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J Appl Physiol 95: 844-853, 2003; doi:10.1152/japplphysiol.00192.2003
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HIGHLIGHTED TOPICS
Airway Hyperresponsiveness: From Molecules to Bedside

Invited Review: Do inflammatory mediators influence the contribution of airway smooth muscle contraction to airway hyperresponsiveness in asthma?

Darren J. Fernandes,1,2 Richard W. Mitchell,1 Oren Lakser,3,4 Maria Dowell,1,3 Alastair G. Stewart,2 and Julian Solway1

1Section of Pulmonary and Critical Care Medicine, University of Chicago, and 3Children's Memorial Hospital and 4Northwestern University, Chicago, Illinois 60637; and 2Department of Pharmacology, University of Melbourne, Melbourne, Victoria 3010, Australia


   ABSTRACT
TOP
 ABSTRACT
WHAT MEDIATORS ARE INVOLVED...
 SMOOTH MUSCLE CONTRACTILE...
 DO INFLAMMATORY MEDIATORS...
 SUMMARY
 DISCLOSURES
 REFERENCES
 
It is now accepted that a host of cytokines, chemokines, growth factors, and other inflammatory mediators contributes to the development of nonspecific airway hyperresponsiveness in asthma. Yet, relatively little is known about how inflammatory mediators might promote airway structural remodeling or about the molecular mechanisms by which they might exaggerate smooth muscle shortening as observed in asthmatic airways. Taking a deep inspiration, which provides relief of bronchodilation in normal subjects, is less effective in asthmatic subjects, and some have speculated that this deficiency stems directly from an abnormality of airway smooth muscle and results in airway hyperresponsiveness to constrictor agonists. Here, we consider some of the mechanisms by which inflammatory mediators might acutely or chronically induce changes in the contractile apparatus that in turn might contribute to hyperresponsive airways in asthma.

actin; myosin; airflow obstruction

ASTHMA IS A SYNDROME IN WHICH multiple factors interact to generate the characteristic clinical manifestations of shortness of breath, hyperresponsiveness to nonspecific bronchoconstrictor stimuli, cough, and airway inflammation. Genetic susceptibility, immune deviation toward T helper 2 (Th2) predominance, antigen exposure, and inflammation all are interwoven and interdependent contributing factors. The cytokines, chemokines, growth factors, and other mediators that participate in asthmatic airway inflammation probably influence airway responsiveness in multiple ways. For example, airway wall remodeling resulting from chronic inflammation undoubtedly contributes to the development of airway hyperresponsiveness (AHR) (12, 19, 20, 38, 44, 47, 86, 96) through the excess accumulation of muscle mass and the mechanical augmentation of luminal narrowing promoted by airway submucosal thickening. Although it has long been certain that airway smooth muscle (ASM) contraction plays a principal role in effecting airway narrowing in asthma, there has been little evidence for abnormal sensitivity of airway muscle to contractile stimulation, as assessed during static, isometric force measurements. However, emerging evidence suggests the possibility that dysregulated ASM contraction dynamics might be an important contributor to AHR. We discuss in this review the effects that inflammatory mediators might have on the ASM contractile apparatus and how these changes potentially contribute to AHR.


   WHAT MEDIATORS ARE INVOLVED IN ASTHMA INFLAMMATION?
TOP
 ABSTRACT
 WHAT MEDIATORS ARE INVOLVED...
SMOOTH MUSCLE CONTRACTILE...
 DO INFLAMMATORY MEDIATORS...
 SUMMARY
 DISCLOSURES
 REFERENCES
 
