Winston Y. Lee

JAM-A regulates permeability and inflammation in the intestine in vivo

Recent evidence has linked intestinal permeability to mucosal inflammation, but molecular studies are lacking. Candidate regulatory molecules localized within the tight junction (TJ) include Junctional Adhesion Molecule (JAM-A), which has been implicated in the regulation of barrier function and leukocyte migration. Thus, we analyzed the intestinal mucosa of JAM-A–deficient (JAM-A−/−) mice for evidence of enhanced permeability and inflammation. Colonic mucosa from JAM-A−/− mice had normal epithelial architecture but increased polymorphonuclear leukocyte infiltration and large lymphoid aggregates not seen in wild-type controls. Barrier function experiments revealed increased mucosal permeability, as indicated by enhanced dextran flux, and decreased transepithelial electrical resistance in JAM-A−/− mice. The in vivo observations were epithelial specific, because monolayers of JAM-A−/− epithelial cells also demonstrated increased permeability. Analyses of other TJ components revealed increased expression of claudin-10 and -15 in the colonic mucosa of JAM-A−/− mice and in JAM-A small interfering RNA–treated epithelial cells. Given the observed increase in colonic inflammation and permeability, we assessed the susceptibility of JAM-A−/− mice to the induction of colitis with dextran sulfate sodium (DSS). Although DSS-treated JAM-A−/− animals had increased clinical disease compared with controls, colonic mucosa showed less injury and increased epithelial proliferation. These findings demonstrate a complex role of JAM-A in intestinal homeostasis by regulating epithelial permeability, inflammation, and proliferation.

Interferon-γ Regulates Intestinal Epithelial Homeostasis through Converging β-Catenin Signaling Pathways

Inflammatory cytokines have been proposed to regulate epithelial homeostasis during intestinal inflammation. We report here that interferon-gamma (IFN-gamma) regulates the crucial homeostatic functions of cell proliferation and apoptosis through serine-threonine protein kinase AKT-beta-catenin and Wingless-Int (Wnt)-beta-catenin signaling pathways. Short-term exposure of intestinal epithelial cells to IFN-gamma resulted in activation of beta-catenin through AKT, followed by induction of the secreted Wnt inhibitor Dkk1. Consequently, we observed an increase in Dkk1-mediated apoptosis upon extended IFN-gamma treatment and reduced proliferation through depletion of the Wnt coreceptor LRP6. These effects were enhanced by tumor necrosis factor-alpha (TNF-alpha), suggesting synergism between the two cytokines. Consistent with these results, colitis in vivo was associated with decreased beta-catenin-T cell factor (TCF) signaling, loss of plasma membrane-associated LRP6, and reduced epithelial cell proliferation. Proliferation was partially restored in IFN-gamma-deficient mice. Thus, we propose that IFN-gamma regulates intestinal epithelial homeostasis by sequential regulation of converging beta-catenin signaling pathways.

Annexin I Regulates SKCO-15 Cell Invasion by Signaling through Formyl Peptide Receptors

Annexin 1 (AnxA1) is a multifunctional phospholipid-binding protein associated with the development of metastasis in some invasive epithelial malignancies. However, the role of AnxA1 in the migration/invasion of epithelial cells is not known. In this study, experiments were performed to investigate the role of AnxA1 in the invasion of a model epithelial cell line, SKCO-15, derived from colorectal adenocarcinoma. Small interfering RNA-mediated knockdown of AnxA1 expression resulted in a significant reduction in invasion through Matrigel-coated filters. Localization studies revealed a translocation of AnxA1 to the cell surface upon the induction of cell migration, and functional inhibition of cell surface AnxA1 using antiserum (LCO1) significantly reduced cell invasion. Conversely, SKCO-15 cell invasion was increased by ∼2-fold in the presence of recombinant full-length AnxA1 and the AnxA1 N-terminal-derived peptide mimetic, Ac2-26. Because extracellular AnxA1 has been shown to regulate leukocyte migratory events through interactions with n-formyl peptide receptors (nFPRs), we examined the expression of FPR-1, FPRL-1, and FPRL-2 in SKCO-15 cells by reverse transcriptase-PCR and identified expression of all three receptors in this cell line. Treatment of SKCO-15 cells with AnxA1, Ac2-26, and the classical nFPR agonist, formylmethionylleucylphenylalanine, induced intracellular calcium release consistent with nFPR activation. Furthermore, the nFPR antagonist, Boc2, abrogated the AnxA1 and Ac2-26-induced intracellular calcium release and increase in SKCO-15 cell invasion. Together, these results support an autocrine/paracrine role for membrane AnxA1 in stimulating SKCO-15 cell migration through nFPR activation. The findings in this study suggest that activation of nFPRs stimulates epithelial cell motility important in the development of metastasis as well as wound healing.

