Murine Airway Smooth Muscle Cells Research Articles

Interleukin 31 receptor α promotes smooth muscle cell contraction and airway hyperresponsiveness in asthma

Asthma is a chronic inflammatory airway disease characterized by airway hyperresponsiveness (AHR), inflammation, and goblet cell hyperplasia. Multiple cytokines, including IFNγ, IL-4, and IL-13 are associated with asthma; however, the mechanisms underlying the effects of these cytokines remain unclear. Here, we report a significant increase in the expression of IL-31RA, but not its cognate ligand IL-31, in mouse models of allergic asthma. In support of this, IFNγ, IL-4, and IL-13 upregulated IL-31RA but not IL-31 in both human and mice primary airway smooth muscle cells (ASMC) isolated from the airways of murine and human lungs. Importantly, the loss of IL-31RA attenuated AHR but had no effect on inflammation and goblet cell hyperplasia in mice challenged with allergens or treated with IL-13 or IFNγ. We show that IL-31RA functions as a positive regulator of muscarinic acetylcholine receptor 3 expression, augmenting calcium levels and myosin light chain phosphorylation in human and murine ASMC. These findings identify a role for IL-31RA in AHR that is distinct from airway inflammation and goblet cell hyperplasia in asthma.

Spleen tyrosine kinase inhibition blocks airway constriction and protects from Th2-induced airway inflammation and remodeling.

Spleen tyrosine kinase (Syk) is an intracellular nonreceptor tyrosine kinase, which has been implicated as central immune modulator promoting allergic airway inflammation. Syk inhibition has been proposed as a new therapeutic approach in asthma. However, the direct effects of Syk inhibition on airway constriction independent of allergen sensitization remain elusive. Spectral confocal microscopy of human and murine lung tissue was performed to localize Syk expression. The effects of prophylactic or therapeutic Syk inhibition on allergic airway inflammation, hyperresponsiveness, and airway remodeling were analyzed in allergen-sensitized and airway-challenged mice. The effects of Syk inhibitors BAY 61-3606 or BI 1002494 on airway function were investigated in isolated lungs of wild-type, PKCα-deficient, mast cell-deficient, or eNOS-deficient mice. Spleen tyrosine kinase expression was found in human and murine airway smooth muscle cells. Syk inhibition reduced allergic airway inflammation, airway hyperresponsiveness, and pulmonary collagen deposition. In naïve mice, Syk inhibition diminished airway responsiveness independently of mast cells, or PKCα or eNOS expression and rapidly reversed established bronchoconstriction independently of NO. Simultaneous inhibition of Syk and PKC revealed additive dilatory effects, whereas combined inhibition of Syk and rho kinase or Syk and p38 MAPK did not cause additive bronchodilation. Spleen tyrosine kinase inhibition directly attenuates airway smooth muscle cell contraction independent of its protective immunomodulatory effects on allergic airway inflammation, hyperresponsiveness, and airway remodeling. Syk mediates bronchoconstriction in a NO-independent manner, presumably via rho kinase and p38 MAPK, and Syk inhibition might present a promising therapeutic approach in chronic asthma as well as acute asthma attacks.

Reactive oxygen species induce a Ca2+-spark increase in sensitized murine airway smooth muscle cells

The level of reactive oxygen species (ROS) and the activity of spontaneous, transient, localized Ca2+ increases (known as Ca2+ sparks) in tracheal smooth muscle cells (TSMCs) in an experimental allergic asthma mouse model has not yet been investigated. We used laser confocal microscopy and fluorescent dyes to measure ROS levels and Ca2+ sparks, and we found that both events were significantly increased in TSMCs obtained from ovalbumin (OVA)-sensitized/-challenged mice compared with control mice. ROS levels began to increase in TSMCs after the first OVA challenge, and this increase was sustained. However, this elevation and Ca2+-spark increase was abolished after the administration of the ROS scavenger N-acetylcysteine amide (NACA) for 5days. Furthermore, a similar inhibition was also observed following the direct perfusion of NACA into cells isolated from the (OVA)-sensitized mice that were not treated with NACA. Moreover, we used 0.1-mM caffeine treatment to increase the Ca2+ sparks in single TSMCs and observed cell shortening. In addition, we did not find increases in the mRNA levels of ryanodine (RyRs) and inositol 1,4,5-trisphosphate (IP3Rs) receptors in the tracheal smooth muscle cells of (OVA)-sensitized mice compared with controls. We concluded that ROS and Ca2+ sparks increased in (OVA)-sensitized TSMCs. We found that ROS induces Ca2+ sparks, and increased Ca2+ sparks resulted in the contraction of (OVA)-sensitized TSMCs, resulting in the generation of airway hyperresponsiveness (AHR). This effect may represent a novel mechanism for AHR pathogenesis and might provide insight into new methods for the clinical prevention and treatment of asthma and asthmatic AHR.