Airway inflammation in asthma is a multifactorial process involving the release of many inflammatory mediators. Some of the substances that have already been specifically implicated in asthma pathogenesis include cytokines [interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-11, IL-13, IL-16), granulocyte macrophage colony-stimulating factor, and tumor necrosis factor-{alpha} (TNF-{alpha})]; chemokines [eotaxin, eosinophil cationic protein, macrophage inflammatory protein-1{alpha}, monocyte chemotactic proteins (MCP-1 MCP-3, MCP-5), regulated-upon-activation normal T cell expressed and secreted (RANTES), macrophage-derived chemokine, and thymus and activation-regulated chemokine (TARC)]; growth factors [transforming growth factor-{beta} (TGF-{beta}), connective tissue growth factor, basic fibroblast growth factor, platelet-derived growth factor (PDGF), nerve growth factor, and insulin-like growth factors I and II (IGF-I and IGF-II)]; neurotransmitters (NK-A, NK-B, and substance P); platelet-activating factor; leukotrienes; histamine; endothelin 1 (ET-1); matrix metalloproteinases (MMP-2, MMP-8, MMP-9); and nitric oxide (NO) (the roles of these mediators are reviewed in Refs. 13, 23, 32, 36, 70, 73, 87). This is by no means an exhaustive list. Interaction among these different cytokines and growth factors in airway inflammation forms a complex network because 1) inflammatory mediators (particularly cytokines) have pleiotropic effects, 2) there exist antagonistic and synergistic relationships between different inflammatory mediators, and 3) inflammatory mediators exhibit high levels of redundancy of function (87).


   SMOOTH MUSCLE CONTRACTILE APPARATUS DYSFUNCTION IN ASTHMA
TOP
 ABSTRACT
 WHAT MEDIATORS ARE INVOLVED...
 SMOOTH MUSCLE CONTRACTILE...
DO INFLAMMATORY MEDIATORS...
 SUMMARY
 DISCLOSURES
 REFERENCES
 
Given that smooth muscle contraction is ultimately the major mechanism associated with airway narrowing, it stands to reason that the increased sensitivity and maximum response to contractile agonists might possibly be caused by a "dysfunction" in ASM contraction. A number of studies have tested the hypothesis that nonspecific AHR in asthma might be caused by increased force generation in the smooth muscle itself, due to either increased receptor numbers, increased affinity of ligand-receptor interaction, or modified intracellular contractile signaling pathways. The majority of evidence to date, conducted mostly with the use of isometric force generation in isolated muscle strips, suggests this is not the case. In response to a variety of contractile agonists, such as histamine, muscarinic agonists, cysteinyl leukotrienes, or prostanoids, isometric contraction of smooth muscle from airways of asthmatic subjects appears similar to that of tissues from nonasthmatic subjects, and isometric contraction of airway muscle from allergen-sensitized animals parallels that of ASM from sham-sensitized animals to agonists (reviewed in Ref. 81), although this is not a universal finding (76).

The concept that the "dynamic" component of smooth muscle contraction might be altered in asthma was first proposed by Stephens and colleagues (6). This proposal was confirmed in humans in 1981, when Fish and colleagues (24) demonstrated that a deep inspiration (DI) to total lung capacity produced substantial (and sustained) bronchodilatation after constriction had been acutely induced in nonasthmatic subjects. Importantly, in asthmatic subjects, this bronchodilatory effect was substantially reduced, and, in some asthmatic subjects, DI can even exacerbate bronchoconstriction (50). The importance of DI was further highlighted by the fact that a prolonged period in which DI is voluntarily prohibited significantly enhances airway responsiveness to methacholine in nonasthmatic subjects (62, 78). These studies together suggest that DI is a key physiological way of diminishing the effects of bronchoconstrictor stimuli and that prevention of DI alone can make healthy airways hyperresponsive in a manner similar to that of asthmatic subjects.

Why is the response to DI altered in asthma? One possible explanation is that the airways are stiffer and cannot easily be expanded in response to the increased pull on the airway by the expanding lung parenchyma during DI. Examination of airway dead space volume showed that asthmatic subjects do have reduced distensibility on inspiration (79, 97), suggesting that increased stiffness is evident in bronchodilator-treated asthmatic airways. In addition, Jensen and colleagues (39) concluded that contracted asthmatic ASM is stiffer than contracted healthy ASM because in their study asthmatic airways did not fully distend on DI. However, Brown and colleagues (10) demonstrated that stiffness may not fully explain the response to DI in asthmatic subjects because they measured similar increases in airway diameter on DI in methacholine-contracted asthmatic and healthy airways with high-resolution computed tomography scans. What was clear in the Brown and Jensen studies, however, was that the airways quickly narrowed again after return to normal tidal breathing in the asthmatic patients, in contrast to the normal airways that remained relatively dilated following DI (10, 39).

