RESUMO
Emphysema is the major component of chronic obstructive pulmonary disease (COPD). During emphysema, elastin breakdown in the lung tissue originates from the release of large amounts of elastase by inflammatory cells. Elevated levels of elastin-derived peptides (EP) reflect massive pulmonary elastin breakdown in COPD patients. Only the EP containing the GXXPG conformational motif with a type VIII ß-turn are elastin receptor ligands inducing biological activities. In addition, the COOH-terminal glycine residue of the GXXPG motif seems a prerequisite to the biological activity. In this study, we endotracheally instilled C57BL/6J mice with GXXPG EP and/or COOH-terminal glycine deleted-EP whose sequences were designed by molecular dynamics and docking simulations. We investigated their effect on all criteria associated with the progression of murine emphysema. Bronchoalveolar lavages were recovered to analyze cell profiles by flow cytometry and lungs were prepared to allow morphological and histological analysis by immunostaining and confocal microscopy. We observed that exposure of mice to EP elicited hallmark features of emphysema with inflammatory cell accumulation associated with increased matrix metalloproteinases and desmosine expression and of remodeling of parenchymal tissue. We also identified an inactive COOH-terminal glycine deleted-EP that retains its binding-activity to EBP and that is able to inhibit the in vitro and in vivo activities of emphysema-inducing EP. This study demonstrates that EP are key actors in the development of emphysema and that they represent pharmacological targets for an alternative treatment of emphysema based on the identification of EP analogous antagonists by molecular modeling studies.
Assuntos
Elastina/metabolismo , Enfisema Pulmonar/tratamento farmacológico , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Colágeno/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Elastase Pancreática/metabolismo , Peptídeos/metabolismo , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Receptores de Superfície Celular/antagonistas & inibidoresRESUMO
RATIONALE: Neutrophils play an important role in the inflammatory process associated with chronic obstructive pulmonary disease (COPD). Lung-infiltrating neutrophils secrete elastinolytic proteases that participate in elastin breakdown and the formation of elastin peptides (EPs). OBJECTIVES: We hypothesized that circulating neutrophil-associated immune response may be modulated by EPs during COPD. METHODS: Neutrophils obtained from patients with either stable or exacerbated COPD and controls were cultured with or without EPs. Cell chemotaxis was analysed by the Boyden method and cytokine expression was analysed by ELISA and real-time reverse transcriptase PCR. Bacterial phagocytosis and killing of ingested bacteria were evaluated after incubation with Pseudomonas aeruginosa. Reactive oxygen species (ROS) measurement and elastin receptor expression were determined by flow cytometry. RESULTS: Chemotactic activity of neutrophils from patients with COPD towards the VGVAPG EP was reduced compared with controls. VGVAPG increased proinflammatory cytokine synthesis and bacterial load, but reduced ROS production in neutrophils from controls and from patients with stable COPD. Patients with exacerbated COPD were unresponsive to VGVAPG treatment. These findings were associated with a decreased or almost complete loss of S-Gal elastin receptor expression in neutrophils from patients with stable or exacerbated COPD, respectively. CONCLUSIONS: The study demonstrates that the response of neutrophils from patients with COPD to VGVAPG varied according to COPD phase and critical level of S-Gal expression. S-Gal downregulation could result from a feedback mechanism induced by high levels of EPs.
Assuntos
Citocinas/biossíntese , Ativação de Neutrófilo , Neutrófilos/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptores de Superfície Celular/biossíntese , Idoso , Células Cultivadas , Quimiotaxia , Elastina , Feminino , Citometria de Fluxo , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Neutrófilos/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Espécies Reativas de Oxigênio/metabolismo , Fumar/efeitos adversos , Escarro/citologia , Escarro/metabolismoRESUMO
Staphylococcus aureus is frequently isolated from lungs of patients with cystic fibrosis (CF). Upon lung infection with S. aureus, airway epithelial cells (AEC) produce high levels of chemokines that enhance T-cell chemotaxis. Although the number of lymphocytes is increased in the airways and bronchoalveolar lavage fluid of patients with CF, the mechanisms responsible for their accumulation and the role of S. aureus in this process are largely unknown. This study investigated early S. aureus impact on chemokine secretion by CF epithelial cells and chemotaxis of CF T cells. CF and non-CF AEC were grown in a cell culture model and apically stimulated with S. aureus. Supernatants were quantified for chemokine secretions and assayed for T-cell chemotaxis. CF AEC secreted constitutively larger amounts of IL-8, GROalpha, MIG, MIP-3beta, and MCP-1 than non-CF epithelial cells. S. aureus interaction with epithelial cells increased chemokine production by non-CF cells but had no effect on CF cells. Chemotaxis of T cells derived from patients with CF was greater than that of T cells from subjects without CF. Moreover, there were more CF T cells expressing CXCR1 as compared with non-CF T cells. Under our experimental conditions, inhibition of IL-8 or its receptor CXCR1 resulted in a considerable decrease in T-cell chemotaxis (up to 80%). These data suggest that IL-8 and its receptor CXCR1 are key players in the chemotaxis of CF T cells and could be used as targets to develop therapies for CF.
