Kota Wada

RECOGNITION OF FUNGAL PROTEASE ACTIVITIES INDUCES CELLULAR ACTIVATION AND EOSINOPHIL-DERIVED NEUROTOXIN RELEASE IN HUMAN EOSINOPHILS

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of asthma and in immunity to certain organisms. Associations between exposure to an environmental fungus, such as Alternaria, and asthma have been recognized clinically. Protease-activated receptors (PARs) are G protein-coupled receptors that are cleaved and activated by serine proteases, but their roles in innate immunity remain unknown. We previously found that human eosinophils respond vigorously to Alternaria organisms and to the secretory product(s) of Alternaria with eosinophils releasing their proinflammatory mediators. In this study, we investigated the roles of protease(s) produced by Alternaria and of PARs expressed on eosinophils in their immune responses against fungal organisms. We found that Alternaria alternata produces aspartate protease(s) and that human peripheral blood eosinophils degranulate in response to the cell-free extract of A. alternata. Eosinophils showed an increased intracellular calcium concentration in response to Alternaria that was desensitized by peptide and protease ligands for PAR-2 and inhibited by a PAR-2 antagonistic peptide. Alternaria-derived aspartate protease(s) cleaved PAR-2 to expose neo-ligands; these neo-ligands activated eosinophil degranulation in the absence of proteases. Finally, treatment of Alternaria extract with aspartate protease inhibitors, which are conventionally used for HIV-1 and other microbes, attenuated the eosinophils’ responses to Alternaria. Thus, fungal aspartate protease and eosinophil PAR-2 appear critical for the eosinophils’ innate immune response to certain fungi, suggesting a novel mechanism for pathologic inflammation in asthma and for host-pathogen interaction.

Alternaria Fungus Induces the Production of GM-CSF, Interleukin-6 and Interleukin-8 and Calcium Signaling in Human Airway Epithelium through Protease-Activated Receptor 2

Rationale: Recent studies suggest that host immune responses to environmental fungi may play an important role in the development of allergic diseases, such as human asthma. Epithelium is considered an active participant in allergic inflammation. We previously reported that aspartate protease from Alternaria induces the activation and degranulation of human eosinophils that are mediated through protease-activated receptor 2 (PAR-2). However, our current knowledge on the innate immune responses of epithelium to environmental fungi is very limited. We investigated the responses of epithelium to fungi and the mechanisms of these responses. Methods: Human airway epithelial cell line BEAS-2B and Calu-3 (both from American Type Culture Collection) were incubated with PAR-2 peptides and extracts of various fungi. The cellular responses, including GM-CSF, interleukin (IL)-6, IL-8, eotaxin, eotaxin-2 and RANTES production as well as increases in intracellular calcium concentration ([Ca2+]i), were examined. To characterize the proteases involved in these responses, protease inhibitors such as pepstatin A and alkalo-thermophilic Bacillus inhibitor (ATBI), HIV protease inhibitors and 4-amidinophenylmethanesulfonyl fluoride hydrochloride were used. To investigate the role of PAR-2, PAR-2-agonistic and PAR-2-antagonistic peptides were used. Results: PAR-2-activating peptide, but not the control peptide, induced GM-CSF, IL-6 and IL-8 production; these cellular responses were accompanied by a quick and marked increase in [Ca2+]i. Among 7 common environmental fungi, only Alternaria induced GM-CSF, IL-6 and IL-8 production and increased [Ca2+]i response. Both cytokine production and increased [Ca2+]i were significantly inhibited by PAR-2 antagonist peptide and by aspartate protease inhibitors (pepstatin A, ritonavir, nelfinavir and ATBI), but not by the PAR-2 control peptide or by other protease inhibitors. Conclusions: Aspartate proteases from Alternaria induce cytokine production and calcium response in airway epithelium that is mediated through PAR-2. This protease-mediated activation of airway epithelium may be implicated in the development and exacerbation of airway allergic disease.

Human eosinophil innate response to Alternaria fungus through protease-activated receptor-2.

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of allergic diseases. An association between eosinophilic inflammation and infection or colonization by fungi has also been long recognized. However, the mechanisms underlying how eosinophils are activated and how they release proinflammatory and immunomodulatory mediators such as major basic protein (MBP) and eosinophil-derived neurotoxin remain largely unknown. We used a fungus, i.e. Alternaria, as a model microbe relevant to human asthma and chronic rhinosinusitis (CRS) to investigate the molecular mechanisms involved in the immune recognition of ubiquitous environmental allergens. Human eosinophils are activated by live Alternaria alternata organisms, release their granule proteins, and kill the fungi. Eosinophils, but not neutrophils, respond to secreted products from A. alternata. We found that eosinophils are equipped with innate cellular activation machinery that responds to an extracellular aspartate protease secreted by Alternaria. Aspartate protease activation of protease-activated receptor (PAR)-2 probably involves a novel mechanism different from that for serine protease activation of PAR-2. Thus, human eosinophils may recognize certain danger signals or virulence factors produced by fungi and provoke inflammatory responses against these organisms. Dysregulation of such an innate immune mechanism may be involved in the pathophysiology of certain human inflammatory diseases, including asthma and CRS.

