Mast cell disease

Mast cell microlocalization within the airway smooth muscle bundle is an important determinant of the asthmatic phenotype. We hypothesized that mast cells migrate toward airway smooth muscle in response to smooth muscle-derived chemokines. In this study, we investigated (1) chemokine receptor expression by mast cells in the airway smooth muscle bundle in bronchial biopsies from subjects with asthma using immunohistology, (2) the concentration of chemokines in supernatants from stimulated ex vivo airway smooth muscle cells from subjects with and without asthma measured by enzyme-linked immunosorbent assay, and (3) mast cell migration toward these supernatants using chemotaxis assays. We found that CXCR3 was the most abundantly expressed chemokine receptor on human lung mast cells in the airway smooth muscle in asthma and was expressed by 100% of these mast cells compared with 47% of mast cells in the submucosa. Human lung mast cell migration was induced by airway smooth muscle cultures predominantly through activation of CXCR3. Most importantly, CXCL10 was expressed preferentially by asthmatic airway smooth muscle in bronchial biopsies and ex vivo cells compared with those from healthy control subjects. These results suggest that inhibition of the CXCL10/CXCR3 axis offers a novel target for the treatment of asthma.

Keywords: airway smooth muscle; chemokine receptors; chemokines; chemotaxis; mast cells

Asthma is a common disease and remains a significant cause of morbidity and mortality worldwide. It affects 10% of children and 5% of adults, and its prevalence continues to rise (1). It is characterized by the presence of variable airflow obstruction, airway hyperresponsiveness, and an airway inflammatory response characterized by eosinophilic airway inflammation, Th2 cytokine expression, and reticular basement membrane thickening, features that have been implicated in the development of the disordered airway physiology (2, 3). We have demonstrated recently that many of the immunopathologic features of asthma are also observed in the airways of patients with eosinophilic bronchitis (4-6), a condition that presents with a corticosteroid-responsive chronic cough, but which, in contrast to asthma, is not associated with airway hyperresponsiveness or airflow obstruction (7). The only-but striking-difference we found in the pathology of these two diseases was the infiltration of asthmatic airway smooth muscle (ASM) by mast cells, suggesting that this is a major determinant of the asthmatic phenotype (5). This hypothesis is biologically plausible because mast cells secrete many autacoids, cytokines, and proteases, which have the ability to induce ASM proliferation, hyperresponsiveness, and contraction (8).

A key question is how mast cells accumulate in the airway smooth muscle in asthma. This is a fundamental question, because if this accumulation can be repressed, then the disease may be attenuated. It is likely that this selective mast cell recruitment will require a chemotactic signal arising from the ASM, suggesting that a primary abnormality in asthma may lie within the ASM itself or in its response to an airway insult. The C-C and C-X-C chemokines in particular are attractive candidates as mast cell chemoattractants. These ubiquitous structurally related peptides mediate the chemotaxis of many cell types, and we have found that the chemokine receptors CCR3, CXCR1, CXCR3, and CXCR4 were expressed highly by ex vivo human lung mast cells (HLMC) (9). Whether ligands for these receptors are produced by asthmatic compared with normal ASM is unknown.

In this study, we have tested our hypothesis that human lung mast cells migrate in response to chemokines secreted by ASM. Using a number of approaches, we have defined a major and novel chemotactic mechanism for the recruitment of HLMC to the asthmatic ASM compartment. Some of the results of these studies have been previously reported in the form of an abstract (10).

METHODS

Subjects

Sixteen subjects with asthma and 14 normal control subjects were recruited from Leicester, United Kingdom, and Sydney, Australia, and underwent bronchoscopy. (Further details of the subjects' characteristics are available in the online supplement.) Biopsies from eight of the subjects with asthma and five normal control subjects had been included in a previous study (5), but sections from these biopsies were recut and stained. The study was approved by the Leicestershire and Sydney Ethics Committees, and patients gave written informed consent.

