Adaptation of airway smooth muscle to basal tone: relevance to airway hyperresponsiveness

Lung inflammation and airway hyperresponsiveness (AHR) are hallmarks of asthma, but their interrelationship is unclear. Excessive shortening of airway smooth muscle (ASM) in response to bronchoconstrictors is likely an important determinant of AHR. Hypercontractility of ASM could stem from a change in the intrinsic properties of the muscle, or it could be due to extrinsic factors such as chronic exposure of the muscle to inflammatory mediators in the airways. The latter could be the link between lung inflammation and AHR. The present study was designed to examine the influence of chronic exposure to a contractile agonist on the force-generating capacity of ASM. Force generation in response to electric field stimulation (EFS) was measured in ovine trachealis with or without a basal tone induced by acetylcholine (ACh). While the tone was maintained, the EFS-induced force decreased transiently but increased over time to reach a plateau in approximately 50 minutes. The total force (ACh tone + EFS force) increased monotonically and in proportion to ACh concentration. The results indicate that the muscle adapted to the basal tone and regained its contractile ability in response to a second stimulus (EFS) over time. Analysis suggests that this is due to a cytoskeletal transformation that allows the cytoskeleton to bear force, thus freeing up actomyosin crossbridges to generate more force. Force adaptation in ASM as a consequence of prolonged exposure to the many spasmogens found in asthmatic airways could be a mechanism contributing to AHR seen in asthma.     

Force oscillations simulating breathing maneuvers do not prevent force adaptation

Airway inflammation in patients with asthma exposes the airway smooth muscle (ASM) to a variety of spasmogens. These spasmogens increase ASM tone, which can lead to force adaptation. Length oscillations of ASM, which occur in vivo due to breathing maneuvers, can attenuate force adaptation. However, in the presence of tone, the force oscillations required to achieve these length oscillations may be unphysiologic (i.e., magnitude greater than the ones achieved due to the swings in transpulmonary pressure required for breathing). In the present study, we applied force oscillations simulating the tension oscillations experienced by the wall of a fourth-generation airway during tidal breathing with or without deep inspirations (DI) to ASM. The goal was to investigate whether force adaptation occurs in conditions mimicking breathing maneuvers. Tone was induced by carbachol (average, 20 nM), and the force-generating capacity of the ASM was assessed at 5-minute intervals before and after carbachol administration using electrical field stimulations (EFS). The results show that force oscillations applied before the introduction of tone had a small effect on the force produced by EFS (declined to 96.8% [P > 0.05] and 92.3% [P < 0.05] with and without DI, respectively). The tone induced by carbachol transiently decreased after a DI and declined significantly (P < 0.05) due to tidal breathing oscillations (25%). These force oscillations did not prevent force adaptation (gain of force of 11.2 ± 2.2 versus 13.5 ± 2.7 and 11.2 ± 3.0% in static versus dynamic conditions with or without DI, respectively). The lack of effect of simulated breathing maneuvers on force adaptation suggests that this gain in ASM force may occur in vivo and could contribute to the development of airway hyperresponsiveness.     

The role of airway smooth muscle during an attack of asthma simulated in vitro

Excessive narrowing of airways in response to contractile agonists is a characteristic feature of asthma. We hypothesized that airway smooth muscle (ASM) adaptation to short lengths could contribute to exaggerated airway narrowing during an acute attack of asthma by allowing the muscle to regain its ability to generate maximal force at a shortened length. To test this hypothesis we mimicked, in vitro, the sequence of contractile events that would occur during a spontaneous attack of asthma. Trachealis muscle was challenged with carbachol (300 nM, submaximal dose) and allowed to shorten to approximately half of its original length. After 30 min of adaptation at the shortened length in the presence of carbachol, muscle force, amount and rate of shortening in response to electrical stimulation were compared with corresponding values obtained from control experiments during which the ASM was not adapted to the short length. After adaptation at the shortened length the developed force, amount and rate of shortening increased by 1.93 +/- 0.08-, 1.57 +/- 0.12-, and 1.75 +/- 0.2-fold, respectively. Shortening of ASM in response to contractile agonists can lead to adaptation of the muscle to the shortened length that, in turn, can result in further shortening and the potential for airway closure.     

