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Proteoglycans (PG) have important effects on the mechanical properties of tissues and the phenotype of various structural cells. Little is known about changes in PG deposition in the airways in animal models of asthma. We studied changes in PG in the airway wall of Brown Norway rats sensitized to ovalbumin (OA) and exposed to repeated OA challenge. Control (Sal) animals were sensitized and challenged with saline. After the 3rd challenge, animals were killed and lungs fixed in formalin. Tissue sections were incubated with antibodies to the small, leucine-rich PG, decorin, and biglycan and collagen type I. Airways were classified according to basement membrane perimeter length (≤0.99, 1–2.99, and ≥3 mm). Decorin, biglycan, and collagen type I were increased in the airways of OA vs. Sal rats. Remodeling was most prominent in central airways. The distribution of PG differed with respect to the subepithelial vs. airway smooth muscle (ASM) vs. adventitial layer. Whereas biglycan was readily detected within the ASM, decorin and collagen were detected outside the ASM and especially in the adventitial layer. Differences in the distribution of these molecules within the layers of the airway wall may reflect their specific functional roles.

Complex rheology of airway smooth muscle cells and its dynamic response during contractile stimulation involves many molecular processes, foremost of which are actomyosin cross-bridge cycling and actin polymerization. With an atomic force microscope, we tracked the spatial and temporal variations of the viscoelastic properties of cultured airway smooth muscle cells. Elasticity mapping identified stiff structural elements of the cytoskeletal network. Using a precisely positioned microscale probe, picoNewton forces and nanometer level indentation modulations were applied to cell surfaces at frequencies ranging from 0.5 to 100Hz. The resulting elastic storage modulus (G′) and dissipative modulus (G″) increased dramatically, with hysteresivity (η=G″/G′) showing a definitive decrease after stimulation with the contractile agonist 5-hydroxytryptamine. Frequency-dependent assays showed weak power-law structural damping behavior and universal scaling in support of the soft-glassy material description of cellular biophysics. Additionally, a high-frequency component of the loss modulus (attributed to cellular Newtonian viscosity) increased fourfold during the contractile process. The complex shear modulus showed a strong sensitivity to the degree of actin polymerization. Inhibitors of myosin light chain kinase activity had little effect on the stiffening response to contractile stimulation. Thus, our measurements appear to be particularly well suited for characterization of dynamic actin rheology during airway smooth muscle contraction.

Asthma is characterized by complexity resulting from the interactions among a variety of biomechanical, immunological and biochemical processes that lead to airway narrowing. Multiple models are therefore required to address particular aspects of its molecular pathophysiology. Exploration of the disease and its treatment requires experimental methodologies that include a range of in vitro and in vivo approaches. This article will contextualize recent advances in our understanding of asthma through the development and exploration of various models. The relative strengths and weaknesses of the various models and their utility for target identification will be reviewed.

Increased airway smooth muscle (ASM) mass is perhaps the most important component of the airway wall remodeling process in asthma. Known mediators of ASM proliferation in cell culture models fall into 2 categories: those that activate receptors with intrinsic receptor tyrosine kinase activity and those that have their effects through receptors linked to heterotrimeric guanosine triphosphate-binding proteins. The major candidate signaling pathways activated by ASM mitogens are those dependent on extracellular signal-regulated kinase and phosphoinositide 3'-kinase. Increases in ASM mass may also involve ASM migration, and in culture, the key signaling mechanisms have been identified as the p38 mitogen-activated protein kinase and the p21-activated kinase 1 pathways. New evidence from an in vivo rat model indicates that primed CD4(+) T cells are sufficient to trigger ASM and epithelial remodeling after allergen challenge. Hyperplasia has been observed in an equine model of asthma and may account for the increase in ASM mass. Reduction in the rate of apoptosis may also play a role. beta(2)-Adrenergic receptor agonists and glucocorticoids have antiproliferative activity against a broad spectrum of mitogens, although it has become apparent that mitogens are differentially sensitive. Culture of ASM on collagen type I has been shown to enhance proliferative activity and prevent the inhibitory effect of glucocorticoids, whereas beta(2)-agonists are minimally affected. There is no evidence that long-acting beta(2)-agonists are more effective than short-acting agonists, but persistent stimulation of the beta(2)-adrenergic receptor probably helps suppress growth responses. The maximum response of fluticasone propionate against thrombin-induced proliferation is increased when it is combined with salmeterol.

The purpose of this study was to test the therapeutic potential of monomethoxypolyethylene glycol (mPEG) conjugated-allergen using a rodent model of allergic asthma. Previously, this conjugate has been shown to possess the dual capacity of inducing long-term ovalbumin (OA)-specific suppression of the antibody response and inactivating rat mast cells that have been sensitized with murine IgE to OA. Ovalbumin sensitized and challenged Brown Norway rats were studied. Fourteen days after sensitization, a test group of six rats received mPEG–OA solution intratracheally and were challenged 30 min later with aerosolized OA. Another group of seven sensitized rats was similarly challenged with OA 30 min after intratracheal administration of normal saline. A group of six sensitized rats received mPEG–OA solution intratracheally but were challenged with normal saline. Another group of seven sensitized rats received mPEG–BSA solution intratracheally and were challenged 30 min later with aerosolized OA. A final group of five unsensitized rats were neither challenged nor medicated intratracheally. Pulmonary resistance was measured before and for 8 h following inhalation challenge. mPEG–OA treatment had an inhibitory effect on the allergic late airway response, but the early response was not significantly altered. Both mPEG–OA and mPEG–BSA reduced the total cells, eosinophils and neutrophils, in bronchoalveolar lavage and decreased the expression of IL-4, IL-5 and IFN-γ mRNA. In conclusion, mPEG–OA can prevent the development of allergen-induced late airway responses and reduce airway Th2-type cytokine expression whereas mPEG conjugated to an irrelevant antigen (BSA) is anti-inflammatory but does not affect the late response.

Airway smooth muscle maintains airway tone and may assist in adjusting ventilation distribution within the normal lung. Alterations in the properties or the quantity of ASM are likely responsible for some instances of airways hyperresponsiveness to bronchoconstrictive stimuli that is a characteristic of diseases such as asthma. Morphometric studies have shown an increase in the mass of ASM in human asthmatic airways. Animal models have been developed that confirm that ASM can be induced to grow by allergic sensitization and challenge. Growth is in large part by hyperplasia as measured by incorporation of bromodeoxyuridine as a marker of the S-phase of the cell cycle. T cells, in particular CD4+ cells, may participate in the stimulation of growth of ASM by allergen challenge. The growth factors responsible for the increase in ASM are as yet unidentified but two mediators associated with allergic airway responses, cysteinyl leukotrienes and endothelin, have been implicated using specific receptor antagonists. The links between T cells and the biochemical mediators of growth have not been established.

The leukotriene modifiers are a novel generation of therapeutic agents in the treatment of allergic asthma. However, the mechanisms by which the cysteinyl (cys) leukotrienes (LTs) participate in allergen-induced airway eosinophilia and airway hyperresponsiveness (AHR) are still unclear. In the present study, we have investigated the role of cys-LTs in ovalbumin (OVA)-induced airway responses in a murine model of asthma. Montelukast (3 or 10 mg/kg), a selective cys-LT1 receptor antagonist, reduced airway eosinophilia and AHR after OVA challenge. The levels of interleukin (IL)-5 and eotaxin in the bronchoalveolar lavage fluid (BALF) from montelukast-treated (3 mg/kg) mice were unaffected, although a decrease in IL-5 was observed with a dose of 10 mg/kg. LTD4 (50 ng) instilled intranasally to immunized mice augmented macrophages in the BALF, but in conjunction with OVA challenge it caused BALF eosinophilia and neutrophilia when given before challenge and BALF neutrophilia but not eosinophilia when given 2 h after challenge. However, there were no increases of IL-5 or eotaxin in BALF following LTD4 treatment. Repeated instillations of LTD4 to immunized mice, mimicking allergen challenge, did not induce AHR but in conjunction with OVA challenge LTD4 enhanced AHR. These results indicate that allergen-induced eosinophilia and AHR are in part mediated by the cys-LT1 receptor, and that, although LTD4 alone has no effect on airway eosinophilia, in conjunction with antigenic stimulation it potentiates the degree of airway inflammation and AHR.

