Pseudomonas aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells.

BACKGROUND: Pseudomonas aeruginosa causes chronic respiratory disease, and the elastase enzyme that it produces increases the permeability of airway epithelial cells owing to the disruption of tight junctions. P. aeruginosa is also implicated in prolonged chronic rhinosinusitis. However, the effects of P. aeruginosa elastase (PE) against the barrier formed by human nasal epithelial cells (HNECs) remain unknown. METHODS: To investigate the mechanisms involved in the disruption of tight junctions by PE in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were used. The hTERT-HNECs were pretreated with inhibitors of various signal transduction pathways, PKC, MAPK, p38MAPK, PI3K, JNK, NF-κB, EGF receptor, proteasome, COX1 and COX2 before treatment with PE. Some cells were pretreated with siRNA and agonist of protease activated receptor-2 (PAR-2) before treatment with PE. Expression and structures of tight junctions were determined by Western blotting, real-time PCR, immunostaining and freeze-fracture. Transepithelial electrical resistance (TER) was examined as the epithelial barrier function. RESULTS: PE treatment transiently disrupted the epithelial barrier and downregulated the transmembrane proteins claudin-1 and -4, occludin, and tricellulin, but not the scaffold PDZ-expression proteins ZO-1 and -2 and adherens junction proteins E-cadherin and ß-catenin. The transient downregulation of tight junction proteins was controlled via distinct signal transduction pathways such as the PKC, MAPK, PI3K, p38 MAPK, JNK, COX-1 and -2, and NF-κB pathways. Furthermore, treatment with PE transiently decreased PAR-2 expression, which also regulated the expression of the tight junction proteins. Treatment with a PAR-2 agonist prevented the downregulation of the tight junction proteins after PE treatment in HNECs. CONCLUSIONS: PE transiently disrupts tight junctions in HNECs and downregulates PAR-2. The transient disruption of tight junctions by PE might occur repeatedly during chronic rhinosinusitis.

[Post-operative functional evaluation of accessory nerve reconstruction].

A nerve reconstruction was performed in 20 patients whose spinal accessory nerve was resected during total neck dissection. Re-anastomosis or a cable graft was performed between both cut ends of the accessory nerve in 14 patients (accessory nerve reconstruction group), and between the peripheral cut end of the accessory nerve and the central cut end of the cervical nerve (C2 or C3) in 6 patients (cervical/accessory nerve reconstruction group). There was no difference in the postoperative shoulder functions between the reconstruction groups, and both groups were significantly better than the group without reconstruction (n = 13), although they tended to be poorer than the nerve preservation group (n = 41).

A Novel Drug Delivery System for the Human Nasal Epithelium.

The epithelium of upper respiratory tissues such as the human nasal mucosa forms a continuous barrier via tight junctions (TJs). The development of a drug delivery system for use across the nasal mucosa is being reconsidered. In intranasal administration across the nasal mucosa, the paracellular pathway regulated by TJs is extremely important. It is known that the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds the TJ protein claudin and disrupts the tight junctional barrier without inducing a cytotoxic effect. We investigated the effects of C-CPE mutants on the function of TJs of human nasal epithelial cells (HNECs) and on the permeability of human recombinant insulin across HNECs treated with C-CPE 194 and C-CPE m19. We recently reported that C-CPE mutants 194 and m19 can regulate the permeability of insulin across HNECs via the MAPK pathway and may play a crucial role in therapy for various diseases via direct intranasal insulin administration. On the other hand, microRNAs (miRNAs) are known to regulate the expression of TJs as direct or indirect targets in genes to maintain barrier function. We investigated the effects of miRNAs on the epithelial barrier of HNECs and found that miRNA-146a plays crucial roles in the maintenance of the TJ barrier and innate immune response against invading pathogens. This chapter reviews a novel drug delivery system across the nasal mucosa from the point of view of the epithelial barrier function.