Assuntos
Quimiotaxia de Leucócito , Fibrose Cística/imunologia , Interleucina-8/imunologia , Mucosa Respiratória/imunologia , Staphylococcus aureus/patogenicidade , Linfócitos T/imunologia , Adulto , Anticorpos Monoclonais , Complexo CD3/imunologia , Estudos de Casos e Controles , Linhagem Celular , Fibrose Cística/microbiologia , Impedância Elétrica , Feminino , Humanos , Interleucina-8/metabolismo , Masculino , Receptores de Interleucina-8A/imunologia , Proteínas Recombinantes/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/microbiologia , Linfócitos T/microbiologia , Fatores de Tempo , Adulto JovemRESUMO
According to the widely accepted view, neutrophil elastase (NE), a neutrophil-specific serine protease, is a major contributor to Pseudomonas aeruginosa infection-associated host tissue inflammation and damage, which in severe cases can lead to death. Herein, we provide for the first time compelling evidence that the host rather employs NE to protect itself against P. aeruginosa infection. Using a clinically relevant model of pneumonia, targeted deficiency in NE increased the susceptibility of mice to P. aeruginosa. We found that NE was required for maximal intracellular killing of P. aeruginosa by neutrophils. In investigating the mechanism of NE-mediated killing of P. aeruginosa, we found that NE degraded the major outer membrane protein F, a protein with important functions, including porin activity, maintenance of structural integrity, and sensing of host immune system activation. Consistent with this, the use of an isogenic mutant deficient in outer membrane protein F negated the role of NE in host defense against P. aeruginosa infection.
Assuntos
Imunidade Inata , Elastase de Leucócito/fisiologia , Infecções por Pseudomonas/enzimologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/fisiologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Imunidade Inata/genética , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/deficiência , Elastase de Leucócito/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Pneumonia Bacteriana/enzimologia , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genéticaRESUMO
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by abnormal lung inflammation that exceeds the protective response. Various culture models using epithelial cell lines or primary cells have been used to investigate the contribution of bronchial epithelium in the exaggerated inflammation of COPD. However, these models do not mimic in vivo situations for several reasons (e.g, transformed epithelial cells, protease-mediated dissociation of primary cells, etc.). To circumvent these concerns, we developed a new epithelial cell culture model. METHODS: Using non transformed non dissociated bronchial epithelium obtained by bronchial brushings from COPD and non-COPD smokers, we developed a 3-dimensional culture model, bronchial epithelial spheroids (BES). BES were analyzed by videomicroscopy, light microscopy, immunofluorescence, and transmission electron microscopy. We also compared the inflammatory responses of COPD and non-COPD BES. In our study, we chose to stimulate BES with lipopolycaccharide (LPS) and measured the release of the pro-inflammatory mediators interleukin-8 (IL-8) and leukotriene B4 (LTB4) and the anti-inflammatory mediator prostaglandin E2 (PGE2). RESULTS: BES obtained from both COPD and non-COPD patients were characterized by a polarized bronchial epithelium with tight junctions and ciliary beating, composed of basal cells, secretory cells and ciliated cells. The ciliary beat frequency of ciliated cells was not significantly different between the two groups. Of interest, BES retained their characteristic features in culture up to 8 days. BES released the inflammatory mediators IL-8, PGE2 and LTB4 constitutively and following exposure to LPS. Interestingly, LPS induced a higher release of IL-8, but not PGE2 and LTB4 in COPD BES (p < 0.001) which correlated with lung function changes. CONCLUSION: This study provides for the first time a compelling evidence that the BES model provides an unaltered bronchial surface epithelium. More importantly, BES represent an attractive culture model to investigate the mechanisms of injuring agents that mediate epithelial cell inflammation and its contribution to COPD pathogenesis.