To the Editor: Asthma affects all age groups and presents itself as a spectrum of severity and symptoms. Reactive oxidative species (ROS) play a pivotal role in asthma pathogenesis. Exhaled levels of mediators associated with ROS positively correlate with asthma severity.(1) Autophagy, the process of cellular waste disposal through lysosomal -dependent pathways, is induced by ROS to remove oxidized proteins or organelles to minimize tissue damage.(2) Although autophagy is augmented in the lungs of COPD patients compared to healthy control subjects (3), evidence for autophagy in asthma, particularly moderate-to-severe asthma, has not been reported. We hypothesize that autophagy is associated with asthma pathogenesis, and sought to detect its presence using both genetic and histological approaches. We conducted a genetic association study to investigate whether single nucleotide polymorphisms (SNPs) in genes of the autophagy pathway are associated with asthma. We selected 5 genes of the autophagy pathway (unc-51-like kinase 1(ULK1), sequestosome 1 (SQSTM1), microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), beclin 1 (BECN1) and autophagy related 5 homolog (ATG5)). (2) We tested for genetic associations in an asthma family-based study from northeastern Quebec, Canada (The Saguenay-Lac-Saint-Jean (SLSJ) asthma Study) using the family based association test (FBAT) statistic and UNPHASED software for an odds ratio estimate.(4, 5) Patient recruitment has previously been described.(6) These SNPs have been genotyped previously in a genome-wide association study.(6) To reduce the likelihood of false positive findings, we reset the statistical significance threshold from p=0.05 to p=0.001 according to the Bonferroni method. To confirm our positive findings, we tested the association in a second family based population; the non-Hispanic Caucasian participants of the Childhood Asthma Management Program (CAMP), and patient recruitment has previously been described. (7) The SLSJ local ethics committee and the Institutional Review Board for CAMP approved the study, and all subjects gave informed consents. In the SLSJ asthma study a total of 1338 individuals (483 nuclear families) with known asthma status were included in the analysis, and 336 individuals were either probands or affected siblings. Of this group, the male:female ratio was 0.83. The mean age was 16.45 (standard deviation (SD) +/− 9.43) years. 77.1% were atopic. The mean (SD) forced expiratory volume in 1 second (FEV1)% predicted was 94.1(20.1)%. A total of 39 SNPs were tested, and after Bonferroni correction, SNP rs12212740 G>A of ATG5 remained statistically significant (Table 1). Allele G with allele frequency of 0.88 is over - transmitted to asthmatic offspring (p=0.0002), (Odds ratio = 1.35, (95% confidence interval = 1.01 and 1.89). SNP rs12212740 was not associated with asthma in CAMP, however, it was associated with pre - bronchodilator FEV1 (pre-FEV1) (adjusted for age, sex and height) (p=0.04). In the SLSJ cohort, rs12212740 was associated with pre-FEV1 (p=0.007). In both populations, allele G was negatively associated with adjusted pre-FEV1. SNP rs12212740 is located in intron 3 of ATG5, 7kb downstream and 8kb upstream of exon 3 and 4, respectively. At present, the functional consequence of SNP rs12212740 is unknown, and it is probable that the association is due to the linkage disequilibrium between SNP rs12212740 and the true causative variant. Nevertheless, the association between a genetic variant of ATG5 and pre-FEV1 in both study populations suggest that autophagy is associated with reduced lung function in asthmatic subjects. Table 1 Association of SNPs and asthma in the SLSJ asthma study To determine if autophagy is present in the airways of asthmatic individuals, bronchial biopsy stored at the Tissue Bank of the Respiratory Health Network of the Fonds de la Recherche en Sante du Quebec (McGill University Health Centre site) were obtained. Patient recruitment and bronchoscopy have previously been described.(8) Bronchial biopsy tissue from a moderately severe asthma patient and a healthy control were viewed by electron microscopy (EM) for double membrane autophagosomes. Here, we demonstrated by EM in a tissue sample from a moderately severe asthma subject evidence of autophagy in asthma pathogenesis. Using EM, double membrane autophagosomes were detected in fibroblasts and epithelial cells. A representative fibroblast from a bronchial biopsy tissue of a moderate asthma subject is depicted in Figures 1A-C, and epithelial cells in figures 2A-C. Corresponding fibroblast and epithelial cells from a healthy control are depicted in figures 1D-F and figures 2D-F, respectively, where fewer or no autophagosome was detected. Fig 1 A double membrane autophagosome was detected in a fibroblast from a bronchial biopsy tissue sample of a moderate asthma subject. Tissue was viewed at magnifications of (A) 1480x, (B) 16100x and (C) 62200x. Boxed areas were viewed under higher magnification ... Fig 2 Autophagosomes were detected in bronchial epithelial cells from a moderate asthma subject. Tissue was viewed at magnifications of (A) 4030x, (B) 9760x and (C) 37000x, respectively. Corresponding epithelial cells of a healthy subject was viewed at magnifications ... This is the first report to present genetic and histological evidence of autophagy in asthma pathogenesis. ATG5 is involved in the elongation step of the autophagosome formation. ATG5 forms a complex with ATG12 and ATG16L1 and the complexes are found on the outer membrane of the forming autophagosome.(2) We speculate that the positive association of allele G with asthma and the negative association with pre-FEV1 in asthmatics may be due to the inverse relationship between pre-FEV1 and asthma severity(9). If allele G is a risk factor for low pre-FEV1, given that pre-FEV1 tends to be lower in those with more severe forms of asthma, the allele would also increase the risk of developing moderate-to-severe asthma. The genetic association of ATG with pre-FEV1 in asthmatics and the detection of autophagosomes in fibroblasts and epithelial cells in tissues from a moderately severe asthma patient suggest an association between autophagy and reduced lung function in asthmatic subjects. At present the mechanistic pathway of autophagy in asthma is unclear, but it opens up a new avenue to explore the mechanism of the chronic nature of asthma pathogenesis.

Airway hyperresponsiveness is a characteristic feature of asthma, and its measurement is an important tool in its diagnosis. With a few caveats, methacholine bronchial provocation by a 2-minute tidal breathing method is highly sensitive; a negative test result (PC20 > 16 mg/mL, PD20 > 400 μg) rules out current asthma with reasonable certainty. A PC20 value of less than 1 mg/mL/PD20 value of less than 25 μg is highly specific (ie, diagnostic) but quite insensitive for asthma. For accurate interpretation of the test results, it is important to control and standardize technical factors that have an impact on nebulizer performance. In addition to its utility to relate symptoms such as cough, wheeze, and shortness of breath to variable airflow obstruction (ie, to diagnose current asthma), the test is useful to make a number of other clinical assessments. These include (1) evaluation of patients with occupational asthma, (2) evaluation of patients with exercise-induced respiratory symptoms, (3) evaluation of novel asthma medications, (4) evaluation of relative potency of inhaled bronchodilators, (5) as a biomarker to adjust anti-inflammatory therapy to improve clinical outcomes, and (6) in the evaluation of patients with severe asthma to rule out masqueraders such as laryngeal dysfunction. The actual mechanism of altered smooth muscle behavior in asthma that is assessed by direct (eg, methacholine) or indirect (eg, allergen) bronchial provocation remains one of the most fundamental questions related to asthma that needs to be determined. The test is underutilized in clinical practice.

The type I interferon pathway plays a critical role in both host defense and tolerance against viral infection and thus requires refined regulatory mechanisms. RIPK3-mediated necroptosis has been shown to be involved in anti-viral immunity. However, the exact role of RIPK3 in immunity to Influenza A Virus (IAV) is poorly understood. In line with others, we, herein, show that Ripk3-/- mice are highly susceptible to IAV infection, exhibiting elevated pulmonary viral load and heightened morbidity and mortality. Unexpectedly, this susceptibility was linked to an inability of RIKP3-deficient macrophages (Mφ) to produce type I IFN in the lungs of infected mice. In Mφ infected with IAV in vitro, we found that RIPK3 regulates type I IFN both transcriptionally, by interacting with MAVS and limiting RIPK1 interaction with MAVS, and post-transcriptionally, by activating protein kinase R (PKR)—a critical regulator of IFN-β mRNA stability. Collectively, our findings indicate a novel role for RIPK3 in regulating Mφ-mediated type I IFN anti-viral immunity, independent of its conventional role in necroptosis., Author summary Influenza A virus (IAV) is a pulmonary pathogen that presents a significant threat to human health through seasonal epidemics and occasional, highly lethal pandemics. Type I IFN is an essential component of anti-viral immunity to influenza infection and pulmonary macrophages are the major source of type I IFN. Recently, we have shown that programmed macrophage cell death (apoptosis) plays a key role in immunity to influenza infection. Interestingly, another cell death program, termed necroptosis, has been also implicated in control of viral infection via receptor-interacting serine/threonine protein kinase 3 (RIPK3). In the present study, we define a novel role of RIPK3 in regulating the type I IFN pathway to protect against lethal IAV infection, which is independent of necroptosis. RIPK3 regulates type I IFN signaling at both the transcriptional and post-transcriptional levels to sequester viral replication and protect the host against influenza infection. Thus, manipulation of the RIPK3 pathways poses a novel avenue in the development of anti-IAV treatments.

