Shino Shimizu

Role of the coagulation system in allergic inflammation in the upper airways

Thrombin has been detected and demonstrated to play a role in the airways of patients with bronchial asthma, but its role in the upper airways including during allergic rhinitis is unknown. This study was conducted to explore whether thrombin is presence in the upper airways and, if so, whether it affects mucin secretion. Fifteen patients with allergic rhinitis were enrolled in the clinical study; primary nasal septum epithelial cells and normal bronchial epithelial cells were used for in vitro evaluation, and rats as animal models. Significant concentrations of thrombin were found in nasal secretion after allergic provocation in allergic patients, and thrombin and its agonistic receptor peptide induced significant secretion of mucin in primary nasal cells and normal bronchial epithelial cells as compared to non-stimulated cells. Increased mucosubstance secretion in septum epithelial cells was also induced after nasal instillation of thrombin in rats. Further, the anticoagulant, activated protein C, significantly inhibited thrombin-induced mucin secretion from septum epithelial cells in rats. The results of this study suggest that activation of the coagulation system occurs during the allergic response and that thrombin plays a crucial role in the regulation of mucin production in the upper airways.

Role of thrombin in chronic rhinosinusitis-associated tissue remodeling.

Background Thrombin, the effector enzyme of the coagulation system, has been reported to promote inflammatory responses in nasal diseases through its protease-activated receptors (PARs). Chronic rhinosinusitis (CRS) is characterized by increased deposition of extracellular matrix proteins, tissue remodeling, and formation of nasal polyps. The role of thrombin in chronic nasal inflammation–associated tissue remodeling still has not been appraised. This study was conducted to elucidate the role of thrombin in the pathogenesis of CRS. Methods Nasal secretion was collected from patients with CRS with nasal polyp (CRSwNP) with asthma (n = 9), CRSwNP without asthma (n = 10), allergic rhinitis (n = 7), and control patients (n = 3). The concentrations of thrombin, thrombin–antithrombin (TAT) complex, and vascular endothelial growth factor (VEGF) were evaluated by enzyme immunoassays. The concentration of thrombin and TAT complex was measured in nasal secretion from each group of patients, and VEGF was measured in culture medium from airway epithelial cells treated with thrombin or thrombin receptor agonist peptide. Results Thrombin and TAT complex were significantly increased in nasal secretion of patients with CRSwNPs with asthma compared with the control group. Thrombin and PAR-1 agonist peptide significantly stimulated VEGF secretion from cultured human airway epithelial cells. Conclusion The results of this study showed that there is increased activation of the coagulation system in the nasal mucosa of CRS patients and that thrombin may play a role in nasal polyp formation by stimulating VEGF production from airway epithelial cells.

Eosinophil-epithelial cell interactions stimulate the production of MUC5AC mucin and profibrotic cytokines involved in airway tissue remodeling.

Background Predominant eosinophil infiltration and tissue remodeling are common characteristics of chronic airway inflammation such as nasal polyposis and bronchial asthma. This study was designed to elucidate the role of eosinophils in tissue remodeling of chronic airway inflammation; eosinophil–epithelial interactions were examined by the coculture of airway epithelial cell line NCI-H292 with the eosinophilic cell line EoL-1 or with human blood eosinophils. Methods The coculture-induced production of MUC5AC mucin, platelet-derived growth factor AB (PDGF-AB), vascular endothelial growth factor (VEGF), transforming growth factor (TGF) beta1, and interleukin-8 (IL-8) were evaluated by enzyme-linked immunosorbent assay and reverse transcription–polymerase chain reaction. Results Eosinophil–epithelial interactions significantly stimulated the secretion of MUC5AC, PDGF-AB, VEGF, TGF-beta1, and IL-8 in culture supernatants. The epidermal growth factor receptor tyrosine kinase inhibitor AG1478 inhibited the coculture-induced secretion of MUC5AC, PDGF-AB, VEGF, and IL-8. Neutralizing antibodies directed against TGF-alpha or amphiregulin and pan-metalloproteinase inhibitor GM6001 inhibited the coculture-induced secretion of MUC5AC and amphiregulin from the cocultured NCI-H292 cells. Coculture of NCI-H292 cells with peripheral blood eosinophils also significantly stimulated MUC5AC production. Conclusion The results of this study indicate that eosinophil–epithelial cell interactions are important in the pathogenesis of tissue remodeling of eosinophil-predominant airway inflammation such as occurs in nasal polyposis and bronchial asthma.

