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1.
Front Allergy ; 4: 1296601, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38106504

RESUMO

Background: Regulatory T cells (Tregs) contribute to the maintenance of immunological tolerance. There is evidence of impaired function of these cells in people with asthma and allergy. In this study, we evaluated and compared the function of Tregs in allergic asthmatic and allergic non-asthmatic patients, both before and after low-dose allergen challenges. Methods: Three groups of subjects were recruited for a baseline evaluation: healthy controls without allergy or asthma, allergic asthmatic subjects, and allergic non-asthmatic subjects. All of them were subjected to expiratory flow measurements, sputum induction, and blood sampling. In addition, both groups of allergic subjects underwent low-dose allergen challenges. Tregs were isolated from whole blood using CD4+CD25high and CD127low staining. The suppression function was measured by flow cytometry. The levels of IL-10, IFN-γ, IgG4, IgA, and TGF-ß were measured using ELISA, and sputum Foxp3 was evaluated using qRT-PCR. Results: The suppressive function of Tregs in healthy controls was significantly higher than in allergic asthmatic or allergic non-asthmatic subjects. Repeated exposure to low doses of allergen increased the suppressor function of Tregs in allergic non-asthmatic subjects but decreased it in allergic asthmatic subjects. Foxp3 gene expression was increased in induced sputum in allergic non-asthmatic subjects, whereas it did not change in asthmatic subjects. Serum IL-10 level was decreased in allergic asthmatic subjects after allergen challenge but not in allergic non-asthmatic subjects. IFN-γ level increased upon allergen challenge in allergic non-asthmatic subjects. IgG4 level was higher in allergic non-asthmatic subjects than in allergic asthmatic subjects. Conclusions: Low-dose allergen challenges stimulate the suppressor function of Tregs in non-asthmatic allergic subjects but not in allergic asthmatic subjects.

2.
Allergy Asthma Clin Immunol ; 18(1): 24, 2022 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-35296352

RESUMO

BACKGROUND: Allergic rhinitis is a risk factor for asthma development. In asthma, fibroblast progenitors, fibrocytes, are increased in the blood and bronchial mucosa following allergen exposure. These cells may play a role in lower airways remodeling as observed in non-asthmatic subjects with allergic rhinitis. OBJECTIVE: To determine the influence of seasonal allergen exposure on blood circulating fibrocytes in allergic rhinitic subjects without asthma. METHODS: Non-asthmatic subjects with seasonal allergic rhinitis had blood sampling at baseline and at the peak of rhinitis symptoms. Cells were stained for fibrocyte markers (CD34, CD45, CXCR4, collagen I) and analyzed by flow cytometry. RESULTS: Data from 26 subjects (11M:15F) aged 29 ± 8 years were analysed. Compared to baseline, there was a significant decrease in blood fibrocytes during the pollen season in subjects sensitized to trees [median (25-75 percentile), 9.3 (6.4-20.7)% vs 7.0 (4.2-10.1)%, P = 0.007] and a significant increase in subjects sensitized to grass [12.7 (9.9-23.1)% vs 64.0 (57.6-73.6)%, P < 0.001] and ragweed [8.0 (7.4-10.8)% vs 48.2 (43.5-52.6)%, P < 0.001]. A significant decrease in CXCR4 mean fluorescence was also observed between the two visits [1814 (1261-2235) vs 1352 (814-1796) (arbitrary units), P = 0.02]. CONCLUSIONS AND CLINICAL RELEVANCE: These results contribute to document dynamic variations in blood fibrocytes' activation and migration into the airways following natural exposure to allergens. These findings may help identify one of the potential factors involved in the development of asthma in allergic rhinitic subjects.

