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PubMed1998

MAP-kinase dependent induction of monocytic procoagulant activity by beta2-integrins.

beta2-Integrin adhesion molecules play crucial roles in monocyte transmigration and adherence to the inflamed extracellular matrix. While integrin engagement contributes to inflammatory cell activation, little is known about the precise signaling pathways that are important to integrin-dependent monocyte activation. We examined the role of tyrosine phosphorylation and extracellular-signal regulated kinase (ERK) activity in beta2-integrin signaling in monocytes. Cross-linking of the LFA-1 (CD11a/CD18) and MAC-1 (CD11b/CD18) integrins on the surface of THP-1 monocytic cells induced the accumulation of tyrosine phosphoproteins. As part of this signal both ERK-1 and ERK-2 are tyrosine phosphorylated. In vitro kinase assays documented an increase in ERK-2 activity following both LFA-1 and MAC-1 cross-linking. beta2-Integrin cross-linking also led to a marked increase in 4-h procoagulant activity (PCA) in THP-1 cells and purified human monocytes. Inhibition of tyrosine phosphorylation by genistein (10 microg/ml), or selective ERK inhibition with PD98059 (10 microM), was able to block the integrin-dependent induction of PCA in both THP-1 cells and human monocytes. Thus, beta2 integrin signaling in monocytic cells can flow through the tyrosine phosphorylation and activation of the ERK mitogen activated protein kinases, which is essential for the subsequent expression of tissue factor. These results suggest that the ERK proteins likely function to integrate various adhesion-dependent signals during the process of monocyte transmigration.

Blood Coagulation Factors↗
PubMed2002

Soluble interleukin 6 receptor (sIL-6R) mediates colonic tumor cell adherence to the vascular endothelium: a mechanism for metastatic initiation?

The mechanisms by which surgery increases metastatic proliferation remain poorly characterized, although endotoxin and immunocytes play a role. Recent evidence suggests that endothelial adherence of tumor cells may be important in the formation of metastases. Soluble receptors of interleukin-6 (sIL-6R) shed by activated neutrophils exert IL-6 effects on endothelial cells, which are unresponsive under normal circumstances. This study examined the hypothesis that sIL-6R released by surgical stress increases tumor cell adherence to the endothelium. Neutrophils (PMN) were stimulated with lipopolysaccharide, C-reactive protein (CRP), and tumor necrosis factor-alpha. Soluble IL-6R release was measured by enzyme-linked immunosorbent assay. Colonic tumor cells transfected with green fluorescent protein and endothelial cells were exposed to sIL-6R, and tumor cell adherence and transmigration were measured by fluorescence microscopy. Basal release of sIL-6R from PMN was 44.7 +/- 8.2 pg/ml at 60 min. This was significantly increased by endotoxin and CRP (131 +/- 16.8 and 84.1 +/- 5.3, respectively; both P < 0.05). However, tumor necrosis factor-alpha did not significantly alter sIL-6R release. Endothelial and tumor cell exposure to sIL-6R increased tumor cell adherence by 71.3% within 2 h but did not significantly increase transmigration, even at 6 h. Mediators of surgical stress induce neutrophil release of a soluble receptor for IL-6 that enhances colon cancer cell endothelial adherence. Since adherence to the endothelium is now considered to be a key event in metastatic genesis, these findings have important implications for colon cancer treatment strategies.

Cell Adhesion↗
PubMed1995

In vitro invasiveness of DU-145 human prostate carcinoma cells is modulated by EGF receptor-mediated signals.

