Nancy A. Louis

Ecto-5′-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia

Under conditions of limited oxygen availability (hypoxia), multiple cell types release adenine nucleotides in the form of ATP, ADP, and AMP. Extracellular AMP is metabolized to adenosine by surface-expressed ecto-5-nucleotidase (CD73) and subsequently activates surface adenosine receptors regulating endothelial and epithelial barrier function. Therefore, we hypothesized that hypoxia transcriptionally regulates CD73 expression. Microarray RNA analysis revealed an increase in CD73 and ecto-apyrase CD39 in hypoxic epithelial cells. Metabolic studies of CD39/CD73 function in intact epithelia revealed that hypoxia enhances CD39/CD73 function as much as 6 +/- 0.5-fold over normoxia. Examination of the CD73 gene promoter identified at least one binding site for hypoxia-inducible factor-1 (HIF-1) and inhibition of HIF-1alpha expression by antisense oligonucleotides resulted in significant inhibition of hypoxia-inducible CD73 expression. Studies using luciferase reporter constructs revealed a significant increase in activity in cells subjected to hypoxia, which was lost in truncated constructs lacking the HIF-1 site. Mutagenesis of the HIF-1alpha binding site resulted in a nearly complete loss of hypoxia-inducibility. In vivo studies in a murine hypoxia model revealed that hypoxia-induced CD73 may serve to protect the epithelial barrier, since the CD73 inhibitor alpha,beta-methylene ADP promotes increased intestinal permeability. These results identify an HIF-1-dependent regulatory pathway for CD73 and indicate the likelihood that CD39/CD73 protects the epithelial barrier during hypoxia.

Resolvin E1 promotes mucosal surface clearance of neutrophils: a new paradigm for inflammatory resolution

Migration of neutrophils (PMN) across epithelia is a pathological hallmark of numerous muco‐sal diseases. Whereas lesions at mucosal surfaces are generally self‐limiting, endogenous mechanisms of resolution are incompletely understood. Previous studies revealed that resolvins directly act on PMN to attenuate transendothelial migration, less is known about the influence of resolvins on PMN‐epithelial interactions and whether they act on epithelia. We studied the dynamics of resolvin E1 (RvE1) actions on leukocyte transepithelial migration. PMN exposure to RvE1 or chemerin (peptide agonist of ChemR23) reduced trans‐epithelial migration in a concentration‐dependent manner. Conversely, activation of epithelial ChemR23 promoted apical clearance of PMN. A nonbiased screen of known PMN ligands expressed on epithelial cells in response to RvE1 revealed selective induction of CD55, an apically expressed antiadhesive molecule. CD55 promoter analysis demonstrated that both RvE1 and chemerin activate the CD55 promoter. Inhibition of CD55 by neutralizing antibody prevented RvE1‐depen‐dent augmentation of apical PMN clearance. Taken together these findings implicate a “two‐hit” model of inflammatory resolution, whereby activation of the PMN RvE1 receptor attenuates transepithelial migration and subsequent actions on the epithelium promote CD55‐dependent clearance of PMN across the epithelial cell surface promoting active inflammatory resolution.—Campbell, E. L., Louis, N. A., Tomassetti, S. E., Canny, G. O., Arita, M., Serhan, C. N., Colgan, S. P. Resolvin E1 promotes mucosal surface clearance of neutrophils: a new paradigm for inflammatory resolution. FASEB J. 21, 3162–3170 (2007)

Selective induction of mucin-3 by hypoxia in intestinal epithelia.

