RESUMO
Lung injury can release intracellular actin into the alveolar milieu and is also associated with increased susceptibility to secondary infections. We investigated the effect of free (extracellular) actin on lung macrophage host defense functions. Western blot analysis demonstrated free actin release into the lung lavage fluids of mouse models of ozone injury, influenza infection, and secondary pneumococcal pneumonia and in samples from patients following burn and inhalation injury. Using levels comparable with those observed in lung injury, we found that free actin markedly inhibited murine lung macrophage binding and uptake in vitro of S. pneumoniae, S. aureus, and E. coli, (e.g., S. pneumoniae, mean %inhibition, actin vs. vehicle: 85 ± 0.3 (SD); n = 22, P < .001). Similar effects were observed on the ability of primary human macrophages to bind and ingest fluorescent Saureus (~75% inhibition). Plasma gelsolin (pGSN), a protein that functions to bind and cleave actin, restored bacterial binding and uptake by both murine and human macrophages. Scavenger receptor inhibitors reduced binding of fluorescent actin by murine macrophages [fluorescence index (×10-3) after incubation with vehicle, actin, or actin + polyinosinic acid, respectively: 0.8 ± 0.7, 101.7 ± 50.7, or 52.7 ± 16.9; n = 5-6, P < 0.05]. In addition, actin binding was reduced in a MARCO/SR-AI/II-deficient cell line and by normal AMs obtained from MARCO-/- mice. After release from injured cells during lung injury, free actin likely contributes to impaired host defense by blocking scavenger receptor binding of bacteria. This mechanism for increased risk of secondary infections after lung injury or inflammation may represent another target for therapeutic intervention with pGSN.
Assuntos
Actinas/metabolismo , Gelsolina/sangue , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Receptores Imunológicos/metabolismo , Receptores Depuradores/metabolismo , Animais , Bactérias/imunologia , Feminino , Humanos , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Masculino , Camundongos Endogâmicos C57BL , Ligação ProteicaRESUMO
Human macrophages at mucosal sites are essential targets for acute HIV infection. During the chronic phase of infection, they are persistent reservoirs for the AIDS virus. HIV virions gain entry into macrophages following ligation of surface CD4-CCR5 co-receptors, which leads to the release of two copies of HIV ssRNA. These events lead to reverse transcription and viral replication initiation. Toll-like receptors TLR7 and TLR8 recognize specific intracellular viral ssRNA sequences, but in human alveolar macrophages, their individual roles in TLR-mediated HIV ssRNA recognition are unclear. In the current study, HIV-1 ssRNA induced TNFα release in a dose-dependent manner in adherent human macrophages expressing both intracellular TLR7 and TLR8. This response was reduced by inhibiting either endocytosis (50 µm dynasore) or endosomal acidification (1 µg/ml chloroquine). Either MYD88 or TLR8 gene knockdown with relevant siRNA reduced HIV-1 ssRNA-mediated TNFα release, but silencing TLR7 had no effect on this response. Furthermore, HIV-1 ssRNA induced histone 4 acetylation at the TNFα promoter as well as trimethylation of histone 3 at lysine 4, whereas TLR8 gene knockdown reduced these effects. Taken together in human macrophages, TLR8 binds and internalizes HIV ssRNA, leading to endosomal acidification, chromatin remodeling, and increases in TNFα release. Drugs targeting macrophage TLR8-linked signaling pathways may modulate the innate immune response to acute HIV infection by reducing viral replication.
Assuntos
Epigênese Genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/metabolismo , Macrófagos/citologia , RNA/metabolismo , Receptor 7 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Cromatina/química , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina , Infecções por HIV/genética , Humanos , Macrófagos Alveolares/citologia , Oligonucleotídeos/química , RNA Viral/metabolismoRESUMO
Mycobacterium tuberculosis disease represents an enormous global health problem, with exceptionally high morbidity and mortality in HIV-seropositive (HIV(+)) persons. Alveolar macrophages from HIV(+) persons demonstrate specific and targeted impairment of critical host cell responses, including impaired M. tuberculosis-mediated tumor necrosis factor (TNF) release and macrophage apoptosis. Vitamin D may promote anti-M. tuberculosis responses through upregulation of macrophage NO, NADPH oxidase, cathelicidin, and autophagy mechanisms, but whether vitamin D promotes anti-M. tuberculosis mechanisms in HIV(+) macrophages is not known. In the current study, human macrophages exposed to M. tuberculosis demonstrated robust release of TNF, IκB degradation, and NF-κB nuclear translocation, and these responses were independent of vitamin D pretreatment. In marked contrast, HIV(+) U1 human macrophages exposed to M. tuberculosis demonstrated very low TNF release and no significant IκB degradation or NF-κB nuclear translocation, whereas vitamin D pretreatment restored these critical responses. The vitamin D-mediated restored responses were dependent in part on macrophage CD14 expression. Importantly, similar response patterns were observed with clinically relevant human alveolar macrophages from healthy individuals and asymptomatic HIV(+) persons at high clinical risk of M. tuberculosis infection. Taken together with the observation that local bronchoalveolar lavage fluid (BALF) levels of vitamin D are severely deficient in HIV(+) persons, the data from this study demonstrate that exogenous vitamin D can selectively rescue impaired critical innate immune responses in vitro in alveolar macrophages from HIV(+) persons at risk for M. tuberculosis disease, supporting a potential role for exogenous vitamin D as a therapeutic adjuvant in M. tuberculosis infection in HIV(+) persons.