Mast Cell Chemokine Receptor and ASM Chemokine Expression

Bronchial mucosal biopsies from 10 subjects with asthma and 7 normal control subjects, recruited from Leicester, were embedded in glycolmethacrylate (Polysciences, Northampton, UK) (11). Seven subjects with asthma and six normal control subjects had assessable ASM (5). Colocalization of chemokine receptors with mast cells was assessed in the bronchial submucosa and ASM by staining sequential sections (12) with tryptase mAb (Dako, Cambridge, UK) and chemokine receptors: mouse mAb CCR1, 2, 4, 5, and 6; CXCR1, 2, 3, 4, 5, and 6 (R&D Systems, Abingdon, UK); CCR3, 7, 9, and 10 (gift from Millennium, Cambridge, MA) and rabbit polyclonal antibodies CCR8 (AMS Biotechnology, Oxfordshire, UK) and CX^sub 3^CR1 (Chemicon, Hampshire, UK) with appropriate isotype controls. Further sections from six subjects in each group with assessable ASM were available and were stained for the chemokines CXCL10 (BD Biosciences, Oxford, UK) and CXCL12 (R&D). Details on the immunohistochemistry methods are available in the online supplement.

HLMC and ASM Culture

The HLMC were isolated in Leicester and all but one asthmatic ASM cell line were isolated in Sydney. HLMC from normal lung obtained at surgery for carcinoma and ASM bundles in bronchial biopsies were isolated and cultured as previously described (13, 14). ASM cells (passage 3-7) from subjects with asthma (n = 7) and normal control subjects (n = 7) were plated into six-well plates (9.6 × 10^sup 4^ cells/2 ml Dulbecco's modified Eagle medium, 10% fetal calf serum), grown for 1 week, then growth was arrested for 48 hours with serum-deprived medium and stimulated with tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interferon (IFN)-γ (R&D; 10 ng/ml of each) for 24 hours. The supcrnatants were removed and stored at -80°C for later experiments. Cell counts were derived for each well.

Chemokine Concentration in ASM Supernatants

The concentration of CCL11 (Eotaxin), CXCL8 (IL-8), CXCL9 (Mig), CXCL10 (IP-10), CXCL11 (ITAC), and CXCL12 (SDF-1α) were measured in the ASM supernatants by enzyme-linked immunosorbent assay (R&D and BD).

HLMC Chemotaxis

Chemotaxis assays were performed as previously described (9). To assess the role of individual chemokine receptors or chemokines in HLMC migration to stimulated ASM supernatant, we preincubated the cells with receptor-blocking antibodies to CCR3 and CXCR1, 3, or 4, or the supernatants with CXCL10 neutralizing antibody or isotype control (R&D). Further details are available in the online supplement.

Statistical Analysis

Statistical analysis was performed using MINITAB13.31 (Minitab Ltd., Coventry, UK). The number of mast cells and cells that expressed chemokine receptors and the proportion of these cells colocalized to mast cells in bronchial biopsies were not normally distributed and thus were described as the median (interquartile range [IQR]). Chemokine concentrations were log normally distributed and were described as the geometric mean (log SEM). HLMC migration was expressed as the mean (SEM) fold difference in migration compared with control. Nonparametric data were analyzed by Mann-Whitney test and parametric data by t tests. A value of p

RESULTS

Chemokine Receptor Expression by Mast Cells in ASM Bundles

The number of tryptase^sup +^ mast cells in the ASM bundle (cells/mm^sup 2^) was increased in the subjects with asthma (median 12.5, IQR 11.5, n = 7) compared with normal controls (median 0, IQR 0.8) (p = 0.004, n = 6). CXCR3 was the chemokine receptor most abundantly expressed by cells within the ASM, with a marked increase in the number of CXCR3^sup +^ cells/mm^sup 2^ in the ASM of subjects with asthma (median 12.5, IQR 10.8) compared with normal control subjects (median 0, IQR 0.8) (p

We have previously reported that, in addition to CXCR3, the chemokine receptors CCR3, CXCR1, and CXCR4 were also highly expressed by ex vivo HLMC (9). In the current study, we found that all of these chemokine receptors were expressed by some cells in the bronchial submucosa. However, CCR3 was the only other chemokine receptor expressed by mast cells within the ASM, but was only identified in one of the subjects with asthma.

