The squamous cell carcinoma antigen 2 inhibits the cysteine proteinase activity of a major mite allergen, Der p 1

The squamous cell carcinoma antigens 1 (SCCA1) and SCCA2 belong to the ovalbumin-serpin family. Although SCCA1 and SCCA2 are closely homologous, these two molecules have distinct properties; SCCA1 inhibits cysteine proteinases such as cathepsin K, L, and S, whereas SCCA2 inhibits serine proteinases such as cathepsin G and human mast cell chymase. Although several intrinsic target proteinases for SCCA1 and SCCA2 have been found, the biological roles of SCCA1 and SCCA2 remain unknown. A mite allergen, Der p 1, is one of the most immunodominant allergens and also acts as a cysteine proteinase probably involved in the pathogenesis of allergic diseases. We have recently shown that both SCCA1 and SCCA2 are induced by two related Th2-type cytokines, IL-4 and IL-13, in bronchial epithelial cells and that SCCA expression is augmented in bronchial asthma patients. In this study, we explored the possibility that SCCA proteins target Der p 1, and it turned out that SCCA2, but not SCCA1, inhibited the catalytic activities of Der p 1. We furthermore analyzed the inhibitory mechanism of SCCA2 on Der p 1. SCCA2 contributed the suicide substrate-like mechanism without formation of a covalent complex, causing irreversible impairment of the catalytic activity of Der p 1, as SCCA1 does on papain. In addition, resistance to cleavage by Der p 1 also contributed to the inhibitory mechanism of SCCA2. These results suggest that SCCA2 acts as a cross-class serpin targeting an extrinsic cysteine proteinase derived from house dust mites and that it may have a protective role against biological reactions caused by mites.     

哮喘患儿血白细胞介素-10、13检测的临床意义

目的探讨哮喘患儿外周血白细胞介素-10(IL-10)、13的变化及其在哮喘发病机制中的作用。方法用ELISA双抗体夹心法测定20例哮喘患儿及18例正常儿童血浆IL-10、13的含量。结果哮喘组血浆IL-13水平明显高于正常对照组,IL-10水平明显低于正常对照组(p均<0.05)。结论IL-10、13等细胞因子参与儿童哮喘发病的病理生理过程,可为判断病情提供较好的实验室参数。     

Uteroglobin suppresses SCCA gene expression associated with allergic asthma

Uteroglobin (UG), the founding member of the Secretoglobin superfamily, is a potent anti-inflammatory protein constitutively expressed at a high level in the airway epithelia of all mammals. We previously reported that the lungs of UG-knock-out (UG-KO) mice express elevated levels of Th2 cytokines (e.g. interleukin (IL)-4 and IL-13), which are augmented by allergen sensitization and challenge leading to exaggerated airway inflammation. Notably, these responses are suppressed by recombinant UG treatment (Mandal, A. K., Zhang, Z., Ray, R., Choi, M. S., Chowdhury, B., Pattabiraman, N., and Mukherjee, A. B. (2004) J. Exp. Med. 199, 1317-1330). Recent reports indicate that human orthologs of murine squamous cell carcinoma antigen-2 (SCCA-2/serpinb3a), a serine protease-inhibitor, are overexpressed in the airways of asthmatic patients. We report here that compared with wild type littermates, UG-KO mouse lungs express markedly elevated levels of SCCA-2 mRNA and protein, which are augmented by allergen-challenge. Most importantly, these effects are abrogated by recombinant UG treatment. We further demonstrate that treatment of cultured human bronchial epithelial cells with IL-4 or IL-13 stimulates phosphorylation of STAT-1 and STAT-6 leading to SCCA-1 (SERPINB3) and SCCA-2 (SERPINB4) gene expression. We propose that: (i) IL-4- and IL-13-stimulated SCCA gene expression is mediated via STAT-1 and STAT-6 activation, and (ii) by suppressing the production, and most likely by interfering with the signaling of these cytokines, UG inhibits SCCA gene expression associated with airway inflammation in asthma.     

