RESUMO
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
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
Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires the development of T1-type immunity. CCR2-deficient mice infected with C. neoformans develop a non-protective T2 immune response and persistent infection. The mechanisms responsible for this aberrant response are unknown. The objective of this study was to define the number, phenotype, and microanatomic location of dendritic cells (DC) residing within the lung of CCR2+/+ or CCR2-/- mice throughout a time course following infection with C. neoformans. Results demonstrate the CCR2-mediated recruitment of conventional DC expressing modest amounts of costimulatory molecules. DC recruitment was preceded by the up-regulation in the lung of the CCR2 ligands CCL2 and CCL7. Colocalization of numerous DC and CD4+ T cells within bronchovascular infiltrates coincided with increased expression of IL-12 and IFN-gamma. By contrast, in the absence of CCR2, DC recruitment was markedly impaired, bronchovascular infiltrates were diminished, and mice developed features of T2 responses, including bronchovascular collagen deposition and IL-4 production. Our results demonstrate that CCR2 is required for the recruitment of large numbers of conventional DC to bronchovascular infiltrates in mice mounting a T1 immune response against a fungal pathogen. These findings shed new insight into the mechanism(s) by which DC recruitment alters T cell polarization in response to an infectious challenge within the lung.
Assuntos
Brônquios/imunologia , Movimento Celular , Criptococose/imunologia , Cryptococcus neoformans/imunologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Receptores CCR2/imunologia , Animais , Diferenciação Celular/imunologia , Criptococose/genética , Criptococose/metabolismo , Criptococose/patologia , Células Dendríticas/imunologia , Feminino , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-12/genética , Interleucina-12/metabolismo , Cinética , Ligantes , Camundongos , Camundongos Knockout , Fenótipo , RNA Mensageiro/genética , Receptores CCR2/deficiência , Receptores CCR2/genética , Receptores CCR2/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
Nontypeable Haemophilus influenzae (NTHi) is strongly associated with exacerbations of chronic obstructive pulmonary disease, which often coincide with viral respiratory infections. TLR2 contributes importantly to innate immunity to NTHi, but whether this pathway is affected by simultaneous antiviral responses is unknown. To analyze potential interactions, resident murine and human alveolar macrophages (AMphi) were exposed, in the presence or absence of the appropriate rIFN-beta, to synthetic lipopeptides corresponding to the triacylated N-terminal fragments of three outer membrane proteins (OMP) (PCP, P4, and P6) that are highly conserved among different NTHi strains. Synthetic OMP elicited strong release of IL-6, the principal inducer of airway mucin genes, and induced CCL5 and CXCL10 from murine AMphi only when IFN-beta was also present. Surprisingly, combined stimulation by OMPs and IFN-beta also markedly enhanced TNF-alpha release by murine AMphi. Stimulation with PCP plus IFN-beta induced IFN-regulatory factor 1 expression and sustained STAT1 activation, but did not alter the activation of MAPKs or NF-kappaB. AMphi derived from STAT1-deficient mice did not demonstrate increased production of TNF-alpha in response to PCP plus IFN-beta. Analysis of wild-type and STAT1-deficient AMphi using real-time PCR showed that increased TNF-alpha production depended on transcriptional up-regulation, but not on mRNA stabilization. The synergistic effect of synthetic OMP and IFN-beta was conserved between murine AMphi and human AMphi for IL-6, but not for TNF-alpha. Thus, IFN-beta, which is produced by virally infected respiratory epithelial cells, converts normally innocuous NTHi OMP into potent inflammatory stimulants, but does so via different mechanisms in mice and humans.
Assuntos
Adjuvantes Imunológicos/fisiologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Citocinas/biossíntese , Haemophilus influenzae/imunologia , Interferon beta/fisiologia , Lipoproteínas/fisiologia , Macrófagos Alveolares/imunologia , Receptor 2 Toll-Like/metabolismo , Regulação para Cima/imunologia , Adjuvantes Imunológicos/metabolismo , Idoso , Animais , Aderência Bacteriana/genética , Aderência Bacteriana/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sequência Conservada , Feminino , Haemophilus influenzae/classificação , Haemophilus influenzae/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Interferon Tipo I/farmacologia , Lipoproteínas/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/genética , Ligação Proteica/imunologia , Proteínas Recombinantes , Fator de Transcrição STAT1/fisiologia , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Regulação para Cima/genéticaRESUMO
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
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.