Prostate carcinomas often present an autocrine stimulatory loop in which the transformed cells both express the EGF receptor (EGFR) and produce activating ligands (TGF alpha and EGF forms). Up-regulated EGFR signalling has been correlated with tumor progression in other human neoplasia; however, the cell behaviour which is promoted remains undefined. To determine whether an EGFR-induced response contributes to cell invasiveness, we transduced DU-145 human prostate carcinoma cells with either a full-length (WT) or a mitogenically-active but motility-deficient truncated (c'973) EGFR. The DU-145 Parental and two transgene sublines all produced EGFR and TGF alpha, but the transduced WT and c'973 EGFR underwent autocrine downregulation to a lesser degree, with more receptor remaining intact. DU-145 cells transduced with WT EGFR transmigrated a human amniotic basement membrane matrix (Amgel) to a greater extent than did Parental DU-145 cells (175 +/- 22%). Cells expressing the c'973 EGFR invaded through the Amgel only to about two thirds the extent of the Parental cells (62 +/- 23%). A monoclonal antibody which prevents ligand-induced activation of EGFR decreased the invasiveness of WT-expressing cells by half and Parental cells by a fifth, but had little effect on the invasiveness of c'973-expressing cells; with the result that in the presence of antibody, all three cell lines transmigrated the Amgel to the same extent. The different levels of invasiveness between the three sublines were independent of cell proliferation. These findings demonstrated that EGFR-mediated signals increase tumor cell invasiveness and suggested that domains in the carboxy-terminus are required to signal invasiveness. As an initial investigation into the mechanisms underlying the EGFR-mediated enhanced invasiveness, we determined whether these cells presented different collagenolytic activity, as the major constituents of Amgel are collagen types I and IV. All three sublines secreted easily detectable levels of gelatin-directed proteases and TIMP-1, with WT cells secreting equivalent or lower levels of proteases. The proteolytic balance in these cells did not correlate with invasiveness. These data suggest that the TGF alpha-EGFR autocrine loop promotes invasiveness and that this is accomplished by signalling cell properties other than differential secretion of collagenolytic activity.

Carcinoma↗
PubMed1991

Morphologic assessment of leukocyte-endothelial cell interactions in mesenteric venules subjected to ischemia and reperfusion.

Intravital microscopic studies of the mesenteric microcirculation have demonstrated that leukocyte adherence and emigration in postcapillary venules are a characteristic feature of tissues exposed to ischemia-reperfusion. The objectives of this study were to determine whether: (1) neutrophils are the predominant leukocytes that adhere and emigrate in postischemic mesenteric venules, and (2) leukocyte adherence and/or emigration are a prerequisite for reperfusion-induced increases in venular permeability. Leukocyte kinetics in cat mesenteric venules (25-35 microns diameter) were evaluated using both intravital microscopy and quantitative morphometry. The intestine and mesentery were exposed to 60 min of ischemia, followed by 60 min reperfusion. Some animals were pretreated with a monoclonal antibody (MoAb IB4) against the leukocyte adhesion glycoprotein, CD11/CD18. Vessels observed by intravital microscopy and adjacent venules of similar diameter were excised and processed for light (LM) and electron microscopy (EM). Horseradish peroxidase (HRP), administered intravenously, was used to assess vascular permeability by EM. By LM, the control (nonischemic) mesentery is sparsely populated by plasma cells, mast cells, and leukocytes; 30-50% of the resident population is neutrophils. Ischemia-reperfusion led to a significant increase in the number of extravascular cells, with neutrophils accounting for greater than 80% of the total cell population. Control and ischemic venules demonstrated no leakage of HRP into the interstitium. However, venules exposed to ischemia and reperfusion demonstrated HRP leakage between endothelial cells and into the surrounding interstitium; neutrophils were adherent to the luminal surface of the endothelium, transmigrating the vessel wall, and in the surrounding interstitium. Animals pretreated with MoAb IB4 presented the same cell profile as nonischemic controls, with no adherent or transmigrating neutrophils. However, some HRP leakage was noted following reperfusion in venules treated with MoAb IB4. The results of this study indicate that: (1) neutrophils are the predominate leukocytes that adhere and emigrate in postischemic venules, and (2) inhibition of leukocyte adhesion does not completely prevent the venular dysfunction associated with ischemia-reperfusion.

Animals↗
PubMed2004

Production of matrix metalloproteinase-9 by cord blood CD34+ cells and its role in migration.