Mechanical determinants of airways hyperresponsiveness

Asthmatic individuals typically experience exaggerated decrements in their ability to breathe after receiving standardized doses of smooth muscle agonist, a phenomenon known as airways hyperresponsiveness (AHR). Breathing difficulties are caused by excessive narrowing of the pulmonary airways, which is instigated by shortening of the airway smooth muscle (ASM). Exactly why many asthmatic individuals are hyperresponsive, however, remains controversial because of the many varied mechanisms that could possibly be involved. Nevertheless, much of the understanding of AHR comes down to a matter of considering the spatial configuration of the components that make up the airway, and the static and dynamic physical forces these components experience. In this review, we consider these mechanical factors, which are conveniently subdivided into three groups involving (i) the active forces construing to narrow the airways, (ii) the mechanical loads against which these forces must work, and (iii) the geometric transformation of a given degree of ASM shortening into airway narrowing. Each of these groups of factors has potent potential to influence AHR. It is likely, however, that they operate together to produce the AHR characteristic of severe asthma.     

Role of mast cells in airway remodeling

The extent of airway remodeling correlates with severity of asthma. Persistent airway hyperresponsiveness (AHR) is associated with airway remodeling, but not with inflammation. The increase in ASM mass is recognized as one of the most important factors related to AHR and to the severity of asthma. The infiltration of ASM by mast cells (MCs) is associated with the disordered airway function. The mediators such as tryptase and cytokines from MCs can modulate ASM cell function and induce goblet cell hyperplasia. MCs were found to contribute to the development of multiple features of chronic asthma in MC-deficient mice. Therefore, MCs play an important role not only in immediate hypersensitivity and late phase inflammation but also in tissue remodeling in the airway.     

Maturational changes in airway smooth muscle shortening and relaxation. Implications for asthma

Greater airway responsiveness in healthy juveniles is considered a factor in the higher asthma prevalence at a young age compared with adults. Several studies on the contractile response of airway smooth muscle (ASM) from birth to adulthood have addressed the hypothesis that a maturation of ASM plays a role in juvenile airway hyperresponsiveness. Maturation of distinct ASM properties, i.e. force generation, shortening, and relaxation, has been reported, although the majority of the studies have focused on maturation of maximum force and/or sensitivity to contractile agonists. However, in most animal species maturation of the ability to generate force does not correlate with maturation of airway responsiveness. Ontogenesis of ASM shortening has been less extensively studied and the existing reports emphasize an increase during maturation of tissue passive forces opposing shortening. ASM spontaneous relaxation has been very minimally investigated. We have recently demonstrated that the ability of ASM to spontaneously relax during stimulation is sharply reduced in juvenile airway tissue. It remains to be determined the role of these ASM properties in the onset of childhood asthma and whether specific alterations are induced by the occurrence of obstructive airway diseases in young individuals.     

Autocrine regulation of airway smooth muscle responsiveness

Bronchial asthma is characterized by airway inflammation, exaggerated airway narrowing to bronchoconstrictor agonists, and attenuated beta-adrenoceptor-mediated airway relaxation. Various cytokines/chemokines have been implicated in the pathogenesis of the airway inflammatory response, and certain cytokines, most notably including specific Th2-type cytokines and IL-1beta, have been shown to directly regulate airway smooth muscle (ASM) responsiveness. Recent evidence supports the concept that the ASM itself has the capacity to endogenously express a number of these cytokines under specific conditions of ASM sensitization. Moreover, these cytokines were found to act in an autocrine manner on the ASM to evoke the 'pro-asthmatic' phenotype of altered airway responsiveness. This cytokine-driven autocrine signaling mechanism in ASM may be triggered by either Fc receptor activation in the atopic (IgE-mediated) sensitized state or by ASM exposure to specific viral respiratory pathogens, most notably including rhinovirus. Furthermore, the autocrine-induced changes in ASM responsiveness are attributed to altered receptor-coupled transmembrane signaling in the sensitized ASM, resulting in perturbed expression and release of second messenger molecules that regulate ASM contraction and relaxation. Collectively, this evidence identifies mechanisms intrinsic to the ASM itself, including autocrine pro-inflammatory signaling and altered receptor/G protein-coupled second messenger activation, that importantly contribute to phenotypic expression of the changes in ASM responsiveness that characterize the asthmatic state.     