Assuntos
Brônquios/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/patologia , Esferoides Celulares , Técnicas de Cultura de Células/métodos , Humanos , Pneumonia/patologiaRESUMO
In inflammatory diseases, strong release of elastinolytic proteases results in elastin fiber degradation generating elastin peptides (EPs). Chemotactic activity for inflammatory cells was, among wide range of properties, the former identified biological activity exerted by EPs. Recently, we demonstrated the ability of EPs to favor a Th1 cytokine (IL-2, IFN-gamma) cell response in lymphocytes and to regulate IL-1beta expression in melanoma cells. We hypothesized that EPs might also influence inflammatory cell properties by regulating cytokine expression by these cells. Therefore, we investigated the influence of EPs on inflammatory cytokine synthesis by human monocytes. We evidenced that EPs down-regulated both at the mRNA and protein levels the proinflammatory TNF-alpha, IL-1beta, and IL-6 expression in LPS-activated monocytes. Such negative feedback loop could be accounted solely for EP-mediated effects on proinflammatory cytokine production because EPs did not affect anti-inflammatory IL-10 or TGF-beta secretion by LPS-activated monocytes. Furthermore, we demonstrated that EP effect on proinflammatory cytokine expression by LPS-stimulated monocytes could not be due either to a decrease of LPS receptor expression or to an alteration of LPS binding to its receptor. The inhibitory effects of EPs on cytokine expression were found to be mediated by receptor (spliced galactosidase) occupancy, as being suppressed by lactose, and to be associated with the decrease of NF-kappaB-DNA complex formation. As a whole, these results demonstrated that EP/spliced galactosidase interaction on human monocytes down-regulated NF-kappaB-dependent proinflammatory cytokine expression and pointed out the critical role of EPs in the regulation of inflammatory response.
Assuntos
Citocinas/biossíntese , Regulação para Baixo/efeitos dos fármacos , Elastina/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/metabolismo , NF-kappa B/metabolismo , Receptores de Superfície Celular/metabolismo , Células Cultivadas , DNA/metabolismo , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Monócitos/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Superfície Celular/genética , Transdução de Sinais , Receptor 4 Toll-Like/metabolismoRESUMO
In the present study, we investigated the implication of NF-kappaB in the production of pro-inflammatory cytokine IL-18 by human keratinocytes stimulated by UVB. We demonstrated that NCTC 2544 keratinocyte cell line irradiated by UVB enhanced the IL-18 mRNA and protein secretion under its bioactive form. Overexpression of IL-18 by UVB irradiation was accompanied by NF-kappaB transcription factor activation using specific IL-18 gene sequence corresponding to NF-kappaB DNA binding site. The relationship between these transcription factors and IL-18 expression was confirmed using curcumin and PDTC, two inhibitors of NF-kappaB. Our results show that UVB and curcumin or PDTC co-treatment led to a down-regulation of IL-18 expression associated with an inhibition of NF-kappaB DNA binding. Hence, our results demonstrated that this transcription factor is implicated in biologically active IL-18 production by human keratinocytes irradiated by UVB.
Assuntos
Interleucina-18/biossíntese , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , NF-kappa B/metabolismo , Raios Ultravioleta , Células Cultivadas , Curcumina/farmacologia , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Interferon gama/biossíntese , Interleucina-18/genética , Leucócitos/metabolismo , Leucócitos/efeitos da radiação , Prolina/análogos & derivados , Prolina/farmacologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Tiocarbamatos/farmacologiaRESUMO
Hydroxyapatite (HA) is widely used to coat metal parts in order to improve their biocompatibility. Analysis of retrieved tissues associated with failed implants, suggest that phagocytosis of HA wear debris by monocytes/macrophages might provide a potent stimulus for the release of a variety of cytokines. Phagocytosis involved a large variety of cellular receptors like toll-like receptors that results in activation of the transcriptional nuclear factor-kappaB (NF-kappaB) via a cell-signalling pathway. In the present paper, we aimed to evaluate the role of the toll-like receptor 4 (TLR4) in the production of inflammatory cytokines induced by HA particles using TLR4(+) and TLR4(-) peritoneal macrophages. We investigated the production of TNF-alpha and the activation of the nuclear transcription factor NF-kappaB. Our data clearly show for the first time that the production of TNF-alpha by macrophages exposed to HA particles was TLR4 dependent but not the activation of NF-kappaB. All these results open future therapies to reduce the inflammatory response induced by HA biomaterials.