The transmigration of hematopoietic progenitor cells is a crucial step in the homing of transplanted stem cells into bone marrow (BM) microenvironment; however, the molecular basis for this is not fully understood. Matrix metalloproteinases (MMPs), which are implicated in the migration of leukocytes, are important in degrading components of extracellular matrix molecules. In this study, using zymographic analysis and enzyme-linked immunosorbent assay (ELISA), we investigated the production of MMP-9 in CD34(+) cells from cord blood (CB) and BM, compared their spontaneous migration across a reconstituted basement membrane-coated filter in transwell, and studied the role of MMP-9 in the transmigration. Zymography and ELISA showed that MMP-9 is produced by freshly isolated CD34(+) stem/progenitor cells obtained from CB. CB CD34(+) cells showed significantly higher migrational capacity than BM CD34(+) cells ( p=0.008). Furthermore, the migrational ability of CB CD34(+) cells over the extracellular matrix (ECM) was significantly inhibited by the inhibitor of MMP, o-phenanthroline and anti-MMP-9 monoclonal antibody (73.3+/-11.8% and 37.5+/-10.4% inhibition, respectively). Our results strengthen the potential role of MMP-9 in the higher migrational capacity of CB CD34(+) cells, which may be beneficial to homing of these cells to the BM environment.

Basement Membrane↗
PubMed2006

Pravastatin in vivo reduces mononuclear cell migration through endothelial monolayers.

The objective was to evaluate pravastatin modulation on peripheral blood mononuclear cell (PBMC) migration across endothelial monolayers. Eleven hypercholesterolaemic patients were treated with pravastatin 20 mg/day. At baseline (T0), after 40 days (T40) and after 6 months (T 6 months) of treatment total serum cholesterol, low-density lipoprotein (LDL), high-density lipoprotein, triglycerides, C-reactive protein, as well as tumour necrosis factor-alpha (TNF-alpha) and metalloproteinases-9 plasma levels were evaluated. At the same time points the effect of pravastatin on migration of PBMCs through a monolayer of murine brain endothelial cells was studied both in basal conditions and after endothelial stimulation with recombinant mouse TNF-alpha 10 ng/ml for 24 h. Seven volunteers were used as healthy controls. Significant decreases in total cholesterol, LDL and triglycerides as well as inhibition of transmigration were observed. PBMCs transmigration in patients prior to pravastatin therapy was higher than in healthy controls. These results suggest that pravastatin could be of benefit in a spectrum of diseases characterised by extravasation of PBMCs into the central nervous system.

Aged↗
PubMed1995

Characterization of transendothelial chemotaxis of T lymphocytes.

We have adapted a chemotaxis assay using human umbilical vein endothelial cell (HUVEC) monolayers on microporous membranes for studying lymphocyte transendothelial chemotaxis in vitro. Supernatants of peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA) were identified as an excellent source of lymphocyte chemoattractant activity. The activity in PHA supernatant typically caused 2-6% of peripheral blood lymphocytes (PBL) to transmigrate compared to 0.1-0.3% to media control. Checkerboard analysis demonstrated that transmigration was directional and not attributable to random locomotion. Purified T lymphocytes also underwent transendothelial chemotaxis to PHA supernatant. Using monoclonal antibodies to several human adhesion receptors, we found that the interaction between LFA-1 and ICAM-1/ICAM-2 was more important for transendothelial lymphocyte chemotaxis than the interaction between VLA-4 and VCAM-1. A monoclonal antibody to the beta 1 integrin subunit inhibited chemotaxis more than antibodies to the VLA alpha 2, alpha 3, alpha 4, or alpha 5 subunits. The transendothelial assay was used to guide purification of the lymphocyte chemoattractant activity, which we reported previously to be monocyte chemoattractant protein-1 (MCP-1) (Carr et al., Proc. Natl. Acad. Sci. USA (1994) 91, 3652). The adhesion molecules required for chemotaxis to MCP-1 were similar to those with PHA supernatant. The use of HUVEC in the assay enhances the signal-to-background ratio of chemotaxis and provides a model that is physiologically relevant to lymphocyte emigration from the bloodstream into sites of inflammation.