Assuntos
Soropositividade para HIV/microbiologia , Macrófagos Alveolares/imunologia , Mycobacterium tuberculosis/imunologia , Receptores Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia , Vitamina D/farmacologia , Líquido da Lavagem Broncoalveolar/imunologia , Linhagem Celular , Soropositividade para HIV/imunologia , Soropositividade para HIV/metabolismo , Humanos , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , Mycobacterium tuberculosis/metabolismo , NF-kappa B/imunologia , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Transdução de Sinais/imunologia , Receptores Toll-Like/metabolismo , Tuberculose/metabolismo , Tuberculose/virologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Células U937 , Regulação para Cima/imunologia , Vitamina D/imunologia , Vitamina D/metabolismoRESUMO
TLR-4-mediated signaling is significantly impaired in macrophages from HIV(+) persons, predominantly owing to altered MyD88-dependent pathway signaling caused in part by constitutive activation of PI3K. In this study we assessed in these macrophages if the blunted increase in TLR-4-mediated TNF-α release induced by lipid A (LA) is associated with PI3K-induced upregulation of mammalian target of rapamycin (mTOR) activity. mTOR inhibition with rapamycin enhanced TLR-4-mediated TNF-α release, but suppressed anti-inflammatory IL-10 release. Targeted gene silencing of mTOR in macrophages resulted in LA-induced TNF-α and IL-10 release patterns similar to those induced by rapamycin. Rapamycin restored MyD88/IL-1R-associated kinase interaction in a dose-dependent manner. Targeted gene silencing of MyD88 (short hairpin RNA) and mTOR (RNA interference) inhibition resulted in TLR-4-mediated 70-kDa ribosomal protein S6 kinase activation and enhanced TNF-α release, whereas IL-10 release was inhibited in both silenced and nonsilenced HIV(+) macrophages. Furthermore, mTOR inhibition augmented LA-induced TNF-α release through enhanced and prolonged phosphorylation of ERK1/2 and JNK1/2 MAPK, which was associated with time-dependent MKP-1 destabilization. Taken together, impaired TLR-4-mediated TNF-α release in HIV(+) macrophages is attributable in part to mTOR activation by constitutive PI3K expression in a MyD88-dependent signaling pathway. These changes result in MAPK phosphatase 1 stabilization, which shortens and blunts MAPK activation. mTOR inhibition may serve as a potential therapeutic target to upregulate macrophage innate immune host defense responsiveness in HIV(+) persons.
Assuntos
Infecções por HIV/metabolismo , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Western Blotting , Ativação Enzimática/imunologia , Ensaio de Imunoadsorção Enzimática , Técnicas de Silenciamento de Genes , Infecções por HIV/imunologia , Humanos , Imunoprecipitação , Macrófagos/imunologia , Macrófagos/virologia , Serina-Treonina Quinases TOR/imunologia , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Alveolar macrophages (AMs) are the predominant effector cell in the lungs and contribute to a critical first line of defense against bacterial pathogens through recognition by pattern recognition receptors such as Toll-like receptor 4 (TLR4). TLR4-mediated tumor necrosis factor alpha (TNFalpha) release is significantly impaired in HIV(+) macrophages, but whether HIV impairs myeloid differentiation factor 88 (MyD88)-dependent and/or MyD-independent TLR4 signaling pathways in human macrophages is not known. Comparing human U937 macrophages with HIV(+) U1 macrophages (HIV-infected U937 subclone), the current study shows that HIV infection is associated with impaired macrophage TLR4-mediated signaling, specifically targeting the MyD88-dependent TLR4-mediated signaling pathway (reduced MyD88-interleukin-1 receptor-associated kinase [IRAK] interaction, IRAK phosphorylation, nuclear factor [NF]-kappaB nuclear translocation, and TNFalpha release) while preserving the MyD88-independent TLR4-mediated signaling pathway (preserved STAT1 phosphorylation, interferon regulatory factor [IRF] nuclear translocation, and interleukin-10 [IL-10] and RANTES release). Extracellular TLR4 signaling complex was intact (similar levels of CD14 and MD2), and similar patterns of response were observed in clinically relevant AMs from healthy and asymptomatic HIV(+) persons at high clinical risk of pneumonia. Taken together, these data support the concept that chronic HIV infection is associated with specific and targeted disruption of critical macrophage TLR4 signaling, which in turn may contribute to disease pathogenesis of bacterial pneumonia.
