RESUMO
Although elevated blood or sputum eosinophils are present in many patients with COPD, uncertainties remain regarding the anatomical distribution pattern of lung-infiltrating eosinophils. Basophils have remained virtually unexplored in COPD. This study mapped tissue-infiltrating eosinophils, basophils and eosinophil-promoting immune mechanisms in COPD-affected lungs.Surgical lung tissue and biopsies from major anatomical compartments were obtained from COPD patients with severity grades Global Initiative for Chronic Obstructive Lung Disease stages I-IV; never-smokers/smokers served as controls. Automated immunohistochemistry and in situ hybridisation identified immune cells, the type 2 immunity marker GATA3 and eotaxins (CCL11, CCL24).Eosinophils and basophils were present in all anatomical compartments of COPD-affected lungs and increased significantly in very severe COPD. The eosinophilia was strikingly patchy, and focal eosinophil-rich microenvironments were spatially linked with GATA3+ cells, including type 2 helper T-cell lymphocytes and type 2 innate lymphoid cells. A similarly localised and interleukin-33/ST2-dependent eosinophilia was demonstrated in influenza-infected mice. Both mice and patients displayed spatially confined eotaxin signatures with CCL11+ fibroblasts and CCL24+ macrophages.In addition to identifying tissue basophilia as a novel feature of advanced COPD, the identification of spatially confined eosinophil-rich type 2 microenvironments represents a novel type of heterogeneity in the immunopathology of COPD that is likely to have implications for personalised treatment.
Assuntos
Basófilos/imunologia , Eosinófilos/imunologia , Macrófagos/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Eosinofilia Pulmonar/etiologia , Adulto , Idoso , Animais , Biomarcadores , Quimiocina CCL11/imunologia , Quimiocina CCL24/imunologia , Feminino , Fator de Transcrição GATA3/imunologia , Humanos , Imunidade Inata , Masculino , Camundongos , Pessoa de Meia-Idade , Fumantes , Adulto JovemRESUMO
BACKGROUND: Chronic rhinosinusitis (CRS) with nasal polyps is a common chronic condition. The exact cause of nasal polyps remains unknown. Recently, we made the novel observation of intracellular localization of Staphylococcus aureus within mast cells in nasal polyps. OBJECTIVE: This follow-up study aimed to further characterize interactions between S aureus and mast cells in this setting and elucidate potential internalization mechanisms with particular emphasis on the role of staphylococcal enterotoxin B (SEB). METHODS: A prospective study was performed using an explant tissue model with ex vivo inferior turbinate mucosa obtained from patients with chronic rhinosinusitis with nasal polyps (n = 7) and patients without CRS (n = 5). Immunohistochemistry was used to characterize S aureus uptake into mast cells and investigate the effects of SEB on this process. An in vitro cell-culture model was used to investigate mast cell-S aureus interactions by using a combination of fluorescent in situ hybridization, confocal laser scanning microscopy, scanning electron microscopy, transmission electron microscopy, and proliferation assays. RESULTS: S aureus was captured by extracellular traps and entered mast cells through phagocytosis. Proliferating intracellular S aureus led to the expansion and eventual rupture of mast cells, resulting in release of viable S aureus into the extracellular space. The presence of SEB appeared to promote internalization of S aureus into mast cells. CONCLUSION: This study provides new insights into the interactions between S aureus and mast cells, including the internalization process, and demonstrates a prominent role for SEB in promoting uptake of the bacteria into these cells.
Assuntos
Enterotoxinas/imunologia , Mastócitos , Pólipos Nasais , Fagocitose , Staphylococcus aureus , Adulto , Idoso , Linhagem Celular , Feminino , Humanos , Masculino , Mastócitos/imunologia , Mastócitos/microbiologia , Mastócitos/ultraestrutura , Pessoa de Meia-Idade , Pólipos Nasais/imunologia , Pólipos Nasais/microbiologia , Pólipos Nasais/ultraestrutura , Estudos Prospectivos , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidade , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: An increased degree of mast cell (MC) degranulation and damage to the epithelial lining are prominent features of bronchial asthma. In asthmatic airways, it seems likely that epithelial cells will be exposed to increased concentrations of proteases from MC, though their actions on the epithelium are still not very clear. METHODS: Bronchial rings from human lung tissue or 16HBE cell monolayer were incubated with MC chymase in different doses or various inhibitors. The sections of paraffin-embedded tissue were haematoxylin-eosin stained and computerized by image analysis for epithelial damage-scale-evaluation; the cell viability, proliferation, adhesion and lactate dehydrogenase activity release were assayed; the expressions of gelatinases, cell junction molecules and structure proteins of 16HBE were examined. RESULTS: Mast cell chymase was found to provoke profound changes in the morphology of bronchi epithelial layer. Following incubation with chymase, there was 40% reduction in the length of epithelium that was intact, with detachment of columnar epithelial cells and basal cells. Chymase reduced epithelial cell proliferation and induced cell detachment, which were associated with the changes in secretion and activation of matrix metalloproteinase-2/9. In intact epithelial cell layers, immunocytochemistry study revealed that chymase reduced the expressions of occludin, claudin-4, ZO-1, E-cadherin, focal adhesion kinase and cytokeratin. Overall data of this study indicated that MC chymase can influence tissue remodelling, disrupt epithelial cell junctions, inhibit wound healing and impair the barrier function of epithelium, resulting in dysfunction of airway wall and ECM remodelling in pathogenesis of asthma. CONCLUSION: Mast cell chymase plays a key role in inducing the damage to bronchial epithelium in asthma.