Antibodies, Monoclonal↗
PubMed2005

Membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation.

Sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and certain signaling molecules segregate from bulk membrane lipids into lateral domains termed lipid rafts, which are often isolated based on their insolubility in cold nonionic detergents. During immunohistological studies of gangliosides, major sphingolipids of the brain, we found that cold Triton X-100 solubility is bidirectional, leading to histological redistribution from gray to white matter. When brain sections were treated with > or =0.25% Triton X-100 at 4 degrees C, ganglioside GD1a, which is normally enriched in gray matter and depleted in white matter, redistributed into white matter tracts. Incubation of brain sections from knockout mice lacking GD1a with wild-type sections in the presence of cold Triton X-100 resulted in GD1a redistribution from wild-type gray matter to knockout white matter. GM1, which is normally enriched in white matter, remained in white matter after cold detergent treatment and did not migrate to knockout mouse brain sections. However, when gray matter gangliosides were enzymatically converted into GM1 in situ, the newly formed GM1 transmigrated to knockout mouse brain sections in the presence of cold detergent. When purified GD1a was added to knockout mouse brain sections in the presence of cold Triton X-100, it preferentially incorporated into white matter tracts. These data demonstrate that brain white matter is a sink for gangliosides, which redistribute from gray matter in the presence of low concentrations of cold Triton X-100. A GPI-anchored protein, Thy-1, also transmigrated from wild-type to Thy-1 knockout mouse brain sections in the presence of detergent at 4 degrees C, although less efficiently than did gangliosides. These data raise technical challenges for using nonionic detergents in certain histological protocols and for isolation of lipid rafts from brain tissue.

Animals↗
PubMed2006

Granulocyte-based immune response against decellularized or glutaraldehyde cross-linked vascular tissue.

Supporting structures derived from biological tissue have been used in numerous tissue-engineering applications. This study focuses on the immune response of human leukocytes toward decellularized or glutaraldehyde (GA) cross-linked vascular tissue in vitro. Porcine and human pulmonary roots were sterilized with antibiotics, decellularized or cross-linked with GA. Proteins of the vascular tissue were extracted and the migratory response of human leukocytes toward protein extracts was examined using an in vitro migration chamber. Transmigrated leukocytes were counted and subsets (lymphocytes, monocytes, granulocytes) analyzed by flow cytometry. Decellularization significantly reduced the migration of monocytes compared to native porcine tissue. Although the proportion of transmigrating lymphocytes was much lower, decellularization again reduced the migratory response. Surprisingly, after decellularization granulocyte migration was still significantly higher than the negative control. Results comparable to those obtained with porcine material were found when human tissue was used for the experiments. Interestingly, migratory behavior toward extracts of GA-fixed porcine tissue was similar to that of decellularized specimens. We have shown that decellularization of vascular tissue reduces lymphocyte and monocyte recruitment comparable to cross-linking treatment. However, the migration of granulocytes, which are also known to be strongly involved in early inflammatory reactions, could be abolished neither by decellularization nor by fixation with GA.

Animals↗
PubMed2005

Astrocytes and microglia differentially regulate trafficking of lymphocyte subsets across brain endothelial cells.

Feline brain endothelial cells (BECs), astrocytes, and microglia were combined in different configurations in a cell culture insert system to assess the effect of different cell types on the trafficking of peripheral blood mononuclear cell (PBMC) subsets in response to feline immunodeficiency virus (FIV). The addition of astrocytes to BECs significantly increased the adherence of PBMCs. This increase in adherence was suppressed by microglia, whereas microglia alone had no effect on PBMC adherence. FIV exposure of the glial cells did not alter PBMC adherence as compared to same configurations with untreated cells. All PBMC subsets showed some level of trafficking across the endothelial cell layer. The level of trafficking of monocytes and B cells was significantly increased if astrocytes were present. The presence of microglia with the astrocytes reduced transmigration across all PBMC subsets. FIV exposure of astrocytes significantly increased the percentage of CD8 T cell transmigration from 24% to 64% of the total CD4 and CD8 numbers. The presence of microglia significantly reversed the preferential trafficking of CD8 cells in the presence of astrocytes. The results suggested that interaction between the triad of endothelial cells, astrocytes, and microglia played an important, but varying, role in the trafficking of different PBMC subsets. In general, astrocytes had a positive effect on trafficking of PBMCs, while microglia had a suppressive effect. Effects of FIV on trafficking were largely restricted to increases seen in CD8 T cells and monocytes.