Assuntos
Infecções por HIV/metabolismo , HIV , Macrófagos Alveolares/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Transporte Ativo do Núcleo Celular/imunologia , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Infecções por HIV/imunologia , Humanos , Fatores Reguladores de Interferon/imunologia , Fatores Reguladores de Interferon/metabolismo , Quinases Associadas a Receptores de Interleucina-1/imunologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Macrófagos Alveolares/imunologia , Masculino , Fator 88 de Diferenciação Mieloide/imunologia , Fosforilação/imunologia , Pneumonia/imunologia , Pneumonia/metabolismo , Risco , Fatores de Risco , Receptor 4 Toll-Like/imunologia , Células U937RESUMO
Tuberculosis (TB) disease remains one of the highest causes of mortality in HIV-infected individuals, and HIV-TB coinfection continues to grow at alarming rates, especially in sub-Saharan Africa. Surprisingly, a number of important areas regarding coinfection remain unclear. For example, increased risk of TB disease begins early in the course of HIV infection; however, the mechanism by which HIV increases this risk is not well understood. In addition, there is lack of consensus on the optimal way to diagnose latent TB infection and to manage active disease in those who are HIV infected. Furthermore, effective point-of-care testing for TB disease remains elusive. This review discusses key areas in the epidemiology, pathogenesis, diagnosis, and management of active and latent TB in those infected with HIV, focusing attention on issues related to high- and low-burden areas. Particular emphasis is placed on controversial areas where there are gaps in knowledge and on future directions of study.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/epidemiologia , Tuberculose Pulmonar/epidemiologia , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/tratamento farmacológico , Fármacos Anti-HIV/uso terapêutico , Antituberculosos/uso terapêutico , Interações Medicamentosas , Humanos , Tuberculose Latente/diagnóstico , Tuberculose Latente/epidemiologia , Mycobacterium tuberculosis/imunologia , Prevalência , Rifamicinas/uso terapêutico , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/etiologia , Tuberculose Pulmonar/imunologiaRESUMO
HIV-1 infection has significant effect on the immune system as well as on the nervous system. Breakdown of the blood-brain barrier (BBB) is frequently observed in patients with HIV-associated dementia (HAD) despite lack of productive infection of human brain microvascular endothelial cells (HBMEC). Cellular products and viral proteins secreted by HIV-1 infected cells, such as the HIV-1 Gp120 envelope glycoprotein, play important roles in BBB impairment and HIV-associated dementia development. HBMEC are a major component of the BBB. Using cocultures of HBMEC and human astrocytes as a model system for human BBB as well as in vivo model, we show for the first time that cannabinoid agonists inhibited HIV-1 Gp120-induced calcium influx mediated by substance P and significantly decreased the permeability of HBMEC as well as prevented tight junction protein down-regulation of ZO-1, claudin-5, and JAM-1 in HBMEC. Furthermore, cannabinoid agonists inhibited the transmigration of human monocytes across the BBB and blocked the BBB permeability in vivo. These results demonstrate that cannabinoid agonists are able to restore the integrity of HBMEC and the BBB following insults by HIV-1 Gp120. These studies may lead to better strategies for treatment modalities targeted to the BBB following HIV-1 infection of the brain based on cannabinoid pharmacotherapies.
