Interleukin-17A and vascular remodelling in severe asthma; lack of evidence for a direct role.

BACKGROUND: Bronchial vascular remodelling may contribute to the severity of airway narrowing through mucosal congestion. Interleukin (IL)-17A is associated with the most severe asthmatic phenotype but whether it might contribute to vascular remodelling is uncertain. OBJECTIVE: To assess vascular remodelling in severe asthma and whether IL-17A directly or indirectly may cause endothelial cell activation and angiogenesis. METHODS: Bronchial vascularization was quantified in asthmatic subjects, COPD and healthy subjects together with the number of IL-17A+ cells as well as the concentration of angiogenic factors in the sputum. The effect of IL-17A on in vitro angiogenesis, cell migration and endothelial permeability was assessed directly on primary human lung microvascular endothelial cells (HMVEC-L) or indirectly with conditioned medium derived from normal bronchial epithelial cells (NHBEC), fibroblasts (NHBF) and airway smooth muscle cells (ASMC) after IL-17A stimulation. RESULTS: Severe asthmatics have increased vascularity compared to the other groups, which correlates positively with the concentrations of angiogenic factors in sputum. Interestingly, we demonstrated that increased bronchial vascularity correlates positively with the number of subepithelial IL-17A+ cells. However IL-17A had no direct effect on HMVEC-L function but it enhanced endothelial tube formation and cell migration through the production of angiogenic factors by NHBE and ASMC. CONCLUSIONS & CLINICAL RELEVANCE: Our results shed light on the role of IL-17A in vascular remodelling, most likely through stimulating the synthesis of other angiogenic factors. Knowledge of these pathways may aid in the identification of new therapeutic targets.

Human bronchial and parenchymal fibroblasts display differences in basal inflammatory phenotype and response to IL-17A.

BACKGROUND: Chronic inflammation, typified by increased expression of IL-17A, together with airway and parenchymal remodelling are features of chronic lung diseases. Emerging evidence suggests that phenotypic heterogeneity of repair and inflammatory capacities of fibroblasts may contribute to the differential structural changes observed in different regions of the lung. OBJECTIVE: To investigate phenotypic differences in parenchymal and bronchial fibroblasts, either in terms of inflammation and remodelling or the ability of these fibroblasts to respond to IL-17A. METHODS: Four groups of primary fibroblasts were used: normal human bronchial fibroblast (NHBF), normal human parenchymal fibroblast (NHPF), COPD human bronchial fibroblast (CHBF) and COPD human parenchymal fibroblast (CHPF). Cytokine and extracellular matrix (ECM) expression were measured at baseline and after stimulation with IL-17A. Actinomycin D was used to measure cytokine mRNA stability. RESULTS: At baseline, we observed higher protein production of IL-6 in NHPF than NHBF, but higher levels of IL-8 and GRO-α in NHBF. IL-17A induced a higher expression of GRO-α (CXCL1) and IL-6 in NHPF than in NHBF, and a higher level of IL-8 expression in NHBF. IL-17A treatment decreased the mRNA stability of IL-6 in NHBF when compared with NHPF. CHPF expressed higher protein levels of fibronectin, collagen-I and collagen-III than CHBF, NHBF and NHPF. IL-17A increased fibronectin and collagen-III protein only in NHPF and collagen-III protein production in CHBF and CHPF. CONCLUSIONS AND CLINICAL RELEVANCE: These findings provide insight into the inflammatory and remodelling processes that may be related to the phenotypic heterogeneity of fibroblasts from airway and parenchymal regions and in their response to IL-17A.

Monocyte-derived fibrocytes induce an inflammatory phenotype in airway smooth muscle cells.

