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

Abstract

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.

Figure 1

A. TER measurements from 12 sets of cultured AFRS and non-inflammatory control primary sinonasal epithelial cells. Cells were paired based on monolayer maturity, which was judged by length of time from development of beating cilia. B. Overall mean TER values calculated from all cell sets. Mean AFRS TER was 296±89 ohms × cm², a decrease of 41% from the control value of 503±134 ohms × cm² (*P<0.01). Data are graphed as mean ± SE (n=12).

Figure 2

A. Western immunoblots for tight and adherens junction-associated proteins in AFRS and non-inflammatory control primary sinonasal epithelial cells. JAM-A, occludin, and E-cadherin are decreased in AFRS compared to control. Claudin-2, a “leaky,” pore-forming claudin is increased in AFRS. B. Densitometric analysis of Western immunoblots. Data were normalized to actin levels and are presented as mean signal intensity ± SE as a percentage of the total signal (n=4).

Figure 3

Representative immunofluorescence staining in AFRS and non-inflammatory control cultured sinonasal epithelial cells. JAM-A, occludin, and E-cadherin signal intensity at cell-cell junctions is diminished in AFRS cell layers compared to controls. Claudin-2 localizes to a nonfunctional cytosolic pool in control cell layers, and to cell-cell junctions in AFRS.