Serum levels of interleukin-13 and interferon-gamma from adult patients with asthma in Mysore

Serum protein analysis for noninvasive quantification of airway inflammation in asthma is a promising research tool in the field of lung diseases. Cytokines are believed to have major role in inflammatory process of the airways of the lung. There is an imbalance between T-helper (Th)-2 cells, which secrete interleukin (IL)-4 and interleukin (IL)-13, and Th1 cells, which secrete interferon (IFN)-gamma in asthma. To test the hypothesis that serum IL-13 and IL-4 levels may be elevated whereas IFN-gamma would be decreased in this cohort of patients, a property that could make them possible candidate biomarkers in determining asthma occurrence and severity, we measured concentrations of IL-4, IL-13 and IFN-gamma in serum samples of 88 subjects (44 normal, 12 with mild asthma, 16 with moderate asthma, and 16 with severe asthma). Serum Levels of IL-4, IL-13, and IFN-gamma were determined by an enzyme-linked immune-sorbent assay (ELISA). Median serum level of IFN-gamma in asthmatic patients was 8.0pg/ml, while it was 11.4pg/ml in healthy controls. However, the difference was not significant. Among the different age groups in whom IFN-gamma was assessed, the highest median value in both cases and controls was observed in the age group of 31-40years. The median serum level of IL-13 was 40.0pg/ml in asthmatic patients and 58.25pg/ml in healthy controls. The difference was not significant. On subgroup analysis, no significant difference of IFN-gamma and IL-13 between asthma of different severities was observed. The study also revealed nonsignificant difference of serum cytokines with the duration of asthma, number of allergens, and severity of sensitization. Normal serum levels of IFN-gamma and IL-13 in asthmatic patients suggest their neutral role in the inflammatory process; however, more studies are required to establish the effect of these cytokines in adulthood asthma in different ethnic populations.     

Co-Culture of Human Bronchial Fibroblasts and CD4+ T Cells Increases Th17 Cytokine Signature

Background

Airway inflammation is an important characteristic of asthma and has been associated with airway remodelling and bronchial hyperreactivity. The mucosal microenvironment composed of structural cells and highly specialised extracellular matrix is able to amplify and promote inflammation. This microenvironment leads to the development and maintenance of a specific adaptive response characterized by Th2 and Th17. Bronchial fibroblasts produce multiple mediators that may play a role in maintaining and amplifying this response in asthma.

Objective

To investigate the role of bronchial fibroblasts obtained from asthmatic subjects and healthy controls in regulating Th17 response by creating a local micro-environment that promotes this response in the airways.

Methods

Human bronchial fibroblasts and CD4⁺T cells were isolated from atopic asthmatics and non-atopic healthy controls. CD4⁺T were co-cultured with bronchial fibroblasts of asthmatic subjects and healthy controls. RORc gene expression was detected by qPCR. Phosphorylated STAT-3 and RORγt were evaluated by western blots. Th17 phenotype was measured by flow cytometry. IL-22, IL17, IL-6 TGF-β and IL1-β were assessed by qPCR and ELISA.

Results

Co-culture of CD4⁺T cells with bronchial fibroblasts significantly stimulated RORc expression and induced a significant increase in Th17 cells as characterized by the percentage of IL-17⁺/CCR6⁺ staining in asthmatic conditions. IL-17 and IL-22 were increased in both normal and asthmatic conditions with a significantly higher amount in asthmatics compared to controls. IL-6, IL-1β, TGF-β and IL-23 were significantly elevated in fibroblasts from asthmatic subjects upon co-culture with CD4⁺T cells. IL-23 stimulates IL-6 and IL-1β expression by bronchial fibroblasts.

Conclusion

Interaction between bronchial fibroblasts and T cells seems to promote specifically Th17 cells profile in asthma. These results suggest that cellular interaction particularly between T cells and fibroblasts may play a pivotal role in the regulation of the inflammatory response in asthma.     