Assuntos
Materiais Biocompatíveis/química , Durapatita/química , Macrófagos/citologia , Monócitos/citologia , Receptor 4 Toll-Like/fisiologia , Animais , Inflamação , Lipopolissacarídeos/química , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C3H , NF-kappa B/metabolismo , Fagocitose , Pós , Propriedades de Superfície , Receptor 4 Toll-Like/metabolismoRESUMO
Bullous pemphigoid is an inflammatory disease of the skin associated with eosinophil infiltration and the presence of high levels of Th2 cytokines in the associated blister fluid. Little is known about the contribution of chemokines in this disease. We found that eotaxin and MCP-4 mRNA and immunoreactivity were expressed in all biopsies of BP patients and were mainly localized to the epidermis and eosinophils. The expression of eotaxin and MCP-4 was enhanced in eosinophils following IL-5 treatment. Subsequent stimulation of IL-5-primed eosinophils with Ig-immune complexes, results in increase secretion of eotaxin and MCP-4 in the supernatants. Using immunostaining, these two chemokines were localized to the granules of eosinophils. BF was found to contain chemotactic activity for eosinophils, neutrophils and T cells. The chemotactic effect of BF for eosinophils was more effective when eosinophils were stimulated with IL-5 or IL-4. We also found that the levels of Th(2)-associated chemokines (eotaxin and MCP-4) in BF were significantly higher than the Th(1)-associated chemokines (MIP-1beta and IP-10). This was consistent with the increased chemotaxis of polarized Th(2) cells toward BF, when compared to Th(1)-differentiated T cells. Our results support the involvement of Th(2)-associated chemokines in the pathogenesis of BP disease.
Assuntos
Quimiocinas CC/metabolismo , Quimiocinas/metabolismo , Eosinófilos/imunologia , Proteínas Quimioatraentes de Monócitos/metabolismo , Penfigoide Bolhoso/imunologia , Células Th2/imunologia , Adulto , Vesícula/imunologia , Quimiocina CCL11 , Quimiocinas/genética , Quimiocinas CC/genética , Quimiotaxia/imunologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Humanos , Interleucina-4/farmacologia , Interleucina-5/farmacologia , Proteínas Quimioatraentes de Monócitos/genética , Neutrófilos/imunologia , RNA Mensageiro/biossínteseRESUMO
In a previous work, we reported the influence of elastin fragments (EFs) on matrix metalloproteinases-2 and -14 expression and activation in melanoma cells in vitro. We hypothesized that EFs might also modulate expression of other mediators involved during melanoma progression. Therefore we investigated the contribution of EFs on IL-1beta expression, a cytokine playing a key role in melanoma cells activation. Our results evidenced that high tumorigenic melanoma cells (M3Da cells) treated with EFs led to IL-1beta mRNA and protein upregulation. The effects of EFs on M3Da cells were found to be mediated by receptor (spliced galactosidase) occupancy, as being suppressed by lactose and reproduced by cell stimulation with the VGVAPG peptide. Binding of EFs to their receptor induced a rapid activation of extracellular signal-regulated kinase 1/2; and p38 mitogen-activated protein kinase pathways. However, these pathways were not associated with IL-1beta mRNA upregulation by EFs. Concomitantly, we demonstrated that EFs stimulation induced NF-kappaB nuclear translocation and DNA binding on IL-1beta promoter region whereas inhibition of NF-kappaB with the specific chemical inhibitor SN-50 or by overexpression of IkappaB, the endogenous inhibitor of NF-kappaB pathway, totally abolished EFs-mediated IL-1beta mRNA overexpression. These results demonstrate that EFs induce NF-kappaB activation, leading to IL-1beta upregulation in invasive melanoma cells.