CXCL10 Expression by ASM

CXCL10 was expressed by ASM in the bronchial biopsies in three of six subjects with asthma and zero of six normal control subjects (p = 0.046) (Figures 4A-4C). CXCL12 was not expressed by ASM in the bronchial biopsies, but was highly expressed in the epithelium in both subjects with asthma and normal control subjects (Figures 4D and 4E). After activation with TNF-α, IFN-γ, and IL-1β (10 ng/ml of each), the CXCR3 ligand CXCL10 (IP-10) geometric mean (log SEM) concentration in ASM supernatants was significantly higher in those subjects with asthma (n = 7)-28.3 (0.2) ng/ml/10^sup 5^ cells-compared with normal control subjects (n = 7)-3.6 (0.3) ng/ml/105 cells (p = 0.014) (Figure 5). The concentration of CXCL10 (IP-10) in the supernatants from subjects with asthma was higher than the other CXCR3 ligands CXCL9 (Mig) or CXCL11 (ITAC) (p = 0.028). There were no differences in the concentration of CXCL11 (ITAC), CXCL9 (Mig), and the CCR3, CXCR1, and CXCR4 ligands CCL11 (eotaxin), CXCL8 (IL-8), and CXCL12 (SDF1α), respectively, in stimulated ASM supernatants from subjects with asthma compared with normal control subjects (Figure 6). There were no differences in the concentration of chemokines released by stimulated ex vivo ASM from subjects with asthma treated with inhaled corticosteroids compared with those who were corticosteroid naive (data not shown).

Asthmatic ASM Demonstrates Enhanced Chemotactic Activity for HLMC via Release of CXCL10

Activated ASM supernatant from subjects with asthma (n = 7) exhibited chemotactic activity for purified HLMC (3.3-fold increase compared with medium alone; 95% CI, 2.8-3.8; p

DISCUSSION

The microlocalization of mast cells within the ASM is a critical step in the development of the asthmatic phenotype (5). We describe for the first time the potential mechanisms by which HLMC are recruited to this tissue compartment in asthma. We have made several novel and important observations. First, CXCR3 was the most abundantly expressed chemokine receptor on human lung mast cells within the ASM, and its expression was increased on mast cells within the ASM compared with those in the bronchial submucosa. Second, HLMC migration was induced by ASM cultures predominantly through activation of CXCR3. Third, CXCL10 was expressed preferentially by asthmatic ASM in bronchial biopsies and by ex vivo ASM cells compared with those from healthy controls. These observations taken together indicate that, in asthma, the interaction between ASM-derived CXCL10 (IP-10) and mast cell-expressed CXCR3 may be the dominant pathway facilitating the migration of mast cells into the ASM bundles.

The marked enrichment of CXCR3^sup +^ mast cells in asthmatic ASM suggests that activation of this receptor is important for mast cell recruitment. This argument is made all the more compelling by the observations that, in vivo, there was increased expression of CXCL10 by ASM in subjects with asthma and that, ex vivo, activated asthmatic ASM conditioned medium produced significantly greater HLMC chemotaxis than that from normal subjects, an effect largely accounted for by the increased production of CXCL10 (IP-10). Thus CXCL10 was produced by asthmatic ASM in concentrations, which induce HLMC chemotaxis and the chemoattractant activity of asthmatic ASM-conditioned medium was largely abolished by blocking CXCR3. The other CXCR3 ligands CXCL11 (ITAC) and CXCL9 (Mig) were also released from stimulated ASM, but in much lower quantities than CXCL10 (IP-10), and HLMC migration to the stimulated asthmatic ASM supernatants was inhibited by specifically blocking CXCL10, suggesting that CXCL10 (IP-10) is the dominant CXCR3 ligand released by asthmatic ASM. There is a precedent for the differential expression of CXCR3 ligands in that vascular smooth muscle preferentially expresses CXCL10 (IP-10) in atherosclerosis (15).