Heparin Inhibits Mucus Hypersecretion in Airway Epithelial Cells

Background Heparin is one of the most important anticoagulant drugs. It has been known that heparin also possesses anti-inflammatory activities. Mucus hypersecretion is an important characteristic of airway inflammation. However, little is known about the regulatory effects of heparin on mucus hypersecretion in airway epithelial cells. To elucidate the anti-inflammatory function of heparin in airway epithelial cells, we examined the in vivo effects of heparin on mucus hypersecretion and neutrophil infiltration in rat nasal epithelium. We also examined the in vitro effects of heparin on mucin production and IL-8 secretion from cultured human airway epithelial cells. Methods We induced hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium by intranasal lipopolysaccharide (LPS) instillation. The effects of intranasal instillation with heparin on mucus production and neutrophil infiltration were examined. In vitro effects of heparin on airway epithelial cells were examined using cultured NCI-H292 cells. Mucus secretion was evaluated by enzyme-linked immunosorbent assay using an anti-MUC5AC monoclonal antibody. Results Intranasal instillation with unfractionated heparin (UFH; 100 IU/0.1 mL) or low molecular weight heparin (LMWH; 100 IU/0.1 mL) at 30 minutes before LPS instillation significantly inhibited LPS-induced mucus production and neutrophil infiltration in rat nasal epithelium. UFH or LMWH inhibited tumor necrosis factor alpha (10 ng/mL)–induced secretion of MUC5AC and IL-8 from NCI-H292 cells in a dose-dependent manner (0.01–10 IU/mL). MUC5AC mRNA expression was also significantly inhibited. Conclusion These results indicate that heparin inhibits airway mucus hypersecretion in airway epithelial cells directly and indirectly through the suppression of IL-8 secretion and neutrophil infiltration.

Epidermal growth factor receptor inhibitor AG1478 inhibits mucus hypersecretion in airway epithelium.

Background Mucus hypersecretion and neutrophil infiltration are important characteristics of airway inflammation. Epidermal growth factor receptor (EGFR) transactivation induces mucus and inflammatory cytokine secretion from airway epithelial cells. To elucidate the roles of EGFR in airway inflammation, the in vitro effects on mucin production and interleukin (IL) 8 secretion from cultured airway epithelial cells and the in vivo effects on mucus hypersecretion and neutrophil infiltration in rat nasal mucosa of the EGFR tyrosine kinase inhibitor AG1478 were examined. Methods The in vitro effects of AG1478 treatment of cultured NCI-H292 cells on lipopolysaccharide (LPS) induced or tumor necrosis factor (TNF) a induced MUC5AC mucin and IL-8 secretion were evaluated. Hypertrophic and metaplastic changes of goblet cells, mucus production and neutrophil infiltration in rat nasal epithelium were induced by intranasal instillation of LPS in vivo, and the inhibitory effects of AG1478 by intraperitoneal injection or intranasal instillation were examined. Results AG1478 (1-1000 nM) significantly inhibited both LPS-induced and TNF-α-induced secretion of MUC5AC and IL-8 from cultured NCI-H292 cells in a dose-dependent manner. The expression of MUC5AC and IL-8 messenger RNAs was also significantly inhibited. Intranasal instillation of AG1478 one hour after intranasal LPS instillation significantly inhibited LPS-induced goblet cell metaplasia, mucus production, and neutrophil infiltration in rat nasal epithelium, as did intraperitoneal injection of AG1478 one hour before LPS instillation. Conclusions These results indicated that EGFR transactivation plays an important role in mucin and IL-8 secretion from airway epithelial cells. Intranasal instillation of an EGFR tyrosine kinase inhibitor may be a new therapeutic approach for the treatment of upper airway inflammation.