Assuntos
Elastina/metabolismo , Interleucina-1/metabolismo , Melanoma/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Neoplasias Cutâneas/metabolismo , Fator de Transcrição RelA/metabolismo , Sequência de Bases , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-1/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Melanoma/fisiopatologia , Melanoma/secundário , Fragmentos de Peptídeos/metabolismo , Regiões Promotoras Genéticas/fisiologia , Receptores de Interleucina-1/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/fisiopatologia , Células Tumorais Cultivadas , Regulação para CimaRESUMO
A possible complication associated with the implantation of hydroxyapatite (HA)-based prosthesis is the release of particles. These particles can be phagocyted by monocytes that are among the first cells to colonize the inflammatory site. The activated monocytes produce inflammatory mediators such as cytokines that cause osteoclasts activation. The present work, describes studies on the effect of sol-gel derived zinc-substituted HA particles with various zinc substitution percentages (0.5-2%) on cytokine production (TNF-alpha, IL-1beta, IL-6, IL-10, and IL-8) by both LPS-stimulated or unstimulated human monocytes. Our data demonstrates that the zinc has an effect on cytokines production. It decreases the production of TNF-alpha and increases the production of IL-8 by unstimulated cells. Using LPS-stimulated cells, it decreases the production of inflammatory cytokines and increases the production of anti-inflammatory cytokine (IL-10), indicating that zinc-substituted hydroxyapatite has favourable effects on the cytokines production by monocytes.
Assuntos
Materiais Biocompatíveis/química , Citocinas/metabolismo , Durapatita/química , Monócitos/metabolismo , Zinco/química , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Inflamação , Interleucina-1/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/metabolismo , Osteoclastos/metabolismo , Transição de Fase , Fator de Necrose Tumoral alfa/metabolismo , Zinco/metabolismoRESUMO
Recently [Jessel N, Schwinte P, Donohue R, Lavalle P, Boulmedais F, Darcy R, et al. Pyridylamino-beta-cyclodextrin as a molecular chaperone for lipopolysaccharide embedded in a multilayered polyelectrolyte architecture. Adv Funct Mater 2004;14:963-9], we demonstrated the biological activity of a lipopolysaccharide from Escherichia coli incorporated into layer-by-layer films made of poly (l-lysine) and poly (l-glutamic acid) and containing a polycationic beta-cyclodextrin (CD) with chaperone properties. Here we develop innovative architectures containing a complex made of a charged beta-cyclodextrin and a lipid A antagonist (LAA) as potential systems for local endotoxin antagonistic activity. We examine the biological activity of these architectures. The CD-LAA complex adsorbed on top, or embedded into the polyelectrolyte films keeps its LPS antagonistic activity on both murine and human macrophages for at least 24h.
Assuntos
Lipídeo A/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Poliaminas/farmacologia , Animais , Células Cultivadas , Humanos , Lipídeo A/química , Camundongos , Camundongos Endogâmicos C3H , Peptídeos/química , Poliaminas/química , Polieletrólitos , Análise Espectral Raman , beta-Ciclodextrinas/químicaRESUMO
In response to bacterial infection, airway epithelium releases inflammatory mediators including cytokines and chemokines that lead to immune cell efflux and could stimulate the adaptive T cell immune response. The aim of our study was to analyze, in a double chamber culture, the chemokine changes in response to Staphylococcus aureus and their consequences for T cells. Our data show that S. aureus stimulates basolateral and apical release of IL-8 and eotaxin by airway epithelial cells. We also observed increased chemokine receptor expression on CD8+ and CD4+ T cells and enhanced chemotaxis of CD4+ T cells toward apical supernatant. Our data strongly suggest that S. aureus interaction with airway epithelium contributes to specific migration of T cells to inflamed sites.
