RESUMO
BACKGROUND: Subpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a "just-in-time" design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument. METHODS: The Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data. RESULTS: Thus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel. CONCLUSIONS: Our study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters. TRIAL REGISTRATION: This study was registered with ClinicalTrials.gov as NCT01969344 .
Assuntos
Líquido da Lavagem Broncoalveolar , Imunofenotipagem/métodos , Doença Pulmonar Obstrutiva Crônica/sangue , Doença Pulmonar Obstrutiva Crônica/metabolismo , Escarro/metabolismo , Biomarcadores , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Separação Celular , Células Dendríticas/citologia , Citometria de Fluxo , Humanos , Leucócitos/citologia , Estudos Longitudinais , Macrófagos/citologia , Fenótipo , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Projetos de Pesquisa , Tamanho da Amostra , Fumar , EspirometriaRESUMO
The lung environment actively inhibits apoptotic cell (AC) uptake by alveolar macrophages (AMøs) via lung collectin signaling through signal regulatory protein α (SIRPα). Even brief glucocorticoid (GC) treatment during maturation of human blood monocyte-derived or murine bone marrow-derived macrophages (Møs) increases their AC uptake. Whether GCs similarly impact differentiated tissue Møs and the mechanisms for this rapid response are unknown and important to define, given the widespread therapeutic use of inhaled GCs. We found that the GC fluticasone rapidly and dose-dependently increased AC uptake by murine AMøs without a requirement for protein synthesis. Fluticasone rapidly suppressed AMø expression of SIRPα mRNA and surface protein, and also activated a more delayed, translation-dependent upregulation of AC recognition receptors that was not required for the early increase in AC uptake. Consistent with a role for SIRPα suppression in rapid GC action, murine peritoneal Møs that had not been exposed to lung collectins showed delayed, but not rapid, increase in AC uptake. However, pretreatment of peritoneal Møs with the lung collectin surfactant protein D inhibited AC uptake, and fluticasone treatment rapidly reversed this inhibition. Thus, GCs act not only by upregulating AC recognition receptors during Mø maturation but also via a novel rapid downregulation of SIRPα expression by differentiated tissue Møs. Release of AMøs from inhibition of AC uptake by lung collectins may, in part, explain the beneficial role of inhaled GCs in inflammatory lung diseases, especially emphysema, in which there is both increased lung parenchymal cell apoptosis and defective AC uptake by AMøs.
Assuntos
Androstadienos/farmacologia , Proteínas Reguladoras de Apoptose/farmacologia , Apoptose/imunologia , Colectinas/fisiologia , Regulação para Baixo/imunologia , Tolerância Imunológica/imunologia , Macrófagos Alveolares/imunologia , Receptores Imunológicos/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/fisiologia , Células Cultivadas , Relação Dose-Resposta Imunológica , Regulação para Baixo/efeitos dos fármacos , Fluticasona , Tolerância Imunológica/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores Imunológicos/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
Recent studies link early rhinovirus (RV) infections to later asthma development. We hypothesized that neonatal RV infection leads to an IL-13-driven asthma-like phenotype in mice. BALB/c mice were inoculated with RV1B or sham on day 7 of life. Viral RNA persisted in the neonatal lung up to 7 d postinfection. Within this time frame, IFN-α, -ß, and -γ peaked 1 d postinfection, whereas IFN-λ levels persisted. Next, we examined mice on day 35 of life, 28 d after initial infection. Compared with sham-treated controls, virus-inoculated mice demonstrated airways hyperresponsiveness. Lungs from RV-infected mice showed increases in several immune cell populations, as well as the percentages of CD4-positive T cells expressing IFN-γ and of NKp46/CD335(+), TCR-ß(+) cells expressing IL-13. Periodic acid-Schiff and immunohistochemical staining revealed mucous cell metaplasia and muc5AC expression in RV1B- but not sham-inoculated lungs. Mucous metaplasia was accompanied by induction of gob-5, MUC5AC, MUC5B, and IL-13 mRNA. By comparison, adult mice infected with RV1B showed no change in IL-13 expression, mucus production, or airways responsiveness 28 d postinfection. Intraperitoneal administration of anti-IL-13 neutralizing Ab attenuated RV-induced mucous metaplasia and methacholine responses, and IL-4R null mice failed to show RV-induced mucous metaplasia. Finally, neonatal RV increased the inflammatory response to subsequent allergic sensitization and challenge. We conclude that neonatal RV1B infection leads to persistent airways inflammation, mucous metaplasia, and hyperresponsiveness, which are mediated, at least in part, by IL-13.