Varying concentrations of lipopolysaccharide (LPS) in ovalbumin (OVA) may influence the airway response to allergic sensitization and challenge. We assessed the contribution of LPS to allergic airway inflammatory responses following challenge with LPS-rich and LPS-free commercial OVA. BALB/c mice were sensitized with LPS-rich OVA and alum and then underwent challenge with the same OVA (10 µg intranasally) or an LPS-free OVA. Following challenge, bronchoalveolar lavage (BAL), airway responsiveness to methacholine and the lung regulatory T cell population (Treg) were assessed. Both OVA preparations induced BAL eosinophilia but LPS-rich OVA also evoked BAL neutrophilia. LPS-free OVA increased interleukin (IL)-2, IL-4 and IL-5 whereas LPS-rich OVA additionally increased IL-1β, IL-12, IFN-γ, TNF-α and KC. Both OVA-challenged groups developed airway hyperresponsiveness. TLR4-deficient mice challenged with either OVA preparation showed eosinophilia but not neutrophilia and had increased IL-5. Only LPS-rich OVA challenged mice had increased lung Tregs and LPS-rich OVA also induced in vitro Treg differentiation. LPS-rich OVA also induced a Th1 cytokine response in human peripheral blood mononuclear cells. We conclude that LPS-rich OVA evokes mixed Th1, Th2 and innate immune responses through the TLR-4 pathway, whereas LPS-free OVA evokes only a Th2 response. Contaminating LPS is not required for induction of airway hyperresponsiveness but amplifies the Th2 inflammatory response and is a critical mediator of the neutrophil, Th1 and T regulatory cell responses to OVA.

To subvert host immunity, influenza A virus (IAV) induces early apoptosis in innate immune cells by disrupting mitochondria membrane potential via its polymerase basic protein 1-frame 2 (PB1-F2) accessory protein. Whether immune cells have mechanisms to counteract PB1-F2-mediated apoptosis is currently unknown. Herein, we define that the host mitochondrial protein nucleotide-binding oligomerization domain-like receptor (NLR)X1 binds to viral protein PB1-F2, preventing IAV-induced macrophage apoptosis and promoting both macrophage survival and type I IFN signaling. We initially observed that Nlrx1-deficient mice infected with IAV exhibited increased pulmonary viral replication, as well as enhanced inflammatory-associated pulmonary dysfunction and morbidity. Analysis of the lungs of IAV-infected mice revealed markedly enhanced leukocyte recruitment but impaired production of type I IFN in Nlrx1(-/-) mice. Impaired type I IFN production and enhanced viral replication was recapitulated in Nlrx1(-/-) macrophages and was associated with increased mitochondrial mediated apoptosis. Through gain- and loss-of-function strategies for protein interaction, we identified that NLRX1 directly bound PB1-F2 in the mitochondria of macrophages. Using a recombinant virus lacking PB1-F2, we confirmed that deletion of PB1-F2 abrogated NLRX1-dependent macrophage type I IFN production and apoptosis. Thus, our results demonstrate that NLRX1 acts as a mitochondrial sentinel protecting macrophages from PB1-F2-induced apoptosis and preserving their antiviral function. We further propose that NLRX1 is critical for macrophage immunity against IAV infection by sensing the extent of viral replication and maintaining a protective balance between antiviral immunity and excessive inflammation within the lungs.

Innate airway hyperresponsiveness (AHR) is well modeled by two strains of rat, the hyperresponsive Fischer 344 rat and the normoresponsive Lewis rat. Arginase has been implicated in AHR associated with allergic asthma models. We addressed the role of arginase in innate AHR using the Fischer-Lewis model. In vivo arginase inhibition with Nω-hydroxy-nor-arginine (nor-NOHA) was evaluated on methacholine-induced bronchoconstriction in the Fischer and the Lewis rats. Arginase activity and mRNA expression were quantified in structural and resident cells of the proximal airway tree. The effect of nor-NOHA was evaluated on cultured tracheal smooth muscle proliferation. Fischer rats exhibited significantly greater changes in respiratory resistance and elastance in response to methacholine compared with Lewis rats. nor-NOHA reduced the methacholine-induced bronchoconstriction in the central airways of Lewis rats, while it did not change the innate AHR of Fischer rats. Lewis rats exhibited greater arginase activity in tracheal smooth muscle but a lower proliferation rate compared with Fischer rats. Smooth muscle proliferation was not affected by nor-NOHA in either strain of rats. The strain-specific arginase expression in the smooth muscle may contribute to the differences in sensitivity of the methacholine challenged airways of Lewis and Fischer rats to inhibition of arginase.

Summary Among patients with cystic fibrosis there is a high prevalence (40–70%) of asthma signs and symptoms such as cough and wheezing and airway hyper-responsiveness to inhaled histamine or methacholine. Whether these abnormal airway responses are due to a primary deficiency in the cystic fibrosis transmembrane conductance regulator (CFTR) or are secondary to the inflammatory environment in the cystic fibrosis lungs is not clear. A role for the CFTR in smooth muscle function is emerging, and alterations in contractile signalling have been reported in CFTR -deficient airway smooth muscle. Persistent bacterial infection, especially with Pseudomonas aeruginosa , stimulates interleukin-8 release from the airway epithelium, resulting in neutrophilic inflammation. Increased neutrophilia and skewing of CFTR -deficient T-helper cells to type 2 helper T cells creates an inflammatory environment characterised by high concentrations of tumour necrosis factor α, interleukin-8, and interleukin-13, which might all contribute to increased contractility of airway smooth muscle in cystic fibrosis. An emerging role of interleukin-17, which is raised in patients with cystic fibrosis, in airway smooth muscle proliferation and hyper-responsiveness is apparent. Increased understanding of the molecular mechanisms responsible for the altered smooth muscle physiology in patients with cystic fibrosis might provide insight into airway dysfunction in this disease.

Morphometric analyses of endobronchial biopsies are commonly performed in asthma research but little is known about the technical and physiological parameters contributing to measurement variability. We investigated factors potentially affecting biopsy size, quality, and airway smooth muscle (ASM) content in heaves, an asthma-like disease of horses. Horses with heaves in clinical exacerbation ( n = 6) or remission ( n = 6) from the disease and six controls were studied using a crossover design. The effect of disease status, age, bronchodilation, biopsy forceps type, and carina size on total biopsy area (Atot), ASM area (AASM), ASM% (AASM/Atot), and histologic quality were assessed. Concordance among different measuring techniques was also assessed. Compared with other groups, horses with heaves in exacerbation yielded larger biopsies ( P < 0.05). Better quality biopsies were obtained from carinae of small size compared with large ones ( P = 0.02), and carina size and forceps type significantly affected the ASM content of the biopsy (interaction, P < 0.05). AASM increased with age only in heaves-affected horses ( r = 0.9, P < 0.05), and ASM% was negatively correlated with pulmonary resistance at 5 Hz in heaves-affected horses ( r = −0.74, P = 0.01), likely because of the increased thickness of the extracellular matrix layer in this group ( P = 0.01). In conclusion, disease status, carina thickness, and the forceps used may significantly affect biopsy size, quality, and ASM content. Endobronchial biopsies are not appropriate samples for ASM quantification in heaves, and studies measuring ASM mass should not be compared when measuring techniques differ.

Heaves in horses shares many similarities with human asthma, including lower airway inflammation, reversible airway obstruction, and bronchial hyperresponsiveness. Extrinsic asthma is an allergic response to environmental allergens and a similar immunologic mechanism may be implicated in heaves. It is now recognized that a Th2 subset of CD4+ lymphocytes is associated with allergic diseases such as atopic asthma. The purpose of this study was to determine whether airway inflammation in heaves is associated with a pattern of expression of cytokine suggestive of a Th2 type response. The expression of mRNA, encoding interleukin (IL)-4, IL-5, and interferon gamma (IFN-gamma) was measured in bronchoalveolar cells from seven horses with heaves and five control horses, using in situ hybridization and radiolabeled equine-specific cRNA probes coding for these cytokines. Bronchoalveolar cells of horses with heaves had an increased expression of IL-4 (p = 0.01) and IL-5 (p = 0.02) mRNA and a decreased expression of INF-gamma (p = 0.01) compared with control horses. Here we show that inflammatory cells in lungs from horses with heaves display a Th2-type cytokine profile that is consistent with the hypothesis that heaves is an allergic condition with similarity to human asthma.

