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Biomedical subjects

S P Colgan

Publications and source records attributed to S P Colgan.

At least 19 recordsLinked to original sources
PubMed2003

Expression of BPI (bactericidal/permeability-increasing protein) in human mucosal epithelia.

Among the antimicrobial proteins and peptides of humans is the cationic 55 kDa bactericidal/permeability-increasing protein (BPI), which possesses antibacterial, endotoxin-neutralizing and opsonic activity against Gram-negative bacteria. Although identified originally as an abundant constituent of neutrophil granules, we have recently identified functional expression of BPI by human mucosal epithelia. BPI expression was markedly up-regulated by exposure of epithelia to lipoxins, endogenous anti-inflammatory eicosanoids that are generated in vivo in the context of aspirin treatment (aspirin-triggered lipoxins). Epithelial BPI was found to be surface expressed and fully functional, as measured by antibacterial activity against Salmonella typhimurium as well as lipopolysaccharide (LPS; endotoxin)-neutralizing activity. These results suggest a role for BPI as an effector of epithelial antibacterial activity and as a modulator of epithelial responses to LPS. Both BPI and the lipoxins are currently the subject of intensive biopharmaceutical development, raising the possibility that therapeutic use of BPI or modulation of epithelial BPI expression may be a useful adjunctive therapy for conditions in which epithelial inflammation is associated with Gram-negative infections and/or endotoxin.

Antimicrobial Cationic Peptides↗
PubMed2003

Implications of hypoxia on mucosal barrier function.

Epithelial cells which line mucosal surfaces (e.g. lung, intestine) critically function as a semi-permeable barrier to the outside world. Mucosal organs are highly vascular with extensive metabolic demands, and for this reason, are particularly susceptible to diminished blood flow and resultant tissue hypoxia. Recent work from a number of groups have defined the critical molecular and cellular determinants of barrier function in hypoxic/ischemic tissues. Here, we will briefly highlight some of these studies from both a basic and clinical viewpoint and provide a perspective on future work related to tigh tjunction function in mucosal hypoxia.

Adherens Junctions↗
PubMed2002

Lipid mediators in epithelial cell-cell interactions.

Epithelial cells which line mucosal surfaces (e.g. lung, intestine) play a central role in the coordination of the inflammatory response. In both the healthy and diseased mucosa, epithelia lie anatomically positioned in close proximity to a number of other cell types, including leukocytes, fibroblasts, smooth muscle cells and vascular endothelia. This complex architecture supports a unique microenvironment for biochemical cell-cell crosstalk. Our previous studies and work by others have elucidated lipid mediator signaling networks emanating from epithelial cell-cell interactive pathways, and have defined a number of targets for development of effective therapeutics. This short review will focus on recently defined pathways of lipid mediator function in the mucosa, particularly with regard to the role of the epithelium.

Animals↗
PubMed2001

Hypoxia-inducible factor 1-mediated inhibition of peroxisome proliferator-activated receptor alpha expression during hypoxia.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone-binding proteins that regulate transcriptional responses to peroxisome proliferators and structurally diverse fatty acids. PPARs have been implicated in a wide variety of functions, including lipid homeostasis and inflammatory responses. In this study, we examined the expression of PPAR-alpha in response to ambient hypoxia. Initial studies using microarray analysis of intestinal epithelial mRNA revealed that hypoxia rapidly down-regulates PPAR-alpha mRNA and protein in epithelial cells in vitro and in vivo. Subsequent studies revealed that the PPAR-alpha gene bears a DNA consensus motif for the transcription factor hypoxia-inducible factor 1 (HIF-1). EMSA analysis revealed that ambient hypoxia induces HIF-1alpha binding to the HIF-1 consensus domain of PPAR-alpha in parallel to HIF-1 nuclear accumulation, and antisense depletion of HIF-1alpha resulted in a loss of PPAR-alpha down-regulation. The PPAR-alpha ligand pirinixic acid (WY14643) functionally promoted IFN-gamma-induced ICAM-1 expression in normoxic epithelia, and this response was lost in cells pre-exposed to ambient hypoxia. Such results indicate that HIF-1-dependent down-regulation of PPAR-alpha may provide an adaptive response to proinflammatory stimuli during cellular hypoxia. These studies provide unique insight into the regulation of PPAR-alpha expression and, importantly, provide an example of a down-regulatory pathway mediated by HIF-1.