Assuntos
Quimiocinas/metabolismo , Quimiotaxia de Leucócito , Células Epiteliais/microbiologia , Mucosa Respiratória/microbiologia , Staphylococcus aureus/fisiologia , Linfócitos T/fisiologia , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Polaridade Celular , Células Cultivadas , Quimiocina CCL11 , Quimiocinas CC/metabolismo , Células Epiteliais/metabolismo , Humanos , Interleucina-8/metabolismo , Receptores de Quimiocinas/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Traqueia/citologiaRESUMO
The interaction of LPS (endotoxin) with the CD14-TLR4 receptor complex modulates the host innate immune response. Several studies using partial structures of LPS have suggested that TLR4 determines the ligand specificity of this complex, and that CD14 indiscriminately serves to deliver the ligand to TLR4. This conclusion has been made despite observations that the response of TLR4(+/+),CD14(-/-) macrophages to LPS is very weak. To determine whether CD14 itself plays a role in specific ligand recognition, the influences of various partial structures of LPS on induction of the proinflammatory cytokine, TNF, by CD14(+/+) and CD14(-/-) macrophages were compared. These studies show that the ligand specificities of CD14(+/+) and CD14(-/-) macrophages are very different. When CD14 is present, the receptor complex shows exquisite specificity for smooth LPS, the major form expressed by Gram-negative bacteria; however, as increasing amounts of carbohydrate are removed from smooth LPS, the sensitivity of CD14(+/+) macrophages decreases as much as 500-fold. In contrast, CD14(-/-) macrophages are unable to distinguish between smooth LPS and its various partial structures. Furthermore, CD14(-/-) macrophages are 150,000-fold less sensitive than CD14(+/+) macrophages to smooth LPS. A similar ability to distinguish the differing LPS structures of various bacteria such as Bacteroides fragilis and Salmonella abortus are observed for CD14(+/+), but not CD14(-/-), macrophages. Thus, CD14(+/+), but not CD14(-/-), macrophages are highly sensitive to stimulation by natural forms of LPS and show the ability to distinguish between various LPS ligands, consistent with CD14 being a highly specific receptor.
Assuntos
Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Animais , Bactérias Gram-Negativas , Ligantes , Lipopolissacarídeos/química , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Estrutura Molecular , Especificidade por Substrato , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Several reports suggest that activated airway smooth muscle (ASM) cells are capable of generating various proinflammatory mediators, including cytokines and chemokines. However, little is known about the mechanism involved in this process. In this regard, we have examined the expression and the role of the high affinity IgE receptor (Fc epsilonRI) by ASM cells. Human ASM cells were found to constitutively express transcripts coding for alpha, beta, and gamma subunits of Fc epsilonRI. Flow cytometry and Western blot analysis confirmed the expression of Fc epsilonRI alpha-chain protein. Interestingly, Fc epsilonRI alpha-chain immunoreactivity was also demonstrated in smooth muscle within bronchial biopsies of asthmatic subjects. Cross-linking of Fc epsilonRI induced mobilization of free calcium in ASM cells, one of the critical signals to trigger smooth muscle contraction. Furthermore, cultured ASM cells released IL-4, IL-13, IL-5, and eotaxin but not IFN-gamma, when sensitized with IgE followed by anti-IgE Ab cross-linking. The addition of anti-Fc epsilonRI alpha-chain Abs directed against IgE binding site inhibited this release. Taken together, these results suggest a potential new and important mechanism by which ASM cells may participate in airway inflammation and bronchoconstriction associated with allergic asthma.