Assuntos
Quimases/metabolismo , Células Epiteliais/metabolismo , Junções Intercelulares/metabolismo , Mastócitos/enzimologia , Mucosa Respiratória/metabolismo , Biomarcadores , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Quimases/genética , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismoRESUMO
AIMS: To identify the presence and geographical distribution of mast cell (MC) subtypes: MCT (tryptase positive-chymase negative) and MCTC (tryptase positive-chymase positive) in bladder tissue. METHODS: Bladder tissue was obtained from patients with painful bladder syndrome/interstitial cystitis (n=14) and normal histology from University Hospital Southampton tissue bank. Sequential tissue slices were immunohistochemically stained for MC subtypes using anti-MC tryptase (for MCT and MCTC) and anti-MC chymase (for MCTC). Stained sections were photographed, and positively stained MCs were quantified using ImageJ. Data were analysed using descriptive statistics and individual paired t-tests. RESULTS: There was a significant difference in the density of MCs between each layer of the disease bladder, with the greatest accumulation within the detrusor (p<0.001). There was a significant increase in MCTC subtype in the lamina (p=0.009) in painful bladder syndrome/interstitial cystitis. CONCLUSIONS: Our results suggest that mastocytosis is present within all layers of disease bladder, especially the muscle layer. The varying increase in MC subtypes in the lamina and mucosa may explain the variability in painful bladder syndrome/interstitial cystitis symptoms. A high influx of MCTC in the mucosa of individuals who also had ulceration noted within their diagnostic notes may be of the Hunner's ulcer subclassification. These findings suggest a relationship between the pathogenesis of MC subtypes and the clinical presentation of painful bladder syndrome/interstitial cystitis. A cohort study would further elucidate the diagnostic and/or therapeutic potential of MCs in patients with painful bladder syndrome/interstitial cystitis.
Assuntos
Cistite Intersticial/patologia , Mastócitos/patologia , Mastocitose/patologia , Bexiga Urinária/patologia , Biomarcadores/análise , Biópsia , Quimases/análise , Cistite Intersticial/enzimologia , Cistite Intersticial/terapia , Humanos , Imuno-Histoquímica , Mastócitos/enzimologia , Mastocitose/enzimologia , Mastocitose/terapia , Valor Preditivo dos Testes , Prognóstico , Triptases/análise , Bexiga Urinária/enzimologiaRESUMO
Dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease, required for activation of serine proteases of granulocytes including mast cells (MCs), neutrophils (NPs) and others, which were found in synovial tissue of patients with rheumatoid arthritis (RA). But, the role of DPPI associated with those cells in RA development is unclear. In this study, the collagen-induced-arthritis (CIA) rat-model was employed to investigate the expression and activity levels of DPPI and its association with RA progress. Primary granulocytes were freshly extracted from bone-marrows of normal or CIA rats, human mast cell line LAD-2 and primary neutrophils, human-recombinant-DPPI, DPPI-inhibitor Gly-Phe-CHN2 , LTB4, anti-IgE antibody, calcium ionophore were used to study the regulatory role of DPPI in cell activations. The increased DPPI activities in synovial fluids, serum, and bone-marrow homogenates of CIA rats associated with RA severities progress were observed after injections. MMP2/9 expressions in SFs and bone-marrow were in different patterns. Regular-Blood-Tests have shown the high leveled DPPI activities associated with granulocytes differentiations in-vivo in blood of CIA rats. In-vitro cell models, DPPI up-regulated the proliferation of primary bone-marrow granulocytes of normal rats, but inhibited that of CIA rats. DPPI up-regulated and Gly-Phe-CHN2 down-regulated MCs intracellular DPPI and chymase activities. Gly-Phe-CHN2 also inhibited the LTB4 -activated-NPs and NP-elastase activities. Following stimulation of calcium ionophore, the net-releases of DPPI and ß-hexosaminidase from MCs were increased over a time-course, while Gly-Phe-CHN2 down-regulated MCs and NPs activation. Our findings demonstrate the role of DPPI in regulating MCs and NPs activation, and modulating proteolysis in the process of RA.