3.
JTO Clin Res Rep ; 2(8): 100212, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34590051

RESUMO

INTRODUCTION: Genotyping circulating tumor DNA (ctDNA) is a promising noninvasive clinical tool to identify the EGFR T790M resistance mutation in patients with advanced NSCLC with resistance to EGFR inhibitors. To facilitate standardization and clinical adoption of ctDNA testing across Canada, we developed a 2-phase multicenter study to standardize T790M mutation detection using plasma ctDNA testing. METHODS: In phase 1, commercial reference standards were distributed to participating clinical laboratories, to use their existing platforms for mutation detection. Baseline performance characteristics were established using known and blinded engineered plasma samples spiked with predetermined concentrations of T790M, L858R, and exon 19 deletion variants. In phase II, peripheral blood collected from local patients with known EGFR activating mutations and progressing on treatment were assayed for the presence of EGFR variants and concordance with a clinically validated test at the reference laboratory. RESULTS: All laboratories in phase 1 detected the variants at 0.5 % and 5.0 % allele frequencies, with no false positives. In phase 2, the concordance with the reference laboratory for detection of both the primary and resistance mutation was high, with next-generation sequencing and droplet digital polymerase chain reaction exhibiting the best overall concordance. Data also suggested that the ability to detect mutations at clinically relevant limits of detection is generally not platform-specific, but rather impacted by laboratory-specific practices. CONCLUSIONS: Discrepancies among sending laboratories using the same assay suggest that laboratory-specific practices may impact performance. In addition, a negative or inconclusive ctDNA test should be followed by tumor testing when possible.

4.
Cancers (Basel) ; 13(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34439288

RESUMO

Lung adenocarcinoma (LUAD) is the most common type of lung cancer and a leading cause of cancer-related deaths worldwide. Despite important recent advances, the prognosis for LUAD patients is still unfavourable, with a 5 year-survival rate close to 15%. Improving the characterization of lung tumors is important to develop alternative options for the diagnosis and the treatment of this disease. Zinc-finger protein 768 (ZNF768) is a transcription factor that was recently shown to promote proliferation and repress senescence downstream of growth factor signaling. Although ZNF768 protein levels were found to be elevated in LUAD compared to normal lung tissue, it is currently unknown whether ZNF768 expression associates with clinicopathological features in LUAD. Here, using tissue microarrays of clinical LUAD surgical specimens collected from 364 patients, we observed that high levels of ZNF768 is a common characteristic of LUAD. We show that ZNF768 protein levels correlate with high proliferative features in LUAD, including the mitotic score and Ki-67 expression. Supporting a role for ZNF768 in promoting proliferation, we report that ZNF768 depletion severely impairs proliferation in several lung cancer cell lines in vitro. A marked decrease in the expression of key proliferative genes was observed in cancer cell lines depleted from ZNF768. Altogether, our findings support a role for ZNF768 in promoting proliferation of LUAD.

5.
J Neurosci Methods ; 345: 108903, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32777310

RESUMO

BACKGROUND: MRI-histology correlation studies of the ex vivo brain mostly employ fresh, extracted (ex situ) specimens, aldehyde fixed by immersion, which has several disadvantages for MRI scanning (e.g. deformation of the organ). A minority of studies are done ex vivo-in situ (unfixed brain), requiring an MRI scanner readily available within a few hours of the time of death. NEW METHOD: We propose a new technique, exploited by anatomists, for scanning the ex vivo brain: fixation by whole body perfusion, which implies fixation of the brain in situ. This allows scanning the brain surrounded by fluids, meninges, and skull, preserving the structural relationships of the brain in vivo. To evaluate the proposed method, five heads perfused-fixed with a saturated sodium chloride solution were employed. Three sequences were acquired on a 1.5 T MRI scanner: T1weighted, T2weighted-FLAIR, and Gradient-echo. Histology analysis included immunofluorescence for myelin basic protein and neuronal nuclei. RESULTS: All MRIs were successfully processed through a validated pipeline used with in vivo MRIs. All cases exhibited positive antigenicity for myelin and neuronal nuclei. COMPARISON WITH EXISTING METHODS: All scans registered to a standard neuroanatomical template in pseudo-Talairach space more accurately than an ex vivo-ex situ scan. The time interval to scan the ex vivo brain in situ was increased to at least 10 months. CONCLUSIONS: MRI and histology study of the ex vivo-in situ brain fixed by perfusion is an alternative approach that has important procedural and practical advantages over the two standard methods to study the ex vivo brain.