Tumor Necrosis Factor-stimulated Gene-6 (TSG-6) Amplifies Hyaluronan Synthesis by Airway Smooth Muscle Cells

We tested the hypothesis that the artificial addition of heavy chains from inter-α-inhibitor to hyaluronan (HA), by adding recombinant TSG-6 (TNF-stimulated gene-6) to the culture medium of murine airway smooth muscle (MASM) cells, would enhance leukocyte binding to HA cables produced in response to poly(I:C). As predicted, the addition of heavy chains to HA cables enhanced leukocyte adhesion to these cables, but it also had several unexpected effects. (i) It produced thicker, more pronounced HA cables. (ii) It increased the accumulation of HA in the cell-associated matrix. (iii) It decreased the amount of HA in the conditioned medium. Importantly, these effects were observed only when TSG-6 was administered in the presence of poly(I:C), and TSG-6 did not exert any effect on its own. Increased HA synthesis occurred during active, poly(I:C)-induced HA synthesis and did not occur when TSG-6 was added after poly(I:C)-induced HA synthesis was complete. MASM cells derived from TSG-6(-/-), HAS1/3(-/-), and CD44(-/-) mice amplified HA synthesis in response to poly(I:C) + TSG-6 in a manner similar to WT MASM cells, demonstrating that they are expendable in this process. We conclude that TSG-6 increases the accumulation of HA in the cell-associated matrix, partially by preventing its dissolution from the cell-associated matrix into the conditioned medium, but primarily by inducing HA synthesis.

Reverse‐mode NCX current in mouse airway smooth muscle: Na+ and voltage dependence, contributions to Ca2+ influx and contraction, and altered expression in a model of allergen‐induced hyperresponsiveness

We examined the electrophysiological properties of reverse-mode Na(+) /Ca(2+) exchange (NCX) in mouse airway smooth muscle (ASM), assessing its contributions to regulation of [Ca(2+) ], and its expression in acute and chronic airway hyperresponsiveness (AHR). Membrane currents were studied in single murine ASM cells under voltage clamp at -60mV using ramp depolarizing commands to +80mV. Confocal fluorimetric and RT-PCR techniques were used to monitor changes in cytosolic [Ca(2+) ] and NCX expression, respectively. With standard KCl-containing electrode, 30μm KB-R7943 (an inhibitor of reverse-mode NCX activity) exhibited variable effects on membrane current, indicating modulation of more than one conductance. KB-R7943 activated outwardly rectifying current that was inhibited by 100μm iberiotoxin (blocker of large-conductance Ca(2+) -dependent K(+) channels), indicating a direct enhancing effect of KB-R7943 on those K(+) channels. After obviating K(+) currents, we found that a current sensitive to 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid (blocker of Ca(2+) -dependent Cl- channels) was markedly increased by elevating [Na(+) ] in the electrode solution to 13, 15.5 and 18mm and suppressed by KB-R7943, indicating Ca(2+) influx via reverse-mode NCX activity. With conditions preventing Ca(2+) influx through voltage-dependent Ca(2+) channels but promoting that through NCX, we found that introduction of Ca(2+) led to marked but transient KB-R7943-sensitive elevation of [Ca(2+) ]. Additionally, KB-R7943 suppressed cholinergically evoked Ca(2+) waves. Finally, NCX1 expression was not significantly changed in allergen-induced AHR acute model but increased approx. 2.5-fold in a chronic model. Reverse-mode NCX activity leads to a physiologically relevant increase in [Ca(2+) ] even under control conditions, and this may be exaggerated in allergen-induced AHR and asthma.

S32 Loss/inhibition of the aVb5 integrin reduces allergen-induced increases in airway smooth muscle mass in in vivo models of asthma

