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Sean P Colgan

Publications and source records attributed to Sean P Colgan.

At least 37 records · Page 2Linked to original sources
PubMed2004

Endogenous adenosine produced during hypoxia attenuates neutrophil accumulation: coordination by extracellular nucleotide metabolism.

Hypoxia is a well-documented inflammatory stimulus and results in tissue polymorphonuclear leukocyte (PMN) accumulation. Likewise, increased tissue adenosine levels are commonly associated with hypoxia, and given the anti-inflammatory properties of adenosine, we hypothesized that adenosine production via adenine nucleotide metabolism at the vascular surface triggers an endogenous anti-inflammatory response during hypoxia. Initial in vitro studies indicated that endogenously generated adenosine, through activation of PMN adenosine A(2A) and A(2B) receptors, functions as an antiadhesive signal for PMN binding to microvascular endothelia. Intravascular nucleotides released by inflammatory cells undergo phosphohydrolysis via hypoxia-induced CD39 ectoapyrase (CD39 converts adenosine triphosphate/adenosine diphosphate [ATP/ADP] to adenosine monophosphate [AMP]) and CD73 ecto-5'-nucleotidase (CD73 converts AMP to adenosine). Extensions of our in vitro findings using cd39- and cd73-null animals revealed that extracellular adenosine produced through adenine nucleotide metabolism during hypoxia is a potent anti-inflammatory signal for PMNs in vivo. These findings identify CD39 and CD73 as critical control points for endogenous adenosine generation and implicate this pathway as an innate mechanism to attenuate excessive tissue PMN accumulation.

Adenosine↗
PubMed2004

Leukocyte adhesion during hypoxia is mediated by HIF-1-dependent induction of beta2 integrin gene expression.

Inflammatory responses are associated with significant changes in tissue metabolism. In particular, metabolic shifts during inflammation can result in significant tissue hypoxia, with resultant induction of hypoxia-responsive genes. Given this association, we hypothesized that leukocyte functional responses are influenced by hypoxia. Initial experiments revealed that exposure of the promonocytic cell line U937 to hypoxia resulted in increased adhesion to activated endothelia. Such increases were transcription-dependent and were blocked by antibodies directed against beta2, but not beta1, integrins. Analysis of beta2 integrin mRNA and protein in U937 cells revealed a 5- to 6-fold increase with hypoxia. Extension of this analysis to hypoxic human whole blood revealed prominent induction of beta2 integrin mRNA and protein ex vivo. Furthermore, murine beta2 integrin mRNA was found to be significantly induced during hypoxia in vivo. Subsequent studies identified a binding site for hypoxia-inducible factor 1 (HIF-1) in the CD18 gene. This gene encodes the subunit common to all four known types of beta2 integrin heterodimer. HIF-1 binding was demonstrated in vivo, and mutational analysis of the HIF-1 site within the CD18 promoter resulted in a loss of hypoxia inducibility. Taken together, these results demonstrate that hypoxia induces leukocyte beta2 integrin expression and function by transcriptional mechanisms dependent upon HIF-1.

Base Sequence↗
PubMed2004

CD1d function is regulated by microsomal triglyceride transfer protein.

CD1d is a major histocompatibility complex (MHC) class I-related molecule that functions in glycolipid antigen presentation to distinct subsets of T cells that express natural killer receptors and an invariant T-cell receptor-alpha chain (invariant NKT cells). The acquisition of glycolipid antigens by CD1d occurs, in part, in endosomes through the function of resident lipid transfer proteins, namely saposins. Here we show that microsomal triglyceride transfer protein (MTP), a protein that resides in the endoplasmic reticulum of hepatocytes and intestinal epithelial cells (IECs) and is essential for lipidation of apolipoprotein B, associates with CD1d in hepatocytes. Hepatocytes from animals in which Mttp (the gene encoding MTP) has been conditionally deleted, and IECs in which Mttp gene products have been silenced, are unable to activate invariant NKT cells. Conditional deletion of the Mttp gene in hepatocytes is associated with a redistribution of CD1d expression, and Mttp-deleted mice are resistant to immunopathologies associated with invariant NKT cell-mediated hepatitis and colitis. These studies indicate that the CD1d-regulating function of MTP in the endoplasmic reticulum is complementary to that of the saposins in endosomes in vivo.