Bronchial macrophages in asthmatics reveal decreased CD16 expression and substantial levels of receptors for IL-10, but not IL-4 and IL-7

The role of different subpopulations of bronchial macrophages (BMs) in asthma pathogenesis has not yet been completely elucidated. In addition, little is known about potential in vivo responsiveness of BMs to pro- and anti-inflam-matory cytokines present in the bronchial milieu. We aimed to characterize asthmatic patients' BM subpopulations delineated by common markers of macrophage/monocyte cells, CD16 and CD14, and subsequently to analyze cytokine receptor expression on those subsets. Subjects included eighteen patients with moderate asthma (six steroid-naive and twelve steroid-treated) and ten healthy control subjects. Flow cytometry was used to analyze phenotypical features of BMs including expression of receptors for IL-10, IL-4 and IL-7. Exhaled nitric oxide analysis and induced sputum eosinophil counts were used to assess airway inflammation. BMs from both steroid-naive and steroid-treated asthmatic patients showed significantly decreased expression of CD16, as compared to healthy subjects' BMs. CD16, but not CD14, expression inversely correlated with exhaled nitric oxide levels and sputum eosinophilia. Short-term administration of inhaled cortiocosteroids (ICS) in steroid-naive asthmatic patients led to significant reduction of CD16 expression and enhancement of CD14 expression. Next, we analyzed the expression of receptors for IL-10, IL-4 and IL-7 on the surface of BM subpopulations characterized by different levels of CD14 and CD16 expression. We observed substantial levels of IL-10R on the surface of BMs collected from asthmatic and healthy subjects. Interestingly, IL-10R was found mostly on those macrophages that co-expressed CD14. In contrast, independently on co-expression of CD14, the levels of IL-4R and IL-7R on BMs were low in both asthmatic and healthy subjects. The results suggest that different BM subsets may be differentially involved in regulating the inflammatory response in allergic asthma.     

Essential role for both CD80 and CD86 costimulation, but not CD40 interactions, in allergen-induced Th2 cytokine production from asthmatic bronchial tissue: role for alphabeta, but not gammadelta, T cells

CD80 and CD86 interact with CD28 and deliver costimulatory signals required for T cell activation. We demonstrate that ex vivo allergen stimulation of bronchial biopsy tissue from mild atopic asthmatic, but not atopic nonasthmatic, subjects induced production of IL-5, IL-4, and IL-13. Explants from both study groups did not produce IFN-gamma, but secreted the chemokine RANTES without any overt stimulation. In addition to allergen, stimulation of asthmatic explants with mAbs to CD3 and TCR-alphabeta but not TCR-gammadelta induced IL-5 secretion. Allergen-induced IL-5 and IL-13 production by the asthmatic tissue was inhibited by anti-CD80 and, to a lesser extent, by anti-CD86 mAbs. In contrast, the production of these cytokines by PBMCs was not affected by mAbs to CD80, was inhibited by anti-CD86, and was strongly attenuated in the presence of both Abs. FACS analysis revealed that stimulated asthmatic bronchial tissue was comprised of CD4+ T cells that expressed surface CD28 (75. 3%) but little CTLA-4 (4.0%). Neutralizing mAbs to CD40 ligand had no effect on the cytokine levels produced by asthmatic tissue or PBMCs. Collectively, these findings suggest that allergen-specific alphabeta T cells are resident in asthmatic bronchial tissue and demonstrate that costimulation by both CD80 and CD86 is essential for allergen-induced cytokine production. In contrast, CD86 appears to be the principal costimulatory molecule required in PBMC responses. Attenuation of type 2 alphabeta T cell responses in the bronchial mucosa by blocking these costimulatory molecules may be of therapeutic potential in asthma.     

Cytokines mRNA in bone marrow-derived dendritic cells in asthmatic mouse

By inducing and amplifying dendritic cells(DCs)derived from the bone marrow of asthma murine in vitro,cytokines mRNA were expressed,and the functions of DCS were investigated.Cells isolated from murine bone marrow have been cultured with rmGM-CSF and rmIL-4,and the expression of cytokines mRNA was determined by ribonuclease protection assay combined with multi-probe templates.Large numbers of DCS have been obtained from bone marrow,and they expressed interleukin-13(IL-13),interleukin-9(IL-9),and interleukin-3(IL-3)mRNA.Moreover.the level of IL-13 mRNA and IL-9 mRNA expressed by DCs in asthmatic mice was significantly higher than those in the control groups(P＜0.05).But,the level of IL-3 mRNA showed no discrepancy between the two groups(P＞0.05).Des are very important in the forming and developing of asthma,which implies that the therapy targeted at DCs will possibly become a new goal.     