Airway remodelling is a common feature of severe asthma. Transforming growth factor-ß (TGF-ß) is a pro-fibrotic, pleiotropic cytokine implicated in airway remodelling. TGF-ß is sequestered in the extracellular matrix as a latent complex and requires activation to function. We have previously shown that contraction agonists cause aVß5-mediated TGF-ß activation by human airway smooth muscle cells. The study aims were to investigate the role of the aVß5integrin in airway remodelling in vivo using two distinct mouse models of asthma. A blocking antibody directed against the aVß5 was used in the ovalbumin (OVA) model of asthma. Mice were sensitised with OVA/Alum on days 0 and 12, then challenged byoropharyngeal administration of OVA 10 times over 2 weeks. The anti-aVß5antibody or an isotype matched control antibody was administered for the duration of the OVA challenges. The second invivo model utilised <i>itgb5^−/−</i>mice. <i>Aspergillous fumigatus</i> antigen preparation was administered intra-nasally (10 μg/mice) to <i>itgb5^−/−</i> and wild type controls 9 times over a 21-day period. a-Smooth muscle actin (a-SMA) was quantified in lung sections from both studies by immunofluorescence. Murine airway smooth muscle cells express aVß5 integrin and can activate TGF-ß in vivo in response to allergen challenge as measured by αvβ5 and phospho-Smad2 immunostaining. Treatment with both OVA and <i>Asp.f</i> resulted in an increase in a-SMA staining around the smaller airways. The aVß5blocking antibody significantly reduced a-SMA staining compared with the isotype control antibody. Furthermore, <i>itgb5^−/−</i>mice had significantly less a-SMA around their airways than wild type control mice in response to <i>Asp.f</i> treatment. However, both <i>itgb5^−/−</i> andanti-aVß5 treated mice had significantly more airway inflammation and more inflammatory cells present in the bronchoalveolar lavage compared with their matched controls. These data provide evidence that airway smooth muscle cells can activate TGF-ß in vivo. Inhibition of, or genetic loss of, the aVß5 integrin significantly reduces allergen-induced increases in airway smooth muscle mass, however, peribronchial inflammation is increased consistent with the known effects of TGFβ. Targeted inhibition of the aVß5 integrin may reduce airway remodelling, but global inhibition is unlikely to be useful due to the enhanced inflammatory response.

Integrin αvβ5-Mediated TGF-β Activation by Airway Smooth Muscle Cells in Asthma

Severe asthma is associated with airway remodeling, characterized by structural changes including increased smooth muscle mass and matrix deposition in the airway, leading to deteriorating lung function. TGF-β is a pleiotropic cytokine leading to increased synthesis of matrix molecules by human airway smooth muscle (HASM) cells and is implicated in asthmatic airway remodeling. TGF-β is synthesized as a latent complex, sequestered in the extracellular matrix, and requires activation for functionality. Activation of latent TGF-β is the rate-limiting step in its bioavailability. This study investigated the effect of the contraction agonists LPA and methacholine on TGF-β activation by HASM cells and its role in the development of asthmatic airway remodeling. The data presented show that LPA and methacholine induced TGF-β activation by HASM cells via the integrin αvβ5. Our findings highlight the importance of the β5 cytoplasmic domain because a polymorphism in the β5 subunit rendered the integrin unable to activate TGF-β. To our knowledge, this is the first description of a biologically relevant integrin that is unable to activate TGF-β. These data demonstrate that murine airway smooth muscle cells express αvβ5 integrins and activate TGF-β. Finally, these data show that inhibition, or genetic loss, of αvβ5 reduces allergen-induced increases in airway smooth muscle thickness in two models of asthma. These data highlight a mechanism of TGF-β activation in asthma and support the hypothesis that bronchoconstriction promotes airway remodeling via integrin mediated TGF-β activation.

Expression Profiling Identifies Klf15 as a Glucocorticoid Target That Regulates Airway Hyperresponsiveness

Glucocorticoids (GCs), which activate GC receptor (GR) signaling and thus modulate gene expression, are widely used to treat asthma. GCs exert their therapeutic effects in part through modulating airway smooth muscle (ASM) structure and function. However, the effects of genes that are regulated by GCs on airway function are not fully understood. We therefore used transcription profiling to study the effects of a potent GC, dexamethasone, on human ASM (HASM) gene expression at 4 and 24 hours. After 24 hours of dexamethasone treatment, nearly 7,500 genes had statistically distinguishable changes in expression; quantitative PCR validation of a 40-gene subset of putative GR-regulated genes in 6 HASM cell lines suggested that the early transcriptional targets of GR signaling are similar in independent HASM lines. Gene ontology analysis implicated GR targets in controlling multiple aspects of ASM function. One GR-regulated gene, the transcription factor, Kruppel-like factor 15 (Klf15), was already known to modulate vascular smooth and cardiac muscle function, but had no known role in the lung. We therefore analyzed the pulmonary phenotype of Klf15(-/-) mice after ovalbumin sensitization and challenge. We found diminished airway responses to acetylcholine in ovalbumin-challenged Klf15(-/-) mice without a significant change in the induction of asthmatic inflammation. In cultured cells, overexpression of Klf15 reduced proliferation of HASM cells, whereas apoptosis in Klf15(-/-) murine ASM cells was increased. Together, these results further characterize the GR-regulated gene network in ASM and establish a novel role for the GR target, Klf15, in modulating airway function.

