Activation of protein tyrosine kinases and matrix metalloproteinases causes blood-brain barrier injury: Novel mechanism for neurodegeneration associated with alcohol abuse.
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Blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC) regulates the passage of molecules and leukocytes in and out of the brain. Activation of matrix metalloproteinases (MMPs) and alteration of basement membrane (BM) associated with BBB injury was documented in stroke patients. While chronic alcoholism is a risk factor for developing stroke, underlying mechanisms are not well understood. We hypothesized that ethanol (EtOH)-induced protein tyrosine kinase (PTK) signaling resulted a loss of BBB integrity via MMPs activation and degradation of BM component, collagen IV. Treatment of BMVEC with EtOH or acetaldehyde (AA) for 2-48 h increased MMP-1, -2 and -9 activities or decreased the levels of tissue inhibitors of MMPs (TIMP-1, -2) in a PTK-dependent manner without affecting protein tyrosine phosphatase activity. Enhanced PTK activity after EtOH exposure correlated with increased phosphorylated proteins of selective receptor and nonreceptor PTKs. Up-regulation of MMPs activities and protein contents paralleled a decrease in collagen IV content, and inhibitors of EtOH metabolism, MMP-2 and -9, or PTK reversed all these effects. Using human BMVEC assembled into BBB models, we found that EtOH/AA diminished barrier tightness, augmented permeability, and monocyte migration across the BBB via activation of PTKs and MMPs. These findings suggest that alcohol associated BBB injury could be mediated by MMPs via BM protein degradation and could serve as a comorbidity factor for neurological disorders like stroke or neuroinflammation. Furthermore, our preliminary experiments indicated that human astrocytes secreted high levels of MMP-1 and -9 following exposure to EtOH, suggesting the role of BM protein degradation and BBB compromise as a result of glial activation by ethanol. These results provide better understanding of multifaceted effects of alcohol on the brain and could help develop new therapeutic interventions. (+info)
Neutrophil interactions with keratocytes during corneal epithelial wound healing: a role for CD18 integrins.
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PURPOSE: To determine the role of keratocytes and leukocyte beta(2) (CD18) integrins in neutrophil (PMN) migration through the corneal stroma after epithelial scrape injury. METHODS: Using C57BL/6 wild-type and CD18(-/-) mice, corneas were excised at 6 hours (wild-type) or 24 hours (CD18(-/-)) after central corneal epithelial abrasion, time points determined previously to have similar levels of emigrated PMNs. Corneas were prepared for ultrastructural morphometric analysis of PMNs, keratocyte networks, and collagen. RESULTS: Transmission electron microscopy revealed intact keratocyte networks within the paralimbus that were morphometrically similar, regardless of epithelial injury or mouse genotype. Secondary to epithelial abrasion, extravasated PMNs within the paralimbus developed close contacts with keratocytes and collagen. In wild-type mice, 40% of the PMN surface was in contact with the keratocyte surface, and this value decreased to 10% in CD18(-/-) mice. PMN contact with collagen was similar in wild-type and CD18(-/-) mice, with approximately 50% of the PMN surface contacting the collagen fibrils. Since corneal edema resulting from scrape injury was similar, regardless of genotype and did not involve structural changes in collagen fibrils, these data favor a direct role for CD18 in mediating PMN contact with keratocytes. CONCLUSIONS: The data show that in response to epithelial scrape injury, PMN migration in the corneal stroma involves close contact between keratocytes and collagen. Although PMN-keratocyte contacts require CD18 integrins, contact with collagen is CD18 independent. Fundamentally, PMN migration along keratocyte networks constitutes the beginning of a new experimental concept for understanding leukocyte migration within the wounded cornea. (+info)
STAT1 signaling modulates HIV-1-induced inflammatory responses and leukocyte transmigration across the blood-brain barrier.
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The relationship among neuroinflammation, blood-brain barrier (BBB) dysfunction, and progressive HIV-1 infection as they affect the onset and development of neuroAIDS is incompletely understood. One possible link is signal transducers and activators of transcription (STATs) pathways. These respond to proinflammatory and regulatory factors and could affect neuroinflammatory responses induced from infected cells and disease-affected brain tissue. Our previous works demonstrated that HIV-1 activates pro-inflammatory and interferon-alpha-inducible genes in human brain microvascular endothelial cells (HBMECs) and that these genes are linked to the Janus kinase (JAK)/STAT pathway. We now demonstrate that HIV-1 activates STAT1, induces IL-6 expression, and diminishes expression of claudin-5, ZO-1, and ZO-2 in HBMECs. The STAT1 inhibitor, fludarabine, blocked HIV-1-induced IL-6, diminished HIV-1-induced claudin-5 and ZO-1 down-regulation, and blocked HIV-1- and IL-6-induced monocyte migration across a BBB model. Enhanced expression and activation of STAT1 and decreased claudin-5 were observed in microvessels from autopsied brains of patients with HIV-1-associated dementia. These data support the notion that STAT1 plays an integral role in HIV-1-induced BBB damage and is relevant to viral neuropathogenesis. Inhibition of STAT1 activation could provide a unique therapeutic strategy to attenuate HIV-1-induced BBB compromise and as such improve clinical outcomes. (+info)
Immunomodulation by alpha(1)-proteinase inhibitor: lack of chemotactic effects of recombinant human alpha(1)-proteinase inhibitor from yeast on human peripheral blood granulocytes.