Assuntos
Catepsina C/metabolismo , Granulócitos/enzimologia , Animais , Anticorpos Anti-Idiotípicos , Artrite Experimental/enzimologia , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Catepsina C/sangue , Modelos Animais de Doenças , Progressão da Doença , Granulócitos/imunologia , Granulócitos/metabolismo , Masculino , Mastócitos/metabolismo , Neutrófilos/metabolismo , Ratos , Ratos Wistar , Líquido Sinovial/enzimologia , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismoRESUMO
BACKGROUND: Asthma is a chronic inflammatory disease involving diverse cells and mediators whose interconnectivity and relationships to asthma severity are unclear. OBJECTIVE: We performed a comprehensive assessment of TH17 cells, regulatory T cells, mucosal-associated invariant T (MAIT) cells, other T-cell subsets, and granulocyte mediators in asthmatic patients. METHODS: Sixty patients with mild-to-severe asthma and 24 control subjects underwent detailed clinical assessment and provided induced sputum, endobronchial biopsy, bronchoalveolar lavage, and blood samples. Adaptive and invariant T-cell subsets, cytokines, mast cells, and basophil mediators were analyzed. RESULTS: Significant heterogeneity of T-cell phenotypes was observed, with levels of IL-13-secreting T cells and type 2 cytokines increased at some, but not all, asthma severities. TH17 cells and γδ-17 cells, proposed drivers of neutrophilic inflammation, were not strongly associated with asthma, even in severe neutrophilic forms. MAIT cell frequencies were strikingly reduced in both blood and lung tissue in relation to corticosteroid therapy and vitamin D levels, especially in patients with severe asthma in whom bronchoalveolar lavage regulatory T-cell numbers were also reduced. Bayesian network analysis identified complex relationships between pathobiologic and clinical parameters. Topological data analysis identified 6 novel clusters that are associated with diverse underlying disease mechanisms, with increased mast cell mediator levels in patients with severe asthma both in its atopic (type 2 cytokine-high) and nonatopic forms. CONCLUSION: The evidence for a role for TH17 cells in patients with severe asthma is limited. Severe asthma is associated with a striking deficiency of MAIT cells and high mast cell mediator levels. This study provides proof of concept for disease mechanistic networks in asthmatic patients with clusters that could inform the development of new therapies.
Assuntos
Imunidade Adaptativa , Asma/imunologia , Imunidade Inata , Células Th17/imunologia , Células Th2/imunologia , Adolescente , Corticosteroides/uso terapêutico , Adulto , Idoso , Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Asma/genética , Asma/patologia , Basófilos/imunologia , Basófilos/patologia , Teorema de Bayes , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Estudos de Casos e Controles , Feminino , Expressão Gênica , Humanos , Interleucina-13/genética , Interleucina-13/imunologia , Masculino , Mastócitos/imunologia , Mastócitos/patologia , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Índice de Gravidade de Doença , Escarro/química , Escarro/citologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Células Th17/patologia , Células Th2/patologiaRESUMO
Real-world evaluation studies have shown that many patients with asthma remain symptomatic despite treatment with inhaled corticosteroids (ICSs). As conventional ICSs have poor access to the peripheral airways, the aim of the present paper was to study the relationship between peripheral airway inflammation and clinical control in allergic asthma. Consequently, bronchial and transbronchial biopsies were obtained from patients with poorly controlled asthma [n=12, asthma control test (ACT) score<20], patients with well-controlled asthma (n=12, ACT score≥20) and healthy controls (n=8). Tissue sections were immunostained to assess multiple leucocyte populations. To determine the degree of T-helper type-2 (Th2) immunity, the logarithmic value of the ratio between Th2 cells/mm2 and Th1 cells/mm2 was used as a surrogate score for Th2-skewed immunity. In the bronchi, the leucocyte infiltration pattern and the Th2-score were similar between patients with well-controlled asthma and those with poorly controlled asthma. In contrast, in the alveolar parenchyma, the expression of T-helper cells was significantly higher in patients with poorly controlled asthma than in patients with well-controlled asthma (P<0.01). Furthermore, the alveolar Th2-score was significantly higher in patients with poorly controlled asthma (median 0.4) than in the controlled patients (median -0.10, P<0.05). In addition, in contrast with bronchial Th2-score, the alveolar Th2-score correlated significantly with ACT score (rs=-0.62, P<0.01) in the pooled asthma group. Collectively, our data reveal an alveolar Th2-skewed inflammation, specifically in asthmatic patients who are poorly controlled with ICSs, and suggest that pharmacological targeting of the peripheral airways may be beneficial in this large patient category.