The negative-feedback regulation of the IL-13 signal by the IL-13 receptor alpha2 chain in bronchial epithelial cells

Bronchial asthma is a complex disease characterized by airway inflammation involving Th2 cytokines. Among Th2 cytokines, the significance of IL-13 in the pathogenesis of bronchial asthma has recently emerged. Particularly, the direct action of IL-13 on bronchial epithelial cells (BECs) is critical for generation of airway hyperresponsiveness. IL-13 has two binding units; the IL-13 receptor alpha1 chain transduces the IL-13 signal comprising a heterodimer with the IL-4R alpha chain, whereas the IL-13 receptor alpha2 chain (IL-13Ralpha2) is thought to act as a decoy receptor. However, it remains obscure how expression of these molecules is regulated in each cell. In this article, we analyzed the expression of these components in BECs. Either IL-4 or IL-13 induced intracellular expression of IL-13Ralpha2 in BECs, which was STAT6-dependent and required de novo protein synthesis. IL-13Ralpha2 expressed on the cell surface as a monomer inhibited the STAT6-dependent IL-13 signal. Furthermore, expression of IL-13Ralpha2 was induced in lung tissues of ovalbumin-induced asthma model mice. Taken together, our results suggested the possibility that IL-13Ralpha2 induced by its ligand is transferred to the cell surface by an unknown mechanism, and it down-regulates the IL-13 signal in BECs, which functions as a unique negative-feedback system for the cytokine signal.     

Nasal Epithelial Cells Can Act as a Physiological Surrogate for Paediatric Asthma Studies

Introduction

Differentiated paediatric epithelial cells can be used to study the role of epithelial cells in asthma. Nasal epithelial cells are easier to obtain and may act as a surrogate for bronchial epithelium in asthma studies. We assessed the suitability of nasal epithelium from asthmatic children to be a surrogate for bronchial epithelium using air-liquid interface cultures.

Methods

Paired nasal and bronchial epithelial cells from asthmatic children (n = 9) were differentiated for 28 days under unstimulated and IL-13-stimulated conditions. Morphological and physiological markers were analysed using immunocytochemistry, transepithelial-electrical-resistance, Quantitative Real-time-PCR, ELISA and multiplex cytokine/chemokine analysis.

Results

Physiologically, nasal epithelial cells from asthmatic children exhibit similar cytokine responses to stimulation with IL-13 compared with paired bronchial epithelial cells. Morphologically however, nasal epithelial cells differed significantly from bronchial epithelial cells from asthmatic patients under unstimulated and IL-13-stimulated conditions. Nasal epithelial cells exhibited lower proliferation/differentiation rates and lower percentages of goblet and ciliated cells when unstimulated, while exhibiting a diminished and varied response to IL-13.

Conclusions

We conclude that morphologically, nasal epithelial cells would not be a suitable surrogate due to a significantly lower rate of proliferation and differentiation of goblet and ciliated cells. Physiologically, nasal epithelial cells respond similarly to exogenous stimulation with IL-13 in cytokine production and could be used as a physiological surrogate in the event that bronchial epithelial cells are not available.     

IL-17 Enhances Chemotaxis of Primary Human B Cells during Asthma

IL-17 is a pro-inflammatory mediator that is believed to play a critical role in regulating tissue inflammation during asthma, COPD, as well as other inflammatory disorders. The level of expression of IL-17 has been shown to be upregulated in lung bronchial tissue of asthmatic patients. Several reports have provided further evidence that this cytokine could play a key role in enhancing the migration of inflammatory as well as structural cells of the bronchial lung tissue during asthma and COPD. B cell infiltration to sites of inflammation during inflammatory disorders such as bowel disease, asthma and COPD has been reported. Accordingly, in this study we hypothesized that IL-17 may exert a chemotactic effect on primary B cells during asthma. We observed that B cells from asthmatic patients expressed significantly higher levels of IL-17RA and IL-17RC, compared to those of healthy subjects. Using an in-vitro migration assay, B cells were shown to migrate towards both IL-17A and IL-17F. Interestingly, blocking IL-17A and IL-17F signaling using either anti-IL-17R antibodies or MAP kinase inhibitors prevented in vitro migration of B cell towards IL-17. These observations indicate a direct chemotactic effect of IL-17 cytokines on primary peripheral blood B cells with higher effect being on asthmatic B cells. These findings revealed a key role for IL-17 in enhancing the migration of B cells to the lung tissue during asthma or COPD.     