Claudin-binder C-CPE mutants enhance permeability of insulin across human nasal epithelial cells.

OBJECTIVE: Intranasal insulin administration has therapeutic potential for Alzheimer's disease and in intranasal administration across the nasal mucosa, the paracellular pathway regulated by tight junctions is important. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds the tight junction protein claudin and disrupts the tight junctional barrier without a cytotoxic effect. The C-CPE mutant called C-CPE 194 binds only to claudin-4, whereas the C-CPE 194 mutant called C-CPE m19 binds not only to claudin-4 but also to claudin-1. METHODS: In the present study, to investigate the effects of C-CPE mutants on the tight junctional functions of human nasal epithelial cells (HNECs) and on the permeability of human recombinant insulin across the cells, HNECs were treated with C-CPE 194 and C-CPE m19. RESULTS: C-CPE 194 and C-CPE m19 disrupted the barrier and fence functions without changes in expression of claudin-1, -4, -7, and occludin or cytotoxicity, whereas they transiently increased the activity of ERK1/2 phosphorylation. The disruption of the barrier function caused by C-CPE 194 and C-CPE m19 was prevented by pretreatment with the MAPKK inhibitor U0126. Furthermore, C-CPE 194 and C-CPE m19 significantly enhanced the permeability of human recombinant insulin across HNECs and the permeability was also inhibited by U0126. CONCLUSION: These findings suggest that C-CPE mutants 194 and m19 can regulate the permeability of insulin across HNECs via the MAPK pathway and may play a crucial role in therapy for the diseases such as Alzheimer's disease via the direct intranasal insulin administration.

Regulation of tight junctions in upper airway epithelium.

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.

Regulation of interleukin-33 and thymic stromal lymphopoietin in human nasal fibroblasts by proinflammatory cytokines.

OBJECTIVES/HYPOTHESIS: Epithelial-derived interleukin (IL)-33 and thymic stromal lymphopoietin (TSLP) are critical regulators of innate and adaptive immune responses associated with Th2 cytokine-mediated inflammation, including allergic rhinitis. IL-33 and TSLP are expressed not only in epithelial cells but also fibroblasts, endothelial cells, and smooth muscle cells at nasal mucosal sites. However, the role and the regulation of IL-33 and TSLP in nasal fibroblasts remain unknown. We investigated the signal transduction regulation of IL-33 and TSLP induced by proinflammatory cytokines in nasal fibroblasts. STUDY DESIGN: In vitro, prospective study. METHODS: Nasal fibroblasts were derived from human nasal mucosa without allergic rhinitis. Expression of IL-33 and TSLP was examined in nasal fibroblasts treated with proinflammatory cytokines IL-1ß or tumor necrosis factor (TNF)-α after pretreatment with or without various inhibitors of signal transduction pathways. RESULTS: In nasal fibroblasts, both Western blotting and reverse transcriptase-polymerase chain reaction demonstrated that expression of mRNAs and proteins of IL-33 and TSLP was increased by treatment with IL-1ß or TNF-α. Immunostaining revealed that IL-33-positive nuclei were markedly increased by the treatment with IL-1ß or TNF-α. Enzyme-linked immunosorbent assay showed that fibroblast-released TSLP was significantly increased by treatment with IL-1ß or TNF-α. The upregulation of both IL-33 and TSLP proteins by treatment with IL-1ß was prevented by inhibitors of pan- protein kinase C (PKC), p38 mitogen-activated protein kinase, and nuclear factor (NF)-κB. In the cells treated with TNF-α, upregulation of IL-33 protein was prevented by inhibitors of phosphoinositide 3-kinase (PI3K), c-Jun N-terminal kinase (JNK), and NF-κB, whereas upregulation of TSLP protein was prevented by inhibitors of pan-PKC, PI3K, JNK, and NF-κB. CONCLUSIONS: Expression of IL-33 and TSLP in nasal fibroblasts was regulated via distinct signal transduction pathways including NF-κB.