Assuntos
Antiasmáticos/uso terapêutico , Asma/imunologia , Células Th2/imunologia , Adulto , Asma/tratamento farmacológico , Asma/patologia , Broncoscopia , Estudos de Casos e Controles , Feminino , Humanos , Imunidade Celular/imunologia , Imunidade Celular/fisiologia , Masculino , Pessoa de Meia-Idade , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/patologia , Células Th2/fisiologia , Falha de Tratamento , Adulto JovemRESUMO
Chronic myeloid leukemia (CML) is a hematopoietic neoplasm characterized by the Philadelphia chromosome and the related BCR-ABL1 oncoprotein. Acceleration of CML is usually accompanied by basophilia. Several proangiogenic molecules have been implicated in disease acceleration, including the hepatocyte growth factor (HGF). However, little is known so far about the cellular distribution and function of HGF in CML. We here report that HGF is expressed abundantly in purified CML basophils and in the basophil-committed CML line KU812, whereas all other cell types examined expressed only trace amounts of HGF or no HGF. Interleukin 3, a major regulator of human basophils, was found to promote HGF expression in CML basophils. By contrast, BCR-ABL1 failed to induce HGF synthesis in CML cells, and imatinib failed to inhibit expression of HGF in these cells. Recombinant HGF as well as basophil-derived HGF induced endothelial cell migration in a scratch wound assay, and these effects of HGF were reverted by an anti-HGF antibody as well as by pharmacologic c-Met inhibitors. In addition, anti-HGF and c-Met inhibitors were found to suppress the spontaneous growth of KU812 cells, suggesting autocrine growth regulation. Together, HGF is a BCR-ABL1-independent angiogenic and autocrine growth regulator in CML. Basophils are a unique source of HGF in these patients and may play a more active role in disease-associated angiogenesis and disease progression than has so far been assumed. Our data also suggest that HGF and c-Met are potential therapeutic targets in CML.
Assuntos
Basófilos/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Basófilos/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Crizotinibe , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-3/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Células-Tronco Neoplásicas/metabolismo , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Pirazóis , Piridinas/farmacologiaRESUMO
BACKGROUND: Little is known about the effect of neuropeptides on basophils, which are important effector cells in immune and allergic responses. OBJECTIVE: This study aimed at revealing the role of α-melanocyte-stimulating hormone (α-MSH) on basophil function. METHODS: Expression of melanocortin receptors and proopiomelanocortin (POMC) was analyzed by means of RT-PCR, Western immunoblotting, fluorescence-activated cell sorting, and double-immunofluorescence analysis. Signal transduction studies included cyclic AMP and Ca(2+) mobilization assays. Basophil activity was assessed based on CD63 surface expression and cytokine release. RESULTS: MC-1R expression was detectable in basophils isolated from human peripheral blood, as well as in basophils within nasal tissue. In isolated basophils from human blood, truncated POMC transcripts were present, but there was no POMC protein. Treatment of basophils with α-MSH increased intracellular Ca(2+) but not cyclic AMP levels. α-MSH at physiologic doses potently suppressed basophil activation induced by N-formyl-methionyl-leucyl-phenylalanine, phorbol 12-myristate 13-acetate, or grass pollen allergen in whole blood of healthy or allergic subjects, respectively. The effect of α-MSH on basophil activation was MC-1R mediated (as shown by blockade with a peptide analogue of agouti-signaling protein) and imitated by adrenocorticotropic hormone but not elicited by the tripeptides KPV and KdPT, both of which lack the central pharmacophore of α-MSH. Moreover, α-MSH at physiologic doses significantly suppressed secretion of 3 proallergic cytokines, IL-4, IL-6, and IL-13, in basophils stimulated with anti-IgE, N-formyl-methionyl-leucyl-phenylalanine, or phorbol 12-myristate 13-acetate. CONCLUSION: Our findings highlight a novel functional activity of α-MSH, which acts as a natural antiallergic basophil-response modifier. These findings might point to novel therapeutic strategies in treating allergic diseases.