Assuntos
Fármacos Anti-HIV/farmacologia , Ácidos Araquidônicos/farmacologia , Encéfalo/irrigação sanguínea , Encéfalo/virologia , Moduladores de Receptores de Canabinoides/farmacologia , Endotélio Vascular/virologia , Glicerídeos/farmacologia , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Complexo AIDS Demência/enzimologia , Complexo AIDS Demência/patologia , Complexo AIDS Demência/prevenção & controle , Complexo AIDS Demência/virologia , Amidoidrolases/antagonistas & inibidores , Fármacos Anti-HIV/uso terapêutico , Ácidos Araquidônicos/fisiologia , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Moduladores de Receptores de Canabinoides/agonistas , Moduladores de Receptores de Canabinoides/fisiologia , Carbamatos/farmacologia , Carbamatos/uso terapêutico , Linhagem Celular , Técnicas de Cocultura , Endocanabinoides , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiologia , Glicerídeos/fisiologia , Proteína gp120 do Envelope de HIV/fisiologia , HIV-1/fisiologia , Humanos , Microcirculação/efeitos dos fármacos , Microcirculação/fisiologia , Receptor CB1 de Canabinoide/fisiologiaRESUMO
TRPV4 is a non-selective cation channel with moderate calcium permeability, which is activated by exposure to hypotonicity. Such a stress induces regulatory volume decrease (RVD) behavior in human corneal epithelial cells (HCEC). We hypothesize that TRPV4 channel mediates RVD in HCEC. Immunohistochemistry revealed centrally and superficially concentrated TRPV4 localization in the corneal tissue. Immunocytochemical and fluorescence activated cell sorter (FACS) analyses identified TRPV4 membrane surface and cytosolic expression. RT-PCR and Western blot analyses identified TRPV4 gene and protein expression in HCEC, respectively. In addition, 4alpha-PDD or a 50% hypotonic medium induced up to threefold transient intracellular Ca2+ ([Ca2+]i) increases. Following TRPV4 siRNA HCEC transfection, its protein expression level declined by 64%, which abrogated these [Ca2+]i transients. Similarly, exposure to either ruthenium red or Ca(2+)-free Ringer's solution also eliminated this response. In these transfected cells, RVD declined by 51% whereas in the non-transfected counterpart, ruthenium red and Ca(2+)-free solution inhibited RVD by 54 and 64%, respectively. In contrast, capsazepine, a TRPV1 antagonist, failed to suppress [Ca2+]i transients and RVD. TRPV4 activation contributes to RVD since declines in TRPV4 expression and activity are associated with suppression of this response. In conclusion, there is TRPV4 functional expression in HCEC.
Assuntos
Cálcio/metabolismo , Epitélio Corneano/metabolismo , Canais de Cátion TRPV/metabolismo , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Linhagem Celular , Corantes/farmacologia , Epitélio Corneano/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Soluções Hipotônicas , Soluções Isotônicas/farmacologia , RNA Interferente Pequeno/metabolismo , Solução de Ringer , Rutênio Vermelho/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/efeitos dos fármacosRESUMO
Alveolar macrophages are "professional phagocytes" and critical effector cells that protect the lungs from a broad array of microbes that can cause severe respiratory tract infections such as pneumonia. The molecular mechanisms that mediate microbial phagocytosis in alveolar macrophages are not fully known, and the specific role of small Rho GTPases has not been established. Most studies of Rho GTPase and phagocytosis focus on cell lines and transfected cells, and results may not accurately represent mechanisms operant in the lungs of humans. The use of clinically relevant primary human lung macrophages to examine phagocytosis in the context of host defense function may provide data that translate more readily to human conditions in health and disease. This chapter provides a description of methods and techniques for isolating, culturing, and assaying human alveolar macrophages for studies of small Rho GTPases and phagocytosis in the context of host defense. Data support the concept that different macrophage phagocytic receptors may exhibit distinct molecular mechanisms of small Rho GTPase activation that mediate phagocytosis.
Assuntos
Macrófagos Alveolares/fisiologia , Fagocitose/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Adolescente , Adulto , Líquido da Lavagem Broncoalveolar/citologia , Humanos , Pessoa de Meia-Idade , Transfecção , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologiaRESUMO
Interaction with the unique fungus Pneumocystis (Pc) promotes IL-8 release by human alveolar macrophages (AM), although the receptor(s) mediating IL-8 release have not been identified. TLR2 recognizes fungal components and mediates release of host defense cytokines and chemokines, although whether TLR2 mediates signaling in response to Pc is not known. In the current study, Pc induced IL-8 release by human AM, and AM pretreatment with anti-TLR2 neutralizing antibody reduced IL-8 release. However, in nonphagocytic human embryonic kidney (HEK)293 cells transfected with human TLR2 cDNA, incubation with Pc did not induce IL-8 release, whereas these same cells released IL-8 in response to the TLR2 agonist lipoteichoic acid. Targeted gene silencing of AM mannose receptors (MR; phagocytic receptors for Pc) using small interfering RNA also reduced Pc-mediated IL-8 release in human AM. However, HEK293 cells transfected with human MR cDNA alone did not release IL-8 in response to Pc. In contrast, HEK293 cells cotransfected with human TLR2 and human MR cDNA released IL-8 in response to Pc. In human AM, Pc promoted direct interaction of MR and TLR2, IL-8 release was reduced markedly upon simultaneous blocking of TLR2 and gene silencing of MR, and IL-8 release was dependent in part on transcription factor NF-kappaB and ERK1/2 and JNK MAPKs. These studies demonstrate that Pc-mediated IL-8 release by human AM requires the coexpression of MR and TLR2 and further supports the concept that combinatorial interactions of macrophage innate receptors provide specificity of host defense cell responses to infectious challenge.