Assuntos
Brônquios/imunologia , Brônquios/metabolismo , Músculo Liso/imunologia , Músculo Liso/metabolismo , Receptores de IgE/fisiologia , Traqueia/imunologia , Traqueia/metabolismo , Asma/imunologia , Asma/metabolismo , Asma/patologia , Brônquios/patologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/metabolismo , Hiper-Reatividade Brônquica/patologia , Sinalização do Cálcio/imunologia , Células Cultivadas , Quimiocinas/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Citocinas/metabolismo , Humanos , Imunoglobulina E/metabolismo , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Contração Muscular/imunologia , Músculo Liso/citologia , Ligação Proteica/imunologia , Isoformas de Proteínas/biossíntese , RNA Mensageiro/biossíntese , Receptores de IgE/biossíntese , Receptores de IgE/genética , Receptores de IgE/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Traqueia/citologiaRESUMO
OBJECTIVE: Increased level of elastin-derived peptides (EDPs) is observed in the serum of patients with manifestations of arterial diseases. We here investigated whether EDPs might exert, at systemic level, a regulatory role for the T-helper type 1 (Th-1)/Th-2 cellular immune response by human peripheral blood lymphocytes (PBLs) expressing the spliced-galactosidase (S-gal)-elastin receptor. METHODS AND RESULTS: Using flow cytometry and Western blot analysis, we demonstrated that EDPs led to an activation of the S-gal-elastin receptor associated with cytokine production on PBLs and CD4+ T cell subpopulations. The constitutive expression of the S-gal-elastin receptor at the surface of human PBLs was upregulated at the mRNA (RT-PCR) and protein (ELISA) levels on cell activation. In nonactivated and phytohemagglutinin-activated conditions, expressions of the predominant Th-2 cytokine interleukin-5 (IL-5) and IL-10 were reduced, whereas those of the major Th-1 cytokines interferon-gamma and IL-2 were enhanced by EDPs. Furthermore, we evidenced that EDPs could not only potentiate the IL-12-induced Th-1 profile but also could reverse the Th-2 (over Th-1) profile induced by IL-4. Finally, Th-1 cytokine upregulation was associated to an increased activator protein-1 DNA binding and enhanced pro-matrix metalloproteinase-9 secretion. CONCLUSIONS: This study highlights the importance of EDPs as stimuli for Th-1 differentiation, whether T cells are in an inactivated state or already orientated toward a Th-1 (IL-12) or Th-2 (IL-4) response.
Assuntos
Polaridade Celular/imunologia , Elastina/metabolismo , Peptídeos/metabolismo , Células Th1/metabolismo , Células Th2/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/metabolismo , Galactosídeos/metabolismo , Expressão Gênica/imunologia , Humanos , Sistema de Sinalização das MAP Quinases/imunologia , Metaloproteinase 9 da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Células Th1/citologia , Células Th1/imunologia , Células Th2/citologia , Células Th2/imunologia , Fator de Transcrição AP-1/metabolismo , Umbeliferonas/metabolismoRESUMO
We have previously demonstrated that treatment of the human keratinocyte cell line NCTC 2544 with a UVB dose equivalent to 1h exposure (100 mJ/cm2) results in a significant increase of IL-8 production. In this study, we use specific inhibitors to investigate the role of both PKA- and PKC-mediated pathways in the regulation of UVB-induced IL-8 expression in NCTC 2544 cell line. We show here that the treatment of irradiated human keratinocytes with PKA inhibitors [H89 and PKA inhibitor (PKAi)] induced a significant decrease of IL-8 production at both mRNA and protein levels. However, the regulation of IL-8 production seems to be mediated via a cAMP-independent PKA pathway, since drugs known to enhance cAMP concentrations [PGE2, cholera toxin and dibutyryl cAMP] decrease IL-8 production in irradiated cells by down-regulating NF-kappa B activation in response to UVB radiation. Using PMA (a potent pharmacological activator of PKC) and calphostin C (a specific PKC inhibitor), we demonstrated an up-regulation of IL-8 in NCTC 2544 cells and a down-regulation of the cytokine in UVB-irradiated cells, respectively. We also observed that in our experimental conditions, staurosporine, an inhibitor of both PKC and PMA-stimulated cellular responses, does not involve PKC inhibition in irradiated cells and significantly decreased NF-kappa B activity in response to UVB radiation. Finally, we concluded that a cAMP-independent PKA activation and a PKC-associated pathway are probably involved in the regulation of UVB-induced IL-8 synthesis in human keratinocytes.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos da radiação , Interleucina-8/metabolismo , Queratinócitos/efeitos da radiação , Proteína Quinase C/metabolismo , Transdução de Sinais , Raios Ultravioleta , Linhagem Celular , Toxina da Cólera/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Dinoprostona/farmacologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/enzimologia , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estaurosporina/farmacologiaRESUMO
A possible complication associated with the implantation of hydroxyapatite (HA)-based prosthesis is the release of particles. Those particles can be phagocyted by monocytes that are among the first cells to colonize the inflammatory site. The activated monocytes produce inflammatory mediators, such as cytokines, which cause osteoclasts activation. It has previously been demonstrated using a surface area ratio (ratio of the total surface of the given particles to the surface area of cells) of 1 to 1 that there was a correlation between the expression and production of cytokines induced by HA. The present work studies the effect of physical characteristics of HA particles on the production of various inflammatory cytokines (tumour necrosis factor-alpha, interleukin (IL)-6, and IL-8) and anti-inflammatory cytokine (IL-10). However, the experiments were performed using a surface area ratio of 10 to 1. Our data demonstrate that all the particles, whatever their characteristics, induced a high expression of cytokines but the production was different, meaning that there was a post-transcriptional regulation. The size and sintering temperature seemed to be a characteristics that were less important compared to the shape; the needle particles appeared to induce the most important production of all the cytokines studied.
