (+info)
Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation.
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Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFalpha). Release of soluble tie by tumor necrosis factor alpha (TNFalpha) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TNFalpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNFalpha-treated cells, and (3) inhibited by BB-24. Additionally, proliferation of endothelial cells in response to VEGF, but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease. (+info)
Protein kinase C mediates experimental colitis in the rat.
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Protein kinase C (PKC) plays an important role in the cell signal transduction of many physiological processes. In contrast to these physiological responses, increases in PKC activity have also been associated with inflammatory disease states, including ulcerative colitis. The objective of this study was to examine the role of PKC as a causative mediator in initiation of experimentally induced colitis in the rat. Colitis was induced in rats by intrarectal (0.6 ml) instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS; 75 mg/kg in 50% ethanol) or the PKC activator phorbol 12-myristate 13-acetate (PMA; 1.5-3.0 mg/kg in 20% ethanol). Gross and histological mucosal damage, mucosal neutrophil infiltration, mucosal PKC activity, and PKC protein content for PKC isoforms alpha, beta, delta, and epsilon were assessed 2 h to 14 days after an inflammatory challenge. Both PKC activity and mucosal injury increased significantly within 4 h of TNBS treatment. PKC activity was maximal at 7 days and declined at 14 days, whereas mucosal damage became maximal at 1 day and declined after 7 days. In contrast, neutrophil infiltration as assessed by myeloperoxidase activity only increased 12 h after TNBS treatment, became maximal 1 day after TNBS administration, and declined thereafter. PKCbeta, -delta, and -epsilon were increased in response to TNBS, whereas PKCalpha protein content was decreased. The PKC antagonists staurosporine and GF-109203X (25 ng/kg iv) reduced TNBS-induced changes in mucosal PKC activity and the degree of mucosal damage. In contrast, neutropenia induced by antineutrophil serum treatment did not significantly affect the degree of injury or mucosal PKC activity. Furthermore, activation of mucosal PKC activity with PMA also induced mucosal damage, which was also inhibited by pretreatment with a PKC antagonist. In conclusion, these results suggest that increases in PKC activity play a causative role in TNBS-induced colitis. The PKC-mediated response to TNBS does not appear to involve neutrophil infiltration. (+info)
Cyclooxygenase-2 expression is up-regulated in human pancreatic cancer.
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A large body of evidence suggests that cyclooxygenase-2 (COX-2) is important in gastrointestinal cancer. The purpose of this study was to determine whether COX-2 was expressed in adenocarcinoma of the human pancreas. Quantitative reverse transcription-PCR, immunoblotting, and immunohistochemistry were used to assess the expression of COX-2 in pancreatic tissue. Levels of COX-2 mRNA were increased by >60-fold in pancreatic cancer compared to adjacent nontumorous tissue. COX-2 protein was present in 9 of 10 cases of adenocarcinoma of the pancreas but was undetectable in nontumorous pancreatic tissue. Immunohistochemical analysis showed that COX-2 was expressed in malignant epithelial cells. In cultured human pancreatic cancer cells, levels of COX-2 mRNA and protein were induced by treatment with tumor-promoting phorbol esters. Taken together, these results suggest that COX-2 may be a target for the prevention or treatment of pancreatic cancer. (+info)
Antigen receptor-induced signal transduction imbalances associated with the negative selection of immature B cells.
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Signals transduced through the B cell Ag receptor (BCR) drive B cell development. However, BCR-induced responses are developmentally regulated; immature B cells are tolerized following antigenic exposure while mature B cells are triggered to proliferate and differentiate. This differential responsiveness allows for the negative selection of self-reactive immature B cells while simultaneously allowing for clonal expansion of mature B cells in response to foreign Ags. Intrinsic differences in BCR-induced signal transduction at various stages of development may account for this functional dichotomy. We had previously demonstrated that the BCR-induced proliferation of mature B cells is accompanied by an increase in intracellular calcium levels and polyphosphoinositide bis phosphate (PIP2) hydrolysis. In contrast, immature B cells that undergo BCR-induced apoptosis increase intracellular calcium in the relative absence of PIP2 hydrolysis. Since PIP2 hydrolysis leads to the generation of diacylglycerol, a cofactor for protein kinase C (PKC) activation, these data suggested that an "imbalance" in BCR-induced signal transduction resulting from a relative inability to activate PKC may play a role in the susceptibility of immature B cells to BCR-induced apoptosis. In support of this hypothesis, we demonstrate that PKC activation can rescue immature B cells from BCR-induced apoptosis. Furthermore, the susceptibility of immature B cells to BCR-induced apoptosis is recapitulated in mature B cells that are either PKC depleted or are stimulated in the presence of PKC inhibitors, suggesting that an uncoupling of PKC activation from BCR-induced signaling is responsible for the apoptotic response of immature B cells. (+info)
Affinity modulation of very late antigen-5 through phosphatidylinositol 3-kinase in mast cells.