The blockade of alpha4 integrins with a monoclonal antibody (TA-2) decreases late airway responses (LR) in ovalbumin (OVA)-sensitized and challenged rats. In this study, we used a model of CD4+ cell-driven LR to test the hypothesis that alpha4-integrin blockade involves interference with T-cell activation in the inhibition of LR. Purified CD4+ cells from OVA-sensitized rats were transferred to unsensitized recipients, which received either TA-2 or a control antibody (cAb), and were OVA-challenged. A sham-challenged group was also studied. LR, calculated from pulmonary resistance after challenge, were reduced in the TA-2 group compared with the cAb group (p = 0.015). Total cell counts, macrophages, neutrophils, and lymphocytes in bronchoalveolar lavage (BAL), and CD3+ cells in airway sections, were unaffected. The cAb group had higher numbers of cells expressing interleukin-5 (IL-5) mRNA (55.2 +/- 3.39 cells/1,000, mean +/- SEM) and major basic protein (MBP) (6.2 +/- 0.4/100 cells) in bronchoalveolar lavage (BAL), than the TA-2 group (25.37 +/- 2.41 IL-5+ and 2.7 +/- 0.2 MBP+) and the sham group (12.37 +/- 0.96 IL-5+, 1.7 +/- 0.1 MBP+). Interferon gamma (IFN-gamma) mRNA+ cells were downregulated in both OVA-challenged groups, compared with the sham group. Our results suggest that the attenuation of LR and eosinophilia by alpha4-integrin blockade may involve interference with CD4+ cell activation and IL-5 expression.

Background Allergic asthma is characterized by airway inflammation in response to antigen exposure, leading to airway remodeling and lung dysfunction. Epithelial-mesenchymal transition (EMT) may play a role in airway remodeling through the acquisition of a mesenchymal phenotype in airway epithelial cells. TGF-β1 is known to promote EMT; however, other cytokines expressed in severe asthma with extensive remodeling, such as IL-22, may also contribute to this process. In this study, we evaluated the contribution of IL-22 to EMT in primary bronchial epithelial cells from healthy and asthmatic subjects. Methods Primary bronchial epithelial cells were isolated from healthy subjects, mild asthmatics and severe asthmatics (n=5 patients per group). The mRNA and protein expression of epithelial and mesenchymal cell markers and EMT-associated transcription factors was evaluated following stimulation with TGF-β1, IL-22 and TGF-β1+IL-22. Results Primary bronchial epithelial cells stimulated with TGF-β1 underwent EMT, demonstrated by decreased expression of epithelial markers (E-cadherin and MUC5AC) and increased expression of mesenchymal markers (N-cadherin and vimentin) and EMT-associated transcription factors. IL-22 alone had no effect on epithelial or mesenchymal gene expression. However, IL-22+TGF-β1 promoted the expression of some EMT transcription factors (Snail1 and Zeb1) and led to a more profound cadherin shift, but only in cells obtained from severe asthmatics. Conclusion The impact of IL-22 on airway epithelial cells depends on the cytokine milieu and the clinical phenotype of the patient. Further studies are required to determine the molecular mechanism of IL-22 and TGF-β1 cooperativity in driving EMT in primary human bronchial epithelial cells.

Background: Little is known about the comparative kinetics of eosinophil recruitment after exposure to low- and high-molecular-weight sensitizers in subjects with occupational asthma (OA). Objectives: The aims of the study were to investigate the kinetics of changes in inflammatory mediators associated with eosinophil infiltration (IL-5 and eotaxin) and to examine the nature of the airway inflammation induced in response to different types of occupational agents. Methods: We investigated 15 subjects with OA caused by high- and low-molecular-weight agents. The subjects were exposed to increasing doses of the relevant occupational agent over 3 to 4 days until a 20% fall in FEV 1 occurred. Methacholine challenge and sputum induction were performed at the end of each day of exposure. Sputum samples were assessed for differential cell counts, including eosinophils, IL-5, and eotaxin messenger RNA. Results: There was an increase in sputum eosinophils, eotaxin, and IL-5 on the day preceding the occurrence of asthmatic reaction, although there was no change in functional parameters (FEV 1 and PC 20 ). Increase in sputum eosinophils was more prominent in subjects exposed to low-molecular-weight agents than to high-molecular-weight agents. Conclusion: Changes in eosinophils, IL-5, and eotaxin precede functional changes after exposure to occupational agents in subjects with OA. Eosinophil inflammation is a feature of exposure to both high- and low-molecular-weight agents. Induced sputum may be a useful tool in the early diagnosis of OA. (J Allergy Clin Immunol 2000;106:1163-70.)

Airway smooth muscle (ASM) mass is likely to be an important determinant of airway responsiveness. Highly inbred Fisher rats model innate hyperresponsiveness, and also have more ASM in vivo than control Lewis rats. Platelet derived growth factor (PDGF) is an important endogenous growth factor for ASM, and partially purified PDGF-AB causes enhanced growth of Fisher rat ASM cells, compared to Lewis cells. The aim of the present study was to determine the mitogenic effects of all three recombinant PDGF isoforms on ASM cells, and investigate the mechanisms of enhanced Fisher ASM growth responses. The potential mechanisms assessed include PDGF receptor expression and activation (tyrosine phoshorylation), and intracellular calcium (Ca2+) responses to PDGF isoforms. Fisher ASM cells had a greater mitogenic response to PDGF-AB and -AA, and a greater Ca2+response to -BB than Lewis ASM cells. A Ca2+response was not necessary for a mitogenic response, and the effects of PDGF isoforms on Ca2+were not associated with their effects on growth. Therefore, we suggest that enhanced Fisher mitogenic response to PDGF-AA and -AB is not mediated by differences in Ca2+signalling. Western analysis of the PDGF receptor indicated a similar expression of β-PDGF receptor in ASM cells from the two rat strains, but a greater expression of α-PDGF receptor in Fisher cells; however, phosphorylation of the PDGF receptor following growth stimulation did not differ between strains. This suggests a role for post-receptor signals, in addition to enhanced receptor expression, in the enhanced growth response of Fisher ASM cells to PDGF-AA and -AB.Key words: PDGF receptors, tyrosine phosphorylation, intracellular calcium, proliferation, airway smooth muscle cells.

Neurokinins (NKs), which include substance P (SP) and neurokinin A (NKA), act through NK-1 and NK-2 receptors. There is considerable evidence of interaction between the neurogenic and the immune systems, and NKs are candidates for mediating such interactions. We hypothesized that selective inhibition of pulmonary NK-1 or NK-2 receptors may modulate immune responses so as to prevent the development of allergic airway responses in the atopic BN rat sensitized to ovalbumin (OA). To address this hypothesis, we have validated our animal model by showing that NK-1 and NK-2 receptors are expressed in the lungs, and that SP is released in the airways after allergen challenge. The selective NK-1 (CP-99,994) or NK-2 (SR-48968) antagonists before allergen challenge failed to reduce the allergic early airway responses. In contrast, both neurokinin antagonists decreased allergen-induced late airway responses in OA-challenged animals. However, only the NK-2 antagonist decreased the eosinophil numbers in the bronchoalveolar lavage (BAL). Likewise, the NK-2, but not NK-1, antagonist decreased both Th1 (INF-gamma) and Th2 (IL-4 and -5) cytokine expression in BAL cells by in situ hybridization. These results provide initial in vivo evidence linking neurokinins to the regulation of cytokine expression in cells without discrimination as to their phenotype. We conclude that there is a dichotomy between NK receptors in the modulation of the allergic airway inflammation, which has important implications for future therapeutic strategies for asthma using the NK antagonists.

Tachykinins (TK) have been implicated in both bradykinin-(BK) and hyperpnea-induced broncho-constriction (HIB) in the guinea-pig. However, TKs appear to have an indirect effect in HIB by releasing leukotriene (LT)D(4). We postulated that BK may cause bronchoconstriction through a cascade involving TK and LTD(4). We examined the role of TK and LTD(4)in BK-induced bronchoconstriction in ventilated Hartley guinea-pigs. Respiratory resistance (R(rs)) was monitored for 2 h following insufflation of BK (150 nM). Animals were pretreated with propranolol, then with either neurokinin (NK)1 (CP-99,994)+NK2 (SR-48,968) receptor antagonists or pranlukast (90 microg or 900 microg), an LTD(4)antagonist. Control animals received no pretreatment. BK-induced bronchoconstriction was significantly lower in NK1/NK2 (128%+/-6% baseline R(rs)SEM) and pranlukast (90 microg; 205+/-22, 900 microg; 169+/-20) animals compared to controls (284+/-22), P

Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 μM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 μM). Genistein and tyrphostin 23 (40 and 10 μM, respectively) significantly decreased 5-HT-evoked peak Ca2+responses, and the effect of genistein could be observed in the absence of extracellular Ca2+. The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 μM) had no significant effect on peak Ca2+levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of ∼70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the γ isoform of phospholipase C (PLC-γ). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 μM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-β activity. It is unlikely that PLC-γ or the mitogen-activated protein kinase pathway is involved in Ca2+signaling to 5-HT.