How might the augmented post-DI contraction seen in asthma influence airway reactivity? As Solway and Fredberg (83) postulated previously, if reshortening after airway muscle stretching happened slowly, then the bronchodilatory effect of the DI would persist, as it does in healthy airways. This would reduce the net effect of the constrictor agonist used to induce airway narrowing (Fig. 1). If in asthma the airways recontract far more rapidly after DI than in healthy airways, then at the end of expiration the airways would be substantially narrower. In this case, the bronchodilatory effect of the DI would then become transient, indeed perhaps unnoticeable.



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  		Fig. 1. Schematic illustration of relative changes in lung volume and airway diameter during and after 2 deep inhalations. Deep inspiration (DI) in airways with slow shortening velocity or exhibiting plastic behavior (healthy airways?) results in bronchodilation of contracted airways, which persists beyond the end of the DI (top). In contrast, constricted airways with faster smooth muscle shortening velocity or exhibiting elastic behavior (asthmatic airways?) effectively dilate during DI but quickly return to their constricted state during the following return to normal tidal breathing (bottom). In these airways, DI has little net effect on airway diameter (Reproduced from Ref. 18.)

 

The mechanisms leading to the increased reshortening following DI in asthma are not currently known. Is it simply because asthmatic ASM shortens faster after DI? Alternatively, is it due to a dysfunction in the ability of DI to fully disturb the ability of the contractile apparatus to generate force? Or are both mechanisms involved? These hypotheses will be discussed below (presented in summary form in Fig. 2). An additional possibility is that asthmatic airway remodeling alone might explain the increased reshortening following DI: mathematical modeling studies have demonstrated that, for a given degree of ASM shortening, the resistance increase would be far higher in a remodeled airway than in a normal healthy airway (38). Thus, after DI, the asthmatic ASM might reshorten at the same speed as healthy ASM, but the resulting airway narrowing would be exaggerated because of amplification of the smooth muscle contraction caused by the increased airway thickness. Furthermore, at a given level of contractile stimulation, the increased mass of ASM might generate more total force, thereby promoting excessive airway narrowing. The precise contribution to AHR of each of these potential mechanisms remains unknown. However, recent evidence suggests that alterations to the smooth muscle cells per se are likely to contribute to the post-DI contraction response (see below).



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  		Fig. 2. Schematic illustration of potential contributions of different pathways contributing to airway hyperresponsiveness. Intrinsic alterations in the smooth muscle contractile apparatus (such as increases in filament length, tethering to the cell membrane, or increased velocity of contraction) cause exaggerated airway narrowing to bronchoconstrictor stimuli because they hamper the ability of periodic DI to stretch the muscle cells into a more relaxed state. Remodeling of the airway also contributes to exaggerated airway narrowing; however, thickening of the different airway compartments causes increased responsiveness through alternative mechanisms. ASM, airway smooth muscle; MLCK, myosin light chain kinase; Vshortening, shortening velocity.

 

Smooth muscle shortening velocity. Myosin light chain kinase (MLCK) phosphorylation of the regulatory myosin light chain (MLC20) is an essential step during activation of the actin and myosin cross-bridge cycling that shortens the myocyte (80, 91). The phosphorylation state of MLC20 is known to regulate the speed of cross-bridge cycling on addition of a contractile stimulus; thus the level of phosphorylation determines to some extent the velocity of muscle contraction (16). The possibility that velocity of shortening (and consequently the total extent of shortening during contraction of a fixed period) might be abnormally increased in asthma was first championed by Newman Stephens, based on studies in an allergen-sensitized dog model (6, 40, 41). Ma and colleagues (52) have since demonstrated that the velocity of shortening of isolated asthmatic ASM is increased relative to ASM from healthy subjects and identified that MLCK levels and activity are increased in the asthmatic muscle, providing a regulatory protein-mediated explanation for increased shortening velocity. Factors that might influence the level of myosin phosphorylation in asthma are considered later.

Smooth muscle mechanical plasticity. The term "plasticity"has been applied to smooth muscle behavior in at least three different contexts. First, smooth muscle cells appear to be able to acquire different biochemical and functional phenotypes in culture (reviewed in Ref. 30) and in tissue pathology (64). Because contractile filament content and cytoskeletal organization vary among phenotypically diverse myocytes, their mechanical function could also be expected to vary widely. Such "phenotypic plasticity" could conceivably be manifest in asthma, although this has not been established to date.