Epithelial cells line mucosal surfaces (e.g., lung, intestine) and critically function as a semipermeable 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. Here, we pursue the hypothesis that intestinal barrier function is regulated in a protective manner by hypoxia responsive genes. We demonstrate by PCR confirmation of microarray data and by avidin blotting of immunoprecipitated human Mucin 3 (MUC3), that surface MUC3 expression is induced in T84 intestinal epithelial cells following exposure to hypoxia. MUC3 RNA is minimally detectable while surface protein expression is absent under baseline normoxic conditions. There is a robust induction in both the mRNA (first evident by 8 h) and protein expression, first observed and maximally expressed following 24 h hypoxia. This is followed by a subsequent decline in protein expression, which remains well above baseline at 48 h of hypoxia. Further, we demonstrate that this induction of MUC3 protein is associated with a transient increase in the barrier restorative peptide, intestinal trefoil factor (ITF). ITF not only colocalizes with MUC3, by confocal microscopy, to the apical surface of T84 cells following exposure to hypoxia, but is also found, by co‐immunoprecipitation, to be physically associated with MUC3, following 24 h of hypoxia. In exploration of the mechanism of hypoxic regulation of mucin 3 expression, we demonstrated by luciferase assay that the full‐length promoter for mouse Mucin 3 (Muc3) is hypoxia‐responsive with a 5.08u2009±u20091.76‐fold induction following 24 h of hypoxia. Furthermore, analysis of both the human (MUC3A) and mouse (Muc3) promoters revealed potential HIF‐1 binding sites which were shown by chromatin immunoprecipitation to bind the pivotal hypoxia‐regulating transcription factor HIF‐1α. Taken together, these studies implicate the HIF‐1α mediated hypoxic induced expression of mucin 3 and associated ITF in the maintenance of intestinal barrier function under hypoxic conditions. J. Cell. Biochem. 99: 1616–1627, 2006.

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.

HIF-dependent induction of apical CD55 coordinates epithelial clearance of neutrophils

Sites of inflammation are associated with dramatic shifts in tissue metabolism. Inflammation can result in significant tissue hypoxia, with resultant induction of hypoxia‐responsive genes. Given this association, we hypothesized that neutrophil (PMN) ligands expressed on epithelial cells may be regulated by hypoxia. Initial studies confirmed earlier results that epithelial hypoxia enhances PMN transepithelial migration and promotes apical clearance of PMN from the epithelial surface. A screen of known PMN ligands revealed a surprisingly stable expression pattern in hypoxia. However, this screen identified one gene, CD55, as a highly hypoxia‐inducible molecule expressed on the apical membrane of mucosal epithelia. Subsequent studies verified the induction of CD55 mRNA and protein expression by hypoxia. Overexpression of CD55 by transfection in nonhypoxic epithelia resulted in a similar pattern of apical PMN clearance, and peptide mimetics corresponding to the PMN binding site on DAF blocked such apical clearance of PMN. Studies directed at understanding molecular pathways of hypoxia inducibility revealed that a ∼200 bp region of the CD55 gene conferred hypoxia inducibility for CD55. These studies identified a functional binding site for the transcriptional regulator hypoxia‐inducible factor (HIF). Taken together, these results identify HIF‐dependent induction of epithelial CD55 in the resolution of ongoing inflammation through clearance of apical PMN. Louis, N. A., Hamilton, K E., Kong, T., Colgan, S. P. HIF‐dependent induction of apical CD55 coordinates epithelial clearance of neutrophils. FASEB J. 19, 950–959 (2005)

The role of polymorphonuclear leukocyte trafficking in the perpetuation of inflammation during inflammatory bowel disease

Abstract:The inflammatory bowel diseases (IBDs; Crohn’s disease, and ulcerative colitis) are chronically relapsing inflammatory disorders of the intestine and/or colon. The precise etiology of IBD remains unclear, but it is thought that a complex interplay between various factors including genetic predisposition, the host immune system, and the host response to luminal microbes play a role in disease pathogenesis. Furthermore, numerous lines of evidence have implicated the accumulation of large numbers of polymorphonuclear leukocyte (PMN) in the mucosa and epithelial crypts of the intestine as a hallmark of the active disease phase of IBD. Massive infiltration of PMNs is thought to be instrumental in the pathophysiology of IBD with the degree of PMN migration into intestinal crypts correlating with patient symptoms and mucosal injury. Specifically, migrated PMN have been implicated in the impairment of epithelial barrier function, tissue destruction through oxidative and proteolytic damage, and the perpetuation of inflammation through the release of inflammatory mediators. This review highlights the multifactorial role of PMN egress into the intestinal mucosa in the pathogenesis of IBD because it represents an important area of research with therapeutic implications for the amelioration of the symptoms associated with IBD.

Model systems to investigate neutrophil adhesion and chemotaxis.