Assuntos
Basófilos/efeitos dos fármacos , Basófilos/metabolismo , alfa-MSH/farmacologia , Alérgenos/imunologia , Basófilos/imunologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , AMP Cíclico/metabolismo , Citocinas/metabolismo , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Pró-Opiomelanocortina/genética , Receptores de Melanocortina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição GênicaRESUMO
BACKGROUND: Systemic mastocytosis (SM) is a heterogeneous group of disorders with distinct clinical and biological behavior. Despite this, little is known about the immunophenotypic features of the distinct diagnostic categories of SM. OBJECTIVE: To analyze the immunophenotypic characteristics of bone marrow (BM) mast cells (MCs) of different subtypes of SM. METHODS: Bone marrow samples from 123 patients with different subtypes of SM and 92 controls were analyzed for a broad panel of immunophenotypic markers by flow cytometry. RESULTS: Three clearly different maturation-associated immunophenotypic profiles were found for BMMCs in SM. These different profiles were associated with both genetic markers of the disease and its clinical behavior. BMMCs from poor-prognosis categories of SM (aggressive SM and MC leukemia) typically showed an immature phenotype with clonal involvement of all myeloid lineages by the D816V stem cell growth factor receptor gene (KIT) mutation. In turn, a mature activated versus resting BMMC immunophenotype was commonly found among patients with good-prognosis subtypes of SM depending on whether they carried (indolent SM and clonal MC activation disorders) or not (well differentiated SM) the D816V KIT mutation. CONCLUSION: Bone marrow MCs from SM show 3 different maturation-related immunophenotypic profiles that are associated with both the genetic markers of the disease and its clinical behavior.
Assuntos
Mastócitos/imunologia , Mastocitose/genética , Mastocitose/imunologia , Fator de Células-Tronco/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/imunologia , Separação Celular , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase , Prognóstico , Adulto JovemRESUMO
Honey has been used since ancient times as a remedy in wound healing. However, even though the results from randomized clinical trials document that honey accelerates wound healing, no study dealing with its influence on human skin cells (epidermal keratinocytes and dermal fibroblast) has been performed. We demonstrate that keratinocytes, which are known to be involved in wound healing, are responsible for elevated production of mediators including cytokines (TNF-alpha, IL-1beta and TGF-beta) and matrix metalloproteinase-9 (MMP-9) after incubation with honey. Real-time PCR was performed for the quantification of mRNA level of selected cytokines and MMP-9. Furthermore, we show that the increased level of MMP-9 in the epidermis following incubation with honey leads to degradation of type IV collagen in the basement membrane. These data indisputably demonstrate that honey activates keratinocytes and support the findings that honey may accelerate wound healing process.
Assuntos
Citocinas/metabolismo , Ácidos Graxos/farmacologia , Mel , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , RNA Mensageiro/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Citocinas/genética , Relação Dose-Resposta a Droga , Humanos , Interleucina-1beta/metabolismo , Queratinócitos/citologia , L-Lactato Desidrogenase/metabolismo , Masculino , Metaloproteinase 9 da Matriz/genética , RNA Mensageiro/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
Angiogenesis is a multistep complex phenomenon critical for several inflammatory and neoplastic disorders. Basophils, normally confined to peripheral blood, can infiltrate the sites of chronic inflammation. In an attempt to obtain insights into the mechanism(s) underlying human basophil chemotaxis and its role in inflammation, we have characterized the expression and function of vascular endothelial growth factors (VEGFs) and their receptors in these cells. Basophils express mRNA for three isoforms of VEGF-A (121, 165, and 189) and two isoforms of VEGF-B (167 and 186). Peripheral blood and basophils in nasal polyps contain VEGF-A localized in secretory granules. The concentration of VEGF-A in basophils was 144.4 +/- 10.8 pg/10(6) cells. Immunologic activation of basophils induced the release of VEGF-A. VEGF-A (10-500 ng/ml) induced basophil chemotaxis. Supernatants of activated basophils induced an angiogenic response in the chick embryo chorioallantoic membrane that was inhibited by an anti-VEGF-A Ab. The tyrosine kinase VEGFR-2 (VEGFR-2/KDR) mRNA was expressed in basophils. These cells also expressed mRNA for the soluble form of VEGFR-1 and neuropilin (NRP)1 and NRP2. Flow cytometric analysis indicated that basophils express epitopes recognized by mAbs against the extracellular domains of VEGFR-2, NRP1, and NRP2. Our data suggest that basophils could play a role in angiogenesis and inflammation through the expression of several forms of VEGF and their receptors.