In addition to CXCR3, we have previously shown that HLMC expressed the chemokine receptors CCR3, CXCR1, and CXCR4 in more than 10% of cells (9). However, in the bronchial biopsies, mast cells in the ASM bundles in asthma did not express CXCR1 or CXCR4 and only a very small proportion expressed CCR3. Stimulated ASM released ligands for each of these chemokine receptors. CXCL12 (SDF-1α) and CXCL8 (IL-8) have been shown to be chemotactic for cord blood-derived mast cells (16-18) and HLMC (9). We found that blocking these receptors inhibited mast cell migration, but to a lesser extent than for CXCR3 blockade. Thus CXCL12 (SDF-1α) and CXCL8 (IL-8) also contribute to mast cell migration to ASM. Blocking CCR3 did not affect mast cell migration even though CCL11 (eotaxin) was expressed at high concentrations and is known to be chemotactic for HLMC (9, 19, 20). However, CXCL10 (IP-10) is a natural antagonist for CCL11 (eotaxin) (21) and may therefore inhibit CCL11 (eotaxin)-mediated migration.

Although we found that activation of CXCR3 was the most important mechanism in promoting HLMC migration, blocking CXCR3 incompletely inhibited chemotaxis. Therefore, other chemotaxins that are not chemokines may play an additional, albeit minor role, in HLMC migration. For example, using the poorly differentiated human mast cell line HMC-1, it has been shown recently that activated ASM from normal subjects induced mast cell chemotaxis after the production of transforming growth factor β1 and stem cell factor (22). It remains a possibility that it is mast cell precursors that are recruited by the ASM rather than mature airway cells, but this is a far more difficult question to address because their identification and purification for chemotaxis assays is not straightforward. Nevertheless, expression of CXCR3 on human bone marrow-derived mast cells is low (9), suggesting that expression becomes prominent after the cells are in the tissue.

Asthma is generally regarded as a Th2-driven disease, although there is sufficient evidence to suggest the immunology is far more complex and that Th1 cytokines play a role (23, 24). Indeed, CXCL10 (IP-10) is regarded as a marker of Th1 activity because its expression is induced by IFN-γ. Our view that CXCL10 (IP-10) is important in asthma is consistent with the observations that CXCL10 (IP-10) concentrations were elevated in the bronchoalveolar lavage of patients with stable asthma, and its mRNA and protein expression were increased in the asthmatic bronchial mucosa when compared with normal subjects (25). In addition, after segmental allergen challenge in subjects with asthma, there was a marked increase in broncho-alveolar lavage concentration of CXCL10 (IP-10), but not CXCL9 (Mig) or CXCL11 (ITAC) (26, 27). Similarly, in a mouse model of asthma, CXCL10 (IP-10) expression increased after allergen challenge, and CXCL10 (IP-10) transgenic mice develop a Th2 inflammatory response and airway hyperresponsiveness (28). That ASM secretes CXCL10 (IP-10) is not only of interest with respect to this study, but raises several intriguing possibilities relating to ASM function in asthma. For example, is CXCL10 (IP-10) expression increased during viral infection, and, if so, does further mast cell migration into the ASM contribute to asthma exacerbations? This is certainly plausible because rhinovirus, a common cause of exacerbations, induces lymphocyte activation with increased release of IFN-γ in an in vitro model (29), and, more importantly, during episodes of upper respiratory infections, children with virus-induced wheezing had higher concentrations of IFN-γ than did children without wheeze (30). In addition to activation by IFN-γ, expression of CXCL10 (IP-10) is enhanced in several cell types, including ASM, after exposure to TNF-α alone (31, 32) and after activation with TNF-α and or IFN-γ in combination with the Th2 cytokine IL-4 (32, 33). Because there is increased expression of TNF-α (34) and IL-4 (12, 35) in asthmatic airways, this provides an alternative pathway for the upregulation of CXCL10 in this disease.