Fischer rat airway smooth muscle (ASM) models two potential risk factors for asthma: hyperresponsiveness to contractile agonists and to growth stimuli. The aim of this study was to identify the mechanisms responsible for enhanced ASM mitogenic response in Fischer rats compared with the control Lewis strain. The enhanced Fischer ASM cell growth response to fetal bovine serum (FBS) could not be accounted for by phospholipase C, mitogen-activated protein kinases, or tyrosine kinase activities as assessed by pharmacological inhibition and Western blotting. In contrast, depletion of phorbol ester-sensitive isoforms of the serine/threonine kinase protein kinase C (PKC) removed the difference in growth response between the rat strains. Additionally, FBS selectively induced serine/threonine phosphorylation of a 115-kDa protein in Fischer ASM cells. Enhanced activation of PKC-βI and decreased activation of PKC-δ in Fischer compared with Lewis cells following FBS stimulation were suggested by Western blotting of membrane and cytosolic fractions. The data are consistent with a role for PKC in the enhanced ASM cell growth of hyperresponsive rats.

Isocapnic dry gas hyperpnea-induced bronchoconstriction (HIB) in the guinea-pig is mediated by both tachykinin release from airway sensory nerve C-type fiber terminals and secondary synthesis of cysteinyl-leukotrienes, in particular LTD 4 . Beta ( β ) 2 -agonists are potent bronchodilators but potentially could also inhibit the airway response to hyperpnea challenge via effects on the release of LTD 4 from airway cells in vivo. The purpose of this study was to test the hypothesis that β 2 agonists attenuate HIB in guinea-pigs, in part, by reduction in LTD 4 release in vivo. Twenty-six guinea-pigs (400–550 g) were anesthetized with xylazine (7 mg/kg) and pentobarbital (65 mg/kg), tracheotomized and mechanically ventilated with a small animal ventilator using a tidal volume of 3 ml and a breathing frequency of 60 breaths/min. Dry gas (95%O 2 /5%CO 2 ) with a 4 ml tidal volume and a breathing frequency of 150/min was used for hyperpnea challenge. Challenge with isocapnic dry gas triggered a significant increase in pulmonary resistance (0.3 ± 0.02 vs. 0.57 ± 0.06 cmH 2 O/ml per s; P=0.017; n=13) and excretion of LTD 4 in the bile (baseline: 2.43 vs. HIB: 4.66 pmol/h; P=0.04). Salbutamol pretreatment completely blocked the airway response to the challenge (0.3±0.02 vs. 0.3±0.05 cmH 2 O/ml per s; n=13) and reduced the biliary excretion of LTD 4 (baseline: 2.42 pmol/h; vs. HIB: 2.40 pmol/h). We conclude that salbutamol inhibited the airway responses to dry gas hyperpnea challenge and LTD 4 synthesis by the airway cells.

The effects of concomitant P1-receptor stimulation on peak intracellular Ca2+ release by extracellular adenosine 5'-triphosphate (ATP) and 5-hydroxytryptamine (5-HT) were investigated in cultured airway smooth-muscle (ASM) cells. The results show that peak Ca2+ release to ATP is enhanced by preincubation with adenosine (ADO) and with the specific A3 receptor agonist 1-Deoxy-1-(6-([(3-iodophenyl)methyl] amino)-9H-purin-9-yl)-N-methyl-beta-D-ribofuranuronamide (1B-MECA). The response to 5-HT, a smooth-muscle contractile agonist, was also enhanced after preincubation with ADO. Further measurements showed that this enhancement of the response to ATP was dependent on extracellular calcium because it was abolished by the removal of Ca2+ from the extracellular fluid and by incubation with the calcium channel blocker nifedipine. In addition, there was no difference between the levels of total inositol phosphates measured in the presence of ATP alone or of ADO + ATP. AACOCF3, a specific blocker of phospholipase A2, decreased the peak Ca2+ response to ATP and abolished the enhanced response to ATP and 5-HT produced by ADO. We conclude that stimulation of P1 and P2 receptors in ASM cells activates not only phospholipase C but also phospholipase A2. The enhancement of ATP-induced and 5-HT-induced Ca2+ release is due to Ca2+ influx from the extracellular fluid through a Ca2+ channel presumably modulated by arachidonic acid. These data show that endogenous ADO may modulate airway hyperresponsiveness by enhancing the ASM response to contractile agonists.

The aim of the study was to investigate strain dependence and mechanisms of airway responses to dry-gas hyperpnea challenge in the rat. We studied responses in a strain that is hyperresponsive to methacholine, Fischer 344 (F-344); in two normoresponsive strains, Lewis and ACI; and in an atopic but normoresponsive strain, Brown Norway (BN). We examined the effects of a neurokinin (NK) 1-receptor (CP-99994), an NK2-receptor (SR-48968), and a leukotriene D4(LTD4)-receptor antagonist (pranlukast) on responses to hyperpnea challenge in BN rats. The animals were ventilated with a tidal volume of 8 ml/kg and a frequency of 150 breaths/min with either a dry or humidified mixture of 5% CO2-95% O2 for 5 min for hyperpnea challenge, whereas responses to challenge were measured during spontaneous breathing. Pulmonary resistance increased after dry-gas challenge in BN and ACI but not in F-344 and Lewis rats. CP-99994, SR-48968, and pranlukast significantly attenuated the increase in pulmonary resistance after dry-gas challenge. There were no significant differences in responsiveness to airway challenge with LTD4 among the BN, F-344 and ACI rats. We conclude that responses to dry-gas hyperpnea challenge are strain dependent in rats and are mediated by NKs and LTD4.

Antibodies against integrins have been shown to inhibit allergic airway responses. The purpose of this study was to test the hypothesis that the beta1 integrin, very late antigen-4 (VLA-4), is involved in mast cell activation triggered by allergen exposure in sensitized animals. To do this we studied Brown Norway rats that were sensitized to ovalbumin (OA; 1 mg subcutaneously) using Bordetella pertussis as an adjuvant. Two weeks later rats were challenged with OA, pulmonary resistance (RL) was determined, and the concentrations of histamine and tryptase in bronchoalveolar lavage fluid and N-acetyl-leukotriene (LT)E4 in bile were measured. Pretreatment with a monoclonal antibody against VLA-4 (TA-2) attenuated the early response after OA challenge (342.9 +/- 24.4% baseline RL versus 153.3 +/- 19.4%; p < 0.01). There were significantly lower concentrations of histamine (67.11 +/- 11.90 microgram/ml versus 26.69 +/- 1.84; p < 0.01) and tryptase (0.143 +/- 0. 035 microgram/ml versus 0.053 +/- 0.022 microgram/ml; p < 0.01) in TA-2-treated animals. The increases in the concentrations of biliary N-acetyl-LTE4 after OA challenge were also significantly lower in TA-2-treated animals. These data suggest that a selective anti-VLA-4 monoclonal antibody prevents early responses through inhibition of mast cell activation.

The history of respiratory medicine and research at McGill University (Montreal, Quebec) is tightly linked with the growth of academic medicine within its teaching hospitals. Dr Jonathan Meakins, a McGill medical graduate, was recruited to the Royal Victoria Hospital in 1924; as McGill’s first full-time clinical professor and Physician-in-Chief at the Royal Victoria Hospital. His focus on respiratory medicine led to the publication of his first book, Respiratory Function in Disease, in 1925. Meakins moved clinical laboratories from the Department of Pathology and placed them within the hospital. As such, he was responsible for the development of hospital-based research.

Airway smooth muscle (ASM) mass appears to be increased in the bronchi of patients with chronic severe asthma. Although the precise mechanisms that induce these changes are unknown, increases in ASM mass are caused, in part, by ASM cell proliferation. After allergen challenge in rats, it has been possible to demonstrate an increase in ASM mass by morphometric techniques. To examine whether hyperplasia is involved in ASM cell growth in vivo, we investigated whether repeated allergen challenges in sensitized Brown Norway rats stimulated DNA synthesis in airway epithelial and ASM cells. Animals that were actively sensitized to ovalbumin (OA) received either three aerosolized OA or saline challenges at 5-day intervals. DNA synthesis was measured by indirect immunohistochemical techniques with an anti-bromodeoxyuridine (BrdU) antibody. OA inhalations increased ASM mass as determined by morphometry and also induced DNA synthesis in both airway epithelial and ASM cells in the airways of sensitized animals compared with saline-challenged control animals. ASM mass was increased in large- and medium-sized airways but not in small airways. However, the number of BrdU-positive ASM cells normalized to basement membrane length was also greater in the large- and medium-sized airways compared with that in the small airways. When the number of BrdU-positive epithelial cells was normalized to basement membrane length, there was no difference among airway sizes and the number of BrdU-positive epithelial cells. These data suggest that DNA synthesis is induced in both airway epithelial and ASM cells after inhalational antigen challenge.