Second, smooth muscle strips can generate force when activated over a very wide range of precontraction lengths; that is, the active-force vs. passive-precontraction-length relationship of smooth muscle is very flat. Pratusevich and colleagues (67) identified this mechanical property of normal smooth muscle as plasticity of the contractile apparatus and proposed that addition or removal of contractile units in series, possibly by formation or dissolution of myosin filaments (so-called "myosin evanescence"), could explain this adaptation of the contractile apparatus to length change. Seow and colleagues provided evidence for myosin evanescence, by demonstrating myosin filament lability in relaxed muscle, partial dissolution of thick filaments by during oscillatory strain of relaxed muscle (45), and increased thick filament density in activated muscle (33) that might stem from regulatory MLC phosphorylation (69). Along with the observation that shortening velocity changes (without change in isometric force) when smooth muscle is adapted to different lengths (75), these results suggested that a series-to-parallel rearrangement of contractile units could occur during contraction. Gunst and colleagues (27, 28, 55) have highlighted the potential complementary role of rearrangement of connections between focal adhesion complexes and cytoskeletal elements (especially actin filaments) in contributing to this behavior.

A third type of mechanical plasticity has also been identified in normal smooth muscle during contraction, that is, plastic deformation. To illustrate this behavior, consider the difference between stretching and releasing a rubber band vs. stretching and releasing a taffy candy bar. When stretched and released, the rubber band retains a restoring force that promotes reshortening. During stretch, the rubber band has undergone "elastic" deformation. In contrast, when stretched and released, the taffy may reshorten a little, but most of its restoring force has been lost, and it remains stretched beyond its original length. The taffy has undergone "plastic" deformation. It is now well established that healthy ASM exhibits taffylike behavior while contracted; that is, after stretch and release, it loses much of the original force of contraction present before the externally imposed length deformation (25, 26, 56). Clearly, if ASM deformed plastically during the stretch imposed by a DI in normal individuals but elastically during a DI in asthmatic individuals, then loss of plastic deformation in asthmatic individuals could account for their lack of net bronchodilation induced by DI (Fig. 1).

What is the molecular basis of plastic deformation of contracted airway muscle? Fredberg and colleagues (26) demonstrated that imposition of gradually increasing sinusoidal load fluctuations (similar to tidal breathing) on bovine trachealis strips resulted in smooth muscle lengthening. In a proposed mechanism termed "perturbed equilibrium of myosin binding," they suggested that ASM length is not governed by the isometric force-generating capacity of the muscle but by a dynamically equilibrated steady state. This perturbed state is characterized by reduced actomyosin bridges and a faster rate of cross-bridge turnover. Load fluctuations act to perturb the binding of myosin and actin, resulting in reduced muscle stiffness and muscle lengthening. They also found that reducing the force fluctuations back down to a lower amplitude resulted in muscle reshortening, although not all the way back to the original length. This suggested that the load fluctuations resulted in a plastic deformation in the muscle. Furthermore, they concluded that perturbation of myosin binding equilibrium is a self-reinforcing mechanism that limits contraction: the more the muscle stretches (and becomes less stiff), the easier it becomes to stretch with an externally applied force. Such a response might explain the observation that asthmatic airways do not fully distend after DI (39). Conversely, if a contracted muscle strip is allowed to shorten without ever being stretched, then this self-reinforcing mechanism would be lost and the muscle would remain "so stiff that it would be virtually frozen at its static equilibrium length" (26). Presently, it remains unknown whether the factors that determine myosin binding equilibrium differ between normal and asthmatic muscle. However, a recent study by Naghshin and coworkers (63) suggests that chronic adaptation of smooth muscle to passive short lengths renders the muscle bundle refractory to stretch-induced relaxation and increases its force generation capacity at shorter length. It is tempting to speculate that this effect of chronic passive shortening might even interact with the enhanced stiffness of "frozen" contracted unstretched muscle; together, these mechanisms might explain the increased stiffness of chronically contracted asthmatic airways to DI, such as observed by Jensen and colleagues (39).

A second potential molecular mechanism for stretch-induced plastic lengthening of contracted smooth muscle is suggested by the observation of Kuo et al. (45) that mechanical strain decreases the ability of the muscle to generate force (and shortening) due to partial dissolution of myosin thick filaments. Presently, it remains unknown whether this phenomenon is deranged in asthma.