Assuntos
Infecções por Picornaviridae/complicações , Infecções por Picornaviridae/patologia , Hipersensibilidade Respiratória/virologia , Mucosa Respiratória/patologia , Animais , Animais Recém-Nascidos , Separação Celular , Citocinas/biossíntese , Citocinas/imunologia , Citometria de Fluxo , Imuno-Histoquímica , Inflamação/imunologia , Inflamação/patologia , Inflamação/virologia , Metaplasia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Picornaviridae/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Mucosa Respiratória/imunologiaRESUMO
Nontypeable Haemophilus influenzae (NTHi) is an important bacterial pathogen associated with lower respiratory tract colonization and with acute exacerbations and disease progression in chronic obstructive pulmonary disease (COPD). Why the immune system fails to eliminate NTHi and the exact contribution of the organism to COPD progression are not well understood, in part because we lack an animal model that mimics all aspects of COPD. For this study, we used an established murine model that exhibits typical features of COPD. Elastase/LPS-exposed mice infected with NTHi showed persistence of bacteria up to 5 days after infection, whereas mice exposed to elastase, LPS, or PBS cleared all bacteria by 3 days. Elastase/LPS-exposed mice also showed sustained lung neutrophilic inflammation, goblet cell metaplasia, airway hyperresponsiveness, and progression of emphysema at 15 days after infection. Alveolar macrophages isolated from elastase/LPS-exposed mice showed impaired bacterial phagocytosis, reduced expression of MARCO and of mannose receptor, and absent expression of scavenger receptor-A (SR-A). Neutralization of SR-A significantly decreased phagocytosis of NTHi by normal alveolar macrophages. Our results suggest that elastase/LPS-exposed mice show impaired bacterial clearance and sustained lung inflammation. Lack of SR-A expression may, in part, be responsible for impaired phagocytosis of bacteria by alveolar macrophages of elastase/LPS-exposed mice. These data validate the suitability of elastase/LPS model for investigating NTHi pathogenesis and progression of disease in COPD.
Assuntos
Infecções por Haemophilus/imunologia , Imunidade Inata/fisiologia , Lipopolissacarídeos/farmacologia , Elastase Pancreática/farmacologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Receptores Depuradores Classe A/fisiologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Linhagem Celular , Citocinas/metabolismo , Infecções por Haemophilus/fisiopatologia , Haemophilus influenzae/imunologia , Medidas de Volume Pulmonar , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Muco/metabolismo , Fagocitose/imunologia , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/microbiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Enfisema Pulmonar/imunologia , Enfisema Pulmonar/microbiologiaRESUMO
BACKGROUND: Cigarette smoking is associated with increased frequency and duration of viral respiratory infections, but the underlying mechanisms are incompletely defined. We investigated whether smoking reduces expression by human lung macrophages (Mø) of receptors for viral nucleic acids and, if so, the effect on CXCL10 production. METHODS: We collected alveolar macrophages (AMø) by bronchoalveolar lavage of radiographically-normal lungs of subjects undergoing bronchoscopies for solitary nodules (n = 16) and of volunteers who were current or former smokers (n = 7) or never-smokers (n = 13). We measured expression of mRNA transcripts for viral nucleic acid receptors by real-time PCR in those AMø and in the human Mø cell line THP-1 following phorbol myristate acetate/vitamin D3 differentiation and exposure to cigarette smoke extract, and determined TLR3 protein expression using flow cytometry and immunohistochemistry. We also used flow cytometry to examine TLR3 expression in total lung Mø from subjects undergoing clinically-indicated lung resections (n = 25). Of these, seven had normal FEV1 and FEV1/FVC ratio (three former smokers, four current smokers); the remaining 18 subjects (14 former smokers; four current smokers) had COPD of GOLD stages I-IV. We measured AMø production of CXCL10 in response to stimulation with the dsRNA analogue poly(I:C) using Luminex assay. RESULTS: Relative to AMø of never-smokers, AMø of smokers demonstrated reduced protein expression of TLR3 and decreased mRNA for TLR3 but not TLR7, TLR8, TLR9, RIG-I, MDA-5 or PKR. Identical changes in TLR3 gene expression were induced in differentiated THP-1 cells exposed to cigarette smoke-extract in vitro for 4 hours. Among total lung Mø, the percentage of TLR3-positive cells correlated inversely with active smoking but not with COPD diagnosis, FEV1% predicted, sex, age or pack-years. Compared to AMø of never-smokers, poly(I:C)-stimulated production of CXCL10 was significantly reduced in AMø of smokers. CONCLUSIONS: Active smoking, independent of COPD stage or smoking duration, reduces both the percent of human lung Mø expressing TLR3, and dsRNA-induced CXCL10 production, without altering other endosomal or cytoplasmic receptors for microbial nucleic acids. This effect provides one possible mechanism for increased frequency and duration of viral lower respiratory tract infections in smokers. TRIAL REGISTRATION: ClinicalTrials.gov NCT00281190, NCT00281203 and NCT00281229.