Regulation of Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycan Biosynthesis by 4-Fluoro-glucosamine in Murine Airway Smooth Muscle Cells

The importance of the pathological changes in proteoglycans has driven the need to study and design novel chemical tools to control proteoglycan synthesis. Accordingly, we tested the fluorinated analogue of glucosamine (4-fluoro-N-acetyl-glucosamine (4-F-GlcNAc)) on the synthesis of heparan sulfate (HS) and chondroitin sulfate (CS) by murine airway smooth muscle (ASM) cells in the presence of radiolabeled metabolic precursors. Secreted and cell-associated CS and HS were assessed for changes in size by Superose 6 chromatography. Treatment of ASM cells with 4-F-GlcNAc (100 microM) reduced the quantity (by 64.1-76.6%) and decreased the size of HS/CS glycosaminoglycans associated with the cell layer (K(av) shifted from 0.30 to 0.45). The quantity of CS secreted into the medium decreased by 65.7-73.0%, and the size showed a K(av) shift from 0.30 to 0.50. Treatment of ASM cells with 45 microM and 179 microM 4-F-GlcNAc in the presence of a stimulator of CS synthesis, 4-methylumbelliferyl-beta-D-xyloside, reduced the amount of the xyloside-CS chains by 65.4 and 87.0%, respectively. The size of xyloside-CS chains synthesized in the presence of 4-F-GlcNAc were only slightly larger than those with xyloside treatment alone (K(av) of 0.55 compared with that of 0.6). The effects of 4-F-GlcNAc to inhibit CS synthesis were not observed with equimolar concentrations of glucosamine. We propose that 4-F-GlcNAc inhibits CS synthesis by inhibiting 4-epimerization of UDP-GlcNAc to UDP-GalNAc, thereby depleting one of the substrates required, whereas HS elongation is inhibited by truncation when the nonreducing terminus of the growing chain is capped with 4-F-GlcNAc.

Primary Murine Airway Smooth Muscle Cells Exposed to Poly(I,C) or Tunicamycin Synthesize a Leukocyte-adhesive Hyaluronan Matrix

Asthmatic attacks often follow viral infections with subsequent airway smooth muscle cell proliferation and the formation of an abnormal hyaluronan extracellular matrix with infiltrated leukocytes. In this study, we show that murine airway smooth muscle cells (MASM) treated with polyinosinic acid-polycytidylic acid (poly(I,C)), a double-stranded RNA that simulates a viral infection, synthesize an abnormal hyaluronan matrix that binds leukocytes (U937 cells). Synthesis of this matrix is initiated rapidly and accumulates linearly for approximately 10 h, reaching a plateau level approximately 7-fold higher than control cultures. MASM cells treated with tunicamycin, to induce endoplasmic reticulum stress, also rapidly initiate synthesis of the abnormal hyaluronan matrix with linear accumulation for approximately 10 h, but only reach a plateau level approximately 2-fold higher than control cultures. In contrast to poly(I,C), the response to tunicamycin depends on cell density, with pre-confluent cells producing more abnormal matrix per cell. Furthermore, U937 cell adhesion per hyaluronan content is higher in the sparse matrix produced in response to tunicamycin, suggesting that the structure in the poly(I,C)-induced matrix masks potential binding sites. When MASM cells were exposed to tunicamycin and poly(I,C) at the same time, U937 cell adhesion was partially additive, implying that these two toxins stimulate hyaluronan synthesis through two different pathways. We also characterized the size of hyaluronan produced by MASM cells, in response to poly(I,C) and tunicamycin, and we found that it ranges from 1500 to 4000 kDa, the majority of which was approximately 4000 kDa and not different in size than hyaluronan made by untreated cells.

Ginger attenuates acetylcholine-induced contraction and Ca2+ signalling in murine airway smooth muscle cells.