Assuntos
Basófilos/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Fator B de Crescimento do Endotélio Vascular/biossíntese , Fator B de Crescimento do Endotélio Vascular/genética , Adulto , Animais , Anticorpos Monoclonais/farmacologia , Basófilos/citologia , Basófilos/imunologia , Inibição de Migração Celular , Quimiotaxia de Leucócito/imunologia , Embrião de Galinha , Citometria de Fluxo , Liberação de Histamina/imunologia , Humanos , Cinética , Família Multigênica , Pólipos Nasais/imunologia , Pólipos Nasais/metabolismo , Pólipos Nasais/patologia , Neuropilina-1/biossíntese , Neuropilina-1/genética , Neuropilina-2/biossíntese , Neuropilina-2/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , RNA Mensageiro/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular/imunologia , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Fator A de Crescimento do Endotélio Vascular/sangue , Fator A de Crescimento do Endotélio Vascular/fisiologia , Fator B de Crescimento do Endotélio Vascular/fisiologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossínteseRESUMO
CCR5 is one of the major inflammatory chemokine receptors with potential therapeutical applications in humans. However, the redundancy of chemokines and their receptors, and the species specificity of chemokine receptor antagonists pose challenges to understanding of the role they play in pharmacological situations. To address this question, we used a humanized severe combined immunodeficient mouse model grafted with human skin and autologous leukocytes, and evaluated the effect of a blocking antibody against human CCR5, on CCL5-induced cutaneous leukocyte recruitment in vivo. At baseline, CCL5 induced a significant recruitment of T cells mainly of the memory phenotype, of monocytes/macrophages, eosinophils, and IFN-gamma(+) but not IL-4(+) and IL-5(+) cells. In vivo, anti-CCR5 antibody was able to almost completely inhibit the recruitment of monocytes/macrophages and T-helper (Th)1-type cells to inhibit partially the attraction of memory T cells, but had no effect on eosinophil infiltration, although all these cell types express other CCL5 binding chemokine receptors than CCR5. These results indicate that the in vivo environment regulates target cell specificity of CCL5 leading to differential cell recruitment, suggesting that antagonizing CCR5 receptor may be of therapeutic value in diseases such as acquired immuno deficiency syndrome, where CCL5/CCR5, monocytes, and Th1-type cells play a predominant role.
Assuntos
Movimento Celular/imunologia , Quimiocinas CC/imunologia , Imunoterapia/métodos , Receptores CCR5/imunologia , Transplante de Pele/imunologia , Células Th1/imunologia , Animais , Anticorpos/farmacologia , Quimiocina CCL5 , Modelos Animais de Doenças , Eosinófilos/citologia , Eosinófilos/imunologia , Humanos , Memória Imunológica , Interferon gama/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos SCID , Monócitos/citologia , Monócitos/imunologia , Mutação , Receptores CCR5/genética , Células Th1/citologia , Células Th1/metabolismo , Células Th2/citologia , Células Th2/imunologia , Transplante Heterólogo/imunologiaRESUMO
Protease-activated receptors (PARs) have been implicated in the development of acute and chronic inflammatory responses. We have examined the expression of mRNA for PARs and their regulation by growth factors and cytokines in synovial fibroblasts derived from patients with rheumatoid arthritis (RA). Messenger RNA for PAR-1, -2 and -3 was detected in these cells, but not that for PAR-4. Expression of mRNA for PAR-2 was up-regulated by bFGF in a concentration-dependent manner, whereas expression of mRNA for PAR-1 and PAR-3 was not affected. Levels of mRNA encoding PAR-1, PAR-2 and PAR-3 did not increase in response to IL-1beta and TNF-alpha. Expression of mRNA for PAR-2 was maximal 12 h after addition of bFGF, and maximal levels of immunoreactive PAR-2 were reached after 24 h. Furthermore, PAR-2 agonist peptide (SLIGKV-NH(2)), but not the inactive reverse peptide (VKGILS-NH(2)), induced transitory cytosolic Ca(2+) mobilization in cells, and its response was increased by pretreatment with bFGF. An important role could be played by bFGF in the regulation of functional PAR-2 expression in cultured RA synovial fibroblasts.
Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/metabolismo , Receptor PAR-2/biossíntese , Líquido Sinovial/citologia , Cálcio/metabolismo , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Interleucina-1/farmacologia , RNA Mensageiro/biossíntese , Receptor PAR-1/biossíntese , Receptor PAR-2/agonistas , Receptores de Trombina/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
In myeloproliferative disorders (MPDs), basophils typically increase in number in the bone marrow (BM) and blood. In chronic myeloid leukemia (CML), basophilia is a diagnostic and prognostic variable. However, no reliable approach for routine detection and enumeration of basophils in BM sections is available. We applied the antibasogranulin antibody BB1 on paraffin-embedded BM sections in 21 control samples (normal BM), 45 patients with CML, 9 with chronic idiopathic myelofibrosis, 11 with polycythemia vera, 19 with essential thrombocythemia, and 7 with indolent systemic mastocytosis. As assessed by immunostaining of serial BM sections, BB1+ cells coexpressed myeloperoxidase, histidine decarboxylase, and leukosialin but did not express B- or T-cell-restricted antigens. BB1+ BM cells were found to be highly elevated in patients with CML compared with normal BM or other MPDs, with maximum counts found in accelerated phase CML (median, 160 cells/mm(2)). In summary, BB1 (basogranulin) is a new immunohistochemical basophil marker that should allow quantification of basophils in CML at diagnosis and during therapy.