Junctional Adhesion Molecule A Interacts with Afadin and PDZ-GEF2 to Activate Rap1A, Regulate β1 Integrin Levels, and Enhance Cell Migration

Junctional adhesion molecule-A (JAM-A) is a transmembrane tight junction protein that has been shown to regulate barrier function and cell migration through incompletely understood mechanisms. We have previously demonstrated that JAM-A regulates cell migration by dimerization of the membrane-distal immunoglobulin-like loop and a C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding motif. Disruption of dimerization resulted in decreased epithelial cell migration secondary to diminished levels of beta1 integrin and active Rap1. Here, we report that JAM-A is physically and functionally associated with the PDZ domain-containing molecules Afadin and PDZ-guanine nucleotide exchange factor (GEF) 2, but not zonula occludens (ZO)-1, in epithelial cells, and these interactions mediate outside-in signaling events. Both Afadin and PDZ-GEF2 colocalized and coimmunoprecipitated with JAM-A. Furthermore, association of PDZ-GEF2 with Afadin was dependent on the expression of JAM-A. Loss of JAM-A, Afadin, or PDZ-GEF2, but not ZO-1 or PDZ-GEF1, similarly decreased cellular levels of activated Rap1, beta1 integrin protein, and epithelial cell migration. The functional effects observed were secondary to decreased levels of Rap1A because knockdown of Rap1A, but not Rap1B, resulted in decreased beta1 integrin levels and reduced cell migration. These findings suggest that JAM-A dimerization facilitates formation of a complex with Afadin and PDZ-GEF2 that activates Rap1A, which regulates beta1 integrin levels and cell migration.

Annexin A1 Regulates Intestinal Mucosal Injury, Inflammation, and Repair

During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (−/−) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (−/−) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.

Endothelial CD47 interaction with SIRPγ is required for human T-cell transendothelial migration under shear flow conditions in vitro

Leukocyte transendothelial migration (TEM) is a critical event during inflammation. CD47 has been implicated in myeloid cell migration across endothelium and epithelium. CD47 binds to signal regulatory protein (SIRP), SIRPalpha and SIRPgamma. So far, little is known about the role of endothelial CD47 in T-cell TEM in vivo or under flow conditions in vitro. Fluorescence-activated cell sorting and biochemical analysis show that CD3(+) T cells express SIRPgamma but not SIRPalpha, and fluorescence microscopy showed that CD47 was enriched at endothelial junctions. These expression patterns suggested that CD47 plays a role in T-cell TEM through binding interactions with SIRPgamma. We tested, therefore, whether CD47-SIRPgamma interactions affect T-cell transmigration using blocking mAb against CD47 or SIRPgamma in an in vitro flow model. These antibodies inhibited T-cell TEM by 70% plus or minus 6% and 82% plus or minus 1%, respectively, but had no effect on adhesion. In agreement with human mAb studies, transmigration of murine wild-type T helper type 1 cells across TNF-alpha-activated murine CD47(-/-) endothelium was reduced by 75% plus or minus 2% even though murine T cells appear to lack SIRPgamma. Nonetheless, these findings suggest endothelial cell CD47 interacting with T-cell ligands, such as SIRPgamma, play an important role in T-cell transendothelial migration.

Pathophysiology of nasal polyposis: the role of desmosomal junctions.

Background Many mucosal inflammatory conditions are associated with alterations in epithelial intercellular junctions and barrier function; however, little is known about the role of intercellular junctions in inflammatory diseases of the upper airways. In this study, we examined nasal polyps for altered intercellular junctions and protein expression. Methods Biopsy specimens of nasal polyps and normal tissue were obtained intraoperatively from 11 patients and 6 controls. Tissue was analyzed for expression of intercellular junctional proteins by immunofluorescence. In parallel, cultured human bronchial epithelial (HBE) cells were treated with tumor necrosis factor (TNF) alpha, interferon (IFN) gamma, and IL-13 to simulate inflammatory conditions followed by assessment for changes in junctional proteins by immunofluorescence and Western blot. Results Of the intercellular junctional proteins analyzed, including proteins comprising tight and adherens junctions, the only alterations observed were in desmosomal proteins in nasal polyp epithelium compared with normal controls. Specifically, expression of desmosomal proteins DSG2 and DSG3 were significantly decreased in polyps versus controls (0.53 pixel/μm2 versus 1.09 pixel/μm2 [p = 0.009], and 0.29 pixel/μm2 versus 1.11 pixel/μm2 [p = 0.0078], respectively). In vitro experiments involving exposure of cultured HBE cells with inflammatory cytokines revealed that TNF-alpha treatment resulted in internalization and decreased expression of DSG2 by immunofluorescence and Western blotting. Treatment with IFN-gamma resulted in increased expression of DSG2 and evidence of protein cleavage by Western blot. IL-13 exposure resulted in down-regulation of DSG2 expression and evidence of protein cleavage. Conclusion These results indicate that nasal polyps express decreased levels of DSG2 and DSG3 components of desmosomal junctions. This is likely linked to the mucosal inflammatory response. Exposure of a respiratory cell line to Th1/Th2 cytokines results in similar expressional alterations in DSG2, suggesting protein internalization and cleavage. We speculate that weakened desmosomal junctions in nasal mucosa secondary to inflammatory cytokines may contribute to the formation of nasal polyposis.

