RESUMO
BACKGROUND: Mast cell localization within the airway smooth muscle (ASM)-bundle plays an important role in the development of airway hyper-responsiveness (AHR). Genomewide association studies implicate the 'alarmin' IL-33 in asthma, but its role in mast cell-ASM interactions is unknown. OBJECTIVES: We examined the expression and functional role of IL-33 in bronchial biopsies of patients with and without asthma, ex vivo ASM, mast cells, cocultured cells and in a mouse model system. METHODS: IL-33 protein expression was assessed in human bronchial tissue from 9 healthy controls, and 18 mild-to-moderate and 12 severe asthmatic patients by immunohistochemistry. IL-33 and ST2 mRNA and protein expression in human-derived ASM, epithelial and mast cells were assessed by qPCR, immunofluorescence and/or flow cytometry and ELISA. Functional assays were used to assess calcium signalling, wound repair, proliferation, apoptosis and contraction. AHR and inflammation were assessed in a mouse model. RESULTS: Bronchial epithelium and ASM expressed IL-33 with the latter in asthma correlating with AHR. ASM and mast cells expressed intracellular IL-33 and ST2. IL-33 stimulated mast cell IL-13 and histamine secretion independent of FcεR1 cross-linking and directly promoted ASM wound repair. Coculture of mast cells with ASM activated by IL-33 increased agonist-induced ASM contraction, and in vivo IL-33 induced AHR in a mouse cytokine installation model; both effects were IL-13 dependent. CONCLUSION: IL-33 directly promotes mast cell activation and ASM wound repair but indirectly promotes ASM contraction via upregulation of mast cell-derived IL-13. This suggests that IL-33 may present an important target to modulate mast cell-ASM crosstalk in asthma.
Assuntos
Asma/imunologia , Interleucina-13/imunologia , Interleucina-33/imunologia , Mastócitos/imunologia , Receptor Cross-Talk/imunologia , Adulto , Animais , Hiper-Reatividade Brônquica/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Músculo Liso/imunologia , Músculo Liso/metabolismo , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Activated mast cell densities are increased on the airway smooth muscle in asthma where they may modulate muscle functions and thus contribute to airway inflammation, remodelling and airflow obstruction. OBJECTIVES: To determine the effects of human lung mast cells on the secretory and proliferative functions of airway smooth muscle cells from donors with and without asthma. METHODS: Freshly isolated human lung mast cells were stimulated with IgE/anti-IgE. Culture supernatants were collected after 2 and 24 h and the mast cells lysed. The supernatants/lysates were added to serum-deprived, subconfluent airway smooth muscle cells for up to 48 h. Released chemokines and extracellular matrix were measured by ELISA, proliferation was quantified by [(3) H]-thymidine incorporation and cell counting, and intracellular signalling by phospho-arrays. RESULTS: Mast cell 2-h supernatants reduced CCL11 and increased CXCL8 and fibronectin production from both asthmatic and nonasthmatic muscle cells. Leupeptin reversed these effects. Mast cell 24-h supernatants and lysates reduced CCL11 release from both muscle cell types but increased CXCL8 release by nonasthmatic cells. The 24-h supernatants also reduced asthmatic, but not nonasthmatic, muscle cell DNA synthesis and asthmatic cell numbers over 5 days through inhibiting extracellular signal-regulated kinase (ERK) and phosphatidylinositol (PI3)-kinase pathways. However, prostaglandins, thromboxanes, IL-4 and IL-13 were not involved in reducing the proliferation. CONCLUSIONS: Mast cell proteases and newly synthesized products differentially modulated the secretory and proliferative functions of airway smooth muscle cells from donors with and without asthma. Thus, mast cells may modulate their own recruitment and airway smooth muscle functions locally in asthma.