Assuntos
Basófilos/química , Medula Óssea/química , Proteínas de Ligação a DNA/análise , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Fosfoproteínas/análise , Fatores de Transcrição/análise , Adulto , Biomarcadores , Feminino , Humanos , Imuno-Histoquímica , Imunofenotipagem , Masculino , Pessoa de Meia-IdadeRESUMO
Basophils, which are normally confined to the circulation, can migrate to sites of allergic inflammation. Using the specific mAb, BB1, we detected basophil infiltration of the gastric mucosa of Helicobacter pylori-infected patients affected by moderate and severe gastritis. Basophils were not found in H. pylori-free individuals or in subjects with mild gastritis. The H. pylori-derived peptide, Hp(2-20), was a potent basophil chemoattractant in vitro, whereas the control peptide, Hp1, was ineffective. Basophils from peripheral blood of healthy volunteers expressed mRNA for the formyl peptide receptors, N-formyl-peptide receptor (FPR), FPR-like (FPRL)1, and FPRL2. Preincubation of basophils with FMLP or Hp(2-20) caused complete desensitization to a subsequent challenge with homologous stimulus. Incubation of basophils with a low concentration of FMLP, which binds with high affinity to FPR, but not to FPRL1 or FPRL2, did not affect the chemotactic response to Hp(2-20). In contrast, a high concentration of FMLP, which binds to FPRL1 and FPRL2, reduced the chemotactic response to Hp(2-20). The FPR antagonist, cyclosporin H, prevented chemotaxis induced by FMLP, but not by Hp(2-20). Hp(2-20) could be responsible, at least in part, for basophil infiltration of the gastric mucosa of H. pylori-infected patients presumably through the interaction with FPRL1 and FPRL2.
Assuntos
Basófilos/fisiologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/imunologia , Fragmentos de Peptídeos/farmacologia , Proteínas de Bactérias/farmacologia , Biópsia , Estudos de Casos e Controles , Mucosa Gástrica/citologia , Gastrite/microbiologia , Helicobacter pylori/química , Humanos , RNA Mensageiro/análise , Receptores de Formil Peptídeo/genética , Receptores de Formil Peptídeo/fisiologiaRESUMO
BACKGROUND: Nasal polyposis (NP) is frequently associated with asthma. In this disease, asymptomatic bronchial hyperresponsiveness (BHR) is thought to precede the development of asthma. IL-9 and its receptor have been reported as candidate genes for asthma and to be associated with BHR. OBJECTIVE: The objective of this study was to assess the contribution of 11-9 to the pathogenesis of BHR in NP by comparing the expression of IL-9 and its receptor in bronchial biopsy specimens from three groups of patients with NP: NP without BHR, NP with asymptomatic BHR, and NP with BHR and asthma. METHODS: Bronchial biopsy specimens were examined in terms of cellular infiltration and in terms of expression of IL-9 protein and mRNA as well as of its receptor by using immunohistochemistry and in situ hybridization. RESULTS: Patients with NP with asthma as compared with the two other groups exhibited an increased bronchial infiltration of basophils, eosinophils, and T cells that correlated with the asthma score. The two groups of patients with NP with BHR showed an increased expression in IL-9 protein and mRNA as well as an increase in the expression of IL-9R mRNA at the epithelial level. These modifications were inversely correlated with the airway responsiveness to methacholine, producing a 20% fall in FEV1. There was a close association between IL-9+ cells, IL-5 mRNA expression, and eosinophil infiltration that correlated with each other. CONCLUSIONS: These results suggest an important role for IL-9 in the pathogenesis of BHR and a causal relation between IL-9 and the development of bronchial eosinophilia in asthma.