Mechanisms of airway hyperresponsiveness

Airway hyperresponsiveness (AHR) to direct (histamine and methacholine) and indirect (exercise, cold air, hyperventilation, AMP) challenges is a universal and defining feature of asthma. One component of AHR is transient or inducible and occurs after allergen exposure, for example, and improves occasionally rapidly after inhaled corticosteroids or environmental control. This transient airway hyperresponsiveness is more marked to the indirect stimuli. There are convincing data linking this component of AHR to airway inflammation; however, the precise mechanisms linking airway inflammation and hyperresponsiveness of the airway smooth muscle are not clear. The other component of AHR is more persistent and is relatively refractory to environmental control and inhaled corticosteroids. This is likely secondary to structural airway changes, which are collectively referred to as airway remodeling, and which are a result of the chronic (rather than the acute) effects of airway inflammation. This persistent AHR is best reflected by airway hyperresponsiveness to direct stimuli such as methacholine. The mechanisms are also uncertain, but reduced airway caliber, increased airway wall thickness, increased airway smooth muscle mass, and perhaps contractility likely all play a role.     

Airway smooth muscle: a modulator of airway remodeling in asthma

Asthma is a disease characterized, in part, by airway hyperresponsiveness and inflammation. Although asthma typically induces reversible airway obstruction, in some patients with asthma, airflow obstruction can become irreversible. Such obstruction might be a consequence of persistent structural changes in the airway wall caused by the frequent stimulation of airway smooth muscle (ASM) by contractile agonists, inflammatory mediators, and growth factors. Traditional concepts concerning airway inflammation have focused on trafficking leukocytes and on the effects of inflammatory mediators, cytokines, and chemokines secreted by these cells. Recent studies suggest that ASM cells might modulate airway remodeling by secreting cytokines, growth factors, or matrix proteins and by expressing cell adhesion molecules and other potential costimulatory molecules. These ASM cell functions might directly or indirectly modulate submucosal airway inflammation and promote airway remodeling.     

Essential role of IFNbeta and CD38 in TNFalpha-induced airway smooth muscle hyper-responsiveness

We recently identified autocrine interferon (IFN)beta as a novel mechanism mediating tumor necrosis factor (TNF)alpha-induced expression of inflammatory genes in airway smooth muscle (ASM) cells, including CD38, known to regulate calcium signaling. Here, we investigated the putative involvement of IFNbeta in regulating TNFalpha-induced airway hyper-responsiveness (AHR), a defining feature of asthma. Using our pharmacodynamic model to assess ex vivo AHR isolated murine tracheal rings, we found that TNFalpha-induced enhanced contractile responses to carbachol and bradykinin was abrogated by neutralizing anti-IFNbeta antibody or in tracheal rings deficient in CD38. In cultured human ASM cells, where CD38 has been involved in TNFalpha-induced enhanced calcium signals to carbachol and bradykinin, we found that neutralizing anti-IFNbeta prevented TNFalpha enhancing action only on carbachol responses but not to that induced by bradykinin. In a well-characterized model of allergic asthma (mice sensitized and challenged with Aspergillus fumigatus (Af)), we found heightened expression of both IFNbeta and CD38 in the airways. Furthermore, allergen-associated AHR to methacholine, assessed by lung resistance and dynamic compliance, was completely suppressed in CD38-deficient mice, despite the preservation of airway inflammation. These data provide the first evidence that ASM-derived IFNbeta and CD38 may play a significant role in the development of TNFalpha-associated AHR.     