We have postulated (18) an alternative basis for plastic vs. elastic deformation of contracted smooth muscle during stretch. We speculate that actin filaments might differ in length between normal and asthmatic smooth muscle, with longer actin filaments in asthmatic ASM than in normal ASM. This difference might exist before contraction, in resting muscle, or might arise after contractile activation, as actin polymerization is stimulated by contractile activation (55). As illustrated in Fig. 3, the contractile units of a myocyte with shorter actin filaments might necessarily undergo a parallel-to-series rearrangement during stretch if reshortening is to occur after release. Such rearrangement reduces the number of force-generating units in parallel and results in plastic deformation-like behavior; i.e., stretch and release reduce generated force. In contrast, contractile units containing longer actin filaments need not undergo parallel-to-series rearrangement during stretch and thus might not lose force on release. Thus myocytes with longer actin filaments could be expected to exhibit more elastic deformation during stretch. Perhaps the greater elasticity would even limit the lengthening of muscle during stretch, as has been suggested by Jensen et al. (39). At present, it is unknown whether this potential mechanism operates in smooth muscle at all or whether differences in actin filament length account for a potential difference in plastic vs. elastic deformation in normal or asthmatic ASM, respectively. Below, we consider factors that might promote actin filament elongation in airway inflammation.



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  		Fig. 3. Schematic illustration of the hypothesized influence of actin filament length and rearrangement during stretch of contracted airway smooth muscle. Orange lines, actin filaments; red ovals, dense bodies; green crescents, barbed-end actin-capping proteins; blue shapes, myosin filaments. A: inspiration-induced stretch of smooth muscle cells with short actin filaments exerts a parallel-to-series rearrangement of the filament lattice, resulting in a reduction of force generation and plastic behavior. B: inspiration-induced stretch of smooth muscle cells with longer actin filaments would be less likely to exert the parallel-to-series rearrangement of the filament lattice shown in A. As such, the capacity for maximal force generation is maintained and the muscle would behave more elastically. (Reproduced from Ref. 18.)

 


   DO INFLAMMATORY MEDIATORS DIRECTLY INCREASE NONSPECIFIC AIRWAY RESPONSIVENESS?
TOP
 ABSTRACT
 WHAT MEDIATORS ARE INVOLVED...
 SMOOTH MUSCLE CONTRACTILE...
 DO INFLAMMATORY MEDIATORS...
SUMMARY
 DISCLOSURES
 REFERENCES
 
Although there is little evidence that asthmatic airway strips show greater isometric force generation to nonspecific contractile agonists, several studies report that cytokines and mediators potentiate contractile agonist-stimulated force generation of normal ASM. As little as 1-h exposure to atopic serum is sufficient to cause increased responsiveness to histamine in isolated canine tracheal smooth muscle strips (58). Human seventh-generation bronchial rings exposed overnight to serum from asthmatic subjects demonstrated increased responsiveness to histamine (95), increased myogenic responses to quick stretch (37), and increased shortening velocity and capacity (38). These data clearly indicate that hyperresponsiveness can be mediated directly by relatively acute exposure to the inflammatory mediators in the serum. Although Mitchell and colleagues (60) demonstrated that exogenous IgE addition was able to increase responsiveness to a quick stretch provocation (DI?), they indicated that other mediators (potentially cytokine, inflammatory, and so forth in origin) were also likely to be involved in the response to atopic serum. Other organ bath studies have indicated that preexposure of human bronchial or tracheal smooth muscle strips to TNF-{alpha} increases responsiveness to muscarinic agonists, bradykinin, and thrombin (3, 5, 71, 88). Conversely, the response to IL-1{beta} is more variable, in that IL-1{beta} has agonist-specific influence on airway responsiveness of human isolated bronchi (8, 68, 88). There are a number of studies in human airway cell culture and in isolated airways of other species demonstrating that cytokines (including other Th2 cytokines and inflammatory factors) directly contribute to AHR (77). Thus it appears that 1) acute exposure of cytokines can itself induce AHR and 2) more than one mediator is implicated in this effect. However, as discussed above, isometric force generation to contractile agonists is thought to be similar in asthmatic and healthy airways (or sensitized animals vs. sham animals), suggesting that some caution must be applied to these results from studies incorporating experimental cytokine exposure ex vivo. Perhaps exposing normal ASM to "asthma-like environment" (IgE, atopic serum, TNF-{alpha}, IL-1{beta}, and so forth) does not actually reproduce the environment of the disease, or maybe once asthmatic ASM is removed from its natural environment it reverts to normal (as regards isometric force generation).