Abetalipoproteinemia↗
PubMed2004

A molecular defect in intracellular lipid signaling in human neutrophils in localized aggressive periodontal tissue damage.

Host defense mechanisms are impaired in patients with congenital neutrophil (polymorphonuclear neutrophils (PMN)) defects. Impaired PMN chemotaxis is observed in localized aggressive periodontitis (LAP), a familial disorder characterized by destruction of the supporting structures of dentition. In the present studies, we sought evidence for molecular events underlying this aberrant human PMN phenotype. To this end, PMN transendothelial migration and superoxide anion generation were assessed with LAP patients and asymptomatic family members, as well as patients with other chronic mucosal inflammation. PMN from LAP patients showed decreased transmigration across vascular endothelial monolayers (18 +/- 12% of control, n = 4) and increased superoxide anion generation (358 +/- 37%, p = 0.003). Gene expression was analyzed using oligonucleotide microarrays and fluorescence-based kinetic PCR. cDNA microarray and kinetic-PCR analysis revealed diminished RNA expression of leukocyte-type diacylglycerol (DAG) kinase alpha in PMN from LAP patients (4.6 +/- 1.7 relative units, n = 6, p = 0.007) compared with asymptomatic individuals (51 +/- 27 relative units, n = 7). DAG kinase activity was monitored by DAG phosphorylation and individual DAG molecular species were quantified using liquid chromatography and tandem mass spectrometry-based lipidomics. DAG kinase activity was also significantly decreased (73 +/- 2%, p = 0.007) and correlated with increased accumulation of 1,2-diacyl-sn-3-glycerol substrates (p = 0.01). These results implicate defects in both PMN transendothelial migration and PMN DAG kinase alpha signaling as disordered functions in LAP. Moreover, they identify a potential molecular lesion in PMN signal transduction that may account for their aberrant responses and tissue destruction in this disease.

Adolescent↗
PubMed2004

Epithelial hypoxia-inducible factor-1 is protective in murine experimental colitis.

Mucosal epithelial cells are uniquely equipped to maintain barrier function even under adverse conditions. Previous studies have implicated hypoxia in mucosal tissue damage resulting from both acute and chronic inflammation. Given the importance of the transcriptional regulator hypoxia-inducible factor-1 (HIF-1) for adaptive hypoxia responses, we hypothesized that HIF-1 may serve as a barrier-protective element during mucosal inflammation. Initial studies of hapten-based murine colitis revealed extensive mucosal hypoxia and concomitant HIF-1 activation during colitis. To study this in more detail, we generated 2 mouse lines with intestinal epithelium-targeted expression of either mutant Hif1a (inability to form HIF-1) or mutant von Hippel-Lindau gene (Vhlh; constitutively active HIF-1). Studies of colitis in these mice revealed that decreased HIF-1 expression correlated with more severe clinical symptoms (mortality, weight loss, colon length), while increased HIF levels were protective in these parameters. Furthermore, colons with constitutive activation of HIF displayed increased expression levels of HIF-1-regulated barrier-protective genes (multidrug resistance gene-1, intestinal trefoil factor, CD73), resulting in attenuated loss of barrier during colitis in vivo. Taken together, these studies provide insight into tissue microenvironmental changes during model inflammatory bowel disease and identify HIF-1 as a critical factor for barrier protection during mucosal insult.

Animals↗
PubMed2003

Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators.

