The epithelial barrier of the upper respiratory tract plays a crucial role in host defense. In this study, to elucidate whether there is antigen monitoring by dendritic cells (DCs) beyond the epithelial tight-junction barrier in allergic rhinitis, we investigated the expression and function of tight junctions and characterized DCs in the epithelium of nasal mucosa from patients with allergic rhinitis. In reverse transcription-polymerase chain reaction, mRNAs of tight-junction proteins occludin, JAM-1, ZO-1, and claudin-1, -4, -7, -8, -12, -13, and -14 were detected in the nasal mucosa. Occludin, JAM-1, and ZO-1 were colocalized in the uppermost layer in the pseudostratified epithelium of the nasal mucosa, whereas claudin-1, -4, and -7 were found throughout the epithelium. In freeze-fracture replicas of the nasal mucosa, continuous tight-junction strands formed well-developed networks. Epithelial barrier function measured by a dye tracer was well maintained in occludin-positive tight junctions in the epithelium of the nasal mucosa. HLA-DR- and CD11c-positive DCs expressed claudin-1 and penetrated beyond occludin in the epithelium of the nasal mucosa with, but not without, allergic rhinitis. These results indicate that DCs may easily access antigens beyond epithelial tight junctions in the human nasal mucosa of allergic rhinitis.

The mucosal barrier of the upper respiratory tract including the nasal cavity, which is the first site of exposure to inhaled antigens, plays an important role in host defense in terms of innate immunity and is regulated in large part by tight junctions of epithelial cells. Tight junction molecules are expressed in both M cells and dendritic cells as well as epithelial cells of upper airway. Various antigens are sampled, transported, and released to lymphocytes through the cells in nasal mucosa while they maintain the integrity of the barrier. Expression of tight junction molecules and the barrier function in normal human nasal epithelial cells (HNECs) are affected by various stimuli including growth factor, TLR ligand, and cytokine. In addition, epithelial-derived thymic stromal lymphopoietin (TSLP), which is a master switch for allergic inflammatory diseases including allergic rhinitis, enhances the barrier function together with an increase of tight junction molecules in HNECs. Furthermore, respiratory syncytial virus infection in HNECs in vitro induces expression of tight junction molecules and the barrier function together with proinflammatory cytokine release. This paper summarizes the recent progress in our understanding of the regulation of tight junctions in the upper airway epithelium under normal, allergic, and RSV-infected conditions.

The IL-33 and its receptor ST2 play important roles in allergic rhinitis. The IL-33-mediated inflammatory responses via ST2 are regulated by distinct signalling pathways in HNECs and the IL-33/ST2 pathway may provide new therapeutic targets for allergic rhinitis.

Epithelial-derived thymic stromal lymphopoietin (TSLP) triggers dendritic cell (DC)-mediated Th2-type inflammatory responses and is a master switch for allergic inflammatory diseases. In the present study, the expression and induction of TSLP and the effects of TSLP on the tight-junctional barrier of human nasal epithelial cells (HNECs) have been investigated in order to elucidate the role of TSLP in allergic rhinitis. We have found high expression of TSLP in the epithelium from patients with allergic rhinitis with recruitment and infiltration of DCs. In vitro, TSLP is significantly produced in HNECs after treatment with a toll-like receptor 2 (TLR2) ligand, Pam(3)Cys-Ser-(Lys)(4), and a mixture of interleukin-1beta and tumor necrosis factor-alpha. Treatment with TSLP rapidly enhances the barrier function of cultured HNECs, together with an increase of tight-junction proteins claudin-1, -4, -7, and occludin. The nasal-epithelial-derived TSLP thus not only activates DCs but also preserves the epithelial barrier via the upregulation of tight-junction proteins, thereby regulating antigen sensitization during the early stage of allergic rhinitis.

Connexins (Cx) are considered to play a crucial role in the differentiation of epithelial cells and to be associated with adherens and tight junctions. This review describes how connexins contribute to the induction and maintenance of tight junctions in epithelial cells, hepatic cells and airway epithelial cells. Endogenous Cx32 expression and mediated intercellular communication are associated with the expression of tight junction proteins of primary cultured rat hepatocytes. We introduced the human Cx32 gene into immortalized mouse hepatic cells derived from Cx32-deficient mice. Exogenous Cx32 expression and the mediated intercellular communication by transfection could induce the expression and function of tight junctions. Transfection also induced expression of MAGI-1, which localized at adherens and tight junction areas in a gap junctional intercellular communication (GJIC)-independent manner. Furthermore, expression of Cx32 was related to the formation of single epithelial cell polarity of the hepatic cells. On the other hand, Cx26 expression, but not mediated intercellular communication, contributed to the expression and function of tight junctions in human airway epithelial cells. We introduced the human Cx26 gene into the human airway epithelial cell line Calu-3 and used a model of tight junction disruption by the Na(+)/K(+)-ATPase inhibitor ouabain. Transfection with Cx26 prevented disruption of both tight junction functions, the fence and barrier, and the changes of tight junction proteins by treatment with ouabain in a GJIC-independent manner. These results suggest that connexins can induce and maintain tight junctions in both GJIC-dependent and -independent manners in epithelial cells.

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The epithelium of upper respiratory tissues such as human nasal mucosa forms a continuous barrier via tight junctions, which is thought to be regulated in part through a protein kinase C (PKC) signaling pathway. To investigate the mechanisms of the regulation of PKC-mediated tight junction barrier function of human nasal epithelium in detail, primary human nasal epithelial cells were treated with the PKC activator 12-O-tetradecanoylophorbol-13-acetate (TPA). In primary human nasal epithelial cells, treatment with TPA led not only to activation of phosphorylation of PKC, myristoylated alanine-rich C kinase substrate, and mitogenactivated protein kinase but also expression of novel PKC-␦, PKC-, and PKC-. Treatment with TPA increased transepithelial electrical resistance, with tight junction barrier function more than 4-fold that of the control, together with up-regulation of tight junction proteins, occludin, zona occludens (ZO)-1, ZO-2 and claudin-1 at the transcriptional level. Furthermore, it affected the subcellular localization of the tight junction proteins and the numbers of tight junction strands. The up-regulation of barrier function and tight junction proteins was prevented by a pan-PKC inhibitor, and the inhibitors of PKC-␦ and PKC-but not PKC-. In primary human nasal epithelial cells, transcriptional factors GATA-3 and -6 were detected by reverse transcription-polymerase chain reaction. The knockdown of GATA-3 using RNA interference resulted in inhibition of up-regulation of ZO-1 and ZO-2 by treatment with TPA. These results suggest that TPA-induced PKC signaling enhances the barrier function of human nasal epithelial cells via transcriptional up-regulation of tight junction proteins, and the mechanisms may contribute to a drug delivery system.

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