Interleukin-4 and interleukin-13 compromise the sinonasal epithelial barrier and perturb intercellular junction protein expression.

BACKGROUND: Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a "leaky" epithelial barrier phenotype. We hypothesize that T helper 2 (Th2) cytokines interleukin (IL)-4 and IL-13 modulate epithelial junction proteins, thereby contributing to the leaky epithelial barrier. METHODS: Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. RESULTS: IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n = 6) and 68.6% (n = 8) of baseline, respectively. Tight junction protein junctional adhesion molecule-A (JAM-A) expression decreased 42.2% with IL-4 exposure (n = 9) and 37.5% with IL-13 exposure (n = 9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n = 9) and 32.9% with IL-13 exposure (n = 9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or zonula occludens-1 (ZO-1) with IL-4 or IL-13 exposure. CONCLUSION: Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity.

Annexin A2 regulates ß1 integrin internalization and intestinal epithelial cell migration.

The gastrointestinal epithelium functions as an important barrier that separates luminal contents from the underlying tissue compartment and is vital in maintaining mucosal homeostasis. Mucosal wounds in inflammatory disorders compromise the critical epithelial barrier. In response to injury, intestinal epithelial cells (IECs) rapidly migrate to reseal wounds. We have previously observed that a membrane-associated, actin binding protein, annexin A2 (AnxA2), is up-regulated in migrating IECs and plays an important role in promoting wound closure. To identify the mechanisms by which AnxA2 promotes IEC movement and wound closure, we used a loss of function approach. AnxA2-specific shRNA was utilized to generate IECs with stable down-regulation of AnxA2. Loss of AnxA2 inhibited IEC migration while promoting enhanced cell-matrix adhesion. These functional effects were associated with increased levels of ß1 integrin protein, which is reported to play an important role in mediating the cell-matrix adhesive properties of epithelial cells. Because cell migration requires dynamic turnover of integrin-based adhesions, we tested whether AnxA2 modulates internalization of cell surface ß1 integrin required for forward cell movement. Indeed, pulse-chase biotinylation experiments in IECs lacking AnxA2 demonstrated a significant increase in cell surface ß1 integrin that was accompanied by decreased ß1 integrin internalization and degradation. These findings support an important role of AnxA2 in controlling dynamics of ß1 integrin at the cell surface that in turn is required for the active turnover of cell-matrix associations, cell migration, and wound closure.

Sinonasal epithelial wound resealing in an in vitro model: inhibition of wound closure with IL-4 exposure.

BACKGROUND: Prolonged healing and persistent inflammation following surgery for rhinosinusitis impacts patient satisfaction and healthcare resources. Cytokines interleukin (IL)-4, IL-5, and IL-13 are important mediators in T-helper 2 (Th2) inflammatory rhinosinusitis. Decreased wound healing has been demonstrated with Th2 cytokine exposure, but this has not been extensively studied in sinonasal epithelium. We hypothesized that in vitro exposure of primary sinonasal epithelial cell cultures to Th2 inflammatory cytokine IL-4 and IL-13 would impair wound resealing and decrease expression of annexin A2 at the wound edge. METHODS: Following 24-hour exposure to IL-4, IL-5, or IL-13 vs controls, sterile linear mechanical wounds were created in primary sinonasal epithelial cultures (n = 12 wounds per condition). Wounds were followed for 36 hours or until complete closure, and residual wound areas were calculated by image analysis. Group differences in annexin A2 were assessed by immunofluorescence labeling, confocal microscopy, and Western blots. RESULTS: Significant wound closure differences were identified across cytokine exposure groups (p < 0.001). Mean percentage wound closure at the completion of the 36-hour time course was 98.41% ± 3.43% for control wounds vs 85.02% ± 18.46% for IL-4 exposed wounds. IL-13 did not significantly impair sinonasal epithelial wound resealing in vitro. Annexin A2 protein levels were decreased in IL-4 treated wounds when compared to control wounds (p < 0.01). CONCLUSION: Th2 cytokine IL-4 decreases sinonasal epithelial wound closure in vitro. Annexin A2 is also diminished with IL-4 exposure. This supports the hypothesis that IL-4 exposure impairs sinonasal epithelial wound healing and may contribute to prolonged healing in Th2 inflammatory rhinosinusitis.

Epithelial permeability alterations in an in vitro air-liquid interface model of allergic fungal rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is an inflammatory upper-airway disease with numerous etiologies. Patients with a characteristic subtype of CRS, allergic fungal rhinosinusitis (AFRS), display increased expression of T helper 2 (Th2) cytokines and antigen-specific immunoglobulin E (IgE). Various sinonasal inflammatory conditions are associated with alterations in epithelial barrier function. The aim of this study was to compare epithelial permeability and intercellular junctional protein expression among cultured primary sinonasal cells from AFRS patients vs noninflammatory controls. METHODS: Epithelial cells isolated from paranasal sinus mucosa of AFRS and noninflammatory control patients were grown to confluence on permeable supports and transitioned to air-liquid interface (ALI). Transepithelial resistance (TER) was measured with a horizontal Ussing chamber to characterize the functional permeability of each cell type. After TER recordings were complete, a panel of intercellular junctional proteins was assessed by Western blot and immunofluorescence labeling followed by confocal microscopy. RESULTS: After 12 samples were measured from each group, we observed a 41% mean decrease in TER in AFRS cells (296 ± 89 ohms × cm(2) ) compared to control (503 ± 134 ohms × cm(2) , p = 0.006). TER deficits observed in AFRS were associated with decreased expression of the tight junction proteins occludin and junctional adhesion molecule-A (JAM-A), and increased expression of a leaky tight junction protein claudin-2. CONCLUSION: Cultured sinonasal epithelium from AFRS patients displayed increased epithelial permeability and altered expression of intercellular junctional proteins. Given that these cells were not incubated with inflammatory cytokines in vitro, the cultured AFRS epithelial alterations may represent a retained modification in protein expression from the in vivo phenotype.