Assuntos
Regulação para Baixo/genética , Macrófagos Alveolares/metabolismo , RNA de Cadeia Dupla/antagonistas & inibidores , Fumar/metabolismo , Receptor 3 Toll-Like/antagonistas & inibidores , Adulto , Idoso , Linhagem Celular , Células Cultivadas , Estudos de Coortes , Feminino , Humanos , Pulmão/citologia , Pulmão/metabolismo , Pulmão/virologia , Macrófagos Alveolares/virologia , Masculino , Pessoa de Meia-Idade , RNA de Cadeia Dupla/genética , Fumar/genética , Receptor 3 Toll-Like/biossíntese , Receptor 3 Toll-Like/genética , Adulto JovemRESUMO
Lung CD8(+) T cells might contribute to progression of chronic obstructive pulmonary disease (COPD) indirectly via IFN-gamma production or directly via cytolysis, but evidence for either mechanism is largely circumstantial. To gain insights into these potential mechanisms, we analyzed clinically indicated lung resections from three human cohorts, correlating findings with spirometrically defined disease severity. Expression by lung CD8(+) T cells of IL-18R and CD69 correlated with severity, as did mRNA transcripts for perforin and granzyme B, but not Fas ligand. These correlations persisted after correction for age, smoking history, presence of lung cancer, recent respiratory infection, or inhaled corticosteroid use. Analysis of transcripts for killer cell lectin-like receptor G1, IL-7R, and CD57 implied that lung CD8(+) T cells in COPD do not belong to the terminally differentiated effector populations associated with chronic infections or extreme age. In vitro stimulation of lung CD8(+) T cells with IL-18 plus IL-12 markedly increased production of IFN-gamma and TNF-alpha, whereas IL-15 stimulation induced increased intracellular perforin expression. Both IL-15 and IL-18 protein expression could be measured in whole lung tissue homogenates, but neither correlated in concentration with spirometric severity. Although lung CD8(+) T cell expression of mRNA for both T-box transcription factor expressed in T cells and GATA-binding protein 3 (but not retinoic acid receptor-related orphan receptor gamma or alpha) increased with spirometric severity, stimulation of lung CD8(+) T cells via CD3epsilon-induced secretion of IFN-gamma, TNF-alpha, and GM-CSF, but not IL-5, IL-13, and IL-17A. These findings suggest that the production of proinflammatory cytokines and cytotoxic molecules by lung-resident CD8(+) T cells contributes to COPD pathogenesis.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Interleucina-15/imunologia , Interleucina-18/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Subpopulações de Linfócitos T/imunologia , Idoso , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Linfócitos T CD8-Positivos/metabolismo , Separação Celular , Citocinas/biossíntese , Citocinas/imunologia , Citotoxicidade Imunológica , Feminino , Citometria de Fluxo , Volume Expiratório Forçado , Humanos , Lectinas Tipo C/biossíntese , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , RNA Mensageiro/análise , Testes de Função Respiratória , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/metabolismoRESUMO
Innate immune responses are essential for controlling poxvirus infection. The threat of a bioterrorist attack using Variola major, the smallpox virus, or zoonotic transmission of other poxviruses has renewed interest in understanding interactions between these viruses and their hosts. We recently determined that TLR3 regulates a detrimental innate immune response that enhances replication, morbidity, and mortality in mice in response to vaccinia virus, a model pathogen for studies of poxviruses. To further investigate Toll-like receptor signaling in vaccinia infection, we first focused on TRIF, the only known adapter protein for TLR3. Unexpectedly, bioluminescence imaging showed that mice lacking TRIF are more susceptible to vaccinia infection than wild-type mice. We then focused on TLR4, the other Toll-like receptor that signals through TRIF. Following respiratory infection with vaccinia, mice lacking TLR4 signaling had greater viral replication, hypothermia, and mortality than control animals. The mechanism of TLR4-mediated protection was not due to increased release of proinflammatory cytokines or changes in total numbers of immune cells recruited to the lung. Challenge of primary bone marrow macrophages isolated from TLR4 mutant and control mice suggested that TLR4 recognizes a viral ligand rather than an endogenous ligand. These data establish that TLR4 mediates a protective innate immune response against vaccinia virus, which informs development of new vaccines and therapeutic agents targeted against poxviruses.