Mechanisms of inflammation-mediated airway smooth muscle plasticity and airways remodeling in asthma

Recent evidence points to progressive structural change in the airway wall, driven by chronic local inflammation, as a fundamental component for development of irreversible airway hyperresponsiveness. Acute and chronic inflammation is orchestrated by cytokines from recruited inflammatory cells, airway myofibroblasts and myocytes. Airway myocytes exhibit functional plasticity in their capacity for contraction, proliferation, and synthesis of matrix protein and cytokines. This confers a principal role in driving different components of the airway remodeling process, and mediating constrictor hyperresponsiveness. Functional plasticity of airway smooth muscle (ASM) is regulated by an array of environmental cues, including cytokines, which mediate their effects through receptors and a number of intracellular signaling pathways. Despite numerous studies of the cellular effects of cytokines on cultured airway myocytes, few have identified how intracellular signaling pathways modulate or induce these cellular responses. This review summarizes current understanding of these concepts and presents a model for the effects of inflammatory mediators on functional plasticity of ASM in asthma.     

Immune mechanisms of smooth muscle hyperreactivity in asthma

Asthma is characterized by bronchial hyperresponsiveness to a variety of bronchospasmogenic stimuli. To study the pathophysiologic mechanisms underlying the increased sensitivity and degree of maximal airway narrowing, various in vivo and in vitro models have been developed with methods of active and passive sensitization. These studies indicated a major role for alterations in the smooth muscle itself rather than neural dysfunction or airway inflammation as the underlying cause for the development of bronchial hyperresponsiveness. During the last years smooth muscle cells were found to exhibit not only the "classical" contractile phenotype but also a proliferative-synthetic phenotype, which is capable of producing proinflammatory cytokines, chemotaxins, and growth factors. Allergic sensitization can alter both contractile and secretory functions, thereby indicating that the smooth muscle cell could contribute directly to the persistence of airway inflammation in asthma. A better understanding of the changes within the smooth muscle cells and of the mechanisms that lead to their induction could contribute to the development of novel therapeutic approaches for the treatment of asthma.     

Role of the Th2 cytokines in the development of allergen-induced airway inflammation and hyperresponsiveness

Increased production of interleukin (IL)-4 and IL-5 by T-helper cells may be pivotal for the induction and regulation of allergic diseases. We have studied the role of IL-4 and IL-5 in the development of eosinophilic airway inflammation (AI) and airway hyperresponsiveness (AHR) in a mouse model of allergen-induced bronchial asthma. Utilizing different modes of sensitization, we delineated the importance of IL-5-mediated eosinophilic airway infiltration for the development of in vitro and in vivo AHR and demonstrated the inhibition of airway inflammation and AHR by anti-IL-5 antibody treatment. Studies in IL-4- and IL-5 deficient mice revealed the importance of both cytokines for the induction of AI and AHR independently from the production of allergen-specific IgE, and indicated these cytokines as potential targets in novel approaches in the treatment of asthma.     

Prior airway exposure to allergen increases virus-induced airway hyperresponsiveness

BACKGROUND: Respiratory syncytial virus (RSV) bronchiolitis in early life can lead to changes in airway function, but there are likely additional predisposing factors, such as prior allergen exposure, determining which children develop wheezing and asthma. OBJECTIVE: To define the effects of prior airway exposure to sensitizing allergen on the development of airway inflammation and hyperresponsiveness (AHR) to subsequent RSV infection. METHODS: BALB/c mice were exposed to ovalbumin or PBS exclusively through the airways and subsequently infected with RSV or sham-inoculated. AHR, lung inflammation, and the frequency of cytokine-producing T lymphocytes in the lung were determined. RESULTS: In PBS-exposed mice, RSV infection induced AHR and an increased proportion of TH1-type (IFN-gamma and IL-12) cytokine-producing cells in the lungs. However, in mice previously exposed to ovalbumin through the airways and subsequently infected with RSV, the degree of AHR was significantly increased and was associated with an increased proportion of TH2 (IL-4, IL-5) cytokine-producing T lymphocytes. This response was also associated with an increased accumulation of eosinophils, neutrophils, and CD8+ T cells in the lungs. CONCLUSIONS: These data suggest that prior airway exposure to allergen may predispose sensitized hosts to a greater degree of altered airway function upon subsequent respiratory viral infection.     