Assuntos
Materiais Biocompatíveis/efeitos adversos , Citocinas/imunologia , Durapatita/efeitos adversos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Células Cultivadas , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Humanos , Teste de Materiais , Monócitos/metabolismo , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
The use of nanoparticles for gene therapy is gaining more and more interest for medical applications. Chitosan is among the candidate polymers that have a potential application as a gene delivery system. Before using chitosan-DNA nanoparticles in vivo, one must study their interaction and cell's behavior. Since macrophages play an important role in inflammatory processes, this study was performed to investigate the effects of chitosan-DNA nanoparticles on human THP-1 cell line. Cytokine (TNF-alpha, IL-1beta, IL-6 and IL-10) and metalloproteinase (MMP-2 and MMP-9) release as well as their inhibitors (TIMP-1 and TIMP-2) were assessed after time course incubation with different amount of nanoparticles. Their secretion was quantified by enzyme-linked immunosorbent assay. Gelatinolytic activity of MMP-2 and MMP-9 was determined by zymography in cell supernatants and lysates. Cytokine secretion was not detected even in the presence of high amount of nanoparticles. On the contrary, the secretion of MMP-9 in cell supernatants increased significantly after 24 and 48 h in comparison with non-treated cells. MMP-2 secretion was augmented only after 48 h for the highest concentrations of nanoparticles (10 and 20 microg/ml DNA content). However, zymography studies showed that the secreted MMPs were in the proactive forms, while the active form of MMP-9, but not MMP-2, was detected in cell lysates when 10 and 20 microg/ml DNA containing nanoparticles were used. In conclusion, exposure of THP-1 macrophages to Ch-DNA nanoparticles did not induce release of proinflammatory cytokines. The presence of active MMP-9 within the macrophages could possibly be related to nanoparticle phagocytosis and degradation rather than to inflammatory reactions.
Assuntos
Quitosana/farmacocinética , Citocinas/metabolismo , DNA/farmacocinética , Reação a Corpo Estranho/metabolismo , Macrófagos/metabolismo , Metaloproteinases da Matriz/metabolismo , Linhagem Celular , Quitosana/química , Quitosana/imunologia , Citocinas/imunologia , DNA/química , DNA/imunologia , Reação a Corpo Estranho/imunologia , Reação a Corpo Estranho/patologia , Humanos , Macrófagos/imunologia , Metaloproteinases da Matriz/imunologia , Nanotubos/química , Nanotubos/ultraestrutura , Tamanho da PartículaRESUMO
To better understand the role of inflammatory responses in temporal lobe epilepsy, we characterized Interleukin1-beta (IL1-beta), Nuclear Factor-kappaB (NF-kappaB), and Cyclooxygenase-2 (COX-2) expression together with neurodegeneration in the rat lithium-pilocarpine model. The immunohistochemical expression of IL1-beta, NF-kappaB, and COX-2 started by 12 h post-injection, persisted for 24 h (status epilepticus period), and returned to basal levels by 3 and 6 days (latent period). The regional distribution of IL1-beta, NF-kappaB, and COX-2 occurred mainly in structures prone to develop neuronal damage. Using double-staining protocols, we detected IL1-beta expression in glial cells, COX-2 expression in neurons, and NF-kappaB in both cell types. The presence of Fluoro-Jade-B-positive degenerating neurons was associated with IL1-beta, NF-kappaB, and COX-2 proteins expression during status epilepticus but not during the latent period while neurons were still degenerating. These data suggest that seizure-related IL1-beta, NF-kappaB, and COX-2 expression may contribute to the pathophysiology of epilepsy by inducing neuronal death and astrocytic activation.