Increased interleukin-4 and decreased interferon-γ levels in serum of children with asthma

Background and purposeImmune and inflammatory responses, mediated by cytokines, play important roles in the pathophysiology of asthma. These responses are associated with over expression of T helper (Th)-2 cytokine, particularly interleukin (IL)-4 and IL-5, and decreased expression of Th-1 cytokine, IL-2 and IFN-γ. We hypothesized that there would be an imbalance in the levels of circulating IL-4 and IFN-γ in the asthmatic subjects.MethodWe investigated serum levels of IL-4 and IFN-γ among eighty children (18 steroid-naïve, 30 steroid-treated children with asthma and 32 healthy controls) using commercially available ELISA kits.ResultsSerum level of IL-4 was significantly higher in steroid-naïve group of asthmatic children compared to the healthy control subjects and was lower in steroid-treated group though the level was statistically not significant. In contrast, serum levels of IFN-γ were significantly lower in both steroid-naïve and steroid-treated groups of asthmatic children compared to healthy control subjects.ConclusionThe results of our study suggest that serum level of IL-4 may be elevated in concert with decreased level of IFN-γ in asthma. Determination of serum levels of IL-4 and IFN-γ may be a useful tool for understanding the disease processes in asthma.     

Down-regulation of secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), an endogenous allosteric α7 nicotinic acetylcholine receptor modulator, in murine and human asthmatic conditions

Whereas acetylcholine (ACh) acts as a bronchoconstrictor and stimulator of mucus secretion from bronchial epithelium, it acts via α7 nicotinic Ach receptors (nAChRs) on macrophages in the airways to exert anti-inflammatory effects by reducing synthesis of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α). Moreover, the effects of ACh are modified by secreted ly-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of α7 nAChR signaling. Our aim was to explore the roles played by SLURP-1 in the pathophysiology of asthma by assessing SLURP-1 expression in the OVA-sensitized murine asthma model and in cultured human bronchial epithelial cells. Using real-time PCR we found that expression of SLURP-1 mRNA is down-regulated in the lungs of asthmatic model mice, as compared to healthy mice. In addition, immunohistochemical studies confirmed the diminished expression of SLURP-1 in the bronchioles of asthmatic mice, and showed it was due to extensive metaplasia of mucus-secreting cells and the concomitant loss of ciliated epithelial cells. Expression of SLURP-1 mRNA and protein was also significantly down-regulated in human epithelial cells stimulated with the pro-inflammatory cytokine interleukin-13 (IL-13), which is related to asthmatic condition. Thus SLURP-1 appears to be down-regulated in both an animal model of asthma and human epithelial cells treated with an inflammatory cytokine related to asthma. Those findings suggest that diminished expression of SLURP-1 in asthma attenuates its negative regulation of airway inflammation, and that perhaps changes in SLURP-1 expression could serve as a marker of airway damage in asthma.     

Cytokines mRNA in bone marrow-derived dendritic cells in asthmatic mouse

By inducing and amplifying dendritic cells (DCs) derived from the bone marrow of asthma murine in vitro, cytokines mRNA were expressed, and the functions of DCs were investigated. Cells isolated from murine bone marrow have been cultured with rmGM-CSF and rmIL-4, and the expression of cytokines mRNA was determined by ribonuclease protection assay combined with multi-probe templates. Large numbers of DCs have been obtained from bone marrow, and they expressed interleukin-13 (IL-13), interleukin-9 (IL-9), and interleukin-3 (IL-3) mRNA. Moreover, the level of IL-13 mRNA and IL-9 mRNA expressed by DCs in asthmatic mice was significantly higher than those in the control groups (P<0.05). But, the level of IL-3 mRNA showed no discrepancy between the two groups (P>0.05). DCs are very important in the forming and developing of asthma, which implies that the therapy targeted at DCs will possibly become a new goal. __________ Translated from Journal of Nanjing Normal University (Natural Science), 2005, 28(2): 96–100 [译自: 南京师范大学学报 (自然科学版), 2005, 28(2): 96–100]     