Neutrophil Migration across Intestinal Epithelium: Evidence for a Role of CD44 in Regulating Detachment of Migrating Cells from the Luminal Surface

The migration of polymorphonuclear leukocytes (PMNs) across the intestinal epithelium is a histopathological hallmark of many mucosal inflammatory diseases including inflammatory bowel disease. The terminal transmigration step is the detachment of PMNs from the apical surface of the epithelium and their subsequent release into the intestinal lumen. The current study sought to identify epithelial proteins involved in the regulation of PMN migration across intestinal epithelium at the stage at which PMNs reach the apical epithelial surface. A panel of Abs reactive with IFN-γ–stimulated T84 intestinal epithelial cells was generated. Screening efforts identified one mAb, GM35, that prevented PMN detachment from the apical epithelial surface. Microsequencing studies identified the GM35 Ag as human CD44. Transfection studies confirmed this result by demonstrating the loss of the functional activity of the GM35 mAb following attenuation of epithelial CD44 protein expression. Immunoblotting and immunofluorescence revealed the GM35 Ag to be an apically expressed v6 variant exon-containing form of human CD44 (CD44v6). ELISA analysis demonstrated the release of soluble CD44v6 by T84 cells during PMN transepithelial migration. In addition, the observed release of CD44v6 was blocked by GM35 treatment, supporting a connection between CD44v6 release and PMN detachment. Increased expression of CD44v6 and the GM35 Ag was detected in inflamed ulcerative colitis tissue. This study demonstrates that epithelial-expressed CD44v6 plays a role in PMN clearance during inflammatory episodes through regulation of the terminal detachment of PMNs from the apical epithelial surface into the lumen of the intestine.

Novel structural determinants on SIRPα that mediate binding to CD47.

Signal regulatory proteins (SIRP-α, -β, and -γ) are important regulators of several innate immune functions that include leukocyte migration. Membrane distal (D1) domains of SIRPα and SIRPγ, but not SIRPβ, mediate binding to a cellular ligand termed CD47. Because the extracellular domains of all SIRPs are highly homologous, we hypothesized that some of the 16 residues unique to SIRPα.D1 mediate binding to CD47. By site-directed mutagenesis, we determined that SIRPα binding to CD47 is independent of N-glycosylation. We also identified three residues critical for CD47 binding by exchanging residues on SIRPα with corresponding residues from SIRPβ. Cumulative substitutions of the critical residues into SIRPβ resulted in de novo binding of the mutant protein to CD47. Homology modeling of SIRPα.D1 revealed topological relationships among critical residues and allowed the identification of critical residues common to SIRPα and SIRPβ. Mapping these critical residues onto the recently reported crystal structure of SIRPα.D1 revealed a novel region that is required for CD47 binding and is distinct and lateral to another putative CD47 binding site described on that crystal structure. The importance of this lateral region in mediating SIRPα.D1 binding to CD47 was confirmed by epitope mapping analyses of anti-SIRP Abs. These observations highlight a complex nature of the ligand binding requirements for SIRPα that appear to be dependent on two distinct but adjacent regions on the membrane distal Ig loop. A better understanding of the structural basis of SIRPα/CD47 interactions may provide insights into therapeutics targeting pathologic inflammation.

In Vitro Neutrophil Transepithelial Migration

Polymorphonuclear leukocyte (PMN) transmigration into tissues is a highly regulated process and plays a central role in host defense. In inflammatory human diseases such as ulcerative colitis and Crohns disease, the infiltration of intestinal mucosa by large numbers of PMNs contributes to epithelial pathophysiology. The sequence of events that fine-tune PMN migration across epithelial cells is not well-understood. In this chapter, we describe a method to study PMN transmigration across intestinal epithelial T84 monolayers using a modified Boyden chamber system. This in vitro model system consists of three main components: the epithelium, purified PMN, and a chemoattractant gradient. Intestinal epithelial cells are cultured as inverted monolayers on permeable filter supports to facilitate the study of PMN transmigration in the physiologically relevant basolateral-to-apical direction. PMNs are isolated from human blood using dextran sedimentation followed by Ficoll density gradient centrifugation. PMN transmigration is elicited using N-formyl-methionyl-leucyl-phenylalanine gradients and is quantified by assaying for myeloperoxidase activity. The advantages of this model are its reductionist approach and the fact that the system can be easily manipulated. Studies using this model system will shed more light on the mechanisms regulating PMN responses in acute inflammatory diseases.