IL-33, a new member of the IL-1 cytokine family, promotes Th2 inflammation, but evidence on the implications of this cytokine in asthma is lacking. IL-33 would be mainly expressed by structural cells, but whether proinflammatory cytokines modulate its expression in airway smooth muscle cells (ASMC) is unknown. Endobronchial biopsies were obtained from adults with mild (n = 8), moderate (n = 8), severe (n = 9), asthma and from control subjects (n = 5). Immunocytochemistry, laser-capture microdissection, reverse transcriptase, and real-time quantitative PCR were used for determining IL-33 expression in the lung tissues. ASMC isolated from resected lung specimens were cultured with proinflammatory cytokines and with dexamethasone. IL-33 expression by ASMC was determined by PCR, ELISA, and Western blotting. Higher levels of IL-33 transcripts are detected in biopsies from asthmatic compared with control subjects, and especially in subjects with severe asthma. ASMC show IL-33 expression at both protein and mRNA levels. IL-33 and TNF-α transcript levels correlate in the lung tissues, and TNF-α up-regulates IL-33 expression by cultured ASMC in a time- and dose-dependent manner. IFN-γ also increases IL-33 expression and shows synergistic effect with TNF-α. Dexamethasone fails to abolish TNF-α-induced IL-33 up-regulation. IL-33 expression increases in bronchial biopsies from subjects with asthma compared with controls, as well as subjects with asthma severity. ASMC are a source of the IL-33 cytokine. Our data propose IL-33 as a novel inflammatory marker of severe and refractory asthma.

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Asthma is characterized by an increase in airway smooth muscle mass and a decreased distance between the smooth muscle layer and the epithelium. Furthermore, there is evidence to indicate that airway smooth muscle cells (ASMC) express a wide variety of receptors involved in the immune response. The aims of this study were to examine the expression of CCR3 on ASMC, to compare this expression between asthmatic and nonasthmatic subjects, and to determine the implications of CCR3 expression in the migration of ASMC. We first demonstrated that ASMC constitutively express CCR3 at both mRNA and protein levels. Interestingly, TNF-α increases ASMC surface expression of CCR3 from 33 to 74%. Furthermore, using FACS analysis, we found that ASMC CCR3 is expressed to a greater degree in asthmatic vs control subjects (95 vs 75%). Functionality of the receptor was demonstrated by calcium assay; the addition of CCR3 ligand eotaxin to ASMC resulted in an increase in intracellular calcium production. Interestingly, ASMC was seen to demonstrate a positive chemotactic response to eotaxin. Indeed, ASMC significantly migrated toward 100 ng/ml eotaxin (2.2-fold increase, compared with control). In conclusion, the expression of CCR3 by ASMC is increased in asthmatics, and our data show that a CCR3 ligand such as eotaxin induces migration of ASMC in vitro. These results may suggest that eotaxin could be involved in the increased smooth muscle mass observed in asthmatics through the activation of CCR3.

Our data indicate that TNF-␣ upregulates IL-17BR mainly through nuclear factor-B as assessed with the IB kinase 2 inhibitor AS-602868. In addition, both IFN-␥ and dexamethasone are able to antagonize a TNF-␣-induced IL-17BR increase in mRNA expression. The mitogen-activated protein kinase kinase inhibitor U0126 totally reversed the inhibition observed with IFN-␥, suggesting the involvement of the extracellular signal-regulated kinase pathway in this effect. In addition, on stimulation with IL-17E, airway smooth muscle cells increase their expression of ECM components, namely procollagen-␣I and lumican mRNA. Furthermore, immunohistochemical analysis of biopsies from asthmatic subjects reveals that this receptor is abundant in smooth muscle layers. This is the first report showing IL-17BR receptor in structural cells of the airways. Our results suggest a potential proremodeling effect of IL-17E on airway smooth muscle cells through the induction of ECM and that its receptor is upregulated by proinflammatory conditions.