BACKGROUND: Infiltration of fibrocytes (FC) in the airway smooth muscle is a feature of asthma, but the pathological significance is unknown. OBJECTIVE: We sought to explore whether FC modulate the phenotype of airway smooth muscle cells (ASMC) in asthmatic vs. control subjects. METHODS: Fibrocytes were isolated from CD14+ monocytes from asthmatic and normal subjects. Proliferation of ASMC of asthmatic or normal subjects was analysed by (3) H-thymidine incorporation, cell number counting and Ki-67 expression after treatment of ASMC with FC-conditioned medium (FCCM) or co-culture with FC. ASMC-associated cytokines/chemokines implicated in asthma (TGF-ß1, eotaxin, IL-6 and IL-8) were measured in co-culture or transwell culture of ASMC + FC by ELISA. Immunofluorescence staining was performed to localize these cytokines in ASMC. Cytokine secretion was measured in the transwell culture of ASMC + FC, where NF-κB-p65 or ERK1/2 in ASMC was silenced by siRNA. Contractile phenotype of ASMC in transwell culture was assessed by immunoblotting of α-smooth muscle actin (α-SMA) and myosin light chain kinase (MLCK). RESULTS: Fibrocytes did not affect ASMC proliferation and expression of TGF-ß1, eotaxin, α-SMA and MLCK; however, ASMC production of IL-8 and IL-6 was increased in the co-culture and transwell culture by FC. ASMC treated with FCCM were immunopositive for IL-8/IL-6 and produced more IL-8/IL-6. Furthermore, siRNA silencing of NF-κB-p65 or ERK1/2 in transwell cultures of asthmatic ASMC with normal subject FC decreased IL-8 and IL-6 production. CONCLUSIONS AND CLINICAL RELEVANCE: Fibrocytes promoted IL-8 and IL-6 production by ASMC, demonstrating a proinflammatory role for FC and a possible mechanism of the inflammatory phenotype in asthma.

Pathogenesis of severe asthma.

Patients with severe asthma have asthma symptoms which are difficult to control, require high dosages of medication, and continue to experience persistent symptoms, asthma exacerbations or airflow obstruction. Epidemiological and clinical evidences point to the fact that severe asthma is not a single phenotype. Cluster analyses have identified subclasses of severe asthma using parameters such as patient characteristics, and cytokine profiles have also been useful in classifying moderate and severe asthma. The IL-4/IL-13 signalling pathway accounts for the symptoms experienced by a subset of severe asthmatics with allergen-associated symptoms and high serum immunoglobulin E (IgE) levels, and these patients are generally responsive to anti-IgE treatment. The IL-5/IL-33 signalling pathway is likely to play a key role in the disease pathogenesis of those who are resistant to high doses of inhaled corticosteroid but responsive to systemic corticosteroids and anti-IL5 therapy. The IL-17 signalling pathway is thought to contribute to 'neutrophilic asthma'. Although traditionally viewed as players in the defence mechanism against viral and intracellular bacterial infection, mounting evidence supports a role for Th1 cytokines such as IL-18 and IFN-γ in severe asthma pathogenesis. Furthermore, these cytokine signalling pathways interact to contribute to the spectrum of clinical pathological outcomes in severe asthma. To date, glucocorticoids are the most effective anti-asthma drugs available, yet severe asthma patients are typically resistant to the effects of glucocorticoids. Glucocorticoid receptor dysfunction and histone deacetylase activity reduction are likely to contribute to glucocorticoid resistance in severe asthma patients. This review discusses recent development in different cytokine signalling pathways, their interactions and steroid resistance, in the context of severe asthma pathogenesis.

Induction of glucocorticoid receptor-beta expression in epithelial cells of asthmatic airways by T-helper type 17 cytokines.