Animals↗
PubMed2005

Transmission of Chlamydia pneumoniae infection from blood monocytes to vascular cells in a novel transendothelial migration model.

Chlamydia pneumoniae uses blood monocytes (PBMC) for systemic dissemination, persists in atherosclerotic lesions, and has been implicated in the pathogenesis of atherosclerosis. During transmigration in a newly developed transendothelial migration model (TEM) C. pneumoniae-infected PBMC spread their infection to endothelial cells. Transmigrated PBMC retained their infectivity and transmitted the pathogen to smooth muscle cells in the lower chamber of the TEM. Detection of chlamydial HSP60 mRNA proved pathogen viability and virulence. We conclude that PBMC can spread chlamydial infection to vascular wall cells and we suggest the TEM as a novel tool to analyze host-pathogen interactions in vascular chlamydial infections.

Arteriosclerosis↗
PubMed2006

Gelatinase B/matrix metalloproteinase-9 contributes to cellular infiltration in a murine model of zymosan peritonitis.

Murine zymosan-induced peritonitis represents a well-defined model of acute inflammation. However, the molecular mechanisms by which leukocytes degrade basement membranes during extravasation into the peritoneum are not clear. Gelatinase B (MMP-9) is thought to participate in cellular migration, yet its role in leukocyte transmigration through endothelia during inflammation remains controversial. The aim of the present study was to evaluate the role of MMP-9 in the cell influx during zymosan-induced experimental peritonitis. In zymosan-treated Balb/c mice MMP-9 and its natural inhibitor (tissue inhibitor of metalloproteinase 1 - TIMP-1) were present in the peritoneal fluid and plasma at the time of peritoneal neutrophil (polymorphonuclear leukocyte - PMN) infiltration and persisted there until the time of monocytes/macrophages influx. To probe the function of gelatinases, gelatinase B-deficient mice (MMP-9(-/-)) were used as well as Balb/c mice treated with cyclic CTTHWGFTLC (INH), a specific peptide inhibitor of gelatinases. The studies revealed that in either group of mice deprived of MMP-9 activity, PMN infiltration was impaired at the time of their maximal extravasation (6h) while tumor necrosis factor alpha (TNF-alpha), cytokine-induced neutrophil chemoattractant (KC) and interleukin 10 (IL-10) levels were not changed. At later stages (24 h post-zymosan) a significant increase in PMNs was observed in MMP-9(-/-) mice, but not in the inhibitor-treated mice, in comparison to their respective controls. Moreover, intraperitoneal (i.p.) injection of recombinant mouse pro-MMP-9 induced leukocyte accumulation in peritoneum. Collectively, the findings indicate that gelatinase B participates in leukocyte transmigration; however, its function can be compensated by other mechanisms.

Animals↗
PubMed2006

C5a mediates secretion and activation of matrix metalloproteinase 9 from human eosinophils and neutrophils.

