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- Materials and methods
Background: Airway smooth muscle hyperplasia is a feature of asthma, and increases with disease severity. CCR3-mediated recruitment of airway smooth muscle progenitors towards the airway smooth muscle bundle has been proposed as one possible mechanism involved in airway smooth muscle hyperplasia. Mast cells are microlocalized to the airway smooth muscle bundle and whether mast cells influence CCR3-mediated migration is uncertain.
Methods: We examined the expression of CCR3 by primary cultures of airway smooth muscle cells from asthmatics and nonasthmatics. CCR3 function was examined using intracellular calcium measurements, chemotaxis, wound healing, cell proliferation and survival assays. We investigated the recovery and function of both recombinant and airway smooth muscle-derived CCL11 (eotaxin) after co-culture with β-tryptase and human lung mast cells.
Results: Airway smooth muscle expressed CCR3. Airway smooth muscle CCR3 activation by CCL11 mediated intracellular calcium elevation, concentration-dependent migration and wound healing, but had no effect on proliferation or survival. Co-culture with β-tryptase or mast cells degraded recombinant and airway smooth muscle-derived CCL11, and β-tryptase inhibited CCL11-mediated airway smooth muscle migration.
Conclusions: CCL11 mediates airway smooth muscle migration. However co-culture with β-tryptase or mast cells degraded recombinant and airway smooth muscle-derived CCL11 and inhibited CCL11-mediated airway smooth muscle migration. Therefore these findings cast doubt on the importance of the CCL11/CCR3 axis in the development of airway smooth muscle hyperplasia in asthma.
Asthma is characterized by typical symptoms, airway hyperresponsiveness (AHR) and variable airflow obstruction, which can become fixed in severe disease. In addition there is associated airway inflammation together with features of tissue repair known as remodelling (1). The airway inflammation in asthma is typically eosinophilic with increased expression of Th2 cytokines. Importantly the number of mast cells localized within the airway smooth muscle (ASM) bundle is increased in asthma and is related to the degree of AHR (2–5).
Airway remodelling in asthma encompasses several structural changes in the airway wall including reticular lamina and basement membrane thickening, an increased number of subepithelial myofibroblasts and increased ASM mass (6). This latter feature is due to a combination of both ASM hyperplasia (7) and hypertrophy, which increases with disease severity and is associated with fixed airflow obstruction (6, 8).
The cause of ASM hyperplasia in asthma is unknown and is often attributed to increased proliferation. Indeed ASM proliferation is increased in ex vivo asthmatic ASM (9, 10), but several reports have been unable to demonstrate increased ASM proliferation in vivo (7, 8, 11). An alternative explanation is that ASM progenitors either located within the airway wall or derived from peripheral blood fibroblast progenitors (fibrocytes) (12), migrate to the ASM bundle and differentiate into ASM. In support of this view myofibroblasts expressing fibrocyte markers have been identified following ovalbumin (OVA) challenge in a mouse model of asthma and after allergen challenge in human disease (13). We have demonstrated that mast cell and ASM-derived CCL19, a CCR7 ligand, mediates ASM migration (14). The CCR3 ligand CCL11 (eotaxin) is released by ASM (15, 16) and in bronchial biopsies the intensity of expression increases with disease severity (6) suggesting that CCR3-mediated ASM migration may be important in severe asthma.
We hypothesized that the CCR3/CCL11 axis mediates ASM migration. To test our hypothesis we examined ASM CCR3 expression, function and its modulation by mast cells.
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- Materials and methods
We found that ASM cells express CCR3 and that CCL11 mediates ASM migration. Importantly we found that β-tryptase, and stimulated or unstimulated HLMC degrade recombinant and ASM-derived CCL11 and that β-tryptase inhibited CCL11-mediated ASM migration. In addition HLMC supernatants did not up- or down-regulate CCL11 mRNA expression. We found that the CCR3/CCL11 axis has the potential to mediate ASM migration and repair in asthma, but mast cell localization to the ASM-bundle is a feature of the disease and it is therefore likely that mast cells will have a profound effect on local CCL11 concentrations. Therefore, taken together our findings question the biological importance of ASM-derived CCL11 in ASM hyperplasia in asthma.