BACKGROUND: Corticosteroid insensitivity in asthmatics is associated with an increased expression of glucocorticoid receptor-beta (GR-beta) in many cell types. T-helper type 17 (Th17) cytokine (IL-17A and F) expressions increase in mild and in difficult-to-treat asthma. We hypothesize that IL-17A and F cytokines alone or in combination, induce the expression of GR-beta in bronchial epithelial cells. OBJECTIVES: To confirm the expression of the GR-beta and IL-17 cytokines in the airways of normal subjects and mild asthmatics and to examine the effect of cytokines IL-17A and F on the expression of GR-beta in bronchial epithelial cells obtained from normal subjects and asthmatic patients. METHODS: The expression of IL-17A and F, GR-alpha and GR-beta was analysed in bronchial biopsies from mild asthmatics and normal subjects by Q-RT-PCR. Immunohistochemistry for IL-17 and GR-beta was performed in bronchial biopsies from normal and asthmatic subjects. The expression of IL-6 in response to IL-17A and F and dexamethasone was determined by Q-RT-PCR using primary airway epithelial cells from normal and asthmatic subjects. RESULTS: We detected significantly higher levels of IL-17A mRNA expression in the bronchial biopsies from mild asthmatics, compared with normal. GR-alpha expression was significantly lower in the biopsies from asthmatics compared with controls. The expression of IL-17F and GR-beta in biopsies from asthmatics was not significantly different from that of controls. Using primary epithelial cells isolated from normal subjects and asthmatics, we found an increased expression of GR-beta in response to IL-17A and F in the cells from asthmatics (P< or =0.05). This effect was only partially significant in the normal cells. Dexamethasone significantly decreased the IL-17-induced IL-6 expression in cells from normal individuals but not in those from asthmatics (P< or =0.05). CONCLUSION: Evidence of an increased GR-beta expression in epithelial cells following IL-17 stimulation suggests a possible role for Th17-associated cytokines in the mechanism of steroid hypo-responsiveness in asthmatic subjects.

Localization and distribution of NOS1 in murine airways.

Nitric oxide synthase 1 (NOS1) is a major determinant of bronchial responsiveness in mice and has been proposed as an asthma gene in man. Nevertheless, how nitric oxide production by NOS1 contributes to airway responsiveness remains unclear. Although NOS1 is usually closely associated with nerves, it has also been found in a variety of other cell types, particularly epithelium. We sought to better understand the role of NOS1 by determining its major site of expression in murine airways. Using nicotinamide adenine dinucleotide phosphate-diaphorase (diaphorase), which non-selectively detects nitric oxide synthase (NOS), we found strong evidence of NOS in the airways largely restricted to the airway epithelium and trachea glands. In contrast, diaphorase staining of NOS1-deficient mutant mice demonstrated a marked reduction in epithelial cells of the trachea but not bronchioles, suggesting that the epithelium is the major site of NOS1 expression. This was supported by immunohistochemistry, which also demonstrated significant staining in glands and to a lesser degree in airway smooth muscle. Double immunofluorescence staining of tracheas for NOS1 and the nerve marker PGP 9.5 failed to demonstrate co-localization, indicating that nerves are not an important source of NOS1 in the murine airway wall. Finally, removal of the trachea epithelium by digestion resulted in a marked decrease in NOS1 detection by Western blotting, confirming the epithelium as the major site of NOS1 expression in the murine airway. These findings support the notion that the role of NOS1 in murine bronchial responsiveness involves the epithelium of the central airways.

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma.

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not "cure" asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Analysis of glutathione in rat airway surface liquid by capillary zone electrophoresis with conductivity detection.

Glutathione (GSH) is an important component of antioxidant defenses in airway surface liquid (ASL), a thin layer (10-30 microm) of liquid covering the epithelial cells lining the airways of the lung. Decreased levels of ASL GSH have been reported in cystic fibrosis (CF), potentially contributing to the severe oxidative stress seen in this disease. To help investigate the role of GSH in ASL, we developed a technique suitable for analysis of GSH and its oxidized form (GSSG) in microliter samples using capillary sampling followed by capillary zone electrophoresis (CZE) analysis with conductivity detection. CZE was carried out in 100 mM CHES and 40 mM lithium hydroxide with 5 mM spermine at pH 9.1 under an applied electric field of -416 V cm(-1). To prevent any autooxidation of GSH during sample manipulations, the samples were treated with N-ethylmaleimide (50 mM) to alkylate free thiol (-SH). Under these conditions, GSH and GSSG were cleanly separated without interference from common anions (e.g. Cl(-), PO(4)(3-), HCO(3)(-), etc.) and the limit of detection for ASL analysis was 11 microM for GSH and 8 microM for GSSG (S/N=3). GSH and GSSG were also measured in rat plasma. Baseline values of 897+/-210 microM (GSH) and 215+/-61 microM (GSSG) were obtained for rat ASL (n=8), whereas 12.4+/-2.7 microM (GSH) and 14.8+/-6.7 microM (GSSG) were obtained for rat plasma (n=5).