Assuntos
Asma/imunologia , Pulmão/imunologia , Mastócitos/imunologia , Miócitos de Músculo Liso/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células , Células Cultivadas , Quimiocinas/biossíntese , Matriz Extracelular/metabolismo , Feminino , Humanos , Pulmão/citologia , Masculino , Pessoa de Meia-Idade , Transdução de Sinais , Adulto JovemRESUMO
IL-13 is a T-helper type 2 cytokine. Animal models have implicated IL-13 as a critical cytokine in the development of asthma and chronic obstructive pulmonary disease (COPD). In vitro IL-13 exerts important effects on both structural and inflammatory cells within the airway and has the capacity to drive the clinical features of airways disease. In asthma, this view is strongly supported by associations with IL-13 genetic polymorphisms and increased mRNA and protein expression in blood, sputum and bronchial submucosa. In particular, IL-13 up-regulation is associated with severe disease. Current evidence in COPD is conflicting, with some reports supporting and others refuting a role for IL-13. Early clinical trials of anti-IL-13 therapies in asthma have shown promise, and the results of further efficacy studies are eagerly awaited.
Assuntos
Asma/tratamento farmacológico , Interleucina-13/antagonistas & inibidores , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Animais , Anticorpos Monoclonais/uso terapêutico , Asma/imunologia , Brônquios/imunologia , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Humanos , Interleucina-13/genética , Interleucina-13/imunologia , Polimorfismo Genético , Doença Pulmonar Obstrutiva Crônica/imunologia , Mucosa Respiratória/imunologia , Escarro/imunologia , Regulação para CimaRESUMO
BACKGROUND: Airway smooth muscle (ASM) hyperplasia and mast cell localization within the ASM bundle are important features of asthma. The cause of this increased ASM mass is uncertain and whether it is a consequence of ASM-mast cell interactions is unknown. OBJECTIVE: We sought to investigate ASM proliferation and survival in asthma and the effects of co-culture with mast cells. METHODS: Primary ASM cultures were derived from 11 subjects with asthma and 12 non-asthmatic controls. ASM cells were cultured for up to 10 days in the presence or absence of serum either alone or in co-culture with the human mast cell line-1, unstimulated human lung mast cells (HLMC) or IgE/anti-IgE-activated HLMC. Proliferation was assessed by cell counts, CFSE assay and thymidine incorporation. Apoptosis and necrosis were analysed by Annexin V/propidium iodide staining using flow cytometry and by assessment of nuclear morphology using immunofluorescence. Mast cell activation was confirmed by the measurement of histamine release. RESULTS: Using a number of techniques, we found that ASM proliferation and survival was not significantly different between cells derived from subjects with or without asthma. Co-culture with mast cells did not affect the rate of proliferation or survival of ASM cells. CONCLUSION: Our findings do not support a role for increased airway smooth proliferation and survival as the major mechanism driving ASM hyperplasia in asthma.
Assuntos
Asma/patologia , Mastócitos/fisiologia , Músculo Liso/patologia , Idoso , Apoptose , Asma/imunologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Humanos , Imunoglobulina E/imunologia , Masculino , Mastócitos/imunologia , Pessoa de Meia-Idade , Músculo Liso/metabolismoRESUMO
BACKGROUND: Chemokine receptors play an important role in cell migration and wound repair. In asthma, CCR3 and 7 are expressed by airway smooth muscle (ASM) and CCR7 has been implicated in the development of ASM hyperplasia. The expression profile of other chemokine receptors by ASM and their function needs to be further explored. OBJECTIVE: We sought to investigate ASM chemokine receptor expression and function in asthma. METHODS: ASM cells were derived from 17 subjects with asthma and 36 non-asthmatic controls. ASM chemokine receptor expression was assessed by flow cytometry and immunofluorescence. The function of chemokine receptors expressed by more than 10% of ASM cells was investigated by intracellular calcium measurements, chemotaxis, wound healing, proliferation and survival assays. RESULTS: In addition to CCR3 and 7, CXCR1, 3 and 4 were highly expressed by ASM. These CXC chemokine receptors were functional with an increase in intracellular calcium following ligand activation and promotion of wound healing [CXCL10 (100 ng/mL) 34 +/- 2 cells/high-powered field (hpf) vs. control 29 +/- 1; P=0.03; n=8]. Spontaneous wound healing was inhibited by CXCR3 neutralizing antibody (mean difference 7 +/- 3 cells/hpf; P=0.03; n=3). CXC chemokine receptor activation did not modulate ASM chemotaxis, proliferation or survival. No differences in chemokine receptor expression or function were observed between ASM cells derived from asthmatic or non-asthmatic donors. CONCLUSIONS: Our findings suggest that the chemokine receptors CXCR1, 3 and 4 modulate some aspects of ASM function but their importance in asthma is uncertain.