Endogenous Protease Inhibitors in Airway Epithelial Cells Contribute to Eosinophilic Chronic Rhinosinusitis

Rationale: Cystatin A and SPINK5 are endogenous protease inhibitors (EPIs) that may play key roles in epithelial barrier function. Objectives: To investigate the roles of EPIs in the pathogenesis of chronic rhinosinusitis (CRS). Methods: We examined the expression of cystatin A and SPINK5 in the nasal epithelial cells of patients with CRS. Additionally, the in vitro effects of recombinant EPIs on the secretion of the epithelial‐derived cytokines IL‐25, IL‐33, and thymic stromal lymphopoietin in airway epithelial cells, and the in vivo effects of recombinant EPIs in the nasal epithelium of mice exposed to multiple airborne allergens (MAA) were examined. Measurements and Main Results: Compared with control subjects and patients with noneosinophilic CRS, patients with eosinophilic CRS showed significantly lower protein and mRNA expression of cystatin A and SPINK5 in the nasal epithelium. Allergen‐induced production of IL‐25, IL‐33, and thymic stromal lymphopoietin in normal human bronchial epithelial cells was inhibited by treatment with recombinant cystatin A or SPINK5. Conversely, the production of these cytokines was increased when cystatin A or SPINK5 were knocked down with small interfering RNA. Chronic MAA exposure induced goblet cell metaplasia and epithelial disruption in mouse nasal epithelium and decreased the tissue expression and nasal lavage levels of cystatin A and SPINK5. Intranasal instillations of recombinant EPIs attenuated this MAA‐induced pathology. Conclusions: Cystatin A and SPINK5 play an important role in protecting the airway epithelium from exogenous proteases. The preservation of EPIs may have a therapeutic benefit in intractable airway inflammation, such as eosinophilic CRS.

Valproic acid promotes neural regeneration of olfactory epithelium in adult mice after methimazole-induced damage.

Background Recent experiments have revealed that valproic acid (VPA), a histone deacetylase inhibitor, has neuroregenerative effects in rodent models of spinal cord and optic nerve injury. VPA has a potential to provide a new therapeutic strategy for sensorineural olfactory dysfunction. To elucidate the effects of VPA on regeneration of olfactory sensory neurons, we examined the in vivo effects of oral VPA administration on recovery from methimazole-induced degeneration of olfactory neuroepithelium in mice. Methods Male ICR mice (10 weeks old) were intraperitoneally injected with methimazole (75 mg/kg), an olfactory toxic reagent, to induce degenerative changes in the olfactory neuroepithelium. The effects of daily VPA administration on recovery from methimazole-induced changes were examined histologically. Results Oral VPA administration dose dependently enhanced increases in epithelial thickness and number of olfactory marker protein (OMP) positive cells in the olfactory epithelium during recovery from methimazole-induced degeneration. VPA also enhanced early increases in the number of Ki-67+ and growth-associated protein-43+ cells during the regeneration of olfactory neuroepithelium. Conclusion VPA administration promotes regeneration of olfactory sensory neurons in damaged neuroepithelium by stimulating the proliferation and differentiation of olfactory precursor cells. VPA has been used for several decades to safely treat neurological disorders. VPA may provide a new therapeutic strategy for the treatment of olfactory dysfunction caused by degeneration of the olfactory neuroepithelium.

Azithromycin inhibits mucus hypersecretion from airway epithelial cells.