Assuntos
Técnicas Histológicas , Imageamento por Ressonância Magnética , Encéfalo/diagnóstico por imagem , Humanos
6.
Epigenomics ; 10(12): 1555-1568, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30468398

RESUMO

AIM: This study aimed to characterize DNA methylation (DNA-me) in promoter region of IL33, IL1RL1 and CCL26 in asthma and their impacts on transcriptional activity in bronchial epithelial cells (BECs). PATIENTS & METHODS: We performed bis-pyrosequencing, quantitative real-time PCR and sequencing in BECs from ten asthmatic and ten control individuals. RESULTS: We detected lower DNA-me levels of IL33 and CCL26 in asthmatic than control BECs. No correlation was found between methylation and expression levels. Interestingly, carriers of a mutative allele in a haplotype within the promoter of IL33 had a lower IL33 DNA-me level and CCL26 gene expression correlated with eosinophil count. CONCLUSION: These findings highlight the importance of investigating both epigenetic and genetic mechanisms in understanding the epithelial immune response in asthma.


Assuntos
Asma/genética , Quimiocina CCL26/genética , Metilação de DNA , Regulação da Expressão Gênica/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Interleucina-33/genética , Adulto , Alelos , Asma/imunologia , Eosinófilos/imunologia , Epigenômica , Células Epiteliais/imunologia , Feminino , Haplótipos , Humanos , Pulmão/imunologia , Masculino , Pessoa de Meia-Idade , Mutação , Regiões Promotoras Genéticas/genética , Adulto Jovem
7.
Sci Immunol ; 3(20)2018 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-29475849

RESUMO

The key factors underlying the development of allergic diseases-the propensity for a minority of individuals to develop dysfunctional responses to harmless environmental molecules-remain undefined. We report a pathway of immune counter-regulation that suppresses the development of aeroallergy and shrimp-induced anaphylaxis. In mice, signaling through epithelially expressed dectin-1 suppresses the development of type 2 immune responses through inhibition of interleukin-33 (IL-33) secretion and the subsequent recruitment of IL-13-producing innate lymphoid cells. Although this homeostatic pathway is functional in respiratory epithelial cells from healthy humans, it is dramatically impaired in epithelial cells from asthmatic and chronic rhinosinusitis patients, resulting in elevated IL-33 production. Moreover, we identify an association between a single-nucleotide polymorphism (SNP) in the dectin-1 gene loci and reduced pulmonary function in two cohorts of asthmatics. This intronic SNP is a predicted eQTL (expression quantitative trait locus) that is associated with reduced dectin-1 expression in human tissue. We identify invertebrate tropomyosin, a ubiquitous arthropod-derived molecule, as an immunobiologically relevant dectin-1 ligand that normally serves to restrain IL-33 release and dampen type 2 immunity in healthy individuals. However, invertebrate tropomyosin presented in the context of impaired dectin-1 function, as observed in allergic individuals, leads to unrestrained IL-33 secretion and skewing of immune responses toward type 2 immunity. Collectively, we uncover a previously unrecognized mechanism of protection against allergy to a conserved recognition element omnipresent in our environment.