Inositol (1,4,5)trisphosphate metabolism and enhanced calcium mobilization in airway smooth muscle of hyperresponsive rats

Airway hyperresponsiveness (AHR) is a phenotype of asthma and can be modeled by the inbred Fisher strain of rat, which is hyperresponsive in vivo relative to the Lewis strain. Enhanced airway smooth muscle (ASM) contractility and Ca(2+) mobilization are associated with the AHR observed in Fisher rats. In this study, we investigated whether the interstrain differences in Ca(2+) mobilization to serotonin (5HT) result from differences in inositol (1,4,5)trisphosphate (IP(3)) metabolism and/or IP(3) receptor (IP(3)R) sensitivity. Ca(2+) mobilization by 5HT in cultured ASM cells from both rat strains was phospholipase C (PLC) dependent. Inositol polyphosphate accumulation, and hence PLC activity, was similar in both rat strains, but a specific IP(3) transient was detectable only in Fisher myocytes in response to 5HT. These findings suggested that IP(3) degradation rather than production differed between the two strains. The Vmax and Michaelis constant (K(m)) of IP(3)-specific 5-phosphatase activity were higher in the particulate fraction of Lewis than in Fisher ASM cell homogenates and appeared to be related to a greater expression of two isoforms of 5-phosphatase (type I and type II) in Lewis cells as shown by Western blot analysis. The sensitivity of the IP(3)R to IP(3) was similar between Fisher and Lewis ASM cells, indicating that the interstrain intracellular Ca(2+) differences were unrelated to IP(3)R function. We propose that interstrain variations in 5-phosphatase activity and expression may give rise to the interstrain differences in IP(3)-mediated Ca(2+) release in ASM and may be a determinant of AHR.     

Schnurri-2 regulates Th2-dependent airway inflammation and airway hyperresponsiveness

Schnurri (Shn)-2 is a large zinc finger-containing protein, which plays a critical role in cell growth, signal transduction and lymphocyte development. In Shn-2-deficient (Shn-2(-/-)) CD4 T cells, the activation of nuclear factor-kappaB is up-regulated and their ability to differentiate into Th2 is enhanced. Here, we extend our investigation and demonstrate that Shn-2 regulates Th2 responses in vivo using an ovalbumin-induced allergic asthma model. Eosinophilic inflammation, mucus hyperproduction and airway hyperresponsiveness (AHR) were all enhanced in Shn-2(-/-) mice. Moreover, eosinophilic infiltration and AHR were enhanced in mice given a transfer of Shn-2(-/-) effector Th2. Shn-2 in Th2 is thus considered to play an important role as a negative regulator in allergic airway inflammation.     

TNF-alpha induces the late-phase airway hyperresponsiveness and airway inflammation through cytosolic phospholipase A(2) activation

BACKGROUND: Late-phase airway hyperresponsiveness (AHR) in asthma is considered the event leading to persistent inflammation in the lungs, but the molecular mechanisms involved in this process are poorly understood. OBJECTIVE: To examine the role of TNF-alpha in the development of a late AHR and airway inflammation in asthma. METHODS: We established a murine model of asthma with not only biphasic AHR to methacholine but also airway eosinophilia. The effect of TNF-alpha blockade was determined by using anti-TNF-alpha antibody and TNF-alpha knockout mice. Cytosolic phospholipase A(2) (cPLA(2)) mRNA expression and activity were assessed by using RT-PCR and 1-stearoyl-2-[1-(14)C] arachidonyl-sn-glycero-3-phosphocholine as the substrate, respectively. RESULTS: TNF-alpha blockade resulted in significant inhibition of the late AHR without affecting the early AHR, and reduction in airway eosinophilia and inflammation. cPLA(2) activity was increased in asthmatic lungs in a TNF-alpha-dependent way, and cPLA(2) inhibitor blocked late AHR and airway eosinophilia. TNF-alpha also stimulated the synthesis of cPLA(2) metabolites such as leukotriene B(4) and platelet-activating factor in the airway. Specific inhibitors of cPLA(2) metabolites inhibited the late AHR and airway eosinophilia. CONCLUSIONS: TNF-alpha is the proximal key cytokine capable of developing late-phase AHR and subsequent airway inflammation through expression/activation of cPLA(2).     

