Chronic airways diseases, including asthma, are associated with an increased airway smooth muscle (ASM) mass, which may contribute to chronic airway hyperresponsiveness. Increased muscle mass is due, in part, to increased ASM proliferation, although the precise molecular mechanisms for this response are not completely clear. Caveolae, which are abundant in smooth muscle cells, are membrane microdomains where receptors and signaling effectors can be sequestered. We hypothesized that caveolae and caveolin-1 play an important regulatory role in ASM proliferation. Therefore, we investigated their role in p42/p44 MAPK signaling and proliferation using human ASM cell lines. Disruption of caveolae using methyl-beta-cyclodextrin and small interfering (si)RNA-knockdown of caveolin-1 caused spontaneous p42/p44 MAPK activation; additionally, caveolin-1 siRNA induced ASM proliferation in mitogen deficient conditions, suggesting a key role for caveolae and caveolin-1 in maintaining quiescence. Moreover, caveolin-1 accumulates twofold in myocytes induced to a contractile phenotype compared with proliferating ASM cells. Caveolin-1 siRNA failed to increase PDGF-induced p42/p44 MAPK activation and cell proliferation, however, indicating that PDGF stimulation actively reversed the antimitogenic control by caveolin-1. Notably, the PDGF induced loss of antimitogenic control by caveolin-1 coincided with a marked increase in caveolin-1 phosphorylation. Furthermore, the strong association of PDGF receptor-beta with caveolin-1 that exists in quiescent cells was rapidly and markedly reduced with agonist addition. This suggests a dynamic relationship in which mitogen stimulation actively reverses caveolin-1 suppression of p42/p44 MAPK signal transduction. As such, caveolae and caveolin-1 coordinate PDGF receptor signaling, leading to myocyte proliferation, and inhibit constitutive activity of p42/p44 MAPK to sustain cell quiescence.

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Thymic stromal lymphopoietin (TSLP) is a novel cytokine that triggers dendritic cell-mediated T helper (Th)-2 inflammatory responses. Previous studies have demonstrated that human airway smooth muscle cells (HASMC) play a critical role in initiating or perpetuating airway inflammation by producing chemokines and cytokines. In this study, we first evaluated the expression of TSLP in primary HASMC and investigated how proinflammatory cytokines (TNF-alpha and IL-1beta) and Th-2 cytokines (IL-4, IL-9) regulate TSLP production from HASMC. TSLP mRNA and protein were assessed by real-time RT-PCR, ELISA, and immunofluorescence from primary HASMC cultures. Primary HASMC express constitutive level of TSLP. Incubation of HASMC with IL-1 or TNF-alpha resulted in a significant increase of TSLP mRNA and protein release from HASMC. Furthermore, combination of IL-1beta and TNF-alpha has an additive effect on TSLP release by HASMC. Primary HASMC pretreated with inhibitors of p38 or p42/p44 ERK MAPK, but not phosphatidylinositol 3-kinase, showed a significant decrease in TSLP release on IL-1beta and TNF-alpha treatment. Furthermore, TSLP immunoreactivity was present in ASM bundle from chronic obstructive pulmonary disease (COPD) and to lesser degree in normal subjects. Taken together, our data provide the first evidence of IL-1beta- and TNF-alpha-induced TSLP expression in HASMC via (p38, p42/p44) MAPK signaling pathways. Our results raise the possibility that HASMC may play a role in COPD airway inflammation via TSLP-dependent pathway.

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Parasitic infections are often associated with eosinophilia and high levels of immunoglobulin E (IgE). This observation has led to speculation that eosinophils and IgE may act together in the immune response against parasites. In support of this hypothesis, IgE and eosinophils participate in cytotoxic reactions directed against Schistosoma mansoni larvae in vitro. Furthermore, epidemiological studies have shown an inverse correlation between levels of specific IgE and rates of infection with Schistosoma. The low-affinity IgE receptor (Fc epsilon RII/CD23) was first incriminated in eosinophil activation. The fact that the high-affinity IgE receptor (Fc epsilon RI) is not only expressed on mast cells and basophils but also on Langerhans cells led us to investigate the presence of Fc epsilon RI on eosinophils. Here we show that Fc epsilon RI is expressed on eosinophils from hypereosinophilic patients, is involved in eosinophil degranulation, and participates in eosinophil-mediated cytotoxicity against S. mansoni. Our results indicate that Fc epsilon RI may play a major part in immune defence against parasites.