Assuntos
Asma/imunologia , Suscetibilidade a Doenças , Lectinas Tipo C/imunologia , Tropomiosina/imunologia , Animais , Asma/induzido quimicamente , Células Cultivadas , Feminino , Humanos , Lectinas Tipo C/genética , Camundongos , Camundongos Knockout , Polimorfismo de Nucleotídeo Único/genética
8.
Am J Respir Cell Mol Biol ; 58(1): 99-106, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28853918

RESUMO

Activated bronchial epithelial cells (BEC) release various alarmins, including thymic stromal lymphopoietin (TSLP), that drive type 2 inflammation. We hypothesize that BEC-derived factors promote in situ eosinophil differentiation and maturation, a process that is driven by an IL-5-rich microenvironment in asthmatic airways. To assess the eosinophilopoietic potential of epithelial-derived factors, eosinophil/basophil colony forming units (Eo/B-CFU) were enumerated in 14-day methylcellulose cultures of blood-derived nonadherent mononuclear cells incubated with BEC supernatants (BECSN) from healthy nonatopic controls (n = 8), mild atopic asthmatics (n = 9), and severe asthmatics (n = 5). Receptor-blocking antibodies were used to evaluate the contribution of alarmins. Modulation of the mRNA expression of transcription factors that are crucial for eosinophil differentiation was evaluated. BECSN stimulated the clonogenic expansion of eosinophil progenitors in vitro. In the presence of IL-5, Eo/B-CFU numbers were significantly greater in cocultures of BESCN from severe asthmatics compared with other groups. This was attenuated in the presence of a TSLP (but not an IL-33) receptor-blocking antibody. Recombinant human TSLP (optimal at 100 pg/ml) stimulated Eo/B-CFU growth, which was significantly enhanced in the presence of IL-5 (1 ng/ml). Overnight culture of CD34+ cells with IL-5 and TSLP synergistically increased GATA-binding factor 2 and CCAAT/enhancer-binding protein α mRNA expression. The eosinophilopoietic potential of factors derived from BEC is increased in severe asthma. Our data suggest that TSLP is a key alarmin that is produced by BECs and promotes in situ eosinophilopoiesis in a type 2-rich microenvironment.


Assuntos
Asma/metabolismo , Brônquios/metabolismo , Diferenciação Celular , Citocinas/metabolismo , Eosinófilos/metabolismo , Células Epiteliais/metabolismo , Adulto , Asma/patologia , Brônquios/patologia , Microambiente Celular , Meios de Cultivo Condicionados/farmacologia , Eosinófilos/patologia , Células Epiteliais/patologia , Feminino , Humanos , Masculino , Adulto Jovem , Linfopoietina do Estroma do Timo
9.
Cell Signal ; 43: 47-54, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29242170

RESUMO

BACKGROUND: Bronchial fibroblasts are the main structural cells responsible for extracellular matrix production and turnover in lung tissue. They play a key role in airway remodelling in asthma through different cytokines including interleukin (IL-6). OBJECTIVE: To decipher IL-6 signalling in bronchial fibroblasts obtained from severe eosinophilic asthmatics compared to mild asthmatics and healthy controls. METHODS: Human bronchial fibroblasts were isolated from bronchial biopsies of mild and severe eosinophilic asthmatics and non-atopic healthy controls. IL-6 was assessed by qRT-PCR and ELISA. Phosphorylated STAT3, SHP2 and p38/MAPK were evaluated by Western blot. Chemical inhibitors for SHP2 and p38 were used. Fibroblast proliferation was evaluated by BrdU incorporation test. RESULTS: IL-6 release was significantly increased in fibroblasts from mild and severe asthmatics compared to healthy controls. Fibroblasts from severe asthmatics showed a reduced STAT3 activation compared to mild asthmatics and healthy controls. Constitutive activation of phosphatase SHP2 was found to negatively regulate IL-6 induced STAT3 phosphorylation in fibroblasts from severe asthmatics. This effect was accompanied by a decrease in fibroblast proliferation rate due to the activated p38/mitogen-activated protein kinase. SHP2 and p38/MAPK specific inhibitors (PHPS1 and SB212190) significantly induce a restoration of STAT3 phosphorylation, IL-6 target gene expression and cell proliferation. CONCLUSION: These data show dysregulated IL-6 signalling in bronchial fibroblasts derived from severe eosinophilic asthmatic subjects involving the protein tyrosine phosphatase SHP2 and p38MAPK. Collectively, our data provides new insights into the mechanisms by which bronchial fibroblasts regulate airway remodelling in severe asthma.