Assuntos
Pneumopatias/imunologia , Receptor 4 Toll-Like/imunologia , Vacínia/imunologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Células Cultivadas , Imunidade Inata , Pneumopatias/virologia , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Camundongos Knockout , Receptor 4 Toll-Like/deficiência , Vaccinia virus/imunologiaRESUMO
Urease, a major virulence factor for Cryptococcus neoformans, promotes lethal meningitis/encephalitis in mice. The effect of urease within the lung, the primary site of most invasive fungal infections, is unknown. An established model of murine infection that utilizes either urease-producing (wt and ure1::URE1) or urease-deficient (ure1) strains (H99) of C. neoformans was used to characterize fungal clearance and the resultant immune response evoked by these strains within the lung. Results indicate that mice infected with urease-producing strains of C. neoformans demonstrate a 100-fold increase in fungal burden beginning 2 weeks post-infection (as compared with mice infected with urease-deficient organisms). Infection with urease-producing C. neoformans was associated with a highly polarized T2 immune response as evidenced by increases in the following: 1) pulmonary eosinophils, 2) serum IgE levels, 3) T2 cytokines (interleukin-4, -13, and -4 to interferon-gamma ratio), and 4) alternatively activated macrophages. Furthermore, the percentage and total numbers of immature dendritic cells within the lung-associated lymph nodes was markedly increased in mice infected with urease-producing C. neoformans. Collectively, these data define cryptococcal urease as a pulmonary virulence factor that promotes immature dendritic cell accumulation and a potent, yet non-protective, T2 immune response. These findings provide new insights into mechanisms by which microbial factors contribute to the immunopathology associated with invasive fungal disease.
Assuntos
Cryptococcus/enzimologia , Células Dendríticas/imunologia , Urease/farmacologia , Animais , Ensaio de Unidades Formadoras de Colônias , Criptococose/imunologia , Cryptococcus neoformans/enzimologia , Células Dendríticas/efeitos dos fármacos , Proteínas Fúngicas/farmacologia , Humanos , Hospedeiro Imunocomprometido , Leucócitos/patologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Urease/deficiência , Urease/genéticaRESUMO
Viral infection is associated with approximately one-half of acute exacerbations of chronic obstructive pulmonary disease (COPD), which in turn, accelerate disease progression. In this study, we infected mice exposed to a combination of elastase and LPS, a constituent of cigarette smoke and a risk factor for development of COPD, with rhinovirus serotype 1B, and examined animals for viral persistence, airway resistance, lung volume, and cytokine responses. Mice exposed to elastase and LPS once a week for 4 wk showed features of COPD such as airway inflammation and obstruction, goblet cell metaplasia, reduced lung elastance, increased total lung volume, and increased alveolar chord length. In general, mice exposed to elastase or LPS alone showed intermediate effects. Compared with rhinovirus (RV)-infected PBS-exposed mice, RV-infected elastase/LPS-exposed mice showed persistence of viral RNA, airway hyperresponsiveness, increased lung volume, and sustained increases in expression of TNFalpha, IL-5, IL-13, and muc5AC (up to 14 days postinfection). Furthermore, virus-induced IFNs, interferon response factor-7, and IL-10 were deficient in elastase/LPS-treated mice. Mice exposed to LPS or elastase alone cleared virus similar to PBS-treated control mice. We conclude that limited exposure of mice to elastase/LPS produces a COPD-like condition including increased persistence of RV, likely due to skewing of the immune response towards a Th2 phenotype. Similar mechanisms may be operative in COPD.
Assuntos
Exposição por Inalação , Lipopolissacarídeos/administração & dosagem , Elastase Pancreática/administração & dosagem , Infecções por Picornaviridae/imunologia , Infecções por Picornaviridae/virologia , Rhinovirus/fisiologia , Animais , Feminino , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/patologia , Interferons/genética , Interferons/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-13/genética , Interleucina-13/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Pulmão/virologia , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Mucina-5AC/genética , Mucina-5AC/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Elastase Pancreática/farmacologia , Infecções por Picornaviridae/complicações , Infecções por Picornaviridae/fisiopatologia , Pneumonia/complicações , Pneumonia/patologia , Pneumonia/fisiopatologia , Pneumonia/virologia , Testes de Função Respiratória , Sus scrofa , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
RATIONALE: Staphylococcus aureus is a major pathogen complicating postsurgical care. OBJECTIVES: To test the effect of sterile laparotomy (LAP) on pulmonary clearance of S. aureus in a murine model. METHODS: Control and LAP mice were infected intranasally with 10(8) cfu of S. aureus. Microbial clearance, pulmonary leukocyte recruitment, and cytokine profiles were compared between the groups. Antibody neutralization or cytokine gene knockout mice were used to evaluate the role of cytokines. MEASUREMENTS AND MAIN RESULTS: Laparotomy resulted in a 10-fold increase in S. aureus lung colony-forming units on Days 2 and 3 postinfection. Both groups cleared the infection by Day 4. No defect in leukocyte recruitment into the lungs was observed in infected LAP animals; however, an increase in the number of Mac-3-positive cells and a significant decrease of cells with high surface expression of Fc-gammaR suggest suboptimal activation of leukocytes in the lungs of infected LAP animals. Infected LAP mice had decreased expression of interferon (IFN)-gamma and increased expression of mRNA for IL-13 in the lungs on Day 1 postinfection and decreased levels of IL-6, keratinocyte-derived chemokine (KC), and macrophage inflammatory protein-2 (MIP-2) in bronchoalveolar lavage at Day 2 postinfection. Neutralization of IFN-gamma mimicked the effect of LAP with impaired clearance on Day 2. CONCLUSIONS: Sterile LAP induced temporary deactivation of innate immune responses to pulmonary S. aureus challenge. Impaired microbial clearance was accompanied by altered cytokine expression and suboptimal activation of pulmonary leukocytes. Lack of early IFN-gamma induction in the infected lungs of LAP animals is a likely mechanism contributing to the observed phenotype.
