Airway eosinophilia is a prominent feature of asthma that is believed to be mediated in part through the expression of specific chemokines such as eotaxin, a potent eosinophil chemoattractant that is highly expressed by epithelial cells and inflammatory cells in asthmatic airways. Airway smooth muscle (ASM) has been identified as a potential source of cytokines and chemokines. The aim of the present study was to examine the capacity of human ASM to express eotaxin. We demonstrate that airway myocytes constitutively express eotaxin mRNA as detected by RT-PCR. Treatment of ASM for 24 h with different concentrations of TNF-alpha and IL-1beta alone or in combination enhanced the accumulation of eotaxin transcripts. Maximal mRNA expression of eotaxin was shown at 12 and 24 h following IL-1beta and TNF-alpha stimulation, respectively. The presence of immunoreactive eotaxin was demonstrated by immunocytochemistry, and constitutive and cytokine-stimulated release of eotaxin was confirmed in ASM culture supernatants by ELISA. Strong signals for eotaxin mRNA and immunoreactivity were observed in vivo in smooth muscle in asthmatic airways. In addition, chemotaxis assays demonstrated the presence of chemoattractant activity for eosinophils and PBMCs in ASM supernatants. The chemotactic responses of eosinophils were partly inhibited with antibodies directed against eotaxin or RANTES, and a combined blockade of both chemokines causes > 70% inhibition of eosinophil chemotaxis. The results of this study suggest that ASM may contribute to airway inflammation in asthma through the production and release of eotaxin.

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.

The involvement of chemokines in eosinophil recruitment during inflammation and allergic reactions is well established. However, a functional role for chemokines in eosinophil differentiation has not been investigated. Using in situ RT-PCR, immunostaining, and flow cytometric analysis, we report that human CD34+ cord blood progenitor cells contain CCR3 mRNA and protein. Activation of CD34+ progenitor cells under conditions that promote Th2 type differentiation up-regulated surface expression of the CCR3. In contrast, activation with IL-12 and IFN-γ resulted in a significant decrease in the expression of CCR3. Eotaxin induced Ca2+ mobilization in CD34+ progenitor cells, which could explain the in vitro and in vivo chemotactic responsiveness to eotaxin. We also found that eotaxin induced the differentiation of eosinophils from cord blood CD34+ progenitor cells. The largest number of mature eosinophils was found in cultures containing eotaxin and IL-5. The addition of neutralizing anti-IL-3, anti-IL-5, and anti-GM-CSF Abs to culture medium demonstrated that the differentiation of eosinophils in the presence of eotaxin was IL-3-, IL-5-, and GM-CSF-independent. These results could explain how CD34+ progenitor cells accumulate and persist in the airways and peripheral blood of patients with asthma and highlight an alternative mechanism by which blood and tissue eosinophilia might occur in the absence of IL-5.

Several reports suggest that activated airway smooth muscle (ASM) cells are capable of generating various proinflammatory mediators, including cytokines and chemokines. However, little is known about the mechanism involved in this process. In this regard, we have examined the expression and the role of the high affinity IgE receptor (FcεRI) by ASM cells. Human ASM cells were found to constitutively express transcripts coding for α, β, and γ subunits of FcεRI. Flow cytometry and Western blot analysis confirmed the expression of FcεRI α-chain protein. Interestingly, FcεRI α-chain immunoreactivity was also demonstrated in smooth muscle within bronchial biopsies of asthmatic subjects. Cross-linking of FcεRI induced mobilization of free calcium in ASM cells, one of the critical signals to trigger smooth muscle contraction. Furthermore, cultured ASM cells released IL-4, IL-13, IL-5, and eotaxin but not IFN-γ, when sensitized with IgE followed by anti-IgE Ab cross-linking. The addition of anti-FcεRI α-chain Abs directed against IgE binding site inhibited this release. Taken together, these results suggest a potential new and important mechanism by which ASM cells may participate in airway inflammation and bronchoconstriction associated with allergic 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.

The existence of a functional receptor for secretory component (SC) on the eosinophil membrane might explain the preferential degranulation induced by secretory IgA (sIgA) when compared to serum IgA. Indeed, flow cytometry analysis revealed that purified human SC could bind to a subpopulation (4-59%) of blood eosinophils purified from 19 patients with eosinophilia. Binding of radiolabeled human SC could be competitively inhibited using unlabeled SC or secretory IgA but not with serum IgA or IgG. Immunoprecipitation and immunosorbent chromatography using human SC revealed the presence of a major component at 15 kDa in eosinophil extracts as well as in culture supernatants but not in neutrophils. The 15-kDa protein eluted from the human SC immunosorbent was able to bind to SC or to sIgA but not to serum IgA. Eosinophils preincubated with human SC or sIgA released eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) after addition of anti-SC or anti-IgA monoclonal antibody as respective cross-linking reagents. These results indicated that binding of free or complexed SC to human eosinophils could induce eosinophil degranulation. Furthermore, the dose-dependent inhibition by SC of mediator release induced by sIgA but not by serum IgA, suggested that the receptor for SC could be involved in the preferential degranulation mediated by sIgA. These results indicate a novel pathway of eosinophil activation and its potential involvement in mucosal immunity, particularly in inflammatory diseases associated with infiltration of eosinophils and the enhanced production of sIgA.