Animals↗
PubMed2001

Phosphoinositide 3-kinase modulation of beta(3)-integrin represents an endogenous "braking" mechanism during neutrophil transmatrix migration.

During episodes of inflammation, neutrophils (polymorphonuclear leukocytes [PMNs]) encounter subendothelial matrix substrates that may require additional signaling pathways as directives for movement through the extracellular space. Using an in vitro endothelial and epithelial model, inhibitors of phosphoinositide 3-kinase (PI3K) were observed to promote chemoattractant-stimulated migration by as much as 8 +/- 0.3-fold. Subsequent studies indicated that PMNs respond in a similar manner to RGD-containing matrix substrates and that PMN-matrix interactions are potently inhibited by antibodies directed against beta(3)- but not beta(1)-integrin antibodies, and that PI3K inhibitors block beta(3)-integrin dependence. Biochemical analysis of intracellular beta(3)-integrin uncoupling by PI3K inhibitors revealed diminished beta(3)-integrin tyrosine phosphorylation and decreased association with p72(syk). Similarly, the p72(syk) inhibitor piceatannol promoted PMN transmatrix migration, whereas HIV-tat peptide-facilitated loading of peptides corresponding to the beta(3)-integrin cytoplasmic tail identified the functional tyrosine residues for this activity. These data indicate that PI3K-regulated beta(3)-integrin represents a natural "braking" mechanism for PMNs during transit through the extracellular matrix.

Androstadienes↗
PubMed2001

Hypoxia-inducible factor 1-dependent induction of intestinal trefoil factor protects barrier function during hypoxia.

Mucosal organs such as the intestine are supported by a rich and complex underlying vasculature. For this reason, the intestine, and particularly barrier-protective epithelial cells, are susceptible to damage related to diminished blood flow and concomitant tissue hypoxia. We sought to identify compensatory mechanisms that protect epithelial barrier during episodes of intestinal hypoxia. Initial studies examining T84 colonic epithelial cells revealed that barrier function is uniquely resistant to changes elicited by hypoxia. A search for intestinal-specific, barrier-protective factors revealed that the human intestinal trefoil factor (ITF) gene promoter bears a previously unappreciated binding site for hypoxia-inducible factor (HIF)-1. Hypoxia resulted in parallel induction of ITF mRNA and protein. Electrophoretic mobility shift assay analysis using ITF-specific, HIF-1 consensus motifs resulted in a hypoxia-inducible DNA binding activity, and loading cells with antisense oligonucleotides directed against the alpha chain of HIF-1 resulted in a loss of ITF hypoxia inducibility. Moreover, addition of anti-ITF antibody resulted in a loss of barrier function in epithelial cells exposed to hypoxia, and the addition of recombinant human ITF to vascular endothelial cells partially protected endothelial cells from hypoxia-elicited barrier disruption. Extensions of these studies in vivo revealed prominent hypoxia-elicited increases in intestinal permeability in ITF null mice. HIF-1-dependent induction of ITF may provide an adaptive link for maintenance of barrier function during hypoxia.

Animals↗
PubMed2001

Neutrophil transepithelial migration: regulation at the apical epithelial surface by Fc-mediated events.