We assessed the effects of dexamethasone on functional and histological changes after acute exposure to a high level of chlorine gas in an animal model of reactive airways dysfunction syndrome (RADS). Sprague-Dawley male rats were exposed to 1500 ppm of chlorine for 5 min and treated with either dexamethasone (dex; 300 micrograms/kg/day) or saline intraperitoneally for 7 days. Lung resistance (RL), airway responsiveness to inhaled methacholine (MCh), airway wall morphometric measurements, and bronchoalveolar lavage (BAL) cells were assessed over a 2-week period after exposure. Dex administration significantly attenuated both chlorine-induced increased RL and chlorine-induced increased responsiveness to methacholine compared with saline: -2.7 +/- 6.8% vs 102.3 +/- 36.6% change from baseline RL (P < 0.01) and 2.5 +/- 0.6 mg/ml vs 1.2 +/- 0.7 mg/ml in the MCh concentration required to double the RL from baseline (P < 0.01). There was a tendency, albeit nonsignificant, for improvement in some indices of epithelial injury. Dex significantly attenuated the postexposure neutrophilic cellular response in BAL 1 day after exposure (15.8 +/- 4.9% neutrophils in the dex group vs 49.8 +/- 2.7% neutrophils in the saline group) (P < or = 0.001). Our results show that dex administration helps maintain pulmonary function, reduces BAL inflammatory cell number, and tends to improve some morphometric airway wall structure parameters in rats exposed to chlorine.

Summary Severe respiratory viral infections in childhood are associated with the development of asthma later in life. Rhinovirus, respiratory syncytial virus and metapneumovirus are of particular importance as triggers of asthma. Effects of virus infection on dendritic cell function in the airways may predispose children to allergic sensitization. Asthmatic subjects may have impaired interferon responses to viral infection that also predispose to allergic sensitization. Difference in Toll-like receptor expression on airway epithelial cells is a potential mechanism for the altered immune responses of asthmatic children.

The impact of oral nutritional supplementation during an acute exacerbation of COPD on functional status was assessed through measuring change in lung function, strength testing, and general well-being. Subjects hospitalized for an acute exacerbation of COPD (n = 33) were randomized to extra nutritional support or the regular hospital care. They consumed an additional 10 kcal/kg/d. Outcome measures were measured at 2 wk as change scores. Forced vital capacity (% predicted) improved in the treatment group as compared with the control group (+8.7% versus -3.5%, p = 0.015), and change in FEV1 was in the same direction but not significantly different (p = 0.099). There were no changes in handgrip strength or respiratory muscle strength, but there was a trend towards more improvement in the general well-being score (+11.96 versus -10.25, p = 0.066). Almost all subjects were in negative nitrogen balance, indicating muscle wasting. The degree of muscle wasting was strongly correlated with the dose of corticosteroids (r = 0.73, p < 0.005). In conclusion, it is difficult to prevent important muscle wasting in patients with COPD treated with corticosteroids, but some small gains were observed with increased dietary intake.

The potential contribution of microvascular leakage to airway narrowing during the early (ER) and late (LR) responses following allergen challenge was determined in Brown Norway (BN) rats actively sensitized with ovalbumin (OA). To study leak into the airway wall, rats (n = 46) were challenged 2 weeks later with aerosolized OA, saline, or serotonin (5-HT) to study either ER (n = 25), LR (n = 15), or 5-HT (n = 6) responses. Pulmonary resistance (RL) was used to measure airway narrowing. Microvascular leak was determined by the Evans blue (EB) technique in the airway tissues, and in another group of BN, by EB in bronchoalveolar lavage (BAL) in the airway lumen (n = 43). EB increased in the airways of 5-HT challenged rats (P < 0.05), but not during ER or LR. EB in BAL increased significantly during the ER (101 +/- 31.35 ng/ml BAL), vs saline challenge (10.82 +/- 1.66; P < 0.05) and during the LR at 3, 4, 5, and 7 h following OA challenge compared with controls (P < 0.05). These results suggest that fluid transits rapidly from the airway microvascular circulation and into the airway lumen following allergen challenge. The measurement of EB in BAL can detect microvascular leak into the airway lumen during the ER and LR. Plasma leak into the airway lumen may contribute to the increase in pulmonary resistance observed during ER and LR in BN rats.

We evaluated the role of C fibers in the development of airway hyperresponsiveness in a rat model of chronic bronchitis. Neonatal rats were treated with capsaicin (50 mg/kg, subcutaneously), a procedure which results in permanent depletion of tachykinins from the lungs and airways as well as degeneration of C fibers. Control rats were treated with the vehicle used to dissolve capsaicin. Three months later, rats from both groups were exposed either to SO2 gas (250 ppm, 5 h/d, 5 d/wk for 4 wk) or to filtered air for the same period of time. One day after the last exposure, rats were anesthetized and instrumented for the measurement of pulmonary resistance (R(L)), dynamic compliance (Cdyn), and airway responsiveness to inhaled aerosolized methacholine. There was a small (30%) but significant increase in R(L) in neonatal capsaicin- but not vehicle-treated rats exposed to SO2. Chronic exposure to SO2 resulted in increased airway responsiveness in both groups of rats, but the effect was more pronounced in the neonatal capsaicin-treated animals in which the doses of methacholine required to double R(L) or decrease Cdyn by 50% decreased 6.3-fold and 4.6-fold, respectively, compared with only 2.2- and 1.3-fold decreases in vehicle-treated rats. Morphometric analysis of histologic sections of airways demonstrated that the average area of smooth muscle in the airway wall, normalized by the length of basement membrane, was significantly greater in SO2 compared with air-exposed capsaicin-treated rats, but not in vehicle-treated control rats (p0.012). The maximal tension generated by tracheal rings in response to cholinergic agonists was also significantly increased by SO2 exposure in neonatal capsaicin-treated, but not vehicle-treated rats (p0.002). These results support the hypothesis that rather than contributing to the pathophysiologic manifestations of bronchitis, C fibers limit the development of airway obstruction and airway hyperresponsiveness during induction of chronic bronchitis by SO2 exposure. The enhanced contractile responses of airways from the SO2-exposed neonatal capsaicin-treated rats may result from increased airway smooth muscle mass and contribute to the increased airway responsiveness observed in these animals.

Airway remodeling comprises the structural changes of airway walls, induced by repeated injury and repair processes. It is characterized by the changes of tissue, cellular, and molecular composition, affecting airway smooth muscle, epithelium, blood vessels, and extracellular matrix. It occurs in patients with chronic inflammatory airway diseases such as asthma, COPD, bronchiectasis, and cystic fibrosis. Airway remodeling is arguably one of the most intractable problems in these diseases, leading to irreversible loss of lung function. Current therapeutics can ameliorate inflammation, but there is no available therapy proven to prevent or reverse airway remodeling, although reversibility of airway remodeling is suggested by studies in animal models of disease. Airway remodeling is often considered the result of longstanding airway inflammation, but it may be present to an equivalent degree in the airways of children with asthma, raising the necessity for early and specific therapeutic interventions. In this review, we consider the factors that may contribute to airway remodeling and discuss the current and potential therapeutic interventions.

Neutrophils are the predominant cells recruited in the airways of horses suffering from heaves. These cells have been shown to express arginase in some species. The metabolism of l-arginine is thought to be involved in chronic inflammation, and airway obstruction and remodeling. The aim of this study was to assess the expression, regulation, activity, and functional role of arginase isoforms in equine neutrophils. Arginase I, arginase II, ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) expression were assessed in resting and stimulated (IL-4, LPS/fMLP, PMA; 5 and 18 h) blood neutrophils using quantitative PCR. Arginase expression was also studied by Western blot and enzyme activity assay. The effect of nor-NOHA (1mM), a specific arginase inhibitor, was assessed on arginase activity in vitro and ex vivo on neutrophil's inflammatory gene expression and viability. Results showed that equine neutrophils constitutively express arginase isoform 2, ODC and OAT. Neutrophil ex vivo stimulation did not induce arginase I or influence arginase II mRNA expression. Ex vivo inhibition of arginase activity by nor-NOHA had no effect on neutrophils inflammatory gene expression induced by LPS/fMLP (5h) but significantly reversed the cell loss observed after this stimulation.

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in naïve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

Allergic asthma is a heterogeneous disease with no curative therapies. T cells infiltrate the airway smooth muscle (ASM) layer and may be implicated in airway remodeling and the increase of ASM mass, a cardinal feature of asthma. The mechanism by which CD4(+) T cells drive airway remodeling remains unknown. This study sought to determine the T cell-mediated mechanism of ASM cell proliferation. We hypothesized that CD4(+) T cells adhere to ASM cells via CD44, and induce ASM cell proliferation through the activation of the epidermal growth factor receptor (EGFR). A coculture model showed that the contact of antigen-stimulated CD4(+) T cells with ASM cells induced high levels of EGFR ligand expression in CD4(+) T cells and the activation of matrix metalloproteinase (MMP)-9, required for the shedding of EGFR ligands. The inhibition of EGFR and MMP-9 prevented the increase of ASM cell proliferation after coculture. The hyaluronan receptor CD44 is the dominant mediator of the tight adherence of T cells to ASM and is colocalized with MMP-9 on the cell surface. Moreover, the neutralization of CD44 prevents ASM cell hyperplasia. These data provide a novel mechanism by which antigen-stimulated CD4(+) T cells induce the remodeling indicative of a direct trophic role for CD4(+) T cells.