PGs and leukotrienes (LTs) mediate cardinal signs of inflammation; hence, their enzymes are targets of current anti-inflammatory therapies. Products of arachidonate 15-lipoxygenases (LO) types I and II display both beneficial roles, such as lipoxins (LXs) that stereoselectively signal counterregulation, as well as potential deleterious actions (i.e., nonspecific phospholipid degradation). In this study, we examined transgenic (TG) rabbits overexpressing 15-LO type I and their response to inflammatory challenge. Skin challenges with either LTB(4) or IL-8 showed that 15-LO TG rabbits give markedly reduced neutrophil (PMN) recruitment and plasma leakage at dermal sites with LTB(4). PMN from TG rabbits also exhibited a dramatic reduction in LTB(4)-stimulated granular mobilization that was not evident with peptide chemoattractants. Leukocytes from 15-LO TG rabbits gave enhanced LX production, underscoring differences in lipid mediator profiles compared with non-TG rabbits. Microbe-associated inflammation and leukocyte-mediated bone destruction were assessed by initiating acute periodontitis. 15-LO TG rabbits exhibited markedly reduced bone loss and local inflammation. Because enhanced LX production was associated with an increased anti-inflammatory status of 15-LO TG rabbits, a stable analog of 5S,6R,15S-trihydroxyeicosa-7E,9E,11Z,13E-tetraenoic acid (LXA(4)) was applied to the gingival crevice subject to periodontitis. Topical application with the 15-epi-16-phenoxy-para-fluoro-LXA(4) stable analog (ATLa) dramatically reduced leukocyte infiltration, ensuing bone loss as well as inflammation. These results indicate that overexpression of 15-LO type I and LXA(4) is associated with dampened PMN-mediated tissue degradation and bone loss, suggesting that enhanced anti-inflammation status is an active process. Moreover, they suggest that LXs can be targets for novel approaches to diseases, e.g., periodontitis and arthritis, where inflammation and bone destruction are features.

Administration, Topical↗
PubMed2003

Antiadhesive role of apical decay-accelerating factor (CD55) in human neutrophil transmigration across mucosal epithelia.

Neutrophil migration across mucosal epithelium during inflammatory episodes involves the precise orchestration of a number a cell surface molecules and signaling pathways. After successful migration to the apical epithelial surface, apically localized epithelial proteins may serve to retain PMN at the lumenal surface. At present, identification of apical epithelial ligands and their PMN counter-receptors remain elusive. Therefore, to define the existence of apical epithelial cell surface proteins involved in PMN-epithelial interactions, we screened a panel of antibodies directed against epithelial plasma membranes. This strategy identified one antibody (OE-1) that both localized to the apical cell membrane and significantly inhibited PMN transmigration across epithelial monolayers. Microsequence analysis revealed that OE-1 recognized human decay-accelerating factor (DAF, CD55). DAF is a highly glycosylated, 70-80-kD, glycosyl-phosphatidyinositol-linked protein that functions predominantly as an inhibitor of autologous complement lysis. DAF suppression experiments using antisense oligonucleotides or RNA interference revealed that DAF may function as an antiadhesive molecule promoting the release of PMN from the lumenal surface after transmigration. Similarly, peptides corresponding to the antigen recognition domain of OE-1 resulted in accumulation of PMN on the apical epithelial surface. The elucidation of DAF as an apical epithelial ligand for PMN provides a target for novel anti-inflammatory therapies directed at quelling unwanted inflammatory episodes.

Amino Acid Sequence↗
PubMed2003

Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B receptors.

Limited oxygen delivery to tissues (hypoxia) is common in a variety of disease states. A number of parallels exist between hypoxia and acute inflammation, including the observation that both influence vascular permeability. As such, we compared the functional influence of activated polymorphonuclear leukocytes (PMN) on normoxic and posthypoxic endothelial cells. Initial studies indicated that activated PMN preferentially promote endothelial barrier function in posthypoxic endothelial cells (>60% increase over normoxia). Extension of these findings identified at least one soluble mediator as extracellular adenosine triphosphate (ATP). Subsequent studies revealed that ATP is coordinately hydrolyzed to adenosine at the endothelial cell surface by hypoxia-induced CD39 and CD73 (>20-and >12-fold increase in mRNA, respectively). Studies in vitro and in cd39-null mice identified these surface ecto-enzymes as critical control points for posthypoxia-associated protection of vascular permeability. Furthermore, insight gained through microarray analysis revealed that the adenosine A2B receptor (AdoRA2B) is selectively up-regulated by hypoxia (>5-fold increase in mRNA), and that AdoRA2B antagonists effectively neutralize ATP-mediated changes in posthypoxic endothelial permeability. Taken together, these results demonstrate transcription coordination of adenine nucleotide and nucleoside signaling at the vascular interface during hypoxia.