Polymorphonuclear neutrophils (PMNs) are important effector cells in host defense and the inflammatory response to antigen. The involvement of PMNs in inflammation is mediated mainly by the Fc receptor family, including IgE receptors. Recently, PMNs were shown to express two IgE receptors (CD23/Fc epsilon RII and galectin-3). In allergic diseases, the dominant role of IgE has been mainly ascribed to its high-affinity receptor, Fc epsilon RI. We have examined the expression of Fc epsilon RI by PMNS: mRNA and cell surface expression of Fc epsilon RI alpha chain was identified on PMNs from asthmatic subjects. Furthermore, preincubation with human IgE Fc fragment blocks completely the binding of anti-Fc epsilon RI alpha chain (mAb15--1) to human PMNS: Conversely, preincubation of PMNs with mAb15--1 inhibits significantly the binding of IgE Fc fragment to PMNs, indicating that IgE bound to the cell surface of PMNs mainly via the Fc epsilon RI. Peripheral blood and bronchoalveolar lavage (BAL) PMNs from asthmatic subjects also express intracellular Fc epsilon RI alpha and beta chain immunoreactivity. Engagement of Fc epsilon RI induces the release of IL-8 by PMNS: Collectively, these observations provide new evidence that PMNs express the Fc epsilon RI and suggest that these cells may play a role in allergic inflammation through an IgE-dependent activation mechanism.

Platelets can be activated by IgE and are therefore involved in IgE-mediated effector mechanisms against parasites and in allergic disorders. Here we show that, besides the low-affinity IgE receptor (Fc epsilon RII/CD23), platelets express the high-affinity receptor for IgE (Fc epsilon RI). Flow cytometry analysis revealed the existence of surface Fc epsilon RI on platelets with a large heterogeneity among individual donors, and a low proportion of platelets co-expressing Fc epsilon RI and FC epsilon RII/CD23. Northern hybridization and reverse transcription polymerase chain reaction confirmed the presence of mRNA encoding the alpha, beta and gamma chains of Fc epsilon RI in platelets and in their megakaryocytic precursors. Cross-linking of Fc epsilon RI with monoclonal antibody (mAb) to alpha chain using either the whole molecule or F(ab')2 triggered platelet cytotoxicity for Schistosoma mansoni larvae. Anti-Fc epsilon RII/CD23 mAb significantly inhibited IgE- or Fc epsilon RI-mediated cytotoxicity, indicating down-regulatory effects of Fc epsilon RII/CD23 on Fc epsilon RI-dependent functions. These results demonstrate functional properties for Fc epsilon RI on platelets and indicate unsuspected interactions between the low- and the high-affinity IgE receptors.

December 22, 2006; doi:10.1152/ajplung.00306.2006.-Recent studies into the pathogenesis of airway disorders such as asthma have revealed a dynamic role for airway smooth muscle cells in the perpetuation of airway inflammation via secretion of cytokines and chemokines. In this study, we evaluated whether IL-17 could enhance IL-1␤-mediated CXCL-8 release from human airway smooth muscle cells (HASMC) and investigated the upstream and downstream signaling events regulating the induction of CXCL-8. CXCL-8 mRNA and protein induction were assessed by real-time RT-PCR and ELISA from primary HASMC cultures. HASMC transfected with site-mutated activator protein (AP)-1/NF-B CXCL-8 promoter constructs were treated with selective p38, MEK1/2, and phosphatidylinositol 3-kinase (PI3K) inhibitors to determine the importance of MAPK and PI3K signaling pathways as well as AP-1 and NF-B promoter binding sites. We demonstrate IL-17 induced and synergized with IL-1␤ to upregulate CXCL-8 mRNA and protein levels. Erk1/2 and p38 modulated IL-17 and IL-1␤ CXCL-8 promoter activity; however, IL-1␤ also activated the PI3K pathway. The synergistic response mediating CXCL-8 promoter activity was dependent on both MAPK and PI3K signal transduction pathways and required the cooperation of AP-1 and NF-B cis-acting elements upstream of the CXCL-8 gene. Collectively, our observations indicate MAPK and PI3K pathways regulate the synergy of IL-17 and IL-1␤ to enhance CXCL-8 promoter activity, mRNA induction, and protein synthesis in HASMC via the cooperative activation of AP-1 and NF-B trans-acting elements.