Assuntos
Asma/metabolismo , Regulação da Expressão Gênica , Receptores de Quimiocinas/biossíntese , Asma/tratamento farmacológico , Asma/patologia , Cálcio/metabolismo , Movimento Celular , Quimiocina CXCL10/biossíntese , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Liso/metabolismo , Músculo Liso/patologiaRESUMO
Airway inflammation is a critical feature of the airway diseases asthma and chronic obstructive pulmonary disease (COPD). There is emerging evidence that structural cells play a key role in the development and perpetuation of the inflammatory response and are pivotal in the development of the changes in the airway structures that lead to airway remodelling. To date, little attention has been given to the localisation of inflammatory cells to airway structures or the potential interactions between these intimately located cells. However, it is likely that interactions between inflammatory and structural cells in the airway contribute enormously to the pathophysiology of asthma and COPD. Indeed, recent evidence suggests that mast cells localised to the airway smooth muscle bundle may be important in the development of airway hyperresponsiveness in asthma. In the present article, the authors aim to summarise: 1) the current understanding of which inflammatory cells locate to airway structures; 2) the proposed mechanisms that may be involved in mediating this microlocalisation; 3) the possible consequences of interactions between inflammatory and structural cells; and 4) the pressing need to investigate whether modulating these interactions is beneficial in asthma and chronic obstructive pulmonary disease.
Assuntos
Movimento Celular , Inflamação/patologia , Sistema Respiratório/fisiopatologia , HumanosRESUMO
BACKGROUND: The Th2 cytokine interleukin (IL)-13 is implicated in the development of various allergic diseases including asthma. The IL-13 receptor, IL-13Ralpha1, is expressed on most leukocytes, except T-cells. Evidence to support IL-13Ralpha1 expression on mast cells is limited. METHODS: We investigated: (i) IL-13Ralpha1 expression by human lung mast cells (HLMC); (ii) the number of IL-13Ralpha1+ bronchial submucosal mast cells in subjects with asthma and normal controls and (iii) the effect of IL-13 priming on HLMC expression of high-affinity IgE receptor (FcepsilonRI), stem cell factor receptor (CD117), histamine release, proliferation, and survival. RESULTS: Human lung mast cell expressed IL-13Ralpha1 mRNA. IL-13Ralpha1 was highly expressed on the surface HLMC (82+/-9%). Bronchial submucosal mast cell IL-13Ralpha1 expression was higher in asthmatics (86+/-2%) than normal controls (78+/-2%; P=0.015). IL-13 priming for 30 min did not increase HLMC histamine release, in the presence or absence of SCF or in response to IgE/anti-IgE activation. IL-13 priming for 5 days upregulated HLMC FcepsilonRI expression (22% increase in fluorescent intensity; P=0.003), increased histamine release following IgE/anti-IgE activation by 56% (P=0.03) and increased proliferation by 50% (P=0.003) without affecting cell survival or CD117 expression. The IL-13 specific neutralizing antibody CAT-354 inhibited all IL-13 mediated effects. CONCLUSION: Human lung mast cell express IL-13Ralpha1 and activation by IL-13 for 5 days increased FcepsilonRI expression and proliferation. Histamine release was not affected by short-term priming with IL-13, but was upregulated by priming for 5 days suggesting that this effect was mediated by the increased FcepsilonRI expression. These data support the view that targeting IL-13 may be beneficial in the treatment of asthma.