Adenine Nucleotides↗
PubMed2003

Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 inhibit human neutrophil migration: comparisons between synthetic 15 epimers in chemotaxis and transmigration with microvessel endothelial cells and epithelial cells.

Lipoxins (LX) are bioactive eicosanoids that can be formed during cell to cell interactions in human tissues to self limit key responses in host defense and promote resolution. Aspirin treatment initiates biosynthesis of carbon 15 epimeric LXs, and both series of epimers (LX and aspirin-triggered 15-epi-LX) display counter-regulatory actions with neutrophils. In this study, we report that synthetic lipoxin A(4) (LXA(4)) and 15-epi-LXA(4) (i.e., 15(R)-LXA(4) or aspirin-triggered LXA(4)) are essentially equipotent in inhibiting human polymorphonuclear leukocytes (PMN) in vitro chemotaxis in response to leukotriene B(4), with the maximum inhibition ( approximately 50% reduction) obtained at 1 nM LXA(4). At higher concentrations, 15-epi-LXA(4) proved more potent than LXA(4) as its corresponding carboxyl methyl ester. Also, exposure of PMN to LXA(4) and 15-epi-LXA(4) markedly decreased PMN transmigration across both human microvessel endothelial and epithelial cells, where 15-epi-LXA(4) was more active than LXA(4) at "stopping" migration across epithelial cells. Differences in potency existed between LXA(4) and 15-epi-LXA(4) as their carboxyl methyl esters appear to arise from cell type-specific conversion of their respective carboxyl methyl esters to their corresponding carboxylates as monitored by liquid chromatography tandem mass spectrometry. Both synthetic LXA(4) and 15-epi-LXA(4) as free acids activate recombinant human LXA(4) receptor (ALXR) to regulate gene expression, whereas the corresponding methyl ester of LXA(4) proved to be a partial ALXR antagonist and did not effectively regulate gene expression. These results demonstrate the potent stereospecific actions shared by LXA(4) and 15-epi-LXA(4) for activating human ALXR-regulated gene expression and their ability to inhibit human PMN migration during PMN vascular as well as mucosal cell to cell interactions.

Aspirin↗
PubMed2003

Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia.

Phosphorylation-dependent ubiquitination combined with proteasomal degradation of transcriptional regulators is a recently appreciated mechanism for control of a number of inflammatory genes. Far less is known about the counterregulatory mechanisms that repress transcriptional activity in these pathways during resolution. Here, we investigated the transient nature of hypoxia-induced tumor necrosis factor (TNF)alpha in T84 cells, a process we have previously shown to involve phosphorylation-dependent degradation of the cAMP-response element-binding protein (CREB). Initial studies indicate hypoxia-induced TNFalpha to be a transient event, the resolution of which is associated with the appearance of a higher molecular weight modified form of CREB. Gene array analysis of mRNA derived from hypoxic cells identified a time-dependent induction of small ubiquitin-related modifier (SUMO)-1 mRNA. In prolonged hypoxia, CREB is posttranslationally modified by SUMO-1. Furthermore, SUMO-1 overexpression stabilizes CREB in hypoxia and enhances CREB-dependent reporter gene activity. Site-directed mutagenesis of lysine residues K285 and K304 identifies them as SUMO acceptors in vivo and in vitro. Mutation of K304 also results in loss of CREB nuclear localization, implying a role for SUMO-1 modification at this site in the subcellular localization of CREB. Thus, in prolonged hypoxia, CREB is modified by association with SUMO-1. Furthermore, we hypothesize that such an event stabilizes and promotes nuclear localization of CREB and thus complements an endogenous resolution phase for hypoxia-induced inflammatory processes.