Greater velocity and magnitude of airway narrowing in immature than in mature rabbit lung explants.

Methacholine (MCh)-induced bronchoconstriction in vivo produces greater maximal increases in pulmonary and airway resistances in immature than in mature rabbits. Our recent findings of lower shear modulus and greater airway distensibility suggest a lower elastic load limiting airway smooth muscle (ASM) shortening in immature rabbit lungs. We hypothesized that a lower elastic load should result in greater velocity of airway narrowing. Lung explants were prepared from three immature and five mature rabbits. Dynamic narrowing of intraparenchymal airways after maximal MCh stimulation was assessed by video microscopy. Immature airways (n = 80) compared with mature airways (n = 110) demonstrated greater peak velocity of shortening (6.98 +/- 0.32 versus 4.22 +/- 0.18% of baseline perimeter/s) and greater maximal airway narrowing, expressed as percentage of baseline area (31.9 +/- 1.6 versus 42.2 +/- 1.8%). For both groups, a greater velocity of shortening resulted in greater airway narrowing. As available data do not support maturational differences in rabbit ASM, our results are consistent with a lower elastic load limiting ASM shortening in the immature rabbit.

Capillary electrophoresis analysis of nitrite and nitrate in sub-microliter quantities of airway surface liquid.

We developed a simple capillary electrophoresis (CE) method to measure nitrite and nitrate concentrations in submicroliter samples of rat airway surface liquid (ASL), a thin (10-30 microm) layer of liquid covering the epithelial cells lining the airways of the lung. The composition of ASL has been poorly defined, in large part because of the small sample volume (approximately 1-3 microl per cm2 of epithelium) and difficulty of harvesting ASL. We have used capillary tubes for ASL sample collection, with microanalysis by CE using a 50 mM phosphate buffer (pH 3), with 0.5 mM spermine as a dynamic flow modifier, and direct UV detection at 214 nm. The limit of detections (LODs), under conditions used, for ASL analysis were 10 microM for nitrate and 30 microM for nitrite (SIN= 3). Nitrate and nitrite were also measured in rat plasma. The concentration of nitrate was 102+/-12 microM in rat ASL and 70+/-1.0 microM in rat plasma, whereas nitrite was 83+/-28 microM in rat ASL and below the LOD in rat plasma. After instilling lipopolysaccharide intratracheally to induce increased NO production, the nitrate concentration in ASL increased to 387+/-16 microM, and to 377+/-88 microM in plasma. The concentration of nitrite increased to 103+/-7.0 microM for ASL and 138+/-17 microM for plasma.

Eosinophil peroxidase mediates protein nitration in allergic airway inflammation in mice.

The eosinophilic inflammatory response in asthma is associated with protein nitration, detected as immunostaining for 3-nitrotyrosine (3NT). As the presence of 3NT is strongly correlated with upregulation of the inducible form of nitric oxide synthase (NOS II), it has been hypothesized that 3NT formation results from the action of peroxynitrite (ONOO-), a highly reactive NO derivative produced from the reaction of molecular NO and O2-. However, recent observations have suggested that the action of peroxidases, including eosinophil peroxidase (EPO), may be responsible for protein nitration. In this study, we used murine models of allergic asthma to address the relative contribution of EPO and NOS II to protein nitration. We studied EPO-deficient New Zealand White (NZW) mice, which were sensitized and challenged intranasally with ovalbumin (OVA). Despite comparable levels of eosinophilia, NO, and superoxide production, NZW mice exhibited markedly decreased 3NT staining around the airways after OVA challenge when compared with two other strains (A/J and C57BL/6J). Immunocytochemical analysis of bronchoalveolar lavage (BAL) cells and lung sections suggested that 3NT staining was largely confined to eosinophils. This was confirmed by Western Blot analysis of proteins from different subsets of BAL cells that demonstrated a marked decrease in 3NT formation in eosinophils from NZW mice. These results contrast with those obtained in OVA-sensitized and -challenged NOS II deficient mice, which despite decreased NO production, exhibited similar 3NT staining in the airways after OVA challenge as in wild-type control mice. In this model, protein nitration was thus not a function of NO production by NOS II. We conclude that in the mouse, 3NT formation after specific allergen challenge is dependent on EPO activity, particularly in eosinophils themselves. In contrast, 3NT formation is not driven by upregulation of NOS II expression in this model and does not appear to depend on increases in the level of NO production.