Asthma is a chronic disease characterized by inflammation and hypersensitivity of airway smooth muscle cells (ASMCs) to different spasmogens. The past decade has seen increased use of herbal treatments for many chronic illnesses. Ginger (Zingiber officinale) is a common food plant that has been used for centuries in treating respiratory illnesses. In this study, we report the effect of its 70% aqueous methanolic crude extract (Zo.Cr) on acetylcholine (ACh)-induced airway contraction and Ca(2+) signalling in ASMCs using mouse lung slices. Airway contraction and Ca(2+) signalling, recorded via confocal microscopy, were induced with ACh, either alone or after pretreatment of slices with Zo.Cr and (or) verapamil, a standard Ca(2+) channel blocker. ACh (10 micromol/L) stimulated airway contraction, seen as decreased airway diameter, and also stimulated Ca(2+) transients (sharp rise in [Ca(2+)]i) and oscillations in ASMCs, seen as increased fluo-4-induced fluorescence intensity. When Zo.Cr (0.3-1.0 mg/mL) was given 30 min before ACh administration, the ACh-induced airway contraction and Ca(2+) signalling were significantly reduced. Similarly, verapamil (1 micromol/L) also inhibited agonist-induced airway contraction and Ca(2+) signalling, indicating a similarity in the modes of action. When Zo.Cr (0.3 mg/mL) and verapamil (1 micromol/L) were given together before ACh, the degree of inhibition was the same as that observed when each of these blockers was given alone, indicating absence of any additional inhibitory mechanism in the extract. In Ca(2+) -free solution, both Zo.Cr and verapamil, when given separately, inhibited Ca(2+) (10 mmol/L)-induced increase in fluorescence and airway contraction. This shows that ginger inhibits airway contraction and associated Ca(2+) signalling, possibly via blockade of plasma membrane Ca(2+) channels, thus reiterating the effectiveness of this age-old herb in treating respiratory illnesses.

Proteoglycans and pathophysiology

ventilation-induced lung injury (VILI) is an important clinical entity whose pathophysiology is incompletely understood. The article by Moriondo and coworkers ([14][1]) in the Journal of Applied Physiology offers new insights into potential mechanistic pathways. These authors report that one of the

Airway smooth muscle cells enhance C3a‐induced mast cell degranulation following cell‐cell contact

Growing evidence suggests that anaphylatoxins, C3a and C5a, play important roles in innate immunity and may also participate in the pathogenesis of asthma. Previous studies with animal models and immunohistochemistry analysis of lung tissue indicated that anaphylatoxins may regulate airway hyperresponsiveness (AHR) in asthma via the activation of their cell surface G protein-coupled receptors (C3aR and C5aR) in airway smooth muscle (ASM) cells. Using RT-PCR, flow cytometry, and confocal microscopy, we made the surprising observation that while C3aR and C5aR were expressed in human mast cells, they were not present in cultured primary human or murine ASM cells. Furthermore, we could not detect C3aR in smooth muscle-positive cells of human trachea or bronchus. Interestingly, incubation of human mast cells with ASM cells, but not its culture supernatant, caused a significant enhancement of C3a-induced mast cell degranulation. Although stem cell factor (SCF) and its receptor c-kit are constitutively expressed on ASM cells and mast cells, respectively, neutralizing antibodies to SCF and c-kit failed to inhibit ASM cell-mediated enhancement of mast cell degranulation. However, dexamethasone-treated ASM cells were normal for cell surface SCF expression but were significantly less effective in enhancing C3a-induced mast cell degranulation when compared with untreated cells. These findings suggest that cell-cell interaction between ASM cells and mast cells, via a SCF-c-kit-independent but dexamethasone-sensitive mechanism, enhances C3a-induced mast cell degranulation, which likely regulates ASM function, thus contributing to the pathogenesis of asthma.

IL-13 may mediate allergen-induced hyperresponsiveness independently of IL-5 or eotaxin by effects on airway smooth muscle

IL-13 is a mediator of allergen-induced airway hyperresponsiveness (AHR). The aim of this study was to evaluate whether eotaxin and IL-5 were implicated in the effects of IL-13 on allergen-induced AHR or whether IL-13 may exert its effects through direct actions on airway smooth muscle (ASM). To study this question airway inflammation and AHR were induced in mice by sensitization and subsequent challenge on three successive days with ovalbumin. A monoclonal anti-IL-13 antibody administered before each challenge significantly reduced AHR without affecting airway eosinophilia. No changes of mRNA in BAL and lung tissues or protein levels in BAL of IL-5 or eotaxin were found following anti-IL-13 treatment. Combined injection of monoclonal anti-IL-5 and antieotaxin antibodies before each antigen challenge blocked airway eosinophilia but failed to reduce AHR. IL-13 induced calcium transients in cultured murine ASM cells and augmented the calcium and contractile responses of these cells to leukotriene D4. These results suggest that IL-13 plays an important role in allergen-induced AHR and is important in the early phases of the inflammatory process. Its effects on AHR are mediated independently of IL-5 and eotaxin and may involve a direct effect on ASM to augment its responsiveness.