Polymorphonuclear neutrophil (PMN) recruitment from the blood stream into surrounding tissues, followed by migration through the tissue with triggered release of oxidative enzymes or eventual clearance from the epithelial surface, involves a regulated series of events central to acute responses in host defense. Accumulations of large numbers of neutrophils within mucosal tissues are pathognomonic features of both acute and chronic inflammatory conditions including sepsis and inflammatory bowel disease, but the precise signals governing neutrophil adhesion and transmigration remain to be fully characterized. Previous chapters examine methods employed for both neutrophil isolation and study of the mechanisms underlying regulation of PMN rolling behavior. Here, we describe in vitro experimental models for the examination of PMN adhesion to endothelial and epithelial monolayers as well as the characterization of signals influencing neutrophil migration, both along acellular matrices and across endothelial and epithelial monolayers, in the physiologically relevant directions. Studies employing these model systems allow further elucidation of the mechanisms governing PMN adhesion and transmigration.

Cell-Cell Interactions on Solid Matrices

Models to study molecular, biochemical, and functional responses in vitro generally incorporate an individual cell type or group of cells organized in a random fashion. Normal physiological responses in vivo require that individual cell types be oriented in an organized fashion with three-dimensional architecture and appropriately positioned cellular interfaces. Much recent progress has been made in the development and implementation of models to study cell-cell contact using substrate grown cells. Here, we summarize the use of membrane permeable supports to study functional responses in appropriately positioned cell types. These models incorporate two or more different cells cultured in physiologically positioned locales on solid substrates. Models incorporating nonadherent cells (e.g., leukocytes) in co-culture with such models also are discussed. Such models have been used extensively to discovery both cell-bound as well as soluble mediators of physiological and pathophysiological processes.

The Fucosyltransferase 3 Driven Expression of Sialyl Lewis A on the Intestinal Epithelium Regulates PMN Clearance During Inflammation: P-206 YI

IBD pathogenesis. While epidemiological evidence demonstrates vitamin D-deficiency is highly prevalent in IBD and vitamin D status is inversely associated with disease severity, the mechanistic impact(s) of vitamin D deficiency remains unclear. Vitamin D hormone activity is mediated by the vitamin D receptor (VDR). Previous studies using IL-10(-/-)/VDR(-/-) mice concluded that VDR signaling in immune cells inhibits colonic inflammation and colitis development. In addition, our investigations of VDR(-/-) mice suggested that the epithelial VDR attenuates experimental colitis by protecting the mucosal epithelial barrier and that colonic epithelial VDR levels were markedly reduced in the biopsies from patients with IBD. Aims: To identify the anti-colitic role of the colonic epithelial vs. immune VDR signaling in the development of experimental colitis. METHODS: Transgenic (Tg) mice were generated in which villin promoter was used to target Flag-tagged human (h)VDR to intestinal epithelial cells (IEC). Transgenic-VDR(-/-) (Tg-KO) mice were also produced through breeding in which only the IECs were reconstituted with hVDR in the VDR(-/-) background. Tg, Tg-KO, VDR(-/-) and wild-type (WT) mice were studied in parallel using TNBSand DSScolitis models. RESULTS: In both TNBS and DSS models, Tg mice were highly resistant to colitis induction compared to WT mice, manifested by lower clinical and histological scores and marked reduction in colonic epithelial damage and inflammatory cytokine production. Colonic epithelial permeability was well preserved in Tg mice. VDR(-/-) mice developed the most severe colitis that led to high mortality in both models, whereas Tg-KO mice were highly resistant to colitis with almost no mortality. Mechanistically, VDR overexpression attenuated TNBSor DSS-induced IEC apoptosis, with marked suppression of PUMA induction and caspase 3 activation. In vitro and in vivo data demonstrated that VDR signaling inhibited PUMA expression by blocking NF-kB activation. CONCLUSION(S): These results demonstrate that intestinal epithelial VDR signaling inhibits colonic inflammation by protecting the integrity of the mucosal epithelial barrier. The observation that the hVDR transgene rescues VDR-null mice from developing severe colitis despite a VDR-null immune system demonstrates that the anticolitic activity of the epithelial VDR is independent of the immune VDR actions.