Matrix metalloproteinase 9 (MMP-9) is a crucial proteinase, utilized by both eosinophils and neutrophils, that mediates transmigration through extracellular basement membranes. We have found that neutralization of MMP-9 by a monoclonal antibody or a chemical inhibitor blocked C5a dependent chemotaxis of these granulocytes in vitro. The levels of MMP-9 secreted by the action of C5a from eosinophils were about 50-fold lower than those from neutrophils, consistent with results from confocal microscopy, where the density of MMP-9 containing granules was fewer within eosinophils than in neutrophils. Zymography indicated gelatin degrading activity of the molecular size of pro MMP-9 in supernatants from eosinophils and neutrophils stimulated by C5a, with no evidence of proteolytic activation. Instead MMP-9 activation appeared oxidative, since inhibition of NADPH oxidase and nitric oxide synthase by DPI or L-NIL abrogated C5a-mediated chemotaxis through basement membranes. In keeping with this mode of activation, C5a, known as an agent of superoxide generation, was also found to induce secretion of nitric oxide from human eosinophils and rat granulocytes and monocytes. In conclusion C5a is an important mediator that brings about secretion and oxidative activation of MMP-9, a requisite protease for transmigration, from both eosinophils and neutrophils.

Animals↗
PubMed2006

Tissue inhibitor of metalloproteinase-2 inhibits T-cell infiltration and preserves pancreatic beta-cell function in an in vitro type 1 diabetes mellitus model.

Type 1 diabetes mellitus (T1DM) results from autoreactive T-cells that attack and destroy insulin producing pancreatic beta-cells. This knowledge has provided a framework for numerous efforts to prevent or mitigate T1DM at various stages of the disease. In this study, we utilized an organ culture model of type 1 diabetes to determine whether tissue inhibitors of metalloproteinases (TIMPs) could block T-cell migration into the pancreas and ultimately preserve beta-cell function. We measured T-cell repertoires, insulin secretion, and performed immunohistochemistry and confocal laser microscopy in order to evaluate the effect of TIMP-1, TIMP-2, and TIMP-3 on our in vitro T1DM organ culture model. TIMP-2 decreased T-cell transmigration and preserved insulin production in our T1DM organ culture model. Moreover, TIMP-2 inhibited transmigration of diabetogenic T-cells across an islet microvascular endothelial cell layer. Our findings suggest that TIMP-2 is effective at blocking infiltration of autoreactive T-cells into target pancreas tissue thereby preserving pancreatic beta-cell mass.

Animals↗
PubMed2004

Monitoring leukocyte traffic in vivo into human delayed-type hypersensitivity reaction.

Leukocyte traffic from blood to sites of inflammation has been elaborately studied in animal models and in vitro. However, to date, little understanding has accumulated on the process in humans in vivo. A noninvasive light confocal microscopy technique has enabled us to image leukocyte rolling, arrest and transmigrated cells in vivo in human tissues. In the current study, a delayed-type hypersensitivity (DTH) reaction was elicited in the lower lip of healthy, Bacille Calmette-Guerin (BCG)-vaccinated volunteers by injection of respective purified protein derivate (PPD), mimicking the classic cutaneous Mantoux reaction. Subjects were imaged with real-time confocal microscopy at baseline and 2, 4, 24 and 48 h after the injection. The number of rolling leukocytes did not increase significantly until at 48 h. Even then, rolling cells were seen in only a minority (23%) of blood vessels. The frequency of engaged blood vessels was, nevertheless, significantly greater than at baseline. As the inflammation generated with this challenge was mild, transmigrated leukocytes could be detected in the confocal microscopy only occasionally. Histology of biopsies taken immediately after imaging at 48 h showed a T cell-dominated leukocyte population at the site of the DTH reaction. We have thus developed a method to monitor noninvasively leukocyte traffic in vivo in human subjects during the classic inflammatory model of delayed-type hypersensitivity reaction.

BCG Vaccine↗
PubMed2006

Functional elements on SIRPalpha IgV domain mediate cell surface binding to CD47.