In bronchial biopsies, cells other than mast cells expressed CXCR3 but were not present in the ASM. This is probably because cell recruitment is a complex process and several conditions need to be met for cell migration, such as the composition of the extracellular matrix (36). Interestingly, the mast cell protease chymase, which is present in most ASM mast cells, degrades human ASM pericellular matrix and inhibits T-cell adhesion to ASM (37). This might therefore contribute to the paucity of T cells within this structure in asthma (5).

One possible criticism of our findings is that our key observation that CXCL10 (IP-10) expression was increased in ASM from subjects with asthma compared with normal subjects was made using ex vivo primary cultures. However, this observation was supported by evidence of increased expression of CXCL10 (IP-10) by ASM in bronchial biopsies in subjects with asthma. Whether ASM cultures maintain an asthmatic phenotype has been questioned, but other important differences between cultured cells from subjects with and without asthma have been reported, such as increased rate of proliferation, increased connective tissue growth factor release in response to transforming growth factor-β activation (14, 38, 39), and decreased prostaglandin E^sub 2^ (PGE^sub 2^) production (40). Therefore, ASM cultured from individuals with asthma differs from ASM from normal subjects with respect to both its synthetic and proliferative responses. HLMC were derived from subjects undergoing lung resection and did not include subjects with asthma. However, our findings in HLMC were supported by chemokine receptor expression by mast cells in bronchial biopsies from well-characterized subjects with asthma. Thus we believe that chemokine receptor expression by HLMC from subjects with asthma is likely to be similar.

In summary, our findings suggest that mast cell microlocalization to the ASM bundles in asthma is predominately mediated by activation of mast cell CXCR3 by ASM-derived CXCL10 (IP-10). Specifically targeting this pathway of mast cell migration into the ASM bundles may provide a novel effective treatment for asthma.

Acknowledgment: The authors are grateful to Millennium for kindly providing some of the chemokine receptor antibodies. They would like to thank Drs. G. King and M. Baraket for subject recruitment and bronchoscopy and Dr. P. Johnson for overseeing the isolation of smooth muscle from bronchial biopsy samples in Australia. The authors thank the theatre and pathology staff of the Sydney metropolitan teaching hospitals and University Hospitals of Leicester for the supply of human lung tissue and the collaborative effort of the cardiopulmonary transplant team at St. Vincent's Hospital.

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Christopher E. Brightling, Alaina J. Ammit, Davinder Kaur, Judith L. Black, Andrew J. Wardlaw, J. Margaret Hughes, and Peter Bradding

Institute for Lung Health, Department of Infection, Inflammation and Immunity, Leicester-Warwick Medical School and University Hospitals of Leicester, Leicester, United Kingdom; and Respiratory Research Croup, Faculty of Pharmacy and Department of Pharmacology, University of Sydney, Australia

(Received in original form September 16, 2004; accepted in final form January 25, 2005)

Supported by Asthma UK, DoH UK Clinician Scientist Scheme, NHMRC Australia.

Correspondence and requests for reprints should be addressed to C. E. Brightling, Ph.D., University Hospitals of Leicester, Groby Road, Leicester LE3 9QP, UK. E-mail: ceb17@le.ac.uk

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Am J Respir Crit Care Med Vol 171. pp 1103-1108, 2005

Originally Published in Press as DOI: 10.1164/rccm.200409-1220OC on February 4, 2005

Internet address: www.atsjournals.org

Conflict of Interest Statement: C.E.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.J.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; D.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.L.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.J.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; P.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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