Airway eosinophilia is not a requirement for allergen-induced airway hyperresponsiveness

BACKGROUND: House dust mites (HDMs) are the major source of perennial allergens causing human allergic asthma. Animal models mimicking as closely as possible the allergic features observed in human asthma are therefore interesting tools for studying the immunological and pathophysiological mechanisms involved. Especially the role of eosinophils and allergen-specific immunoglobulin (Ig) E in the pathophysiology of airway hyperresponsiveness (AHR) remains a subject of intense debate. OBJECTIVE: To develop a mouse model of allergic airway inflammation and hyperresponsiveness based on the use of purified house dust mite allergen (Der p 1) as clinical relevant allergen. Furthermore, we studied the effects of low dose allergen exposure on the airway eosinophilia and AHR. METHODS: On day 0, C57Bl/6 mice were immunized with purified Der p 1 intraperitoneally. From day 14-20, the mice were exposed daily to a 30-min aerosol of different concentrations of house dust mite extract. RESULTS: Mice, actively immunized with Der p 1 and subsequently exposed to HDM aerosols, developed AHR, eosinophil infiltration of the airways and allergen-specific IgE. Moreover, lowering the concentration of the HDM aerosol also induced AHR and IgE without apparent eosinophil influx into the airways. Der p 1-sensitized mice exposed to PBS produced IgE, but did not show AHR or eosinophil influx. CONCLUSION: This in vivo model of HDM-induced allergic airway changes suggests that AHR is not related to either eosinophil influx or allergen-specific serum IgE, thereby reducing the importance of these factors as essential elements for allergic AHR.     

Endotoxin is not essential for the development of cockroach induced allergic airway inflammation

Purpose

Cockroach (CR) is an important inhalant allergen and can induce allergic asthma. However, the mechanism by which CR induces airway allergic inflammation and the role of endotoxin in CR extract are not clearly understood in regards to the development of airway inflammation. In this study, we evaluated whether endotoxin is essential to the development of CR induced airway allergic inflammation in mice.

Materials and Methods

Airway allergic inflammation was induced by intranasal administration of either CR extract, CR with additional endotoxin, or endotoxin depleted CR extract, respectively, in BALB/c wild type mice. CR induced inflammation was also evaluated with toll like receptor-4 (TLR-4) mutant (C3H/HeJ) and wild type (C3H/HeN) mice.

Results

Intranasal administration of CR extracts significantly induced airway hyperresponsiveness (AHR), eosinophilic and neutrophilic airway inflammation, as well as goblet cell hyperplasia in a dose-dependent manner. The addition of endotoxin along with CR allergen attenuated eosinophilic inflammation, interleukin (IL)-13 level, and goblet cell hyperplasia of respiratory epithelium; however, it did not affect the development of AHR. Endotoxin depletion in CR extract did not attenuate eosinophilic inflammation and lymphocytosis in BAL fluid, AHR and IL-13 expression in the lungs compared to CR alone. The attenuation of AHR, eosinophilic inflammation, and goblet cell hyperplasia induced by CR extract alone was not different between TLR-4 mutant and the wild type mice. In addition, heat inactivated CR extract administration induced attenuated AHR and eosinophilic inflammation.

Conclusion

Endotoxin in CR extracts may not be essential to the development of airway inflammation.