Assuntos
Asma/metabolismo , Asma/patologia , Interleucina-6/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Adulto , Asma/enzimologia , Proliferação de Células , Ativação Enzimática , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Modelos Biológicos , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
PLoS One ; 8(12): e81983, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24349168

RESUMO

BACKGROUND: Airway inflammation is an important characteristic of asthma and has been associated with airway remodelling and bronchial hyperreactivity. The mucosal microenvironment composed of structural cells and highly specialised extracellular matrix is able to amplify and promote inflammation. This microenvironment leads to the development and maintenance of a specific adaptive response characterized by Th2 and Th17. Bronchial fibroblasts produce multiple mediators that may play a role in maintaining and amplifying this response in asthma. OBJECTIVE: To investigate the role of bronchial fibroblasts obtained from asthmatic subjects and healthy controls in regulating Th17 response by creating a local micro-environment that promotes this response in the airways. METHODS: Human bronchial fibroblasts and CD4(+)T cells were isolated from atopic asthmatics and non-atopic healthy controls. CD4(+)T were co-cultured with bronchial fibroblasts of asthmatic subjects and healthy controls. RORc gene expression was detected by qPCR. Phosphorylated STAT-3 and RORγt were evaluated by western blots. Th17 phenotype was measured by flow cytometry. IL-22, IL17, IL-6 TGF-ß and IL1-ß were assessed by qPCR and ELISA. RESULTS: Co-culture of CD4(+)T cells with bronchial fibroblasts significantly stimulated RORc expression and induced a significant increase in Th17 cells as characterized by the percentage of IL-17(+)/CCR6(+) staining in asthmatic conditions. IL-17 and IL-22 were increased in both normal and asthmatic conditions with a significantly higher amount in asthmatics compared to controls. IL-6, IL-1ß, TGF-ß and IL-23 were significantly elevated in fibroblasts from asthmatic subjects upon co-culture with CD4(+)T cells. IL-23 stimulates IL-6 and IL-1ß expression by bronchial fibroblasts. CONCLUSION: Interaction between bronchial fibroblasts and T cells seems to promote specifically Th17 cells profile in asthma. These results suggest that cellular interaction particularly between T cells and fibroblasts may play a pivotal role in the regulation of the inflammatory response in asthma.


Assuntos
Asma/genética , Asma/patologia , Brônquios/patologia , Linfócitos T CD4-Positivos/patologia , Fibroblastos/patologia , Interleucina-17/imunologia , Adulto , Asma/imunologia , Brônquios/imunologia , Brônquios/metabolismo , Linfócitos T CD4-Positivos/imunologia , Estudos de Casos e Controles , Microambiente Celular , Técnicas de Cocultura , Feminino , Fibroblastos/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-17/genética , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucinas/genética , Interleucinas/imunologia , Masculino , Pessoa de Meia-Idade , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Fosforilação , Cultura Primária de Células , Receptores CCR6/genética , Receptores CCR6/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Interleucina 22
11.
Respir Res ; 14: 118, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24283210