CCR3 is expressed preferentially, but not exclusively, by Th2-lymphocytes (21), basophils (22) and mast cells (17, 23). The number of CCR3+ cells is increased in bronchial biopsies in asthma (24) and CCR3 is considered an important potential therapeutic target in asthma and other allergic diseases (25). There is increasing recognition that structural cells can express functional chemokine receptors. Indeed bronchial epithelial cells (26), vascular (27) and, in one report, ASM expresses CCR3 (28). Here we confirm that ASM from both asthmatics and nonasthmatics equally express functional CCR3. However we found that CCL11 had no effect on ASM proliferation. There is a paucity of data on the effect of chemokine receptor activation on ASM proliferation with a single report that CCR7 activation did not affect proliferation (14). In contrast there are several studies of chemokine receptor activation modulating vascular smooth muscle proliferation with CCR2 (29), CXCR6 (30) and CX3CR1 (31) activation increasing proliferation and CCR3 having no effect (32). In addition we report for the first time that ASM metabolic activity and survival was not affected by CCR3 activation. In keeping with an earlier (28) report we found that CCL11 mediated increased [Ca2+]i, concentration-dependent migration and extended these findings to support a role in wound healing. The time course for the both assays was 6 h, which is not sufficient time to observe proliferation by cell counts. Therefore proliferation alone cannot explain our observations. Thus CCR3 has the potential to play a role in ASM repair and hyperplasia in asthma.
If CCR3 expression by ASM is important in asthma then the CCR3 ligands should be differentially expressed in health and disease. Primary ASM from asthmatics and nonasthmatics express CCL11, but it is contentious whether the expression is increased in disease with one report supporting (33) and two refuting that CCL11 release is increased in asthma (4, 16) and here we found increased CCL11 mRNA expression in primary ASM from asthmatics compared to nonasthmatics. However perhaps more importantly, CCL11 expression in bronchial biopsies from asthmatics increases with worsening severity of disease (6). Even though CCL11 is expressed by ASM paradoxically there is a marked paucity of eosinophils in the ASM bundle. One possible explanation for this apparent anomaly is that mast cells are microlocalized within the ASM bundle and that mast cell products degrade CCL11.This view is supported by one study suggesting that β-tryptase inactivates CCL11 (34). Here we have extended this earlier observation by examining the effect of mast cell-ASM co-culture upon CCL11 production and recovery. We confirm that β-tryptase, and for the first time show that mast cells degrade recombinant and ASM-derived CCL11. These effects were blocked by leupeptin. Importantly CCL11 mRNA expression was not affected by co-culture confirming that the reduction in CCL11 concentration in ASM supernatants is unlikely to be due to reduced synthesis. β-tryptase attenuated markedly CCL11-mediated ASM migration. These findings support the view that mast cells can modulate the functional consequences of CCL11 released by ASM.
Our observations question the role of the CCL11/CCR3 axis in the recruitment of ASM and its progenitors towards the ASM bundle in asthma. However, the control of the migration of mesenchymal cells within the airway is likely to be under the influence of a variety of mediators, which in turn are themselves controlled in terms of their synthesis, release and degradation. The concept that ASM hyperplasia may be a consequence of fibrocytes trafficking to the airway remains plausible and recent evidence supports a role for CCR7, in mediating ASM migration towards the ASM bundle (14). In contrast to CCL11, CCL19 a ligand for CCR7 is not degraded by mast cells but is expressed and released by ASM and mast cells. In addition recombinant and mast cell-derived CCL19 mediated ASM migration. Future studies that examine the role of other chemokine receptors involved in ASM migration need to consider the complexity of cellular interactions and the microenvironment relevant to the airway compartment to be studied.
One criticism of our study is that the HLMC were derived from lung resection tissue and not asthmatic subjects. Currently we are limited by our inability to isolate sufficient mast cells from the asthmatic airway. However, we feel that using HLMC that are sensitized and IgE/anti-IgE activated is likely to be reflective of asthmatic mast cells. The proportion of mast cells within the ASM-bundle is unknown, but we have estimated the ratio of mast cells : ASM cells based on our experience with the assessment of bronchial biopsies and we are confident that the range we have chosen captures the proportions of these cells in the asthmatic airway.
In conclusion, we have found that ASM express CCR3. The CCR3/CCL11 axis mediated ASM migration. Critically, recombinant and ASM-derived CCL11 was inactivated by β-tryptase and co-culture with mast cells. Mast cells are microlocalized to the ASM-bundle in asthma. Therefore our findings question the importance of ASM-expressed CCR3 in the development of ASM hyperplasia in asthma.