Nitric oxide and protein nitration are eosinophil dependent in allergen-challenged mice.

To explore the possible role of eosinophils in NO-mediated tissue injury, we studied a murine model of allergic asthma. Male A/J mice were sensitized and challenged intranasally with ovalbumin (OVA). Following challenge, the number of eosinophils in bronchoalveolar lavage fluid (BALF) increased from 0.4% of total cells at baseline (0.02 x 10(4) cells/ml) to 60.2% at 48 h after the challenge (9.34 x 10(4) cells/ml). The rise in eosinophil count was accompanied by a 40.3% increase in total NO(2-) plus NO(3-) (NO(x)) in BALF. This in turn was accompanied by expression of inducible NO synthase (NOS II) in airway epithelial and inflammatory cells, as well as by evidence of staining for 3-nitrotyrosine (3NT) in peribronchial inflammatory cells and at the epithelial surface. Both NO(x) production and 3NT were significantly reduced by pretreatment of the challenged mice with the highly specific NOS II inhibitor N-3-aminomethyl-benzyl-acetamidine-dihydrochloride (1400W), as well as by the nonselective NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). L-NAME and 1400W also reduced the number of BALF eosinophils (37.2% and 61.5%, respectively, as compared with the control value), suggesting that NO production by NOS II contributes to eosinophil recruitment. To further examine the role of eosinophils, we pretreated additional mice with an anti-interleukin (IL)-5 antibody, which reduced BALF eosinophilia following OVA challenge by 90.1%. In concert with the decrease in eosinophils, the anti-IL-5 antibody reduced NO(x) in BALF almost to the baseline value, and decreased the number of 3NT-positive cells in the peribronchial region by 74.4%. Western blot analysis of protein extracted from whole lung confirmed the reduction in tyrosine nitration by anti-IL-5 antibody. These findings indicate that NO and eosinophilic inflammation are closely coupled, and suggest that eosinophils are an important source of tyrosine nitration.

Respiratory mechanics and lung development in the rat from early age to adulthood.

The purpose of the present study was to establish how the dependence of respiratory mechanics on lung inflation changes during development. We studied seven groups of rats from 10 days to 3 mo of age at five levels of positive end-expiratory pressure (PEEP) from 0 to 7 hPa (1 hPa = 0.1 kPa approximately 1 cmH(2)O). At each PEEP level, we measured respiratory system resistance and elastance at both 0.9 and 4.8 Hz to partition the mechanical properties into its airway and tissue components. Elastance increased more rapidly with PEEP in the younger animals, which we interpret as reflecting a more pronounced strain stiffening of the younger parenchyma. However, the decrease in airway resistance with PEEP was more pronounced in the older animals. Morphometric analysis showed that mean tissue density decreased and total alveolar surface area increased with age. Our data suggest that the mechanical interdependence between airways and parenchyma is weaker in very young animals compared with mature animals. This may play a role in the hyperresponsiveness of immaturity.

The contribution of airway smooth muscle to airway narrowing and airway hyperresponsiveness in disease.

Airway hyperresponsiveness (AHR), the exaggerated response to constrictor agonists in asthmatic subjects, is incompletely understood. Changes in either the quantity or properties of airway smooth muscle (ASM) are possible explanations for AHR. Morphometric analyses demonstrate structural changes in asthmatic airways, including subepithelial fibrosis, gland hyperplasia/hypertrophy, neovascularization and an increase in ASM mass. Mathematical modelling of airway narrowing suggests that, of all the changes in structure, the increase in ASM mass is the most probable cause of AHR. An increase in ASM mass in the large airways is more closely associated with a greater likelihood of dying from asthma than increases in ASM mass in other locations within the airway tree. ASM contraction is opposed by the elastic recoil of the lungs and airways, which appears to limit the degree of bronchoconstriction in vivo. The cyclical nature of tidal breathing applies stresses to the airway wall that enhance the bronchodilating influence of the lung tissues on the contracting ASM, in all probability by disrupting cross-bridges. However, the increase in ASM mass in asthma may overcome the limitation resulting from the impedances to ASM shortening imposed by the lung parenchyma and airway wall tissues. Additionally, ASM with the capacity to shorten rapidly may achieve shorter lengths and cause a greater degree of bronchoconstriction when stimulated to contract than slower ASM. Changes in ASM properties are induced by the process of sensitization and allergen-exposure such as enhancement of phospholipase C activity and inositol phosphate turnover, and increases in myosin light chain kinase activity. Whether changes in ASM mass or biochemical/biomechanical properties form the basis for asthma remains to be determined.