To examine the in vivo effects of the 15-member macrolide, azithromycin (AZM), on mucus hypersecretion, we induced hypertrophic and metaplastic changes of goblet cells in rat nasal epithelium by intranasal instillation of ovalbumin (OVA) in OVA-sensitized rats, or by intranasal lipopolysaccharides (LPS) instillation. Oral administration of AZM (5–10 mg/kg) or clarithromycin (CAM, 5–10 mg/kg) significantly inhibited OVA- and LPS-induced mucus production, whereas josamycin (JM) or ampicillin (ABPC) showed no effect. In vitro effects of AZM on airway epithelial cells were examined using NCI-H292 cells and human nasal epithelial cells cultured in air-liquid interface. Mucus secretion was evaluated by enzyme-linked immunosorbent assay using an anti-MUC5AC monoclonal antibody. AZM or CAM significantly inhibited tumor necrosis factor-α (TNF-α) (20 ng/mL)-induced MUC5AC secretion from NCI-H292 cells at 10−6–10−7 M, whereas JM or ABPC showed no effect. AZM significantly inhibited TNF-α (20 ng/mL)-induced MUC5AC secretion from human nasal epithelial cells at 10−4 M. MUC5AC mRNA expression was also significantly inhibited. These results indicate that the 15-member macrolide, AZM, exerts direct inhibitory effects on mucus secretion from airway epithelial cells and that it may be useful for the treatment of mucus hypersecretion caused by allergic inflammation and LPS stimulation.

Th2 cytokine inhibitor suplatast tosilate inhibits antigen-induced mucus hypersecretion in the nasal epithelium of sensitized rats.

Objectives: Th2 cytokines such as interleukin (IL) 4 and IL-13 are potential mediators for mucus hypersecretion in allergic inflammation. To elucidate the functions of Th2 cytokines in allergic rhinitis, we examined the in vivo effects of the Th2 cytokine inhibitor suplatast tosilate on mucus hypersecretion and eosinophil infiltration in rat nasal epithelium. Methods: We induced hypertrophic and metaplastic changes in goblet cells in the nasal epithelium of ovalbumin-sensitized rats by intranasal challenge with ovalbumin. The effects of orally administered suplatast tosilate on mucus production and eosinophil infiltration were examined. Results: Suplatast tosilate (30 and 100 mg/kg) dose-dependently inhibited ovalbumin-induced mucus production and eosinophil infiltration. These suppressions of mucus production and eosinophil infiltration were only effective when suplatast tosilate was given in the effector phase; administration in the induction phase resulted in no effect. Conclusions: These results indicate that Th2 cytokines are important mediators of mucus hypersecretion and eosinophil infiltration in allergic rhinitis. Suplatast tosilate may be useful for the treatment of allergic rhinitis by attenuating the inflammation of the effector phase.

HMGB1-TLR4 signaling contributes to the secretion of interleukin 6 and interleukin 8 by nasal epithelial cells.

Background Alarmins play important roles in the pathogenesis of inflammatory and autoimmune diseases. However, the role of the alarmin protein high-mobility group box 1 (HMGB1) in upper airway inflammation is unclear. Objective To determine if HMGB1 is present in the nasal mucosa and, if so, to elucidate its role in upper airway inflammation. Methods Nasal secretions were collected from a total of 32 patients with chronic rhinosinusitis with nasal polyp, allergic rhinitis, and control subjects. The concentration of HMGB1 in nasal secretions and its tissue and cellular localization were examined by enzyme immunoassays and immunofluorescent staining of nasal polyps and cultured nasal epithelial cells. We then examined whether nasal epithelial cells secrete HMGB1 after inflammatory stimulation by tumor necrosis factor (TNF) α. The effects of HMGB1 on the production and secretion of interleukin (IL) 6 and IL-8 were also examined in cultured nasal epithelial cells. Results Significantly higher concentrations of HMGB1 were found in nasal secretions from patients with chronic rhinosinusitis with nasal polyp or allergic rhinitis compared with the control subjects. HMGB1 expression was localized in the nuclei of epithelial cells and other constitutive cells in nasal polyps and in the nuclei of cultured nasal epithelial cells. TNF-α stimulated the production and secretion of HMGB1 by cultured nasal epithelial cells. HMGB1 stimulated the production and secretion of IL-6 and IL-8 by cultured nasal epithelial cells, and anti–toll-like receptor 4 blocking antibody significantly inhibited HMGB1-induced secretion of IL-6 and IL-8. Conclusions Nasal secretions contain substantial amounts of HMGB1. TNF-α stimulates the production of HMGB1, which, in turn, upregulates the production and secretion of IL-6 and IL-8 by nasal epithelial cells via toll-like receptor 4, which indicated that HMGB1 plays an important role in the pathogenesis of upper airway inflammation.