CD8+ (OX-8+) T cells may suppress airway inflammation and airway responsiveness after allergen challenge.We studied the effects of depletion of OX-8+ T cells on allergen-induced lung eosinophilia and airway responsiveness in the Sprague-Dawley rat.Sprague-Dawley rats were sensitized to ovalbumin and challenged by aerosol 14 days later. Test animals received either low-dose (2 mg, n = 9) or high-dose (3 mg, n = 7) OX-8 monoclonal antibody (mAb), whereas controls (n = 8) received BALB/c ascites fluid. A fourth group of animals (n = 10) was not sensitized to ovalbumin and also received ascites fluid. Twenty-four hours after ovalbumin challenge, responsiveness to methacholine was measured, and lung inflammation was assessed in the large airways and small airways and parenchyma.Circulating and airway CD8+ T cells were decreased by OX-8 mAb administration with greatest changes in animals treated with high-dose OX-8 mAb compared with controls (blood: 1.0% +/- 3.6% vs 18.7% +/- 3.9%, p0.05); (large airways: 2.5% +/- 1.2% vs 13.8% +/- 1.2%, p0.05). Ovalbumin challenge resulted in increases in macrophages and neutrophils in the small airways and parenchyma of sensitized compared with unsensitized rats (p0.05). High-dose OX-8 mAb further increased total leukocytes, attributable to increases in neutrophils and eosinophils, retrieved from the large airways and small airways and parenchyma compared with other groups (p0.05). Airway responsiveness to methacholine was not significantly different between control and ovalbumin-challenged animals and was not augmented by OX-8 pretreatment.CD8+ T cells modulate the extent of allergen-induced airway inflammation. However, the enhancement of inflammation was not sufficient to affect airway responsiveness.

Allergen exposures induce growth of airway smooth muscle in the Brown Norway rat.The purpose of the study was to examine the role of mediators associated with the early and late responses in the induction of airway smooth muscle growth.Nedocromil sodium was administered to block early and late responses in ovalbumin-sensitized and ovalbumin-challenged rats undergoing single or multiple challenges (5 times at 5-day intervals) with ovalbumin. Airway smooth muscle was quantitated by morphometry on lungs removed 2 days after the final challenge.Nedocromil sodium administered before ovalbumin challenge blocked both the early and late responses. When administered 2 hours after ovalbumin challenge, it also blocked the late response. Rats undergoing challenge with aerosolized ovalbumin five times at 5-day intervals were also treated with nedocromil before (n = 10) or 2 hours after (n = 10) each ovalbumin inhalation, respectively. The quantity of airway smooth muscle standardized for size was greater after ovalbumin challenge (0.069 +/- 0.005) compared with saline controls (0.033 +/- 0.003, p0.005). Nedocromil significantly reduced the airway smooth muscle (0.036 +/- 0.003, p0.005) when administered before ovalbumin. However, the airway smooth muscle in rats that received nedocromil 2 hours after ovalbumin challenge (0.046 +/- 0.003), although lower than in ovalbumin-challenged rats (p0.01), was still significantly higher than in saline-treated rats (p0.05).Allergen-induced early responses, late responses, and airway inflammation are antagonized by nedocromil. The mediators of both the early and late responses contribute to allergen-induced airway smooth muscle growth, a process that can be prevented by administration of nedocromil.

We examined the relationship between the quantity of smooth muscle in isolated tracheal preparations and their responses to contractile agonists. The responses of two different tracheal preparations, rings and tubes, to carbachol and serotonin were compared both intra-species (Fisher vs. Lewis strain rats) and inter-species (rat vs. guinea-pig). The rank order for carbachol-induced maximal isometric tensions was Fisher>Lewis>guinea-pig and for serotonin Fisher>guinea-pig>Lewis for tracheal rings. The sensitivities to carbachol and serotonin were greater in Fisher than in Lewis rats. Guinea-pig tracheal rings were comparable to Fisher in sensitivity to carbachol, but were more sensitive to serotonin than either Fisher or Lewis rings. In both species, agonist-independent differences were found in the maximal tension of rings taken from different regions of trachea. For whole tracheal tubes under isovolumetric conditions, the rank order for carbachol-induced changes in the intraluminal pressure was guinea-pig>Lewis≥Fisher. The sensitivity to carbachol was greater in guinea-pig tubes than in rat. The quantity of tracheal smooth muscle estimated from myosin was greater in guinea-pigs than in either Fisher or Lewis rats. In addition, the area of cartilage determined by morphometry in guinea-pig trachea was greater than that in the rat. We conclude that a concordance between the magnitude of contraction and the amount of tracheal smooth muscle is obtained only in whole tracheal tubes and not in tracheal rings. Several factors could contribute to the observed discrepancies in tracheal rings, including regional differences in efficacy and sensitivity to contractile agonists and the thickness of cartilage.

The role of nutritional status in the prognosis of subjects with severe chronic obstructive pulmonary disease was studied in a cohort of Canadian men and women followed for 3 to 5 yr. A total of 348 subjects who were recruited for a study of negative pressure ventilation were evaluated for lung function and body weight, and a subset who entered hospital for the study (n=184) had baseline measures of diffusing capacity, maximal inspiratory and expiratory mouth pressure (PImax and PEmax), and blood gases. Predictors of survival were analyzed using Cox regression models. In the total cohort, low body mass index (BMI) and use of home oxygen were independently associated with reduced survival. In the hospitalized group, predictors of respiratory mortality were elevated PaCO2 and low BMI, PImax, and diffusing capacity. PaO2 (measured on oxygen therapy), FEV1, PEmax, age, smoking behavior, and gender were not associated with survival. The predictors of total mortality were similar, except that BMI was no longer significant. In conclusion, low body weight, a potentially modifiable factor, was associated with respiratory mortality, but whether it has a casual effect or is a marker of declining health can only be resolved through an intervention trial.

Activated CD4+ helper T cells have been demonstrated in asthmatic airways and postulated to play a central role in eliciting allergic inflammation; direct evidence of their involvement seems to be lacking. We hypothesized that CD4+ T cells have the potential to induce allergic responses to antigen challenge, and tested this hypothesis in a model of allergic bronchoconstriction, the Brown Norway rat, using the approach of adoptive transfer. Animals were actively sensitized to either ovalbumin (OVA) or BSA and were used as donors of T cells. W3/25(CD4)+ or OX8(CD8)+ T cells were isolated from the cervical lymph nodes of sensitized donors and transferred to naive BN rats. 2 d after adoptive transfer recipient rats were challenged by OVA inhalation, and changes in lung resistance (RL), bronchoalveolar lavage (BAL) cells, and serum levels of antigen-specific IgE were studied. After OVA challenge recipients of OVA-primed W3/25+ T cells exhibited sustained increases in RL throughout the entire 8-h observation period and had significant bronchoalveolar lavage eosinophilia, which was detected by immunocytochemistry using an antimajor basic protein mAb. Recipients of BSA-primed W3/25+ T cells or OVA-primed OX8+ T cells failed to respond to inhaled OVA. OVA-specific immunoglobulin E was undetectable by ELISA or skin testing in any of the recipient rats after adoptive transfer. In conclusion, antigen-induced airway bronchoconstriction and eosinophilia were successfully transferred by antigen-specific W3/25+ T cells in Brown Norway rats. These responses were dependent on antigen-primed W3/25+ T cells and appeared to be independent of IgE-mediated mast cell activation. This study provides clear evidence for T cell mediated immune mechanisms in allergic airway responses in this experimental model.

The aim of the current studies was to investigate the possibility that a decreased relaxant response to nitric oxide (NO) might contribute to strain-related differences in airway responsiveness in the rat. Isolated tracheal rings from hyperresponsive. Fisher rats were confirmed to be more responsive to carbachol [mean effective concentration (EC50) = 2.45 x 10(-7) M] than those from Lewis (EC50 = 3.60 x 10(-7) M, P < 0.03) and ACI (EC50 = 3.85 x 10(-7) M, P < 0.01) rats. Sodium nitroprusside (SNP), a NO donor, caused relaxation of the carbachol (10(-6) M) contracted tracheal rings, but the half-maximal inhibition concentration (IC50) SNP in Fisher rats (5.60 x 10(-6) M) was significantly higher than in Lewis (1.34 x 10(-6) M, P < 0.001) and ACI rats (1.13 x 10(-6) M, P < 0.0005). The inhibitory effect of SNP on airway responsiveness to inhaled methacholine (MCh) in vivo was also less pronounced for Fisher than Lewis rats. SNP induced an accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) in cultured tracheal smooth muscle cells (TSM). Fisher TSM produced less cGMP on exposure to SNP compared with TSM from ACI (P < 0.01) and Lewis (P < 0.0001) rats. A decreased guanylyl cyclase activity may account for the impaired relaxant effect of SNP in Fisher rats.