Amino Acid Motifs↗
PubMed2003

Delineation of a CD1d-restricted antigen presentation pathway associated with human and mouse intestinal epithelial cells.

BACKGROUND & AIMS: CD1d, a major histocompatibility complex (MHC) class I-related molecule that is responsible for the presentation of glycolipid antigens to subsets of natural killer T (NK-T) cells, is expressed by intestinal epithelial cells (IECs). However, CD1d-restricted antigen presentation has not yet been examined on IECs. METHODS: A mouse intestinal epithelial cell line (MODE-K), a human epithelial cell line (T84), T84 cells transfected with CD1d and/or MHC class II, and freshly isolated human IECs were examined for their ability to present model glycolipid antigens to NK-T cells as defined by interleukin (IL)-2 or IL-4 secretion. RESULTS: MODE-K and freshly isolated human IECs exhibited dose-dependent, CD1d-restricted presentation of the functional glycolipid antigen, alpha-galactosylceramide (alpha GalCer), to the mouse NK-T cell hybridoma, DN32.D3. The human IEC line, T84, mainly presented alpha GalCer when transfected with human CD1d. Presentation of alpha GalCer by CD1d-transfected T84 cells (T84d) to DN32.D3 cells was greater along the basal surface in comparison with the apical surface. Induction of the MHC class II antigen presentation machinery by cotransfecting T84d with the MHC class I transactivator (CIITA) did not alter this polarity of presentation. Neither MODE-K nor T84 cells transfected with CD1d, CD1d plus CIITA, or CD1d plus HLA-DR were able to present glycolipid antigens requiring intracellular processing. The MODE-K cell line could also present alpha GalCer to primary mouse NK-T cells. CONCLUSIONS: CD1d is expressed functionally on IECs with a polarity of presentation (basal > apical) predicting a role in presentation of mucosal glycolipid antigens to local CD1d-restricted T cells.

Animals↗
PubMed2003

Intestinal heat shock protein 110 regulates expression of CD1d on intestinal epithelial cells.

CD1d is expressed on the surface of professional and nonprofessional APCs, including intestinal epithelial cells (IECs), for a role in the presentation of glycolipid-based antigens to subsets of T cells. The mechanisms that regulate CD1d expression in any cell type are unknown. To investigate the possibility that expression of CD1d is influenced by exogenous factors present within the intestinal lumen, CD1d expression was analyzed in several IEC lines after culturing in the presence of lumenal contents (LC) of the normal human intestine. Exposure of the colon-derived cell lines T84, HT-29, and Caco-2 to soluble LC resulted in a marked induction of CD1d expression as determined by RT-PCR, confocal microscopy, cell surface ELISA, and Western blot analysis. Similarly, exposure of human IECs to LC isolated from mice bred in both specific pathogen-free and germfree conditions also resulted in the induction of CD1d expression, with the maximum CD1d-inducing activity observed in the small intestine. Biochemical and biophysical characterization of the human CD1d-inducing activity identified heat shock protein 110 (Hsp110) as a major functional component of the LC that contributes to CD1d surface regulation, and immunolocalization studies revealed Hsp110 expression in subsets of human IECs in vivo. These data support the presence of a novel autocrine pathway of CD1d regulation by Hsp110.

Amino Acid Sequence↗
PubMed2002

An aspirin-triggered lipoxin A4 stable analog displays a unique topical anti-inflammatory profile.