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Adhesiveness of integrins is up-regulated rapidly by a number of molecules, including growth factors, cytokines, chemokines, and other cell surface receptors, through a mechanism termed inside-out signaling. The inside-out signaling pathways are thought to alter integrin affinity for ligand, or cell surface distribution of integrin by diffusion/clustering. However, it remains to be clarified whether any physiologically relevant agonists induce a rapid change in the affinity of beta1 integrins and how ligand-binding affinity is modulated upon stimulation. In this study, we reported that affinity of beta1 integrin very late Ag-5 (VLA-5) for fibronectin was rapidly increased in bone marrow-derived mast cells by Ag cross-linking of FcepsilonRI. Ligand-binding affinity of VLA-5 was also augmented by receptor tyrosine kinases when the phospholipase Cgamma-1/protein kinase C pathway was inhibited. Wortmannin suppressed induction of the high affinity state VLA-5 in either case. Conversely, introduction of a constitutively active p110 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) increased the binding affinity for fibronectin. Failure of a constitutively active Akt to stimulate adhesion suggested that the affinity modulation mechanisms mediated by PI 3-kinase are distinct from the mechanisms to control growth and apoptosis by PI 3-kinase. Taken together, our findings demonstrated that the increase of affinity of VLA-5 was induced by physiologically relevant stimuli and PI 3-kinase was a critical affinity modulator of VLA-5. (+info)
Selective regulation of cytokine induction by adenoviral gene transfer of IkappaBalpha into human macrophages: lipopolysaccharide-induced, but not zymosan-induced, proinflammatory cytokines are inhibited, but IL-10 is nuclear factor-kappaB independent.
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Macrophages are the major cytokine producers in chronic inflammatory diseases, but the biochemical pathways regulating cytokine production are poorly understood. This is because genetic tools to dissect signaling pathways cannot be used in macrophages because of difficulties in transfection. We have developed an adenoviral technique to achieve high efficiency gene delivery into macrophages and recently showed that spontaneous TNF-alpha production in rheumatoid arthritis joint cells, chiefly from macrophages, is 75% blocked by adenoviral transfer of IkappaBalpha. In this report we use the same adenovirus to investigate whether the production of a number of proinflammatory cytokines (e.g., TNF-alpha, IL-1beta, IL-6, and IL-8) from human macrophages depends on NF-kappaB. While the cytokine response to certain inducers, such as LPS, PMA, and UV light, is blocked by overexpression of IkappaBalpha, the response to zymosan is not. In contrast, anti-inflammatory mediators (IL-10 and IL-1 receptor antagonist) induced by LPS are only marginally inhibited by IkappaBalpha excess. These studies demonstrate several new points about macrophage cytokine production. First, there is heterogeneity of mechanisms regulating both the proinflammatory and anti-inflammatory cytokines within populations of a single cell type. In addition, the results confirm the utility of the adenoviral technique for functional analysis of cytokine induction. The results also confirm that there are autocrine and paracrine interactions regulating cytokine synthesis within a single cell type. The selectivity of NF-kappaB blockade for proinflammatory but not anti-inflammatory mediators indicates that in macrophages, NF-kappaB may be a good target for the treatment of chronic inflammatory diseases. (+info)
Endogenous platelet-activating factor is critically involved in effector functions of eosinophils stimulated with IL-5 or IgG.
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Eosinophil activation and subsequent release of inflammatory mediators are implicated in the pathophysiology of allergic diseases. Eosinophils are activated by various classes of secretagogues, such as cytokines (e.g., IL-5), lipid mediators (e.g., platelet-activating factor (PAF)), and Ig (e.g., immobilized IgG). However, do these agonists act directly on eosinophils or indirectly through the generation of intermediate active metabolites? We now report that endogenous PAF produced by activated eosinophils plays a critical role in eosinophil functions. Human eosinophils produced superoxide when stimulated with immobilized IgG, soluble IL-5, or PAF. Pretreating eosinophils with pertussis toxin abolished their responses to these stimuli, suggesting involvement of a metabolite(s) that acts on G proteins. Indeed, PAF was detected in supernatants from eosinophils stimulated with IgG or IL-5. Furthermore, structurally distinct PAF antagonists, including CV6209, hexanolamine PAF, and Y-24180 (israpafant), inhibited IgG- or IL-5-induced superoxide production and degranulation. Previous reports indicated that exogenous PAF stimulates eosinophil eicosanoid production through formation of lipid bodies. We found in this study that IgG or IL-5 also induces lipid body formation and subsequent leukotriene C4 production mediated by endogenous PAF. Finally, inhibition of cytosolic phospholipase A2, one of the key enzymes involved in PAF synthesis, attenuated both PAF production and effector functions of eosinophils. These findings suggest that endogenous PAF plays important roles in eosinophil functional responses to various exogenous stimuli, such as cytokines and Igs. Therefore, inhibition of PAF synthesis or action may be beneficial for the treatment of eosinophilic inflammation. (+info)