Assuntos
Interleucina-8/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos Alveolares/metabolismo , Lectinas de Ligação a Manose/metabolismo , Pneumocystis/imunologia , Receptores de Superfície Celular/metabolismo , Receptor 2 Toll-Like/metabolismo , Adulto , Anticorpos/imunologia , Linhagem Celular , Inativação Gênica , Humanos , Imunidade Inata , Lectinas Tipo C/genética , MAP Quinase Quinase 4/metabolismo , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Receptor de Manose , Lectinas de Ligação a Manose/genética , Pessoa de Meia-Idade , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Pneumocystis/fisiologia , Receptores de Superfície Celular/genética , Transdução de Sinais , TransfecçãoRESUMO
Human alveolar macrophages (AMs) phagocytose Pneumocystis (Pc) organisms predominantly through mannose receptors, although the molecular mechanism mediating this opsonin-independent process is not known. In this study, using AMs from healthy individuals, Pc phagocytosis was associated with focal F-actin polymerization and Cdc42, Rac1, and Rho activation in a time-dependent manner. Phagocytosis was primarily dependent on Cdc42 and RhoB activation (as determined by AM transfection with Cdc42 and RhoB dominant-negative alleles) and mediated predominantly through mannose receptors (as determined by siRNA gene silencing of AM mannose receptors). Pc also promoted PAK-1 phosphorylation, which was also dependent on RhoGTPase activation. HIV infection of AMs (as a model for reduced mannose receptor expression and function) was associated with impaired F-actin polymerization, reduced Cdc42 and Rho activation, and markedly reduced PAK-1 phosphorylation in response to Pc organisms. In healthy AMs, Pc phagocytosis was partially dependent on PAK activation, but dependent on the Rho effector molecule ROCK. These data provide a molecular mechanism for AM mannose receptor-mediated phagocytosis of unopsonized Pc organisms that appears distinct from opsonin-dependent phagocytic receptors. Reduced AM mannose receptor-mediated Cdc42 and Rho activation in the context of HIV infection may represent a mechanism that contributes to the pathogenesis of opportunistic pneumonia.
Assuntos
Proteínas de Transporte/metabolismo , Infecções por HIV/imunologia , Lectinas Tipo C/metabolismo , Macrófagos Alveolares/imunologia , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Western Blotting , Líquido da Lavagem Broncoalveolar/citologia , Broncoscopia , Proteínas de Transporte/genética , Células Cultivadas , Proteínas do Citoesqueleto , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Inativação Gênica , Infecções por HIV/complicações , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Humanos , Imunidade Inata , Ativação de Macrófagos , Macrófagos Alveolares/microbiologia , Receptor de Manose , Microscopia Confocal , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Pneumocystis/fisiologia , Testes de Precipitina , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Zimosan/farmacologia , Proteína cdc42 de Ligação ao GTP/genética , Quinases Ativadas por p21 , Proteínas rho de Ligação ao GTP , Proteína rhoB de Ligação ao GTP/genéticaRESUMO
In certain epithelial tissues, activation of transient receptor potential (TRP) vanilloid subtype 1 (TRPV1) by noxious stimuli induces pro-inflammatory cytokine release, which helps to mitigate the challenge. While the corneal epithelium elicits such responses to a variety of challenges, it remains unknown whether TRPV1 mediates pro-inflammatory cytokine secretion. Accordingly, we probed for TRPV1 expression and function in human (HCEC) and rabbit corneal epithelial cell (RCEC) lines, in their primary counterparts, and in human and mouse corneal epithelium in situ. Cell membrane and perinuclear TRPV1 expression was detected in all preparations and its identity verified by Western blot analysis. Capsaicin (CAP) (1-10 microM) increased nonselective cation channel whole cell currents (2.5-fold +/- 0.5-fold between -60 and 130 mV), resulting in calcium transients that were fully blocked by the TRPV1 antagonists capsazepine (CPZ) and ruthenium red, or removal of extracellular calcium. Another signaling event involved transient activation of global mitogen-activated protein kinase (MAPK) superfamily, which was followed by up to 3.3- and 9-fold increases in interleukins (IL)-6 and -8 release, respectively. Such increases in inflammatory mediators' release were suppressed by exposure to CPZ or MAPK inhibitors, or removal of Ca2+. Taken together, TRPV1 receptors may play a role in mediating corneal epithelial inflammatory mediator secretion and subsequent hyperalgesia.