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INTRODUCTION: Recombinant alpha(1)-proteinase inhibitor, clinically developed for inhalative augmentation therapy in patients with alpha(1)-proteinase inhibitor deficiency or cystic fibrosis, may directly contribute to leukocyte accumulation as it may function as a chemoattractant. The migratory effects of yeast-derived human recombinant alpha(1)-proteinase inhibitor on human peripheral blood neutrophils and eosinophils were therefore tested in vitro. MATERIALS AND METHODS: Human peripheral blood leukocytes were prepared from forearm venous blood and tested for migration toward various preparations of yeast-derived recombinant alpha(1)-proteinase inhibitor in modified Boyden-chamber micropore filter assays. RESULTS: No direct effects of yeast-derived recombinant human alpha(1)-proteinase inhibitor on in vitro migration of isolated neutrophils or eosinophils were seen. CONCLUSIONS: The lack of direct chemotactic effects of recombinant human alpha(1)-proteinase inhibitor despite anti-inflammatory effects in other biological activities of leukocytes may contribute to the preserved antibacterial defense mechanisms observed in patients under experimental augmentation therapy with inhaled alpha(1)-proteinase inhibitor. (+info)
C-terminal repeats of Clostridium difficile toxin A induce production of chemokine and adhesion molecules in endothelial cells and promote migration of leukocytes.
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The C-terminal repeating sequences of Clostridium difficile toxin A (designated ARU) are homologous to the carbohydrate-binding domain of streptococcal glucosyltransferases (GTFs) that were recently identified as potent modulins. To test the hypothesis that ARU might exert a similar biological activity on endothelial cells, recombinant ARU (rARU), which was noncytotoxic to cell cultures, was analyzed using human umbilical vein endothelial cells. The rARU could bind directly to endothelial cells in a serum- and calcium-dependent manner and induce the production of interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein 1 in a dose-dependent manner. An oligosaccharide binding assay indicated that rARU, but not GTFC, binds preferentially to Lewis antigens and 3'HSO3-containing oligosaccharides. Binding of rARU to human endothelial or intestinal cells correlated directly with the expression of Lewis Y antigen. Bound rARU directly activated mitogen-activated protein kinases and the NF-kappaB signaling pathway in endothelial cells to release biologically active chemokines and adhesion molecules that promoted migration in a transwell assay and the adherence of polymorphonuclear and mononuclear cells to the endothelial cells. These results suggest that ARU may bind to multiple carbohydrate motifs to exert its biological activity on human endothelial cells. (+info)
Infection of endothelial cells with virulent Rickettsia prowazekii increases the transmigration of leukocytes.
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Comparative study of the usefulness of the drug-induced lymphocyte stimulation test and the leukocyte migration test in drug allergies.
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In 133 patients suspected of hypersensitivity to drugs and 102 control patients without hypersensitivity to drugs, the identification of allergenic drugs was performed by the drug-induced lymphocyte stimulation test (DLST) and the leukocyte migration test (LMT) to compare their usefulness in identifying drug allergies. In the 133 subject patients, the positive rate was 24.8% on the DLST and 60.9% on the LMT (agreement rate; 77.4%); thus, the LMT showed a significantly higher positive rate than the DLST (p<0.000001, chi(2)-test). In the 102 control patients, the positive rates on the DLST and LMT were 6.9%. In addition, the LMT showed a higher positive rate than the DLST for many hypersensitivity symptoms such as skin eruptions and hepatic injury, and for many drug efficacy categories of the suspected drugs such as antibacterial drugs, etc. Furthermore, the positive rate of the DLST did not change when adjusted for the patients' serum and sex, while that of the LMT increased when adjusted for the patients' serum and was found to be higher in females than in males. Our findings indicate that the LMT may be more useful than the DLST in identifying the causative drug in drug allergies and that its interpretation is influenced by the patient's serum and sex. (+info)