RESUMO

BACKGROUND: Allergic asthma is characterized by airway inflammation in response to antigen exposure, leading to airway remodeling and lung dysfunction. Epithelial-mesenchymal transition (EMT) may play a role in airway remodeling through the acquisition of a mesenchymal phenotype in airway epithelial cells. TGF-ß1 is known to promote EMT; however, other cytokines expressed in severe asthma with extensive remodeling, such as IL-22, may also contribute to this process. In this study, we evaluated the contribution of IL-22 to EMT in primary bronchial epithelial cells from healthy and asthmatic subjects. METHODS: Primary bronchial epithelial cells were isolated from healthy subjects, mild asthmatics and severe asthmatics (n=5 patients per group). The mRNA and protein expression of epithelial and mesenchymal cell markers and EMT-associated transcription factors was evaluated following stimulation with TGF-ß1, IL-22 and TGF-ß1+IL-22. RESULTS: Primary bronchial epithelial cells stimulated with TGF-ß1 underwent EMT, demonstrated by decreased expression of epithelial markers (E-cadherin and MUC5AC) and increased expression of mesenchymal markers (N-cadherin and vimentin) and EMT-associated transcription factors. IL-22 alone had no effect on epithelial or mesenchymal gene expression. However, IL-22+TGF-ß1 promoted the expression of some EMT transcription factors (Snail1 and Zeb1) and led to a more profound cadherin shift, but only in cells obtained from severe asthmatics. CONCLUSION: The impact of IL-22 on airway epithelial cells depends on the cytokine milieu and the clinical phenotype of the patient. Further studies are required to determine the molecular mechanism of IL-22 and TGF-ß1 cooperativity in driving EMT in primary human bronchial epithelial cells.


Assuntos
Asma/fisiopatologia , Brônquios/fisiopatologia , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Interleucinas/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Adolescente , Adulto , Idoso , Asma/metabolismo , Asma/patologia , Biópsia , Brônquios/efeitos dos fármacos , Brônquios/patologia , Caderinas/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Técnicas In Vitro , Interleucinas/farmacologia , Masculino , Pessoa de Meia-Idade , Mucina-5AC/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta1/farmacologia , Adulto Jovem , Interleucina 22
12.
Can Respir J ; 19(1): 19-25, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22332127

RESUMO

BACKGROUND: Factors associated with asthma remission need to be determined, particularly when remission occurs in adulthood. OBJECTIVE: To evaluate airway responsiveness and inflammation in adult patients in asthma remission compared with adults with mild, persistent symptomatic asthma. METHODS: Adenosine monophosphate and methacholine responsiveness were evaluated in 26 patients in complete remission of asthma, 16 patients in symptomatic remission of asthma, 29 mild asthmatic patients and 15 healthy controls. Blood sampling and induced sputum were also obtained to measure inflammatory parameters. RESULTS: Perception of breathlessness at 20% fall in forced expiratory volume in 1 s was similar among groups. In subjects with symptomatic remission of asthma, responsiveness to adenosine monophosphate and methacholine was intermediate between mild asthma and complete asthma remission, with the latter group similar to controls. Asthma remission was associated with a shorter duration of disease. Blood immunoglobulin E levels were significantly increased in the asthma group, and blood eosinophils were significantly elevated in the complete asthma remission, symptomatic remission and asthma groups compared with controls. The suppressive function of regulatory T cells was lower in asthma and remission groups compared with controls. CONCLUSION: A continuum of asthma remission was observed, with patients in complete asthma remission presenting features similar to controls, while patients in symptomatic asthma remission appeared to be in an intermediate state between complete asthma remission and symptomatic asthma. Remission was associated with a shorter disease duration. Despite remission of asthma, a decreased suppressor function of regulatory T cells was observed, which may predispose patients to future recurrence of the disease.


Assuntos
Asma/imunologia , Hiper-Reatividade Brônquica/fisiopatologia , Imunoglobulina E/sangue , Linfócitos T/imunologia , Monofosfato de Adenosina , Adulto , Asma/fisiopatologia , Testes de Provocação Brônquica , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Cloreto de Metacolina
13.
Mol Immunol ; 47(11-12): 2112-8, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20471683