Cockroach Induces Inflammatory Responses through Protease-Dependent Pathways

Exposure to cockroaches is a major risk factor for asthma. Products from cockroaches may contain proteases and ligands for pattern recognition receptors. These molecules may activate airway inflammatory cells, such as eosinophils, that are involved in asthma. Among inner-city children, cockroach allergens play an especially important role in increasing asthma morbidity. The molecular mechanism for this association between cockroach exposure and asthma is not fully understood. Enzymatic activities from cockroaches activate inflammatory cells in the airways and may also exacerbate certain human airway diseases, such as asthma. We recently reported that cockroach extracts contain pepstatin A-sensitive proteases that activate PAR-2 and induce activation and degranulation of human eosinophils. This review focuses on the effects of cockroach on various inflammatory cells, including eosinophils, epithelial cells, fibroblasts, dendritic cells, and T cells, in allergic reactions.

Inflammatory responses of human eosinophils to cockroach are mediated through protease-dependent pathways

To the Editor: Exposure to cockroach is one of the major risk factors for developing asthma (1). Among inner-city children, cockroach allergen has an important role in increasing asthma morbidity. The molecular mechanism for this association between cockroach exposure and asthma is unclear. Cockroaches are complex organisms, and the presence and activities of proteases in cockroach extracts have been controversial. In general, the individual allergen proteins, whether purified or recombinant, do not show proteolytic activity (2). However, protease activities were detected in German cockroach frass and whole body extract (3). Eosinophils are likely involved in the pathophysiology of asthma, atopic dermatitis, and certain gastrointestinal diseases (4). Herein, we examined the effects of cockroach products on the activation and effector functions of human eosinophils, and we investigated the biologically active molecules in cockroach extract. As previously described (5), 12.5 μg/ml German or 6.2 μg/ml Oriental cockroach extracts incubated for 3 h with isolated eosinophils released the eosinophil granule protein, eosinophil-derived neurotoxin (EDN), (see Figure E1 in this article’s Online Repository at www.jacionline.org). German cockroach extract induced superoxide anion production after 15 minutes of exposure, and Oriental cockroach extract induced superoxide anion production after 30 minutes (Figure E1). Both German and Oriental cockroach extracts treated at 4 °C or 37 °C potently induced EDN release (Figure 1A). In contrast, inactivating the extracts at 56 °C decreased their ability to stimulate eosinophils (p<0.05, n=4), and treatment at 100 °C almost abolished the activity. To verify the importance of heat-labile molecule(s) in eosinophil activation induced by cockroach extracts and to demonstrate their lack of direct toxicity to eosinophils, we examined IL-8 production. After 24 hr, both German and Oriental cockroach extracts induced IL-8 production by eosinophils (p<0.05, n=4) (Figure 1B); their stimulatory effects were partially heat-inactivated at 56 °C and abolished at 100 °C. Thus, heat-labile molecules, likely proteases, in cockroach extracts are involved in eosinophil activation. Figure 1 The eosinophil-stimulating activities of cockroach extracts are heat-sensitive PARs, in particular PAR-2, likely play a major role in eosinophil activation in response to proteases (6). Therefore, we investigated whether cockroach extracts contain proteolytic activities that activate PAR-2. An authentic ligand for PAR-2, namely trypsin, cleaves the extracellular N-terminus of PAR-2 between R36 and S37 and exposes a tethered “neo-ligand” (i.e. S37LIGKV-) that binds intramolecularly to PAR-2 and triggers cellular activation (7). To mimic the proteolytic activation of PAR-2, we used a fluorogenic peptide substrate, Abz-SKGRSLIGKdD, which encompasses the trypsin cleavage site of human PAR-2 (from Ser33 to Lys41) (8); the Abz group fluoresces only after release of the KdD group, following cleavage of internal peptides. As expected, trypsin cleaved the PAR-2 peptide, increasing the fluorescence intensity over time (Figure 2A). German and Oriental cockroach extracts also cleaved the PAR-2 peptide in a concentration-dependent manner. To characterize the cockroach proteases cleaving this PAR-2, we used protease inhibitors. Pepstatin A specifically inhibits acid proteases, in particular aspartate proteases. Absorption of a complex protease mixture with pepstatin A agarose removes aspartate-like proteases. Treatment of German and Oriental cockroach extracts with pepstatin A agarose decreased