How do these inflammatory mediators act to increase airway responsiveness? It is known that TNF-{alpha} increases human ASM responsiveness in vitro through enhancing contractile agonist-stimulated increases in inositol 1,4,5-trisphosphate and augmentation of peak and sustained intracellular calcium levels, all of which are key components leading to smooth muscle contraction (3, 5, 71, 88). TNF-{alpha}-enhanced force generation takes at least 6 h to appear and could be inhibited by cycloheximide, suggesting that new protein synthesis is required (4).

Besides influencing force generation, passive sensitization of human tracheal smooth muscle with IgE-rich atopic serum, or treatment with individual cytokines such as IL-1{beta} and TNF-{alpha}, can blunt ASM relaxation normally induced by {beta}2-adrenoceptor stimulation (29, 48, 84). This effect is mediated through {beta}2-adrenoceptor phosphorylation (31), Gs protein dysfunction (85), and increased prostanoid (48) and peptidoleukotriene (84) release.

Even less is known of the influence of inflammatory mediators on the dynamic component of smooth muscle contraction, but recent evidence suggests that they may play a significant role.

How might inflammatory mediators influence increased MLCK and shortening velocity? The studies of Stephens and others provide some guide as to how inflammatory mediators might result in increased MLCK activity. Studies in dog (61), guinea pig (59), mouse (22), and human (1) airways show that sensitization of the airways is sufficient to result in increased levels of MLCK or increased shortening velocity. Thus an increase in the activity and/or content of smooth muscle MLCK may be one of the initial events to contribute to the pathogenesis of AHR and may be sustained in the absence of further acute challenge. It should be noted that genetic predisposition might influence the susceptibility to this response. A study comparing two mouse strains, SJL and ASW, demonstrated differences in regulation of shortening velocity on sensitization with ovalbumin (22). The SJL strain exhibited greater splenocyte IL-4 production after specific antigen challenge and greater smooth muscle shortening velocity after sensitization. The ASW strain had diminished IL-4 response to antigen challenge and no increase in maximal ASM shortening velocity. These findings also imply that the change in ASM shortening velocity was dependent on the generation of a substantial immune-based inflammatory response in this model.

What is the time course of change in shortening velocity? Exposure to the serum alone of atopic subjects for as little as 16 h is sufficient to increase the shortening velocity of human ASM (61), indicating that IgE, cytokines, or other inflammatory mediators in the atopic serum might act directly on the smooth muscle to mediate changes in actomyosin dynamics.

Which intracellular signaling pathways are involved? Of the signaling pathways that influence MLC20 phosphorylation (and thus shortening velocity), Rho kinase and increased intracellular calcium appear to be the most important (reviewed in Ref. 81). MLCK activity increases with intracellular free calcium by a calmodulin-dependent mechanism, and it is this pathway by which most agonists stimulate smooth muscle contraction. There was no difference in calmodulin level or activity in the ragweed-sensitized dog model, which showed increased shortening velocity (42), implying that the increase in MLCK levels and activity might be directly due to increased expression. However, a number of cytokines and growth factors such as IL-1{beta}, TNF-{alpha}, and thrombin (2, 65, 71) all activate intracellular calcium in ASM and thus may further potentiate activation of this pathway to contractile agonists in the inflamed airway.

Rho kinase acts to inhibit MLC phosphatase activity, thus promoting the phosphorylated state of MLC20 (43). Rho kinase has also been implicated in the direct phosphorylation of MLC20 in a calcium-independent manner (93). Van Eyk and colleagues showed, in permeabilized cultured canine tracheal myocytes, that a constitutively active form of Rho kinase appeared to phosphorylate the regulatory MLC directly (93). Of particular interest is that RhoA, the GTPase that activates Rho kinase, is increased in ASM of hyperresponsive rats (14). RhoA activity is stimulated by a multitude of mediators such as lysophosphatidic acid TNF-{alpha}, TGF-{beta}, IL-1{beta}, and many growth factors (51, 53, 54). However, in contrast to Van Eyk's results, subsequent studies by Deng et al. (15) demonstrated that integrin-linked kinase was responsible for non-MLCK-mediated MLC20 phosphorylation in smooth muscle. It is not currently known whether changes in integrin activity or extracellular matrix composition can increase MLC20 phosphorylation through this pathway. Thus, in addition to increased MLCK activity, it seems entirely likely that other pathways interact to effect increased MLC20 phosphorylation in the inflamed airway.