Assuntos
Citocinas/metabolismo , Imunidade Inata/fisiologia , Laparotomia , Pneumonia Estafilocócica/imunologia , Pneumonia Estafilocócica/metabolismo , Complicações Pós-Operatórias , Animais , Quimiotaxia de Leucócito/fisiologia , Contagem de Colônia Microbiana , Citocinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Fagócitos/fisiologia , Pneumonia Estafilocócica/microbiologia , RNA Mensageiro/metabolismoRESUMO
Macrophages (Mø) ingest apoptotic cells with unique effects on their cytokine production, but the signaling pathways involved are virtually unknown. Signal transduction in response to recognition of apoptotic thymocytes by resident murine alveolar (AMø) or peritoneal (PMø) Mø was studied by in vitro phagocytosis assay. Phagocytosis was decreased in a dose-dependent and nontoxic manner by inhibiting phosphatidylinositol 3 kinase (wortmannin and LY294002), protein tyrosine phosphorylation (herbimycin A, genistein, piceatannol, and for AMø only, PP2), and protein kinase C (staurosporine, Gö 6976, and calphostin C). Exposure of Mø to apoptotic or heat-killed thymocytes, but not to viable thymocytes, activated ERK1/2 rapidly, as detected by specific phosphorylation, but did not activate NF-kappaB or MAP kinases p38 or JNK. Mø phagocytosis of apoptotic T cells requires tyrosine, serine/threonine, and lipid phosphorylation. Mø recognition of apoptotic T cells triggers rapid but limited MAP kinase activation.
Assuntos
Apoptose , Macrófagos/imunologia , Fagocitose , Transdução de Sinais , Linfócitos T/imunologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Macrófagos Alveolares/imunologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fagocitose/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Proteína Quinase C/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores Fc/fisiologia , Transdução de Sinais/efeitos dos fármacos , Timo/imunologiaRESUMO
Innate immunity is required for effective control of poxvirus infections, but cellular receptors that initiate the host response to these DNA viruses remain poorly defined. Given this information and the fact that functions of TLRs in immunity to DNA viruses remain controversial, we investigated effects of TLR3 on pathogenesis of vaccinia virus, a prototype poxvirus. We used a recombinant strain Western Reserve vaccinia virus that expresses firefly luciferase to infect wild-type C57BL/6 and TLR3-/- mice through intranasal inoculation. Bioluminescence imaging showed that TLR3-/- mice had substantially lower viral replication in the respiratory tract and diminished dissemination of virus to abdominal organs. Mice lacking TLR3 had reduced disease morbidity, as measured by decreased weight loss and hypothermia after infection. Importantly, TLR3-/- mice also had improved survival relative to wild-type mice. Infected TLR3-/- mice had significantly reduced lung inflammation and recruitment of leukocytes to the lung. Mice lacking TLR3 also had lower levels of inflammatory cytokines, including IL-6, MCP-1, and TNF-alpha in serum and/or bronchoalveolar lavage fluid, but levels of IFN-beta did not differ between genotypes of mice. To our knowledge, our findings show for the first time that interactions between TLR3 and vaccinia increase viral replication and contribute to detrimental effects of the host immune response to poxviruses.
Assuntos
Receptor 3 Toll-Like/fisiologia , Vacínia/imunologia , Animais , Citocinas/metabolismo , Feminino , Luciferases/análise , Luciferases/genética , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Knockout , Receptor 3 Toll-Like/genética , Vacínia/patologia , Vacínia/virologia , Vaccinia virus/genética , Vaccinia virus/fisiologia , Replicação ViralRESUMO
Because of concerns about zoonotic transmission of monkeypox to humans and the bioterrorism threat posed by orthopoxviruses, there is renewed interest in probing cellular and molecular mechanisms of host defense to these pathogens. In particular, it is essential to understand viral-host interactions in the respiratory tract, which is the route of infection for smallpox and a likely route of transmission for monkeypox. In this study, we analyze functions of alveolar macrophages in poxvirus infection, using a recombinant vaccinia virus expressing firefly luciferase to quantify infection in mice and cell culture. Depletion of alveolar macrophages with liposomal clodronate worsens the overall severity of infection in mice, including greater replication and systemic dissemination of vaccinia as determined by bioluminescence imaging. Absence of alveolar macrophages increases total numbers of granulocytes and granulocytes/monocyte progenitor cells in the lungs during vaccinia infection, indicating that protective effects of alveolar macrophages may be mediated in part by reducing the host inflammation. Alveolar macrophages also limit vaccinia infection in respiratory epithelium, as shown by a co-culture model of cell lines derived from alveolar macrophages and lung epithelium. Collectively, these data demonstrate that alveolar macrophages are key determinants of host defense against local and systemic infection with poxviruses.