Airway surface liquid composition in mice.

Airway surface liquid (ASL) lines the conducting airways of the respiratory tract. We collected small samples of this liquid from the lower tracheae of anesthetized C57BL/6 mice and determined its ionic composition (in mM: 87.2 Na(+), 4.7 K(+), and 57.0 Cl(-)). Intravenous methacholine produced significant increases in the concentrations of Na(+), K(+), and Cl(-) within ASL. A limited analysis of liquid from cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice revealed no significant differences compared with littermate controls; however, Pseudomonas aeruginosa infection led to an increase in the salt concentration of ASL in cftr(+/+) mice. Morphometric measurements of tracheal submucosal gland volume revealed significant differences between inbred mouse strains, corresponding to ease of ASL collection. We conclude that although submucosal glands may be responsible for the production of some ASL, the ionic composition of this liquid is actively regulated by the underlying epithelial cells.

Upregulation of the transcription factor GATA-3 in upper airway mucosa after in vivo and in vitro allergen challenge.

BACKGROUND: Allergic rhinitis is a complex upper airways disorder characterized by the infiltration of eosinophils and T(H2)-type T lymphocytes. GATA-3 is a novel transcription factor recently shown to regulate IL-5 and, possibly, IL-4 gene expression. We previously reported that GATA-3 is increased within the bronchial mucosa of allergic asthmatic subjects compared with control subjects. OBJECTIVE: In the present study we set out to determine whether there is also an increased number of cells expressing GATA-3 messenger (m)RNA within the nasal mucosa of patients with allergic rhinitis. METHODS: Inferior turbinate biopsy specimens were obtained from patients with allergic rhinitis and nonatopic control subjects before and after local allergen provocation in vivo. To assess the contribution of resident cells expressing GATA-3 mRNA, we also performed isolated explant studies in which nasal mucosal tissue from subjects with allergic rhinitis and nonatopic control subjects was cultured in allergen-treated medium. The presence of mRNA coding for GATA-3, IL-5, IL-4, IL-13, and GM-CSF was assessed by using in situ hybridization. RESULTS: The number of GATA-3 mRNA(+) cells was increased after local allergen provocation in vivo (increase in GATA-3 mRNA(+) cells [mean +/- SEM]: subjects with allergic rhinitis, 11.3 +/- 8.7; control subjects, 1.2 +/- 4.1; P <.05) and in explanted nasal mucosa in vitro (subjects with allergic rhinitis, 10. 2 +/- 3.8; control subjects, 2.7 +/- 4.4; P <.05). The gene expression of GATA-3 was significantly correlated to the numbers of IL-5 (r = 0.87) and GM-CSF (r = 0.79) mRNA(+) cells but not with IL-4 or IL-13 mRNA(+) cells. CONCLUSION: In summary, the expression of the transcription factor GATA-3 was increased after allergen challenge, and this was evident in the absence of de novo inflammatory cell recruitment. GATA-3 may be a potential target in the treatment of allergic diseases, such as rhinitis.

Bronchial responsiveness among inbred mouse strains. Role of airway smooth-muscle shortening velocity.