Neutrophil (PMN) transepithelial migration is a major effector of epithelial defense in inflammatory diseases involving mucosal surfaces. However, major receptor-ligand interactions between epithelial cells and PMN remain incompletely characterized. To better define the molecular events involved in PMN interactions with epithelial cells, we produced a monoclonal antibody called g82 that inhibited PMN transepithelial migration in the physiological basolateral-to-apical direction. The g82 antigen localized to the apical surface of human colonic epithelium and was significantly upregulated under inflammatory conditions. Immunoprecipitation revealed two polypeptides of M(r) 207 and 32 kDa. F(ab')(2) fragments from g82 IgG had no effect on transmigration, suggesting Fc dependence. Further experiments confirmed dependence on the PMN Fc receptor CD32A and that the observed effects were secondary to a failure of PMN to detach from the apical epithelial surface. These Fc-mediated events were epitope specific since binding, isotype-matched antibodies did not affect detachment. These results identify a new mechanism for retention of PMN at the apical epithelial surface following transepithelial migration. This pathway may be important in pathogen clearance and mucosal pathophysiology associated with autoimmunity.

Animals↗
PubMed2000

Regulation of endothelial CD73 by adenosine: paracrine pathway for enhanced endothelial barrier function.

During episodes of inflammation, multiple cell types release adenine nucleotides in the form of ATP, ADP, 5'-AMP, and adenosine. In particular, following activation, polymorphonuclear leukocytes release larger quantities of 5'-AMP. Extracellular 5'-AMP is metabolized to adenosine by surface-expressed 5'-ectonucleotidase (CD73). Adenosine liberated by this process activates surface adenosine A(2B) receptors, results in endothelial junctional reorganization, and promotes barrier function. We hypothesized that adenosine signaling to endothelia provides a paracrine loop for regulated expression of CD73 and enhanced endothelial barrier function. Using an in vitro microvascular endothelial model, we investigated the influence of 5'-AMP; adenosine; and adenosine analogues on CD73 transcription, surface expression, and function. Initial experiments revealed that adenosine and adenosine analogues induce CD73 mRNA (RT-PCR), surface expression (immunoprecipitation of surface biotinylated CD73), and function (HPLC analysis of etheno-AMP conversion to ethenoadenosine) in a time- and concentration-dependent fashion. Subsequent studies revealed that similar exposure conditions increase surface protein through transcriptional induction of CD73. Analysis of DNA-binding activity by EMSA identified a functional role for CD73 cAMP response element and, moreover, indicated that multiple cAMP agonists induce transcriptional activation of functional CD73. Induced CD73 functioned to enhance 5'-AMP-mediated promotion of endothelial barrier (measured as a paracellular flux of 70-kDa FITC-labeled tracer). These results provide an example of transcriptional induction of enzyme (CD73) by enzymatic product (adenosine) and define a paracrine pathway for the regulated expression of vascular endothelial CD73 and barrier function.

5'-Nucleotidase↗
PubMed2000

Phosphorylation-dependent targeting of cAMP response element binding protein to the ubiquitin/proteasome pathway in hypoxia.

Hypoxia activates a number of gene products through degradation of the transcriptional coactivator cAMP response element binding protein (CREB). Other transcriptional regulators (e.g., beta-catenin and NF-kappa B) are controlled through phosphorylation-targeted proteasomal degradation, and thus, we hypothesized a similar degradative pathway for CREB. Differential display analysis of mRNA derived from hypoxic epithelia revealed a specific and time-dependent repression of protein phosphatase 1 (PP1), a serine phosphatase important in CREB dephosphorylation. Subsequent studies identified a previously unappreciated proteasomal-targeting motif within the primary structure of CREB (DSVTDS), which functions as a substrate for PP1. Ambient hypoxia resulted in temporally sequential CREB serine phosphorylation, ubiquitination, and degradation (in vitro and in vivo). HIV-tat peptide-facilitated loading of intact epithelia with phosphopeptides corresponding to this proteasome targeting motif resulted in inhibition of CREB ubiquitination. Further studies revealed that PP1 inhibitors mimicked hypoxia-induced gene expression, whereas proteasome inhibitors reversed the hypoxic phenotype. Thus, hypoxia establishes conditions that target CREB to proteasomal degradation. These studies may provide unique insight into a general mechanism of transcriptional regulation by hypoxia.