Do any inflammatory mediators protect against increased shortening velocity? NO is the only inflammatory agent known to date to "protect" airways via this mechanism, causing elevations in cGMP and cGMP-dependent protein kinase I{alpha} activity, which increase myosin phosphatase activity leading to relaxation (21, 90). The potential for other mediators to protect against increased shortening velocity in asthma has not been studied.

How might inflammatory mediators influence plasticity of smooth muscle? Given the lack of firm evidence that the plasticity-elasticity balance is altered at all in asthma, one can only speculate as to how inflammatory mediators might modulate this balance. However, given the multiple levels of regulation of actin and myosin filament length and tethering of the contractile apparatus to cell membrane adhesion molecules, it seems quite plausible that inflammatory mediators could influence mechanical plasticity (Fig. 4).



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  		Fig. 4. Regulation of actin filament length by cytokines, growth factors, and contractile stimuli. Actin polymerization occurs by profilin binding to G-actin monomers, facilitating aggregation. Profilin activity is regulated by proteins from the Wiscott-Aldrich syndrome (WASp/Scar) family, ezrin, vasodilator-stimulated phosphoprotein (VASP), and the diaphenous-related formin protein (mDia). Extracellular stimuli-driven activation of these profilin-interacting proteins occurs via Rac family GTPases such as RhoA and cdc42. RhoA-GTP also prevents depolymerization of actin filaments by Rho kinase (LIM kinase)-stimulated phosphorylation and inactivation of ADP/cofilin. Actin length is also regulated by capping proteins on both barbed and pointed actin ends. HSP27 (heat shock protein 27), a barbed-end capping protein, binds actin in the unphosphorylated form. Phosphorylation of HSP27 [facilitated through p38 mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) activity], dissociates HSP27 from actin and allows addition of more monomers. Actin filament length is also regulated by severing proteins (like gelsolin) and side-binding proteins (like tropomyosin) that stabilize actin filaments.

 

Fredberg and colleagues (46) provided the first evidence that signaling pathways stimulated by inflammatory mediators might alter the plasticity of ASM. They demonstrated that pharmacological inhibition of p38 mitogen-activated protein kinase (p38 MAPK) activity produced greater smooth muscle tissue lengthening during sinusoidal force oscillation (meant to simulate force fluctuations during tidal breathing). The treated muscle strips also remained at a greater length after the fluctuations had been stopped compared with untreated tissues (via a mechanism independent of MLC20 phosphorylation) (46). Thus p38 MAPK activity was associated with greater tissue stiffness and greater elasticity, whereas p38 MAPK inhibition promoted greater plasticity. The authors speculated that p38 MAPK activity leads to the phosphorylation and inactivation of heat shock protein 27 (HSP27), one of the proteins involved in capping the end of actin filaments (46). As discussed below, by inhibiting actin filament capping, phosphorylation of HSP27 should promote actin filament lengthening and, by the scheme illustrated in Fig. 3, smooth muscle elasticity.

Actin filament length, like myosin filament length, is actively regulated by the contractile state of the cell (55). In addition, many different growth factors, cytokines, and other mediators promote actin polymerization (72, 92) (Fig. 4). The signaling pathways activated by these mediators converge on the RhoA family of proteins, such as RhoA and Cdc42, which facilitate actin polymerization in concert with members of the Wiscott-Aldrich syndrome (WASp/Scar) family (66) and formin homology proteins such as mDia (74). These proteins act through their proline-rich domains to activate profilin, which directly organizes actin monomer addition (11). Other proteins that contribute to actin polymerization by activating profilin include ezrin and vasodilator-stimulated phosphoprotein (VASP). The process of actin polymerization is held in check by proteins that cap the actin ends (and thus slow further monomer addition or prevent monomer removal), such as HSP27, CapZ/Cap{alpha}{beta}, tropomodulin, and SM-Lmod (for review, see Ref. 82).