Assuntos
Macrófagos Alveolares/imunologia , Vaccinia virus/imunologia , Vaccinia virus/fisiologia , Replicação Viral , Animais , Ácido Clodrônico/farmacologia , Técnicas de Cocultura , Leucócitos/imunologia , Pulmão/citologia , Pulmão/imunologia , Macrófagos Alveolares/citologia , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Vacínia/imunologia , Vacínia/virologiaRESUMO
Apoptotic cells must be cleared to resolve inflammation, but few resident alveolar macrophages (AMo) from normal lungs ingest apoptotic cells. We examined how Mo ingestion of apoptotic cells is altered during immune inflammation induced by intratracheal challenge of primed C57BL/6 mice using sheep red blood cells. Resident AMo were labeled in situ before challenge using intravenous PKH26 to distinguish them from recruited monocytes. Using flow cytometry, we identified phagocytosis of fluorescently-labeled apoptotic thymocytes by alveolar mononuclear phagocytes in vitro and in vivo, and measured surface molecule expression. Intratracheal challenge induced rapid recruitment of monocytes, peaking at Day 3 and decreasing thereafter, whereas numbers of resident AMo did not change significantly. At all times, the percentage of phagocytes ingesting apoptotic thymocytes in vitro was greater among resident AMo (28-45%) than among recruited monocytes (9-19%), but was low in both cell types relative to ingestion of immunoglobulin-opsonized targets. There was also a nonsignificant trend toward lower ingestion by monocytes in vivo. MerTK, a receptor tyrosine kinase crucial for apoptotic cell phagocytosis, was expressed by resident AMo, but not by recruited monocytes. Relative to resident AMo, monocytes recruited to the alveolus ingest apoptotic cells meagerly, possibly due to absence of MerTK expression.
Assuntos
Apoptose/imunologia , Movimento Celular/imunologia , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Monócitos/imunologia , Fagocitose/imunologia , Animais , Eritrócitos/imunologia , Regulação da Expressão Gênica/imunologia , Inflamação/imunologia , Inflamação/patologia , Pulmão/patologia , Ativação de Macrófagos/imunologia , Macrófagos Alveolares/patologia , Camundongos , Proteínas Proto-Oncogênicas/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Ovinos , Linfócitos T/imunologia , Linfócitos T/patologia , c-Mer Tirosina QuinaseRESUMO
Dendritic cells (DC) migrate from sites of inflammation to lymph nodes to initiate primary immune responses, but the molecular mechanisms by which DC are replenished in the lungs during ongoing pulmonary inflammation are unknown. To address this question, we analyzed the secondary pulmonary immune response of Ag-primed mice to intratracheal challenge with the particulate T cell-dependent Ag sheep erythrocytes (SRBC). We studied wild-type C57BL/6 mice and syngeneic gene-targeted mice lacking either both endothelial selectins (CD62E and CD62P), or the chemokine receptors CCR2 or CCR6. DC, defined as non-autofluorescent, MHC class II(+)CD11c(mod) cells, were detected in blood, enzyme-digested minced lung, and bronchoalveolar lavage fluid using flow cytometry and immunohistology. Compared with control mice, Ag challenge increased the frequency and absolute numbers of DC, peaking at day 1 in peripheral blood (6.5-fold increase in frequency), day 3 in lung mince (20-fold increase in total DC), and day 4 in bronchoalveolar lavage fluid (55-fold increase in total DC). Most lung DC expressed CD11c, CD11b, and low levels of MHC class II, CD40, CD80, and CD86, consistent with an immature myeloid phenotype. DC accumulation depended in part upon CCR2 and CCR6, but not endothelial selectins. Thus, during lung inflammation, immature myeloid DC from the bloodstream replace emigrating immature DC and transiently increase total intrapulmonary APC numbers. Early DC recruitment depends in part on CCR2 to traverse vascular endothelium, plus CCR6 to traverse alveolar epithelium. The recruitment of circulating immature DC represents a potential therapeutic step at which to modulate immunological lung diseases.