The aim of the study was to evaluate the effects of blocking the integrins VLA-4 and LFA-1 on allergen-induced airway eosinophilia and responsiveness in Brown-Norway rats. Ovalbumin-sensitized rats were exposed to either aerosols of ovalbumin or saline. Airway responsiveness to methacholine (MCh) was determined 8 and 32 h after challenge. Cellular populations in the lung lavage and lung tissues were determined 32 h after allergen challenge. Total numbers of eosinophils were increased in the lung lavage (25 ml) and the small airways/parenchyma in the ovalbumin (OA)-challenged rats (4.37 x 10(6) +/- 0.71 and 15.54 x 10(6) +/- 1.99, respectively) compared with the saline-challenged rats (0.99 x 10(6) +/- 0.81 and 4.84 x 10(6) +/- 2.27; p0.05). Animals treated with both anti-VLA-4 and anti-LFA-1 mAbs and with anti-LFA-1 mAb alone had reduced numbers of eosinophils in the lung lavage (0.76 x 10(6) +/- 0.80 and 0.40 x 10(6) +/- 1.14, respectively; p0.05) and in the small airways/parenchyma (8.64 x 10(6) +/- 2.07 and 4.44 x 10(6) +/- 3.20; p0.05). Anti-VLA-4 mAb treatment alone did not alter the eosinophils recovered from the lung. Airway responsiveness to methacholine increased from 8 to 32 h in all ovalbumin-challenged rats, but treatment with anti-VLA-4, anti-LFA-1, or both mAbs prevented the increase in responsiveness. In conclusion, allergen-induced airway hyperresponsiveness is inhibitable by blocking either VLA-4 or LFA-1 integrins and is associated with a lung eosinophilia that is LFA-1 dependent and VLA-4 independent.(ABSTRACT TRUNCATED AT 250 WORDS)

Background In patients with severe eosinophilic asthma, local maturation rather than systemic recruitment of mature cells might contribute to persistent airway eosinophilia. Group 2 innate lymphoid cells (ILC2s) are a major source of type 2 cytokines (IL-5 and IL-13) and can facilitate eosinophilic inflammatory responses in mouse models of asthma in the absence of CD4 + lymphocytes. This study investigated the potential role of ILC2s in driving chronic airway eosinophilia in patients with severe asthma, despite regular high-dose oral corticosteroid therapy. Methods In a cross-sectional study we enumerated blood and sputum ILC2s (lin − CD45 + 127 + ST2 + ) and levels of intracellular IL-5 and IL-13 in patients with severe asthma (n = 25), patients with steroid-naive mild atopic asthma (n = 19), and nonatopic control subjects (n = 5). Results were compared with numbers of CD4 + lymphocytes, eosinophil lineage–committed progenitors (eosinophilopoietic progenitor cells [EoPs]), and mature eosinophils. Results Significantly greater numbers of total and type 2 cytokine–producing ILC2s were detected in blood and sputum of patients with severe asthma compared to mild asthmatics. In contrast, intracellular cytokine expression by CD4 cells and EoPs within the airways did not differ between the asthmatic groups. In patients with severe asthma, although sputum CD4 + cells were more abundant than ILC2s and EoPs, proportionally, ILC2s were the predominant source of type 2 cytokines. In addition, there were significantly greater numbers of sputum IL-5 + IL-13 + ILC2s in patients with severe asthma whose airway eosinophilia was greater than 3%, despite normal blood eosinophil numbers ( Conclusions Our findings suggest that ILC2s can promote the persistence of airway eosinophilia in patients with severe asthma through uncontrolled localized production of the type 2 cytokines IL-5 and IL-13, despite high-dose oral corticosteroid therapy.

The “fetal origins hypothesis” argued that physiological changes consequent to in utero exposures ultimately contribute to disease susceptibility in later life. The dramatic increase in asthma prevalence is attributed to early exposures acting on preexisting asthma-susceptible genotypes. We showed previously that distinct transcriptome signatures distinguish the developmental respiratory phenotype of atopic (Brown Norway, BN) and normoresponsive (Lewis) rats. We aimed to determine whether maternal allergen exposure would influence asthma pathogenesis by reprogramming primary patterns of developmental lung gene expression. Postnatal offspring of dams sensitized to ovalbumin before mating and challenged during pregnancy were assessed for lung function, inflammatory biomarkers, and respiratory gene expression. Although maternal ovalbumin exposure resulted in characteristic features of an allergic response (bronchoalveolar lavage neutrophils, IgE, methacholine-induced lung resistance) in offspring of both strains, substantial strain-specific differences were observed in respiratory gene expression. Of 799 probes representing the top 5% of transcriptomic variation, only 112 (14%) were affected in both strains. Strain-specific gene signatures also exhibited marked differences in enrichment for gene ontologies, with immune regulation and cell proliferation being prominent in the BN strain, cell cycle and microtubule assembly gene sets in the Lewis strain. Multiple ovalbumin-specific probes in both strains were also differentially expressed in lymphoblastoid cell lines from human asthmatic vs. nonasthmatic sibling pairs. Our data point to the existence of distinct, genetically programmed responses to maternal exposures in developing lung. These different response patterns, if recapitulated in human fetal development, can contribute to long-term pulmonary health including interindividual susceptibility to asthma.

Bioengineered tissue equivalents should provide physiologically relevant biochemical and mechanical cues to support the growth and differentiation of seeded cells. Herein, tubular dense collagen constructs (TDCCs) with collagen content comparable to native extracellular matrix were used to investigate the effect of shear stress alone (i.e. under laminar fluid flow), and shear stress in combination with circumferential strain (i.e. under pulsatile fluid flow) on the proliferation, alignment, and phenotype of three-dimensionally (3D) seeded airway smooth muscle cells (ASMCs). In addition, the effect of ASMC-mediated remodelling on TDCC matrix morphological and mechanical properties was investigated. Compared to static culture, pulsatile flow increased seeded ASMC growth by 70%, improved the homogeneity of cell distribution within the TDCCs and induced differential cellular alignment depending on the primary stimuli. Specifically, within the inner wall, where shear stress is predominant, ASMCs were aligned parallel to fluid flow direction, while within the outer wall ASMCs were aligned parallel to the circumferential strain (perpendicular to fluid flow). In contrast, under laminar flow, ASMCs were aligned parallel to fluid flow direction within both walls. Compared to laminar flow, pulsatile flow resulted in increased positive staining for α-smooth muscle actin, and in up-regulated typical ASMC contractile markers suggesting that circumferential strain modulates ASMC differentiation. Pulsatile flow also caused a 60 and 30% increase in collagen density within both acellular and cellular TDCCs, respectively, which was reflected in an increased apparent modulus. Compared to static culture, pulsatile stimulation of cellular constructs resulted in 70% higher circumferential strength. The TDCCs provide ASMC niche for greater insight into the responses of 3D seeded SMCs to physiologically equivalent in vitro dynamic conditioning.

Recent studies suggest that airway smooth muscle remodeling is an early event in the course of asthma. Little is known of the effects of long-term antigen avoidance and inhaled corticosteroids on chronically established airway remodeling. We sought to measure the effects of inhaled corticosteroids and antigen avoidance on airway remodeling in the peripheral airways of horses with heaves, a naturally occurring asthma-like disease. Heaves-affected adult horses with ongoing airway inflammation and bronchoconstriction were treated with fluticasone propionate (with and without concurrent antigen avoidance) (n = 6) or with antigen avoidance alone (n = 5). Lung function and bronchoalveolar lavage were performed at multiple time points, and peripheral lung biopsies were collected before and after 6 and 12 months of treatment. Lung function improved more quickly with inhaled corticosteroids, but eventually normalized in both groups. Inflammation was better controlled with antigen avoidance. During the study period, corrected smooth muscle mass decreased from 12.1 ± 2.8 × 10(-3) and 11.3 ± 1.2 × 10(-3) to 8.3 ± 1.4 × 10(-3) and 7.9 ± 1.0 × 10(-3) in the antigen avoidance and fluticasone groups, respectively (P = 0.03). At 6 months, smooth muscle mass was significantly smaller compared with baseline only in the fluticasone-treated animals. The subepithelial collagen area was lower at 12 months than at baseline in both groups. During the study period, airway smooth muscle remodeling decreased by approximately 30% in both groups, although the decrease was faster in horses receiving inhaled corticosteroids. Inhaled corticosteroids may accelerate the reversal of smooth muscle remodeling, even if airway inflammation is better controlled with antigen avoidance.