PAR-2 cleavage by 70% and 55 %, respectively (p<0.05, n=5); treatment with control agarose showed no effects (Figure 2B). Pepstatin A treatment showed no effects on trypsin-mediated PAR-2 cleavage, demonstrating the inhibitor’s specificity. A serine protease inhibitor, APMSF, and a cysteine protease inhibitor, E64, did not inhibit PAR-2 cleavage by cockroach extracts (Figure 2C); APMSF significantly inhibited trypsin-induced PAR-2 cleavage. Furthermore, after pretreatment with pepstatin A, both German and Oriental cockroach extracts induced less EDN release compared to the untreated extracts (Figure 2D, p<0.05, n=5). Pretreatment of PMA with pepstatin A did not affect eosinophil degranulation. In addition, preincubating eosinophils with the PAR-2 antagonistic peptide, LSIGKV, before exposure to German cockroach extract inhibited degranulation by an average of 37% (p<0.01, n=9). Overall, the cockroach extracts likely contain pepstatin A-sensitive protease(s) that activate PAR-2 and induce eosinophil degranulation. Figure 2 Cockroach extracts have pepstatin-sensitive aspartate protease activity that is involved in PAR-2 cleavage and eosinophil degranulation In general, trypsin and trypsin-like proteases cleave PAR-2 specifically between Arg36 and Ser37 (7). To investigate the PAR-2 site cleaved by cockroach extracts, we analyzed the peptide fragments by reverse-phase-HPLC-ESI-MS. The same peptide substrate, as used in the PAR-2 cleavage enzymatic assay (Abz-SKGR36S37LIGKdD), was incubated with cockroach extracts, which had been pretreated with medium, pepstatin A agarose or control agarose. As expected, trypsin cleaved the peptide substrate between Arg36 and Ser37 and produced the S37LIGKdD fragment (Figure 3); pretreatment of trypsin with pepstatin A agarose showed no effect on the cleavage (see Table E1 in this article’s Online Repository at www.jacionline.org). Both German and Oriental cockroach extracts cleaved the peptide substrate between Arg36 and Ser37, producing an S37LIGKdD fragment (Figure 3 and Table E1). Pretreatment of German and Oriental cockroach extracts with pepstatin A inhibited production of the S37LIGKdD peptide fragment by about 50~70%. Thus, these cockroach extracts likely contain pepstatin A-sensitive protease(s), which cleaves PAR-2 peptide similarly to the authentic PAR-2 ligand, trypsin. Figure 3 German cockroach extract cleaves PAR-2 peptide substrate between arginine and serine While none of the well-characterized cockroach allergens showed proteolytic activity, the midgut of cockroaches contains trypsin, chymotrypsin, subtilisin, and cysteine protease-like proteases (9). Typically, PAR-2 is cleaved and activated by serine proteases, such as trypsin (7). However, in a human epithelial cell line stimulated with German cockroach extract, the aspartate protease inhibitor, pepstatin A, and the cysteine protease inhibitor, E64, inhibited phospho-p44 MAP kinase levels, suggesting a role for cysteine proteases or aspartate proteases. Furthermore, exogenous chitinase from a bacterium, Streptomyces griseus, cleaved human PAR-2 peptide and induced a PAR-2-dependent [Ca2+]i response. These previous observations and our current findings suggest that PAR-2 recognizes both conventional trypsin-like proteases and perhaps other proteases and glycosidases derived from microbes, fungi and insects. Understanding these cockroach protease molecules and their receptors on immune cells may create novel strategies to prevent and to treat bronchial asthma.

Middle ear mucosa regeneration by grafting of artificial mucosa

Conclusion. Artificial middle ear mucosa (AMEM), a sheet of mucosal cells grown on collagen gel populated with fibroblasts, is useful as graft material that is able to promote mucosal regeneration after middle ear surgery. Objectives. Regeneration of the middle ear mucosa and pneumatization of the mastoid cavity is critical for good prognosis. We examined whether implantation of AMEM into damaged middle ear cavity would promote mucosal regeneration. Materials and methods. AMEM was prepared as described previously using epithelial cells and fibroblasts isolated from the rabbit middle ear. We implanted AMEM into rabbit middle ear from which mucosa had been surgically removed and evaluated its histological and functional recovery 8 weeks later. Three other groups were used for comparison: a normal control group, a mucosa-eliminated group, and a collagen-implanted group. Results. AMEM grew to be morphologically similar to the native middle ear mucosa. Electron microscope studies showed that implanted AMEM has basal lamina and cilia. AMEM implantation suppressed bone hyperplasia and granulation, leading to better mucosal regeneration. Mucosal gas exchange was also significantly improved after implantation.