Assuntos
Diferenciação Celular/imunologia , Movimento Celular/imunologia , Células Dendríticas/imunologia , Selectina E , Eritrócitos/imunologia , Pulmão/imunologia , Receptores de Quimiocinas/fisiologia , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Contagem de Células , Diferenciação Celular/genética , Movimento Celular/genética , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Selectina E/sangue , Selectina E/genética , Selectina E/fisiologia , Feminino , Inflamação/sangue , Inflamação/genética , Inflamação/imunologia , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Receptores CCR2 , Receptores CCR6 , Receptores de Quimiocinas/sangue , Receptores de Quimiocinas/deficiência , Receptores de Quimiocinas/genética , OvinosRESUMO
We showed previously that protein kinase C (PKC) is required for phagocytosis of apoptotic leukocytes by murine alveolar (AMø) and peritoneal macrophages (PMø) and that such phagocytosis is markedly lower in AMø compared with PMø. In this study, we examined the roles of individual PKC isoforms in phagocytosis of apoptotic thymocytes by these two Mø populations. By immunoblotting, AMø expressed equivalent PKC eta but lower amounts of other isoforms (alpha, betaI, betaII, delta, epsilon, mu, and zeta), with the greatest difference in betaII expression. A requirement for PKC betaII for phagocytosis was demonstrated collectively by phorbol 12-myristate 13-acetate-induced depletion of PKC betaII, by dose-response to PKC inhibitor Ro-32-0432, and by use of PKC betaII myristoylated peptide as a blocker. Exposure of PMø to phosphatidylserine (PS) liposomes specifically induced translocation of PKC betaII and other isoforms to membranes and cytoskeleton. Both AMø and PMø expressed functional PS receptor, blockade of which inhibited PKC betaII translocation. Our results indicate that murine tissue Mø require PKC betaII for phagocytosis of apoptotic cells, which differs from the PKC isoform requirement previously described in Mø phagocytosis of other particles, and imply that a crucial action of the PS receptor in this process is PKC betaII activation.
Assuntos
Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Receptores de Superfície Celular/metabolismo , Timo/citologia , Acetofenonas/farmacologia , Animais , Apoptose , Benzopiranos/farmacologia , Western Blotting , Adesão Celular , Relação Dose-Resposta a Droga , Ativação Enzimática , Feminino , Citometria de Fluxo , Immunoblotting , Indóis/farmacologia , Concentração Inibidora 50 , Lipossomos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ácido Mirístico/farmacologia , Fagocitose , Isoformas de Proteínas , Proteína Quinase C/química , Proteína Quinase C beta , Transporte Proteico , Pirróis/farmacologia , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
The innate immune response must be mobilized promptly yet judiciously via TLRs to protect the lungs against pathogens. Stimulation of murine peritoneal macrophage (PMphi) TLR4 or TLR3 by pathogen-associated molecular patterns (PAMPs) typically induces type I IFN-beta, leading to autocrine activation of the transcription factor STAT1. Because it is unknown whether STAT1 plays a similar role in the lungs, we studied the response of resident alveolar macrophages (AMphi) or control PMphi from normal C57BL/6 mice to stimulation by PAMPs derived from viruses (polyriboinosinic:polyribocytidylic acid, specific for TLR3) or bacteria (Pam(3)Cys, specific for TLR2, and repurified LPS, specific for TLR4). AMphi did not activate STAT1 by tyrosine phosphorylation on Y701 following stimulation of any of these three TLRs, but readily did so in response to exogenous IFN-beta. This unique AMphi response was not due to altered TLR expression, or defective immediate-early gene response, as measured by expression of TNF-alpha and three beta chemokines. Instead, AMphi differed from PMphi in not producing bioactive IFN-beta, as confirmed by ELISA and by the failure of supernatants from TLR-stimulated AMphi to induce STAT1 phosphorylation in PMphi. Consequently, AMphi did not produce the microbicidal effector molecule NO following TLR4 or TLR3 stimulation unless exogenous IFN-beta was also added. Thus, murine AMphi respond to bacterial or viral PAMPs by producing inflammatory cytokines and chemokines, but because they lack the feed-forward amplification typically mediated by autocrine IFN-beta secretion and STAT1 activation, require exogenous IFN to mount a second phase of host defense.
Assuntos
Adjuvantes Imunológicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interferon beta/metabolismo , Macrófagos Alveolares/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Diferenciação/metabolismo , Proteínas de Ligação a DNA/efeitos dos fármacos , Feminino , Imunidade Inata/fisiologia , Interferon beta/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Camundongos , Fator 88 de Diferenciação Mieloide , Óxido Nítrico/metabolismo , Fosforilação , Receptores Imunológicos/metabolismo , Fator de Transcrição STAT1 , Receptor 2 Toll-Like , Receptor 3 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Transativadores/efeitos dos fármacosRESUMO
Apoptotic cells must be cleared efficiently by macrophages (Mø) to prevent autoimmunity, yet their ingestion impairs Mø microbicidal function. The principal murine resident lung phagocyte, the alveolar Mø (AMø), is specifically deficient at apoptotic cell ingestion, both in vitro and in vivo, compared with resident peritoneal Mø (PMø). To further characterize this deficiency, we assayed static adhesion in vitro using apoptotic thymocytes and resident AMø and PMø from normal C57BL/6 mice. Adhesion of apoptotic thymocytes by both types of Mø was rapid, specific, and cold-sensitive. Antibody against the receptor tyrosine kinase MerTK (Tyro12) blocked phagocytosis but not adhesion in both types of Mø. Surfactant protein A increased adhesion and phagocytosis by AMø, but not to the levels seen using PMø. Adhesion was largely cation-independent for PMø and calcium-dependent for AMø. Adhesion was not inhibited in either Mø type by mAbs against beta1 or beta3 integrins or scavenger receptor I/II (CD204), but AMø adhesion was inhibited by specific mAbs against CD11c/CD18. Thus, resident murine tissue Mø from different tissues depend on qualitatively disparate receptor systems to bind apoptotic cells. The decreased capacity of murine AMø to ingest apoptotic cells is only partially explained by reduced initial adhesion.