Assuntos
Interleucina-9/metabolismo , Pólipos Nasais/imunologia , Adulto , Asma/etiologia , Asma/genética , Asma/imunologia , Asma/patologia , Basófilos/imunologia , Basófilos/patologia , Brônquios/imunologia , Brônquios/patologia , Hiper-Reatividade Brônquica/etiologia , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/imunologia , Eosinofilia/etiologia , Eosinofilia/genética , Eosinofilia/imunologia , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Interleucina-5/genética , Interleucina-9/genética , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/complicações , Pólipos Nasais/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-9 , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
The mast cell proteases tryptase and chymase are synthesised as inactive precursors, but are stored and secreted as active enzymes. The cysteinyl protease dipeptidyl peptidase I (DPPI, cathepsin C) can activate the corresponding proenzymes in cell-free systems, but it is unknown whether it fulfils this role within the intact cell. We, therefore, tested the effect the DPPI-selective inhibitor Gly-Phe diazomethyl ketone (Gly-Phe-CHN(2)) on the tryptic and chymotryptic activity of the human mast cell-like cell line, HMC-1, and monitored any changes in the amount of immunodetectable enzymes by flow cytometry. Culture in Gly-Phe-CHN(2) produced a significant decrease in tryptase activity in cell lysates within 24hr and further decreases during continued culturing to 216 hr with periodic replenishment of Gly-Phe-CHN(2)-containing media. Flow cytometry showed no significant change in the levels of immunoreactive tryptase. In contrast, chymotryptic activity in treated cells did not differ significantly from untreated cells at any time point. Treatment of 216 hr cell lysates with DPPI revealed significant amounts of activatable protryptase in Gly-Phe-CHN(2)-treated cells, but not in controls, whereas activatable prochymotryptic activity was found in both treated and control cells. Chymase was detected immunologically, though small differences in substrate specificity and molecular mass were observed. These results strongly suggest that DPPI plays a role in the activation of tryptase, but not of the predominant chymotryptic activity of HMC-1 cells. As inhibitors of tryptase have proven efficacious in models of allergic disease, these results also indicate that inhibitors of DPPI might provide an additional point of therapeutic control.
Assuntos
Catepsina C/antagonistas & inibidores , Quimotripsina/metabolismo , Diazometano/análogos & derivados , Diazometano/farmacologia , Dipeptídeos/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/enzimologia , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Animais , Catepsina C/metabolismo , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Coelhos , TriptasesRESUMO
Mast cells accumulate in angiogenesis-dependent situations of lung adenocarcinoma. Human mast cells are divided into two major subsets: MCT (mast cells with immunoreactivity for tryptase but not chymase) and MCTC (reactive for tryptase and chymase). Chymase is an important mediator of tissue remodeling, but research into chymase-containing mast cell subpopulations has been hampered by the lack of reagents suitable for use with formalin-fixed tissue. We stained chymase using CC1 antibody in 66 cases of small sized lung adenocarcinoma as well as CD34 and tryptase. There were significant positive correlations of microvessel counts with MCT-type and MCTC-type mast cell counts in lung adenocarcinomas. When analyzed according to Noguchi's classification, MCT-type and MCTC-type mast cells were significantly increased in Noguchi type-C tumors [localized bronchioloalveolar carcinoma (LBAC) with active fibroblastic proliferation] compared with in Noguchi type-A (LBAC) plus type-B tumors (LBAC with alveolar collapse). Members in the high-count group of MCTC-type but not MCT-type mast cells showed a significantly worse outcome than those in the low-count group in LBACs. Counting chymase-positive (MCTC-type) mast cells in tumor stroma may be a good prognosis predictor for LBACs, especially Noguchi type-C tumors.
Assuntos
Adenocarcinoma/patologia , Neoplasias Pulmonares/patologia , Mastócitos/enzimologia , Serina Endopeptidases/análise , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/enzimologia , Angiotensina II/análise , Contagem de Células , Quimases , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/enzimologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , TriptasesRESUMO
OBJECTIVE: Numbers of mast cells (MCs) of different subpopulations and the extent of eosinophil infiltration were compared in Crohn's disease and ascariasis. These two types of intestinal inflammation are complementary with regard to T cell response (TH1 versus TH2), prevalence and environmental factors. METHODS: Histochemical, immunohistochemical and ultrastructural tools were applied to biopsies of morphologically uninvolved colon, ileum and duodenum from Crohn's and ascariasis patients, as well as resection margins and tissues from an experimental porcine ascariasis model. MC subsets were defined by their dye-binding properties, and their chymase content was analysed using biochemical tools. RESULTS: The TH2 (IgE-mediated) response in ascariasis was characterised by a dramatic increase in mucosal- type MCs (MMCs) and eosinophils in both the mucosa and the deeper layers of the intestinal wall and a simultaneous decrease of connective tissue-type MCs (CTMCs). Uninvolved intestine of Crohn's patients showed moderate proliferation of CTMCs in the deeper layers of the intestinal wall, but a significant decrease of the MMCs, associated with moderate eosinophilia in all layers of the gut. Similar changes were present in the uninvolved duodenum of Crohn's patients. Comparable amounts of chymase could be extracted from mucosal and submucosal duodenum, with similar proportions of its two principal isoforms in each. CONCLUSIONS: Our results indicate that T cell responses (TH1 or TH2) are associated with different MC subsets in intestinal inflammation. Changes remote from the focus of inflammation point to the systemic nature of the different MC responses.