RESUMO

BACKGROUND: Allergic asthma is characterized by infiltration of inflammatory cells into the airways. T cell-derived cytokines regulate both airway inflammation and remodelling. In the human airways, T cell-fibroblast interactions may have a role in regulating inflammation and remodelling. OBJECTIVES: To evaluate the effect of bronchial fibroblast-T cell interaction on profibrogenic cytokine release and determine the nature of the molecules involved in this interaction. METHODS: Human bronchial fibroblasts obtained from healthy and asthmatic donors were co-cultured with purified T cells derived from peripheral blood of the same subjects. IL-6 mRNA and protein levels were measured by real time PCR and ELISA. CD40, CD40L and alpha 5 beta 1 were evaluated by flow cytometry. Bronchial fibroblasts were stimulated with rsCD40L. Neutralisation was performed using neutralizing antibodies anti-CD40L and anti-alpha 5. RESULTS: Contact of T cells with bronchial fibroblasts up-regulated IL-6 at both gene and protein levels. This effect was significantly higher in fibroblasts from asthmatics than those from controls. Blocking CD40L and alpha 5 beta 1 integrin showed a significant inhibition of IL-6 expression in asthmatics but not in healthy controls. Stimulation of fibroblasts with recombinant soluble CD40L up-regulated IL-6 production in asthmatics but not in controls. Adhesion to fibronectin, a alpha 5 beta 1 integrin ligand, is increased in fibroblasts from asthmatics compared to fibroblasts from controls. CONCLUSION: These results showed that interaction of bronchial fibroblasts with T cells increases the production of profibrogenic cytokine IL-6. In asthmatic condition this interaction involves CD40L/alpha 5 beta 1. These results suggest that T cells and structural cells crosstalk in asthma may maintain local mucosal inflammation.


Assuntos
Asma/imunologia , Brônquios/citologia , Ligante de CD40/fisiologia , Comunicação Celular , Fibroblastos/fisiologia , Integrina alfa5beta1/fisiologia , Linfócitos T/fisiologia , Adulto , Antígenos CD40/fisiologia , Adesão Celular , Células Cultivadas , Humanos , Interleucina-6/biossíntese
14.
Am J Respir Cell Mol Biol ; 38(2): 202-8, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17872498

RESUMO

Epithelial damage is an important pathophysiologic feature of asthma. Bronchial epithelium damage results in release of growth factors such as transforming growth factor (TGF)-beta(1) that may affect epithelial cell proliferation. The objective of our study is to evaluate the importance of TGF-beta(1) in regulating epithelial cell repair in asthma. We evaluated the effect of TGF-beta(1) on epidermal growth factor (EGF)-induced proliferation and downstream signaling in epithelial cells obtained from subjects with asthma compared with cells from healthy subjects. Cell proliferation was evaluated by bromodeoxyuridine incorporation. EGF receptor (EGFR), mitogen-activated protein kinase, TGF-beta receptors, Smads, Smad anchor for receptor activation (SARA), and cyclin-dependant kinase inhibitors were evaluated by Western blot. TGF-beta(1) and receptor expression were measured by RT-PCR and by enzyme-linked immunosorbent assay. Proliferation of epithelial cells at baseline and after EGF stimulation was significantly reduced in cells derived from subjects with asthma compared with cells obtained from healthy control subjects. EGF-induced ERK1/2 phosphorylation was reduced in epithelial cells from subjects with asthma compared with cells from healthy control subjects. This was paralleled with a reduced EGFR phosphorylation. Addition of TGF-beta(1) significantly decreased EGF-induced cell proliferation. TGF-beta(1) production was higher in asthmatic epithelial cells compared with normal cells. This was supported by a high expression of pSmad 3 and SARA in cells derived from individuals with asthma compared with normal subjects. Cycline-dependent kinase inhibitors were highly expressed in asthmatic compared with normal cells. Inhibition of TGF-beta(1) signaling in asthmatic epithelial cells restored EGFR, ERK1/2 phosphorylation, and cell proliferation induced by EGF. Our results suggest that TGF-beta restrains EGFR phosphorylation and downstream signaling in bronchial epithelial cells.