IgG4-related disease in the sinonasal cavity accompanied by intranasal structure loss

IgG4-related disease was recently proposed under the classification of systemic chronic inflammatory disease. In the field of otolaryngology, organ-specific diagnostic criteria have been established for the occurrence of this condition in the salivary glands, but not in the sinonasal cavity. Here we report a case involving a 70-year-old man with IgG4-related disease in the sinonasal cavity. The patient, with the chief complaint of nasal bleeding, first visited a physician. However, the patient experienced recurrent bleeding with intranasal structure loss and was subsequently referred to our hospital. His IgG4 level was elevated, and histopathological examination of a tissue sample obtained from the edematous sphenoid sinus showed increased IgG4-positive plasma cells and storiform fibrosclerosis. A definitive diagnosis of IgG4-related rhinosinusitis was made on the basis of comprehensive criteria for IgG4-related disease. The disease showed a progressively destructive course that was clearly different from that of chronic sinusitis and represented a very rare case of IgG4-related rhinosinusitis. IgG4-related disease originating in the sinonasal cavity is rare, and, to the best of our knowledge, this is the first primary case of IgG4-related disease that originated in one side of the sinonasal cavity and showed progressive destruction.

In vitro reconstruction of a three-dimensional middle ear mucosal organ and its in vivo transplantation

Conclusions. These experimental findings suggest the feasibility of artificial middle ear mucosa grafting as an effective treatment for achieving mucosal regeneration after middle ear surgery. Objectives. Postoperative mucosal regeneration of tympanic cavity and mastoid cavity is of great importance after middle ear surgery. We reconstructed in vitro a three-dimensional middle ear mucosal organ, and assessed its feasibility for regenerative medicine of middle ear-related diseases. Materials and methods. Epithelial cells and fibroblasts were isolated from the middle ear mucosa of rats and propagated by subculturing. An artificial middle ear mucosal organ was reconstructed by overlaying the middle ear epithelial cells on three-dimensional lattices of a collagen gel that had been repopulated with the fibroblasts. In addition, the artificial organ was implanted in the middle ear cavity of rats. Results. The artificial middle ear mucosa consisted of the single layer of epithelial cells, the basal membrane, and the underlying connective tissue. Electron microscopy revealed the presence of tight junctions and adherence junctions on the apical side, and adhesion complexes made of desmosomes. The reconstituted mucosa expressed genes of mucin, strongly suggesting that the artificial middle ear mucosa was capable of secreting mucus proteins. The DiI-labeled artificial middle ear mucosa implanted into the middle ear cavity was well engrafted and associated with host tissues.

Increased CXCL10 Expression in Nasal Fibroblasts from Patients with Refractory Chronic Rhinosinusitis and Asthma

BACKGROUND Chronic rhinosinusitis (CRS) is characterized by local inflammation of the sinonasal tissues. CRS patients with nasal polyps and asthma often develop acute exacerbation of sinonasal symptoms after upper respiratory tract infections. However, the influence of concomitant asthma on the nasal immune response to viral infection remains unclear. METHODS Specimens of nasal polyp and mucosal tissues were obtained from 3 groups of CRS patients (n = 14 per group): 1) patients without asthma (CRS group), 2) patients with aspirin-tolerant asthma (ATA group), and 3) patients with aspirin-intolerant asthma (AIA group). Nasal fibroblasts isolated from the specimens were stimulated with poly I:C. CXCL10 expression was analyzed by the quantitative real-time polymerase chain reaction and enzyme-linked immunoadsorbent assay. Biopsy specimens from CRS patients without asthma were subjected to immunohistochemistry for detection of T-bet and GATA-3 expression in CD3+ T cells by double labeling. RESULTS Nasal fibroblasts from the ATA and AIA groups showed significantly enhanced expression of CXCL10 mRNA and protein after poly I:C stimulation compared with cells from the CRS group and the control group (normal nasal mucosa). In addition to T helper (Th)2 cells, there was more abundant infiltration of Th1 cells into tissues from the AIA and ATA groups. CONCLUSIONS Our findings suggest that CRS associated with asthma may become intractable through the over-production of CXCL10 in response to viral infection.

Outcomes of frontal mucocele marsupialization: endonasal and external approaches.

Background Frontal sinus mucoceles are known to undergo repeated recurrences, and the management of this disease constitutes a challenge regarding the ideal treatment. The aim of this study was to evaluate the postoperative results of the endoscopic ventilation approach in the treatment of frontal mucoceles. Methods We retrospectively reviewed the charts of 24 subjects with mucocele who underwent endoscopic marsupialization. The preoperative characteristics of the frontal sinus on computed tomography scans were compared with the postoperative results. Results Postoperative endoscopic examination revealed in 15 nasal sides wide patency of the frontal outflow tract. The ostium was patent but had become slightly narrowed in nine nasal sides. Restenosis of the ostium was found in six cases. Conclusion A membranous type of obstruction in the region of the frontal outflow tract was related to a better outcome.