Identification of pendrin as a common mediator for mucus production in bronchial asthma and chronic obstructive pulmonary disease

Excessive production of airway mucus is a cardinal feature of bronchial asthma and chronic obstructive pulmonary disease (COPD) and contributes to morbidity and mortality in these diseases. IL-13, a Th2-type cytokine, is a central mediator in the pathogenesis of bronchial asthma, including mucus overproduction. Using a genome-wide search for genes induced in airway epithelial cells in response to IL-13, we identified pendrin encoded by the SLC26A4 (PDS) gene as a molecule responsible for airway mucus production. In both asthma and COPD mouse models, pendrin was up-regulated at the apical side of airway epithelial cells in association with mucus overproduction. Pendrin induced expression of MUC5AC, a major product of mucus in asthma and COPD, in airway epithelial cells. Finally, the enforced expression of pendrin in airway epithelial cells in vivo, using a Sendai virus vector, rapidly induced mucus overproduction in the lumens of the lungs together with neutrophilic infiltration in mice. These findings collectively suggest that pendrin can induce mucus production in airway epithelial cells and may be a therapeutic target candidate for bronchial asthma and COPD.     

IL-4 increases type 2, but not type 1, cytokine production in CD8+ T cells from mild atopic asthmatics

Background

Virus infections are the major cause of asthma exacerbations. CD8⁺ T cells have an important role in antiviral immune responses and animal studies suggest a role for CD8⁺ T cells in the pathogenesis of virus-induced asthma exacerbations. We have previously shown that the presence of IL-4 during stimulation increases the frequency of IL-5-positive cells and CD30 surface staining in CD8⁺ T cells from healthy, normal subjects. In this study, we investigated whether excess IL-4 during repeated TCR/CD3 stimulation of CD8⁺ T cells from atopic asthmatic subjects alters the balance of type 1/type 2 cytokine production in favour of the latter.

Methods

Peripheral blood CD8⁺ T cells from mild atopic asthmatic subjects were stimulated in vitro with anti-CD3 and IL-2 ± excess IL-4 and the expression of activation and adhesion molecules and type 1 and type 2 cytokine production were assessed.

Results

Surface expression of very late antigen-4 [VLA-4] and LFA-1 was decreased and the production of the type 2 cytokines IL-5 and IL-13 was augmented by the presence of IL-4 during stimulation of CD8⁺ T cells from mild atopic asthmatics.

Conclusion

These data suggest that during a respiratory virus infection activated CD8⁺ T cells from asthmatic subjects may produce excess type 2 cytokines and may contribute to asthma exacerbation by augmenting allergic inflammation.     

血清IL-17 与IL-23 在支气管哮喘患者中的表达水平及其相关性

目的:探讨IL-17与IL-23在支气管哮喘患者血清中的表达水平及其相关性。方法:选择2010年2月~2015年9月在我院进行诊治的支气管哮喘患者98例,其中包括56例为缓解期组,42例为急性发作期组,对照组为20例体检健康者,观察三组的血清IL-17、IL-23水平及肺功能指标的差异,并分析其相关性。结果:与对照组相比,缓解期组和急性发作期组血清IL-17、IL-23水平均明显升高(P0.05),急性发作期组血清IL-17、IL-23水平均显著高于缓解期组(P0.05);急性发作期组患者的PEF%、FEV1%、V50%、V25%均明显低于缓解期组,差异有统计学意义(P0.05);哮喘患者血清IL-17水平与IL-23水平呈正相关(r=0.685,P=0.000),血清IL-17与FEV1负相关(r=-0.592,P=0.000)、与PEF负相关(r=-0.515,P=0.000),IL-23与FEV1负相关(r=-0.598,P=0.000),与PEF负相关(r=-0.532,P=0.000)。结论:血清IL-17和IL-23的高表达可能参与了支气管哮喘的形成,并能影响疾病的进程,两者表达水平密切相关。