Assuntos
Interleucina-6/metabolismo , Interleucina-8/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Canais de Cátion TRPV/metabolismo , Âmnio/citologia , Animais , Antígenos Transformantes de Poliomavirus/fisiologia , Cálcio/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Transformada , Transformação Celular Viral , Células Cultivadas , Córnea/citologia , Eletrofisiologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Camundongos , Técnicas de Patch-Clamp , CoelhosRESUMO
PURPOSE: In this study, it was determined that human corneal epithelial cells (HCECs) express asialoganglioside ganliotetraosylceramide (asialoGM1) and toll-like receptor (TLR)-5, and their interaction induces interleukin (IL)-8 release through Ca(2+) transient activation and mitogen-activated protein kinase (MAPK) stimulation. METHODS: Expression of asialoGM1 and TLR5 was detected in SV40 HCECs by Western blot and flow cytometry analyses and their association by coimmunoprecipitation. Single-cell fluorescence imaging was used to measure intracellular free Ca(2+) transients in fura-2-loaded cells. The enzyme-linked immunosorbent assay (ELISA) was used to quantify IL-8 production in both cultured and primary HCECs. RESULTS: The HCECs expressed both asialoGM1 and TLR5 receptors. Ligation of asialoGM1 resulted in protein-protein interaction with TLR5, followed by transient increases in Ca(2+) influx through L-type voltage-dependent Ca(2+) channels. This led to P2Y receptor stimulation along with membrane depolarization, resulting from increases in ATP release into the medium. Intracellular Ca(2+) transients led to time-dependent extracellular signal-regulated kinase (ERK) MAPK pathway stimulation, followed by a 9.5-fold increase in IL-8 release. Similarly, in primary HCECs, asialoGM1 receptor stimulation resulted in an 8.1-fold increase. With a TLR5 neutralizing antibody, no asialoGM1-induced increases in IL-8 release occurred, and this response was not suppressed in the presence of a TLR2 neutralizing antibody. CONCLUSIONS: IL-8 release by HCECs is mediated through ligand-induced asialoGM1 protein-protein interactions with TLR5. This response is dependent on ATP efflux into the medium, followed by P2Y receptor stimulation. Such activation, in turn, results in increases in Ca(2+) influx through L-type voltage-dependent Ca(2+) channels, as well as stimulation of the ERK pathway.
Assuntos
Epitélio Corneano/metabolismo , Gangliosídeo G(M1)/metabolismo , Interleucina-8/metabolismo , Receptor 5 Toll-Like/metabolismo , Trifosfato de Adenosina/metabolismo , Western Blotting , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Citometria de Fluxo , Humanos , Imunoprecipitação , Sistema de Sinalização das MAP Quinases/fisiologia , Microscopia de Fluorescência , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Receptores Purinérgicos P2/metabolismoRESUMO
PURPOSE: To test the hypothesis that the protein phosphatases PP2A and MKP-1 are involved in controlling epidermal growth factor (EGF)-induced increases in rabbit corneal epithelial cell (RCEC) migration by mediating crosstalk between signaling pathways eliciting EGF receptor control of migration and proliferation. METHODS: Western blot analysis was used to determine the phosphorylation status of Erk1/2, p38, and the mitogen-activated protein kinase (MAPK) kinase (MEK1/2) using inhibitors of Erk1/2 or p38 and dominant-negative (d/n) Erk1 or d/n p38 cell lines. Coimmunoprecipitation was used to evaluate protein phosphatase (PP)2A and Erk1/2 interaction. Short-interfering RNA (siRNA) transfection was performed to analyze the involvement of MAPK phosphatase (MKP)-1 in crosstalk. Scratch-wound assay was used to determine EGF-dependent effects on cell migration. RESULTS: EGF (10 ng/mL) induced changes in activation of Erk1/2 and p38, which were enhanced by inhibition with 10 microM SB203580 and 10 muM PD98059, respectively. PP inhibition with sodium orthovanadate (100 microM), okadaic acid (10 nM), or Ro 31-8220 (10 microM) resulted in larger and more prolonged increases in the phosphorylation status of Erk1/2 and p38. After 1 hour, EGF induced 14-fold increases in MKP-1 protein expression. After MKP-1 siRNA transfection, EGF had induced a similar pattern of changes in the phosphorylation status in Erk1/2 and p38 following PP inhibition. EGF-induced cell migration was enhanced by Erk1/2 pathway inhibition and was accentuated after PP inhibition. Conversely, p38 pathway inhibition eliminated this response. CONCLUSIONS: EGF-induced changes in Erk1/2 and p38 phosphorylation status are dependent on PP-mediated crosstalk. This control modulates the magnitude of growth factor-induced increases in corneal epithelial cell migration.