Lipoxins and 15-epi-lipoxins are counter-regulatory lipid mediators that modulate leukocyte trafficking and promote the resolution of inflammation. To assess the potential of lipoxins as novel anti-inflammatory agents, a stable 15-epi-lipoxin A(4) analog, 15-epi-16-p-fluorophenoxy-lipoxin A(4) methyl ester (ATLa), was synthesized by total organic synthesis and examined for efficacy relative to a potent leukotriene B(4) (LTB(4)) receptor antagonist (LTB(4)R-Ant) and the clinically used topical glucocorticoid methylprednisolone aceponate. In vitro, ATLa was 100-fold more potent than LTB(4)R-Ant for inhibiting neutrophil chemotaxis and trans-epithelial cell migration induced by fMLP, but was approximately 10-fold less potent than the LTB(4)R-Ant in blocking responses to LTB(4). A broad panel of cutaneous inflammation models that display pathological aspects of psoriasis, atopic dermatitis, and allergic contact dermatitis was used to directly compare the topical efficacy of ATLa with that of LTB(4)R-Ant and methylprednisolone aceponate. ATLa was efficacious in all models tested: LTB(4)/Iloprost-, calcium ionophore-, croton oil-, and mezerein-induced inflammation and trimellitic anhydride-induced allergic delayed-type hypersensitivity. ATLa was efficacious in mouse and guinea pig skin inflammation models, exhibiting dose-dependent effects on edema, neutrophil or eosinophil infiltration, and epidermal hyperproliferation. We conclude that the LXA(4) and aspirin-triggered LXA(4) pathways play key anti-inflammatory roles in vivo. Moreover, these results suggest that ATLa and related LXA(4) analogs may have broad therapeutic potential in inflammatory disorders and could provide an alternative to corticosteroids in certain clinical settings.

Administration, Cutaneous↗
PubMed2002

Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals.

Aspirin (ASA) is unique among current therapies because it acetylates cyclooxygenase (COX)-2 enabling the biosynthesis of R-containing precursors of endogenous antiinflammatory mediators. Here, we report that lipidomic analysis of exudates obtained in the resolution phase from mice treated with ASA and docosahexaenoic acid (DHA) (C22:6) produce a novel family of bioactive 17R-hydroxy-containing di- and tri-hydroxy-docosanoids termed resolvins. Murine brain treated with aspirin produced endogenous 17R-hydroxydocosahexaenoic acid as did human microglial cells. Human COX-2 converted DHA to 13-hydroxy-DHA that switched with ASA to 17R-HDHA that also proved a major route in hypoxic endothelial cells. Human neutrophils transformed COX-2-ASA-derived 17R-hydroxy-DHA into two sets of novel di- and trihydroxy products; one initiated via oxygenation at carbon 7 and the other at carbon 4. These compounds inhibited (IC(50) approximately 50 pM) microglial cell cytokine expression and in vivo dermal inflammation and peritonitis at ng doses, reducing 40-80% leukocytic exudates. These results indicate that exudates, vascular, leukocytes and neural cells treated with aspirin convert DHA to novel 17R-hydroxy series of docosanoids that are potent regulators. These biosynthetic pathways utilize omega-3 DHA and EPA during multicellular events in resolution to produce a family of protective compounds, i.e., resolvins, that enhance proresolution status.

Animals↗
PubMed2002

Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene.