Amino Acid Sequence↗
PubMed2000

Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing.

Aspirin therapy inhibits prostaglandin biosynthesis without directly acting on lipoxygenases, yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins (LXs) epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered LX [ATL]). Here, we report that inflammatory exudates from mice treated with omega-3 polyunsaturated fatty acid and aspirin (ASA) generate a novel array of bioactive lipid signals. Human endothelial cells with upregulated COX-2 treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Each was used by polymorphonuclear leukocytes to generate separate classes of novel trihydroxy-containing mediators, including 5-series 15R-LX(5) and 5,12,18R-triHEPE. These new compounds proved to be potent inhibitors of human polymorphonuclear leukocyte transendothelial migration and infiltration in vivo (ATL analogue > 5,12,18R-triHEPE > 18R-HEPE). Acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on hematologic cells. These findings establish new transcellular routes for producing arrays of bioactive lipid mediators via COX-2-nonsteroidal antiinflammatory drug-dependent oxygenations and cell-cell interactions that impact microinflammation. The generation of these and related compounds provides a novel mechanism(s) for the therapeutic benefits of omega-3 dietary supplementation, which may be important in inflammation, neoplasia, and vascular diseases.

Acetaminophen↗
PubMed2000

Human CD1d associates with prolyl-4-hydroxylase during its biosynthesis.

Recent studies have shown that the CD1 family of proteins present various glycolipid antigens to subsets of T cells. CD1d is expressed on human intestinal epithelial cells (IEC) and exists in two biochemical forms: 37-kDa, beta2-microglobulin (beta2m) independent, nonglycosylated, and 47-kDa, beta2m dependent, glycosylated forms. The biosynthetic pathways and the mechanisms of generation of these two biochemically distinct forms of CD1d in human IEC are unknown. Using a human colonic cell line, T84, transfected with CD1d, the biosynthesis of CD1d was investigated. Pulse-chase metabolic labeling studies of T84 transfected with wild type CD1d demonstrated that CD1d was a stable protein over a 4-day chase period. During the first 24 h of the chase, a novel 65-kDa glycoprotein was co-immunoprecipitated with CD1d. Microsequencing of this protein identified the glycoprotein as the alpha and beta subunits of the resident endoplasmic reticulum protein, prolyl-4-hydroxylase (P4H), an enzyme responsible for hydroxyl modification of proline residues. To study if either one or both biochemical forms of CD1d contained hydroxyproline residues, amino acid composition analysis of the 37 and 48 kDa was performed, and demonstrated that only the 37-kDa, but not the 48-kDa form of CD1d, contained hydroxyproline residues. These studies demonstrate that CD1d exhibits a prolonged association with P4H and that the 37-kDa form contains hydroxyproline residues. This suggests that P4H association with CD1d during its biosynthesis results in a novel post-translational modification of CD1d.

Antigens, CD1↗
PubMed2000

Parallel induction of epithelial surface-associated chemokine and proteoglycan by cellular hypoxia: implications for neutrophil activation.

Neutrophil-induced damage to the protective epithelium has been implicated in mucosal disorders associated with hypoxia, and such damage may be initiated by epithelial-derived chemokines. Because chemokines can bind to membrane proteoglycans, we hypothesized that chemokines may associate with epithelial surfaces and activate polymorphonuclear neutrophils (PMN). Epithelial hypoxia (pO2 20 torr) resulted in a time-dependent induction of interleukin-8 (IL-8) mRNA, soluble protein, as well as surface protein. Such surface IL-8 expression was demonstrated to be dependent on heparinase III expression, and extensions of these experiments indicated that hypoxia induces epithelial perlecan expression in parallel with IL-8. Finally, co-incubation of post-hypoxic epithelia with human PMN induced IL-8-dependent expression of the PMN beta2-integrin CD11b/18. These data indicate that chemokines liberated from epithelia may exist in a surface-bound, bioactive form and that hypoxia may regulate proteoglycan expression.