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Epitélio Corneano/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Receptor Cross-Talk/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/fisiologia , Movimento Celular , Fosfatase 1 de Especificidade Dupla , Epitélio Corneano/metabolismo , Proteínas Imediatamente Precoces/fisiologia , Fosforilação , Proteína Fosfatase 1 , Proteína Fosfatase 2 , Proteínas Tirosina Fosfatases/fisiologia , Coelhos , Transdução de Sinais , TransfecçãoRESUMO
Alveolar macrophages (AM) are critical components of lung innate immunity and contribute to an effective host response to Pneumocystis pneumonia. Recognition of unopsonized Pneumocystis organisms by human AM is mediated predominantly via mannose receptors and results in phagocytosis, release of reactive oxygen species, and activation of the nuclear transcription factor (NF)-kappaB. However, the AM host defense genes activated by Pneumocystis have not been defined. In the present study, incubation of AM with unopsonized Pneumocystis organisms was not associated with release of interleukin (IL)-1beta, IL-6, or tumor necrosis factor (TNF)-alpha (important cytokines in the host response to Pneumocystis) and did not induce IL-1beta, IL-6, or TNF-alpha mRNA transcripts. These findings were not attributed to Pneumocystis-induced cytopathic changes, as these same AM released IL-8 and matrix metalloproteinase-9 in response to Pneumocystis. NF-kappaB-mediated IL-8 release was independent of Pneumocystis phagocytosis. The observed response was specific, as IL-1beta, IL-6, and TNF-alpha release and mRNA induction were preserved in response to lipopolysaccharide or serum-opsonized Pneumocystis. The absence of IL-1beta, IL-6, and TNF-alpha release in response to Pneumocystis was predominately influenced by AM mannose receptors, as blocking mannose receptors or targeted mannose receptor small interfering RNA functional gene silencing resulted in TNF-alpha release in response to unopsonized Pneumocystis organisms. Furthermore, ligation of AM mannose receptors by unopsonized Pneumocystis organisms reduced Toll-like receptor 4-mediated TNF-alpha release. Taken together, these data suggest that mannose receptors on human AM may suppress select proinflammatory cytokine release and may serve to regulate the innate inflammatory responses to infectious challenge in the lungs.
Assuntos
Citocinas/biossíntese , Lectinas Tipo C/imunologia , Macrófagos Alveolares/imunologia , Lectinas de Ligação a Manose/imunologia , Alvéolos Pulmonares/imunologia , Receptores de Superfície Celular/imunologia , Adolescente , Adulto , Animais , Retroalimentação Fisiológica/imunologia , Feminino , Inativação Gênica , Humanos , Técnicas In Vitro , Interleucina-8/biossíntese , Interleucina-8/imunologia , Lectinas Tipo C/genética , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/microbiologia , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/genética , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/imunologia , Pessoa de Meia-Idade , NF-kappa B/imunologia , Pneumocystis/imunologia , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos , Receptores de Superfície Celular/genética , Valores de Referência , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) represents a minimally invasive technique to sample peribronchial and mediastinal lymph nodes for diagnosis of cancer, lymphoma, or sarcoidosis. However, the value of EBUS-TBNA in diagnosis of respiratory infections has not been well explored. Here, microbiologic testing data for EBUS-TBNA samples collected from 82 patients over a 30-month period were retrospectively reviewed. No organisms were identified on Gram, acid-fast, or fungal stains. Bacterial cultures were positive in 52% of samples; however, all but 1 culture were considered contaminants. Mycobacterial cultures yielded Mycobacterium avium-intracellulare not identified in a concurrent bronchoalveolar lavage sample in 1 patient. Fungal cultures were negative. Overall, routine microbiologic tests on EBUS-TBNA samples do not appear sufficiently sensitive to rule out infectious causes of adenopathy. High clinical suspicion for infection may require modification of sampling techniques or more sensitive detection methods.