SIRPalpha and SIRPbeta1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPalpha with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPbeta1 shares highly homologous extracellular IgV structure with SIRPalpha, it does not bind to CD47. Here, we defined key amino acid residues exclusively expressing in the IgV domain of SIRPalpha, but not SIRPbeta1, which determine the extracellular binding interaction of SIRPalpha to CD47. These key residues include Gln67, a small hydrophobic amino acid (Ala or Val) at the 57th position and Met102. We found that Gln67 and Ala/Val57 are critical. Mutation of either of these residues abates SIRPalpha directly binding to CD47. Functional cell adhesion and leukocyte transmigration assays further demonstrated central roles of Gln67 and Ala/Val57 in SIRPalpha extracellular binding mediated cell interactions and cell migration. Another SIRPalpha-specific residue, Met102, appears to assist SIRPalpha IgV binding through Gln67 and Ala/Val57. An essential role of these amino acid residues in SIRPalpha binding to CD47 was further confirmed by introducing these residues into the SIRPbeta1 IgV domain, which dramatically converts SIRPbeta1 into a CD47-binding molecule. Our results thus revealed the molecular basis by which SIRPalpha binds to CD47 and shed new light into the structural mechanisms of SIRP isoform mediated distinctive extracellular interactions and cellular responses.

Amino Acid Sequence↗
PubMed2006

MH2 domain of Smad3 reduces HIV-1 Tat-induction of cytokine secretion.

HIV-1 infection of the central nervous system (CNS) is associated with dysregulation of several important cytokines and chemokines, which are involved in inflammatory process. Earlier studies ascribed a critical role for Tat, a potent viral transcription activator, in this process by enhancing the expression of several immunomodulators including TGFbeta and MCP-1. Investigation of signaling pathways which are controlled by these cytokines led to identification of MH2 domain of Smad3, the downstream activator of TGFbeta pathway, as a modulator of MCP-1 promoter activity. The level of MCP-1 is increased in AIDS patients with neurologic problems, through recruitment of inflammatory cells, which can contribute to neuropathogenesis of AIDS. Therefore, we attempted to investigate the effect of MH2 on expression of MCP-1 and other immunolmodulators in CNS cells. By employing an adenovirus expression vector, we demonstrated that MH2 can decrease the levels of Tat-induced activation of MCP-1 and several other cytokines and chemokines in astrocytic cells. In addition, we showed that MH2 significantly reduced the activity of cytokines produced by cultures of adenovirus-MH2 transduced cells as measured by the transmigration of human PBMC cells. Thus, MH2 domain of Smad3 is a potential agent that may be developed as an inhibitor for the cytokine-mediated inflammatory responses in the brain and may have the potential to prevent transmigration of HIV-1-infected monocytes across the blood brain barrier in AIDS patients.

AIDS Dementia Complex↗
PubMed2005

Transendothelial migration of ferric ion in FeCl3 injured murine common carotid artery.

INTRODUCTION/OBJECTIVES: Adventitial application of FeCl(3) causes endothelial injury, platelet aggregation, and a rapid onset of thrombus formation. The transmigration pathway of the ferric ion has not been definitively identified. Using a combination of TEM and X-ray elemental analysis, this study aims to elucidate the endothelial pathway of ferric ion migration in carotid artery. METHODS AND RESULTS: Vascular injury was induced by placing a Whatman #1 filter paper strip saturated with 10% FeCl(3) over the common carotid artery in male C57BL/6 mice for 3 min. After rinsing in saline, the mice were terminated at 10 or 30 min. The FeCL(3) exposed segments of the common carotid artery were dissected, and processed for TEM. Thrombus formation was observed in all cases. Endothelial and smooth muscle injuries were observed in segments of the vessel in direct contact with the oxidant. The endothelial injury ranged from minimal damage to total denudation. The basal endothelial surface adjacent to the internal elastic lamina showed accumulation of electron opaque vesicles. The membrane enclosed particles transmigrated across the endothelium and exocytosed into the lumen. The nature of the particles shown by STEM/EDS was rich in ferric ion. Elemental analysis also showed that some ferric oxide aggregates formed near the developing thrombus in the vascular lumen. CONCLUSION: Our results showed the ferric ion permeated the endothelial basal lamina before entering the arterial lumen via endocytic-exocytic pathway. This study provides an ultrastructural framework for future analysis of the adluminal and luminal injuries in this model.

Animals↗