CD18 Antigens↗
PubMed2000

Anti-microinflammatory lipid signals generated from dietary N-3 fatty acids via cyclooxygenase-2 and transcellular processing: a novel mechanism for NSAID and N-3 PUFA therapeutic actions.

Aspirin therapy inhibits prostaglandin biosynthesis; yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered lipoxin or ATL). Here, we review our findings indicating that inflammatory exudates from mice treated with omega-3 PUFA and aspirin (ASA) generate a novel array of bioactive lipid signals. Also, human endothelial cells, both HUVEC and microvascular, with upregulated COX-2 and treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Human PMN activated with serum treated zymosan (STZ) utilized each of these R-HEPEs to generate novel classes of trihydroxy-containing mediators including 5-series 15R-LX and 5,12,18R-triHEPE. The novel products were potent inhibitors of human PMN transendothelial migration and infiltration of PMN in dorsal air pouches in vivo. In addition to ASA, both acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant human COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on leukocytes, platelets and endothelial cells. These findings establish new transcellular routes for producing arrays of lipid mediators via COX-2-NSAIDs and cell-cell interactions that impact microinflammation. Moreover, they provide novel mechanism(s) that could underlie the many reported therapeutic benefits of omega-3 dietary supplementation of interest in inflammation, cancer, and vascular disorders.

Animals↗
PubMed1999

Ligation of intestinal epithelial CD1d induces bioactive IL-10: critical role of the cytoplasmic tail in autocrine signaling.

The intestinal epithelium is anatomically positioned to serve as the critical interface between the lumen and the mucosal immune system. In addition to MHC class I and II antigens, intestinal epithelia constitutively express the nonclassical MHC molecule CD1d, a transmembrane molecule with a short cytoplasmic tail expressed as a beta(2)-microglobulin-associated 48-kDa glycoprotein and novel beta(2)-microglobulin-independent 37-kDa nonglycosylated protein on intestinal epithelia. At present, it is not known whether extracellular ligands can signal intestinal epithelial CD1d. To define signaling of CD1d cytoplasmic tail, retrovirus-mediated gene transfer was used to generate stable cell lines expressing wild-type CD1d or a chimeric molecule (extracellular CD1d and cytoplasmic CD1a), and surface CD1d was triggered by antibody crosslinking. Although wild-type CD1d was readily activated (tyrosine phosphorylation), no demonstrable signal was evident in cell lines expressing the chimeric molecule. Subsequent studies revealed that anti-CD1d crosslinking specifically induces epithelial IL-10 mRNA and protein and is blocked by the tyrosine kinase inhibitor genistein. Further studies addressing epithelial-derived IL-10 revealed that anti-CD1d crosslinking attenuates IFN-gamma signaling and that such attenuation is reversed by addition of functionally inhibitory IL-10 antibodies. These results define signaling through surface CD1d, and, importantly, they demonstrate that this pathway may serve to dampen epithelial proinflammatory signals.

Antigens, CD1↗
PubMed1999

Critical role of cAMP response element binding protein expression in hypoxia-elicited induction of epithelial tumor necrosis factor-alpha.