To investigate the relationship between bronchial responsiveness and airway smooth-muscle (ASM) contractile properties, we studied inbred mice with known interstrain differences in airway responsiveness. Using oscillatory mechanics, we confirmed that A/J mice were hyperresponsive to methacholine (MCh) as compared with mice of the C3H/HeJ and C57BL/6J strains. Analysis of respiratory system resistance and elastance at different flow oscillation frequencies indicated that interstrain differences in responsiveness are present in both central and peripheral airways of these mice. We used video microscopy to measure the rate of contraction of explanted airways, and found that the airways of A/J mice contracted more rapidly than those of C3H/HeJ or C57BL/6J mice. In studies of a fourth strain (Balb/C) of mice, we found both bronchial hyperresponsiveness and increased ASM shortening velocity. The rank order of responsiveness among strains was the same as that for shortening velocity (A/J > Balb/C > C3H/HeJ > C57BL/6J). Furthermore, in each strain of mice, shortening velocity correlated with the achieved degree of airway narrowing and with a greater likelihood of airway closure in individual airways. In contrast, generation of isometric tension in trachealis, morphometric measurements of tracheal ASM, tracheal myosin content, and dose-response curves for MCh of explanted intraparenchymal bronchi failed to correspond to the in vivo phenotype of airway reactivity. These results indicate that bronchial responsiveness is related to ASM shortening velocity, and underscore the importance of smooth-muscle dynamics in understanding the mechanisms of bronchial responsiveness.

Enhanced Ca(2+) mobilization in airway smooth muscle contributes to airway hyperresponsiveness in an inbred strain of rat.

The mechanisms underlying airway hyperresponsiveness are still unknown but increased contractility of airway smooth muscle may play a role. This study sought to demonstrate a relationship between in vivo airway responsiveness and a number of measures of airway smooth muscle responsiveness ex vivo, including intracellular Ca(2+) signaling, by comparing three inbred strains of rat with different degrees of airways responsiveness to methacholine. Lewis, ACI, and Fisher strains of rat were characterized for their pulmonary responses to 5-hydroxytryptamine (5HT) in vivo and Fisher rats were found to be hyperresponsive to 5HT compared with ACI and Lewis rats. The responsiveness of the airways from these strains of rat ex vivo revealed that intraparenchymal airways from Fisher rats significantly narrowed to a greater degree and at a faster rate to 5HT than Lewis rat airways, consistent with their differences in vivo. Intraparenchymal ACI airways, however, narrowed to the same degree as Fisher airways but took longer to do so at a high concentration of 5HT. 5HT caused concentration-dependent increases in intracellular Ca(2+) in airway smooth muscle cells from all three strains of rat, but Fisher and ACI displayed higher responses than Lewis airway smooth muscle. Our results demonstrate that the degree of intracellular Ca(2+) mobilization by 5HT in airway smooth muscle parallels the rate and degree of intraparenchymal airway narrowing and suggest that the degree of intracellular Ca(2+) mobilization plays a role in determining airway smooth muscle contractility.

Interleukin-5 expression in the bone marrow of sensitized Balb/c mice after allergen challenge.

Interleukin-5 (IL-5) is a potent eosinophilopoietic factor implicated in the chronic inflammatory cell accumulation accompanying bronchial asthma. However, its role in stimulating eosinophil differentiation within the bone marrow following allergen exposure remains to be elucidated. The aims of our study were to determine the expression of IL-5 within the bone marrow of sensitized and control mice after allergen exposure, and to investigate the cellular phenotype of IL-5-producing cells. Sensitized Balb/c mice were challenged with either ovalbumin (OVA) or sterile saline. After 6 h, the mice were exsanguinated and the bone marrow prepared for cytospins. Bone marrow-derived cells from OVA-sensitized mice exhibited an increase in IL-5 immunoreactivity and mRNA compared with those from nonsensitized control mice (p < 0. 05). After allergen challenge, there was a further increase in IL-5 expression (p < 0.05) within the bone marrow. Both sensitization and allergen challenge resulted in an increase in the number of cells expressing major basic protein (MBP) (p < 0.05). In nonsensitized mice, the IL-5 mRNA was expressed predominantly by CD34-positive (CD34+) progenitor cells. Following sensitization and allergen challenge, CD3-positive (CD3+) T lymphocytes were the major source of this cytokine. These results demonstrate the presence of IL-5 within the bone marrow of normal Balb/c mice. After sensitization and allergen challenge, the increase in IL-5-producing cells within the bone marrow is attributed by T lymphocytes.