Assuntos
Asma/enzimologia , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Asma/metabolismo , Asma/patologia , Asma/fisiopatologia , Sequência de Bases , Western Blotting , Estudos de Casos e Controles , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
J Allergy Clin Immunol ; 117(6): 1321-7, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16750993

RESUMO

BACKGROUND: Asthma is characterized by inflammation and remodeling. Mast cells are generally increased in bronchial mucosa of subjects with asthma. These cells release a wide variety of cytokines and mediators that have the capacity to stimulate other resident cells such as smooth muscle cells and fibroblasts. OBJECTIVE: This study was designed to evaluate whether mast cells modulate collagen production by bronchial fibroblasts isolated from subjects with asthma and normal subjects through cytokine production. METHODS: Human mast cells were cocultured for 72 hours with primary bronchial fibroblasts isolated from bronchial biopsies of subjects with mild asthma and normal controls. Procollagen I (alpha1), IL-4Ralpha, IL-13Ralpha1, and IL-13Ralpha2 gene expression by bronchial fibroblasts and IL-4 and IL-4delta2 gene expression by mast cells were quantified by real-time RT-PCR. IL-4 production was also measured by ELISA in culture supernatants. RESULTS: Procollagen I (alpha1) gene expression by fibroblasts from subjects with asthma was significantly higher compared with cells from normal controls when cocultured with mast cells. Mast cells expressed IL-4 isoform and IL-4delta2, an alternative splice variant of IL-4. Coculture significantly increased the expression of IL-4 but not IL-4delta2 by mast cells when they were cultured with fibroblasts from subjects with asthma compared with cells from normal controls. Neutralization of IL-4 abrogated collagen mRNA expression. There was no significant change in IL-4Ralpha or IL-13Ralpha1. However, IL-13Ralpha2 gene expression was significantly reduced in fibroblasts from subjects with asthma. CONCLUSION: These results suggest that inflammatory process may regulate airway remodelling through crosstalk between inflammatory and structural cells. Targeting this crosstalk may have therapeutic application. CLINICAL IMPLICATIONS: Understanding mechanisms that govern airway remodeling and collagen deposition in asthma is a step toward therapeutic management of this disease. In this work, we found that mast cell-fibroblast crosstalk may be a potential future target to control some aspects of airway remodeling.


Assuntos
Asma/metabolismo , Colágeno Tipo I/biossíntese , Fibroblastos/metabolismo , Interleucina-4/metabolismo , Mastócitos/imunologia , Isoformas de Proteínas/biossíntese , Processamento Alternativo/genética , Asma/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Colágeno Tipo I/antagonistas & inibidores , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Fibroblastos/imunologia , Fibroblastos/patologia , Humanos , Subunidade alfa1 de Receptor de Interleucina-13 , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-13
16.
Respir Res ; 7: 68, 2006 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-16643666

RESUMO

BACKGROUND: Mechanical strain alters protein expression. It results in phosphorylation of MAP kinases and up-regulation of extracellular matrix proteins. We investigated whether phosphorylation of MAP kinase family members was increased in response to mechanical strain in fibroblasts from asthmatic patients (AF) and normal controls (NF), and whether phosphorylation of these signaling molecules would be different in the two cell populations. METHODS: Fibroblasts were obtained from mild, atopic asthmatics and non-atopic volunteers using endobronchial biopsy. Cells were grown on flexible, collagen I-coated membranes, and subjected to mechanical strain (Flexercell). MAP kinase phosphorylation was measured at baseline, and during one hour of strain. We also examined the effect of strain on proteoglycan production. RESULTS: At baseline, there was increased phosphorylation of ERK1/2 and p38, and decreased phosphorylation of JNK in AF vs NF. During strain in NF, p38 phosphorylation was increased. Conversely in AF, strain resulted in an increase in JNK phosphorylation, had no effect on phosphorylation of p38, and resulted in a decrease in ERK1/2 phosphorylation. There was a significant increase in versican protein production after 24 h strain in both AF and NF. JNK inhibition reversed the strain-induced increase in versican in NF, but had no effect in AF. CONCLUSION: These results show that there are phenotypic differences in MAP kinase phosphorylation in AF vs NF, and that different signaling pathways are involved in transducing mechanical stimuli in these two populations of cells.


Assuntos
Asma/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/enzimologia , Adolescente , Adulto , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Estresse Mecânico
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