Tissue hypoxia is intimately associated with a number of chronic inflammatory conditions of the intestine. In this study, we investigated the impact of hypoxia on the expression of a panel of inflammatory mediators by intestinal epithelia. Initial experiments revealed that epithelial (T84 cell) exposure to ambient hypoxia evoked a time-dependent induction of the proinflammatory markers tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), and major histocompatibility complex (MHC) class II (37 +/- 6.1-, 7 +/- 0.8-, and 9 +/- 0.9-fold increase over normoxia, respectively, each p < 0.01). Since the gene regulatory elements for each of these molecules contains an NF-kappaB binding domain, we investigated the influence of hypoxia on NF-kappaB activation. Cellular hypoxia induced a time-dependent increase in nuclear p65, suggesting a dominant role for NF-kappaB in hypoxia-elicited induction of proinflammatory gene products. Further work, however, revealed that hypoxia does not influence epithelial intercellular adhesion molecule 1 (ICAM-1) or MHC class I, the promoters of which also contain NF-kappaB binding domains, suggesting differential responses to hypoxia. Importantly, the genes for TNF-alpha, IL-8, and MHC class II, but not ICAM-1 or MHC class I, contain cyclic AMP response element (CRE) consensus motifs. Thus, we examined the role of cAMP in the hypoxia-elicited phenotype. Hypoxia diminished CRE binding protein (CREB) expression. In parallel, T84 cell cAMP was diminished by hypoxia (83 +/- 13.2% decrease, p < 0.001), and pharmacologic inhibition of protein kinase A induced TNF-alpha and protein release (9 +/- 3.9-fold increase). Addback of cAMP resulted in reversal of hypoxia-elicited TNF-alpha release (86 +/- 3.2% inhibition with 3 mM 8-bromo-cAMP). Furthermore, overexpression of CREB but not mutated CREB by retroviral-mediated gene transfer reversed hypoxia-elicited induction of TNF-alpha defining a causal relationship between hypoxia-elicited CREB reduction and TNF-alpha induction. Such data indicate a prominent role for CREB in the hypoxia-elicited epithelial phenotype and implicate intracellular cAMP as an important second messenger in differential induction of proinflammatory mediators.

Cell Hypoxia↗
PubMed1999

Therapeutic targets for hypoxia-elicited pathways.

Diminished oxygen supply to tissues (hypoxia) can stem from many sources, and is a contributing factor to diverse disease processes. Cell and tissue responses to hypoxia are diverse and include dramatic changes in metabolic demand, regulation of cellular gene products, and release of lipid and protein mediators. Surprisingly little attention has been paid to targeted development of therapeutics for hypoxia-related disease processes. This review will focus on recent advances in cellular and molecular biology pertaining to the hypoxia response, and will discuss paradigms used to study hypoxia and the potential targets for therapeutic intervention.

Animals↗
PubMed1999

Immunolocalization of CD1d in human intestinal epithelial cells and identification of a beta2-microglobulin-associated form.

In order to better understand the role of intestinal CD1d, we sought to define the cellular localization and further characterize the biochemical structure of CD1d in human intestinal epithelial cells (IEC). Using a CD1d-specific rabbit anti-gst-CD1d antibody, immunoprecipitation of radiolabeled cell surface proteins detected a previously identified 37 kDa protein as well as a 48-50 kDa protein which were confirmed by Western blotting with a CD1d-specific mAb, D5. Immunoprecipitation of protein lysates with the CD1d-specific mAb, D5 and 51.1.3, and the beta2-microglobulin (beta2m)-specific mAb, BBM.1, followed by N-glycanase digestion and Western blotting with the D5 mAb showed that the 48-50 kDa protein was a beta2m-associated, CD1d glycoprotein. CD1d was immunolocalized to the apical and lateral regions of native small and large intestinal IEC as defined by confocal laser microscopy using the D5 mAb and the rabbit anti-gst-CD1d antibody. In addition, a large apical intracellular pool of CD1d was identified. Identical observations were made with polarized T84 cells. Selective biotin labeling of apical and basolateral cell surfaces followed by immunoprecipitation with the D5 mAb, N-glycanase digestion and avidin blotting confirmed the presence of glycosylated CD1d on both cell surfaces and immunolocalization of the 37 kDa non-glycosylated form of CD1d to the apical cell surface. These studies show that CD1d is located in an ideal position for luminal antigen sampling and presentation to subjacent intraepithelial lymphocytes.

Antibody Specificity↗