The microenvironment of rapidly growing tumors is associated with increased energy demand and diminished vascular supply, resulting in focal areas of prominent hypoxia. A number of hypoxia-responsive genes have been associated with growing tumors, and here we demonstrate that the multidrug resistance (MDR1) gene product P-glycoprotein, a Mr approximately 170,000 transmembrane protein associated with tumor resistance to chemotherapeutics, is induced by ambient hypoxia. Initial studies using quantitative microarray analysis of RNA revealed an approximately 7-fold increase in MDR in epithelial cells exposed to hypoxia (pO(2) 20 torr, 18 h). These findings were further confirmed at the mRNA and protein level. P-Glycoprotein function was studied by analysis of verapamil-inhibitable efflux of digoxin and rhodamine 123 in intact T84 cells and revealed that hypoxia enhances P-glycoprotein function by as much as 7 +/- 0.4-fold over normoxia. Subsequent studies confirmed hypoxia-elicited MDR1 gene induction and increased P-glycoprotein expression in nontransformed, primary cultures of human microvascular endothelial cells, and analysis of multicellular spheroids subjected to hypoxia revealed increased resistance to doxorubicin. Examination of the MDR1 gene identified a binding site for hypoxia inducible factor-1 (HIF-1), and inhibition of HIF-1 expression by antisense oligonucleotides resulted in significant inhibition of hypoxia-inducible MDR1 expression and a nearly complete loss of basal MDR1 expression. Studies using luciferase promoter constructs revealed a significant increase in activity in cells subjected to hypoxia, and such hypoxia inducibility was lost in truncated constructs lacking the HIF-1 site and in HIF-1 binding site mutants. Extensions of these studies also identified a role for Sp1 in this hypoxia response. Taken together, these data indicate that the MDR1 gene is hypoxia responsive, and such results may identify hypoxia-elicited P-glycoprotein expression as a pathway for resistance of some tumors to chemotherapeutics.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Lipid mediator-induced expression of bactericidal/ permeability-increasing protein (BPI) in human mucosal epithelia.

Epithelial cells which line mucosal surfaces are the first line of defense against bacterial invasion and infection. Recent studies have also indicated that epithelial cells contribute significantly to the orchestration of ongoing inflammatory processes. Here, we demonstrate that human epithelial cells express bactericidal/permeability-increasing protein (BPI), an antibacterial and endotoxin-neutralizing molecule previously associated with neutrophils. Moreover, we demonstrate that such BPI expression is transcriptionally regulated by analogs of endogenously occurring anti-inflammatory eicosanoids (aspirin-triggered lipoxins, ATLa). Initial studies to verify microarray analysis revealed that epithelial cells of wide origin (oral, pulmonary, and gastrointestinal mucosa) express BPI and each is similarly regulated by aspirin-triggered lipoxins. Studies aimed at localization of BPI revealed that such expression occurs on the cell surface of cultured epithelial cell lines and dominantly localizes to epithelia in human mucosal tissue. Functional studies employing a BPI-neutralizing anti-serum revealed that surface BPI blocks endotoxin-mediated signaling in epithelia and kills Salmonella typhimurium. These studies identify a previously unappreciated "molecular shield" for protection of mucosal surfaces against Gram-negative bacteria and their endotoxin.

Anti-Bacterial Agents↗
PubMed2002

Neutrophil-derived glutamate regulates vascular endothelial barrier function.

Endothelial barrier function is altered by the release of soluble polymorphonuclear leukocyte (PMN)-derived mediators during inflammatory states. However, endogenous pathways to describe such changes are only recently appreciated. Using an in vitro endothelial paracellular permeability model, cell-free supernatants from formylmethionylleucylphenylalanine-stimulated PMNs were observed to significantly alter endothelial permeability. Biophysical and biochemical analysis of PMN supernatants identified PMN-derived glutamate in modulating endothelial permeability. Furthermore, novel expression of metabotropic glutamate receptor 1 (mGluR1), mGluR4, and mGluR5 by human brain and dermal microvascular endothelial cells was demonstrated by reverse transcription-PCR, in situ hybridization, immunofluorescence, and Western blot analysis. Treatment of human brain endothelia with glutamate or selective, mGluR group I or III agonists resulted in a time-dependent loss of phosphorylated vasodilator-stimulated phosphoprotein (VASP) and significantly increased endothelial permeability. Glutamate-induced decreases in brain endothelial barrier function and phosphorylated VASP were significantly attenuated by pretreatment of human brain endothelia with selective mGluR antagonists. These observations were extended to an in vivo hypoxic mouse model in which pretreatment with mGluR antagonists significantly decreased fluorescein isothiocyanate-dextran flux across the blood-brain barrier. We conclude that activated human PMNs release glutamate and that endothelial expression of group I or III mGluRs function to decrease human brain endothelial VASP phosphorylation and barrier function. These results identify a novel pathway by which PMN-derived glutamate may regulate human endothelial barrier function.

Base Sequence↗