Assuntos
Apoptose/fisiologia , Adesão Celular/fisiologia , Macrófagos Alveolares/fisiologia , Macrófagos Peritoneais/fisiologia , Proteínas Proto-Oncogênicas , Linfócitos T/fisiologia , Timo/citologia , Animais , Anticorpos Monoclonais/metabolismo , Antígeno CD11c/metabolismo , Antígenos CD18/metabolismo , Cálcio/metabolismo , Cátions/metabolismo , Temperatura Baixa , Feminino , Macrófagos Alveolares/citologia , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína A Associada a Surfactante Pulmonar/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , c-Mer Tirosina QuinaseRESUMO
D10.G4.1 (D10) cells, a murine conalbumin-reactive Th2 cell line, made to overexpress the beta(2) integrin LFA-1 by pharmacological manipulation or by transfection become autoreactive and are capable of inducing in vivo autoimmunity. However, whether this is specific to LFA-1 and whether overexpression of other T cell integrin molecules has the same effect are unknown. We examined the functional consequences of T cell CD49d (alpha(4) integrin) overexpression by transfecting murine CD49d cDNA into D10 cells. Similar to the LFA-1-transfected cells, the CD49d-overexpressing T cells are autoreactive and proliferate in response to APCs in an MHC class II-dependent manner in the absence of nominal Ag. Additionally, CD49d overexpression is associated with increased in vitro adhesion to endothelial cells and increased in vivo splenic homing. However, in contrast to LFA-1 overexpression, increased T cell CD49d expression is not associated with autoreactive cytotoxicity or the ability to induce in vivo autoimmunity. In addition to the novel observation that CD49d overexpression is sufficient to induce T cell autoreactivity, our results also support the hypothesis that the ability to induce in vivo autoimmunity is related to T cell cytotoxicity and not to T cell proliferation function in the D10 murine adoptive transfer model of autoimmunity.
Assuntos
Autoimunidade/genética , Autoimunidade/imunologia , Integrina alfa4/biossíntese , Integrina alfa4/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transfecção , Transferência Adotiva , Animais , Anticorpos Antinucleares/biossíntese , Adesão Celular/genética , Adesão Celular/imunologia , Divisão Celular/genética , Divisão Celular/imunologia , Linhagem Celular/imunologia , Linhagem Celular/metabolismo , Linhagem Celular/transplante , Movimento Celular/genética , Movimento Celular/imunologia , Citotoxicidade Imunológica/genética , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos MRL lpr , Fosforilação , Baço/citologia , Baço/imunologia , Linfócitos T/citologia , Transfecção/métodos , Tirosina/metabolismoRESUMO
We previously demonstrated induction and expression of CD62E and CD62P in the lungs of mice primed and then challenged with intratracheal (i.t.) SRBC. The current study examined accumulation of endogenous lymphocytes in the lungs of endothelial E- and P-selectin-deficient (E(-)P(-)) mice after i.t. SRBC challenge. Compared with syngeneic wild-type (wt) mice, E(-)P(-) mice showed an 85-95% decrease in CD8(+) T cells and B cells in the lungs at both early and late time points. In contrast, CD4(+) T cell accumulation was reduced by approximately 60% early, but equivalent to wt levels later. Surprisingly, many gammadelta T cells were found in lungs and blood of E(-)P(-) mice but were undetectable in the lungs and blood of wt mice. Absolute numbers of peripheral blood CD4, CD8, and B lymphocytes in E(-)P(-) mice equaled or exceeded the levels in wt mice, particularly after challenge. Trafficking studies using alphabeta T lymphoblasts confirmed that the recruitment of circulating cells after challenge was markedly reduced in E(-)P(-) mice. Furthermore, Ag priming occurred normally in both the selectin-deficient and wt mice, because primed lymphocytes from both groups transferred Ag sensitivity into naive wt mice. Lung production of mRNA for six CC and two CXC chemokines after challenge was equivalent by RT-PCR analysis in wt and E(-)P(-) mice. Therefore, reduced lung accumulation of alphabeta T cells and B cells in E(-)P(-) mice did not result from reduced delivery of circulating lymphocytes to the lungs, unsuccessful Ag priming, or defective pulmonary chemokine production. Selectin-dependent lymphocyte recruitment into the lungs following i.t.-SRBC challenge is subset specific and time dependent.