HSP27, when unphosphorylated, caps the barbed end of actin. Phosphorylation of HSP27 disables its capping of actin and thus allows actin polymerization and subsequent filament lengthening. HSP27 is constitutively expressed at high levels in smooth muscle cells, is phosphorylated by p38 MAPK through MAPK-activated protein kinase in ASM (49), and is phosphorylated during contraction, perhaps by protein kinase C (9). Thus HSP27, like p38 MAPK, is modulated in smooth muscle by many inflammatory stressors, including IL-1{beta}, TGF-{beta}, PDGF, angiotensin II, thrombin, and ET-1 (34, 57, 98, 99), providing a mechanism by which these mediators might decrease smooth muscle plasticity.

How is actin depolymerization regulated? Although it also acts as a barbed-end capping protein, gelsolin can sever actin filaments (89), whereas side-binding proteins such as tropomyosin stabilize actin filaments. Depolymerization of filamentous actin is mediated in part by ADP/cofilin binding (7). Smooth muscle relaxants that increase protein kinase A contribute to the depolymerization of actin by phosphorylating and inactivating RhoA (17) and also potentially by VASP phosphorylation (94). Protein kinase A-independent actin depolymerization also occurs through an as yet unknown mechanism (35). Interestingly, RhoA activity (in addition to stimulating actin polymerization), acting via the serine/threonine protein kinase LIM-1 kinase, also inhibits cofilin-induced actin depolymerization in N1E-115 neuroblastoma and HeLa cells (53, 100). Given that {beta}-adrenergic receptor desensitization occurs in asthma, and many mediators stimulate RhoA activity, is prevention of depolymerization also a contributing factor for maintained actin filament length?

Myosin filament length, like actin length, is in a dynamic equilibrium within the ASM cell, with increased filament length during contraction and filament evanescence during stretching (45). During contraction, increased intracellular calcium and MLCK activity appear to be the stimulus for myosin filament formation (33, 69), although resting intracellular calcium levels are also important for the maintenance of myosin filament integrity (33). MLC20 phosphorylation by MLCK is necessary for the unfolding of myosin monomers from the S6 to the S10 conformation for subsequent integration into filaments (37). It is not known whether myosin filament length is different in asthmatic airways, although it is reasonable to suspect that the increased MLCK in ASM from asthmatic subjects might serve to maintain longer myosin filaments. Thus MLCK levels might serve to regulate the plasticity of ASM as well as contraction velocity (similar to RhoA!).


   SUMMARY
TOP
 ABSTRACT
 WHAT MEDIATORS ARE INVOLVED...
 SMOOTH MUSCLE CONTRACTILE...
 DO INFLAMMATORY MEDIATORS...
 SUMMARY
DISCLOSURES
 REFERENCES
 
All evidence suggests that the smooth muscle contractile apparatus is surrounded and bombarded by inflammatory mediators; there is evidence (or at least plausible speculation) that links inflammatory mediators to increased velocity of contraction, increased smooth muscle stiffness, and loss of smooth muscle plasticity. These mechanisms, although not resulting in increased force generation, likely contribute to the altered dynamic smooth muscle response to tidal breathing and the occasional DI (24, 50, 78). Many of the changes in smooth muscle contractile apparatus dynamics can be produced only after short exposures to cytokines or serum from atopic individuals. Thus smooth muscle dysfunction might be one of the first mechanisms by which inflammation causes AHR, which could worsen over time as airway remodeling also makes a contribution. Could it be possible that loss of DI-induced bronchodilatation is one of the first signs that something is wrong in the airways of asthmatic patients?


   DISCLOSURES
TOP
 ABSTRACT
 WHAT MEDIATORS ARE INVOLVED...
 SMOOTH MUSCLE CONTRACTILE...
 DO INFLAMMATORY MEDIATORS...
 SUMMARY
 DISCLOSURES
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants SCOR HL-56399 and HL-64095, the Sandler Program for Asthma Research, and the Thoracic Society of Australia and New Zealand.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Solway, Section of Pulmonary and Critical Care Medicine, Univ. of Chicago, 5841 S. Maryland Ave., MC6026, Chicago, IL 60637 (E-mail: jsolway{at}medicine.bsd.uchicago.edu).


   REFERENCES
TOP
 ABSTRACT
 WHAT MEDIATORS ARE INVOLVED...
 SMOOTH MUSCLE CONTRACTILE...
 DO INFLAMMATORY MEDIATORS...
 SUMMARY
 DISCLOSURES
 REFERENCES
 

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