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Animal studies show that the (ϩ)insert isoform is predominantly expressed in rapidly contracting phasic muscle and the (Ϫ)insert isoform is mostly found in slowly contracting tonic muscle. The expression of the (ϩ)insert isoform has never been demonstrated in human smooth muscle. We hypothesized that the (ϩ)insert isoform is present in humans and that its expression is commensurate with the organ's functional requirements. We report, for the first time, the sequence of the human (ϩ)insert isoform and quantification of its expression by real-time PCR and Western blot analysis in a panel of human organs. The (ϩ)insert isoform mRNA and protein expression levels are significantly greater in small intestine compared with all organs studied except for trachea and are significantly greater in trachea compared with uterus and aorta. To assess the functional significance of this differential myosin isoform expression between organs, we measured the rate of actin filament movement ( max) when propelled by myosin purified from rat organs, because the rat and human inserts are identical and their remaining sequences show 93% identity. max exhibits a rank correlation from the most tonic to the most phasic organ. The selective expression of the (ϩ)insert isoform observed among human organs suggests that it is an important determinant of tissue shortening velocity. A differential expression of the (ϩ)insert isoform could also account for altered contractile properties observed in human pathology. phasic and tonic smooth muscle; real-time polymerase chain reaction; in vitro motility assay SMOOTH MUSCLE IS FOUND in all hollow organs of the mammalian organism, and its function ranges from tone maintenance to content propulsion. Many studies point to the smooth muscle myosin heavy chain (SMMHC) as an important element contributing to these diverse contractile properties (see Ref. 16 for review). SMMHC is made up of a globular head, containing an ATPase site and an actin binding domain, and an ␣-helical tail to which regulatory and essential light chains are bound. SMMHC isoforms are generated by alternative splicing from a single gene (1,9,33,46). Four SMMHC isoforms have been described in various animal species. The first two isoforms identified differ in the carboxy terminus by distinct sequences of 43 (SM1) or 9 (SM2) amino acids (2, 33). The next two isoforms differ in the amino terminus by the presence [(ϩ)insert] or absence [(Ϫ)insert] of a seven-amino acid insert in a surface loop above the ATPase site (18, 46). The (ϩ) and (Ϫ)insert isoforms are also commonly referred to as SM-B and SM-A, respectively. All combinations of these isoforms are possible, i.e., (ϩ)insert SM1, (ϩ)insert SM2, (Ϫ)insert SM1, and (Ϫ)insert SM2. No difference in molecular mechanics has been observed between SM1 and SM2, but, as shown with myosin constructs, the sole presence of the amino-terminal insert doubles the actin-activated ATPase activity and the rate of actin filament movement ( max ) in the in vitro motility assay (17,22,36). Because of t...

C-C chemokines such as CCL11, CCL5, and CCL3 are central mediators in the pathogenesis of asthma. They are mainly associated with the recruitment and the activation of specific inflammatory cells, such as eosinophils, lymphocytes, and neutrophils. It has recently been shown that they can also activate structural cells, such as airway smooth muscle and epithelial cells. The aims of this study were to examine the expression of the CCL3 receptor, CCR1, on human airway smooth muscle cells (ASMC) and to document the regulation of this receptor by cytokines involved in asthma pathogenesis. We first demonstrated that CCR1 mRNA is increased in the airways of asthmatic vs control subjects and showed for the first time that ASMC express CCR1 mRNA and protein, both in vitro and in vivo. Calcium mobilization by CCR1 ligands confirmed its functionality on ASMC. Stimulation of ASMC with TNF-α and, to a lesser extent, IFN-γ resulted in an up-regulation of CCR1 expression, which was totally suppressed by both dexamethasone or mithramycin. Taken together, our data suggest that CCR1 might be involved in the pathogenesis of asthma, through the activation of ASMC by its ligands.

Our results show that ASMC are a potent source of CCL15 in the airways and may directly participate in the recruitment of inflammatory cells to asthmatic airways. Targeting the production of CCL15 by ASMC might reduce the inflammatory response within the airways of asthmatic patients.