Assuntos
Doenças Transmissíveis/diagnóstico , Linfonodos/microbiologia , Doenças Linfáticas/diagnóstico , Doenças do Mediastino/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto JovemRESUMO
STUDY OBJECTIVES: To review our experience with diagnostic bronchoscopy in the evaluation of pulmonary infiltrates in adult hematopoietic stem cell transplantation (HSCT) recipients in the era of Pneumocystis prophylaxis and cytomegalovirus antigen testing. The study focused on diagnostic yields and the influence of bronchoscopic findings on pharmacologic therapy and mortality, comparing allogeneic (allo) HSCT patients to autologous (auto) HSCT patients. DESIGN: Case series review. SETTING: Tertiary care academic urban medical centers. PATIENTS: All adult allo-HSCT and auto-HSCT patients undergoing bronchoscopy for the evaluation of pulmonary infiltrates from January 1997 to September 2001. MEASUREMENTS AND RESULTS: The review identified 169 bronchoscopies that had been performed on HSCT patients, representing 12.5% of all HSCT patients (allo-HSCT patients, 125 bronchoscopies; auto-HSCT patients, 44 bronchoscopies). Bronchoscopy was requested more often in allo-HSCT patients (18.7%) compared to auto-HSCT patients (6.6%). Findings at bronchoscopy provided a specific diagnosis more frequently in allo-HSCT patients (50%) compared to auto-HSCT patients (34%). For both allo-HSCT and auto-HSCT patients, most diagnoses were obtained by BAL alone, whereas transbronchial biopsy (TBBx) provided additional specific information in < 10% of cases. For select patients (n = 27), surgical lung biopsy following bronchoscopy provided unique diagnoses in 47 to 50% of cases. Information from bronchoscopy influenced clinical decisions more often in allo-HSCT patients (50%) than in auto-HSCT patients (36%), and allowed for the discontinuation or addition of antimicrobial, corticosteroid, or antineoplastic agents to treatment. Complications from bronchoscopy occurred in 9% of all HSCT patients (n = 15), and were associated with higher in-hospital mortality rates in allo-HSCT patients (82%; n = 9) compared to auto-HSCT patients (50%; n = 2). The overall in-hospital mortality rates for allo-HSCT and auto-HSCT patients having bronchoscopy was similar (38% vs 27%, respectively; p = 0.25), and establishing a specific diagnosis by bronchoscopy did not improve the in-hospital mortality rate for allo-HSCT or auto-HSCT patients. CONCLUSIONS: Bronchoscopy may provide clinically useful information in the evaluation of adult allo-HSCT and auto-HSCT recipients with pulmonary infiltrates. The results of testing BAL fluid samples alone suggested an etiology in most cases, whereas the findings of TBBx provided unique diagnoses infrequently. Further studies are warranted to improve the utility of diagnostic bronchoscopy in the evaluation of HSCT patients.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Broncoscopia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Pneumopatias/patologia , Adulto , Broncoscopia/efeitos adversos , Feminino , Mortalidade Hospitalar , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
OBJECTIVES: Our current understanding of hormone regulation in lung parenchyma is quite limited. We aimed to quantify a diverse array of biologically relevant protein mediators in alveolar lining fluid (ALF), compared to serum concentrations, and explore factors associated with protein compartmentalization on either side of the air-blood barrier. RESEARCH DESIGN AND METHODS: Participants were 24 healthy adult non-smoker volunteers without respiratory symptoms or significant medical conditions, with normal lung exams and office spirometry. Cell-free bronchoalveolar lavage fluid and serum were analyzed for 24 proteins (including enteric and metabolic hormones, apolipoproteins, adipokines, and cytokines) using a highly sensitive multiplex ELISA. Measurements were normalized to ALF concentrations. The ALF:serum concentration ratios were examined in relation to measures of protein size, hydrophobicity, charge, and to participant clinical and spirometric values. RESULTS: ALF measurements from 24 individuals detected 19 proteins, including adiponectin, adipsin, apoA-I, apoA-II, apoB, apoC-II, apoC-III, apoE, C-reactive protein, ghrelin, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon, insulin, leptin, monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, resistin, and visfatin. C-peptide and serpin E1 were not detected in ALF for any individual, and IL-6, IL-10, and TNF-alpha were not detected in either ALF or serum for any individual. In general, ALF levels were similar or lower in concentration for most proteins compared to serum. However, ghrelin, resistin, insulin, visfatin and GLP-1 had ALF concentrations significantly higher compared to serum. Importantly, elevated ALF:serum ratios of ghrelin, visfatin and resistin correlated with protein net charge and isoelectric point, but not with molecular weight or hydrophobicity. CONCLUSIONS: Biologically relevant enteric and metabolic hormones, apolipoproteins, adipokines, and cytokines can be detected in the ALF of healthy individuals. For the proteins measured, charge may influence trafficking and compartmentalization to the alveolar airspace more than molecular weight or hydrophobicity. These data may have implications for homeostasis and drug delivery to the lung.
Assuntos
Adipocinas/metabolismo , Apolipoproteínas/metabolismo , Citocinas/metabolismo , Hormônios Peptídicos/metabolismo , Alvéolos Pulmonares/metabolismo , Adulto , Feminino , Voluntários Saudáveis , Humanos , Pessoa de Meia-Idade , Testes de Função RespiratóriaRESUMO
Pneumocystis jiroveci (P. carinii) is an opportunistic pathogen that has gained particular prominence since the onset of the AIDS epidemic. Among several important advances in diagnosis and management, appropriately targeting chemoprophylaxis to HIV-infected patients at high clinical risk for P. jiroveci pneumonia and the introduction of effective combination anti-retroviral therapy (including highly active antiretroviral therapy [HAART]) have contributed to the reduced incidence of P. jiroveci pneumonia. Despite the success of these clinical interventions, P. jiroveci pneumonia remains the most common opportunistic pneumonia and the most common life-threatening infectious complication in HIV-infected patients. Trimethoprim/sulfamethoxazole (cotrimoxazole) remains the first-line agent for effective therapy and chemoprophylaxis, and corticosteroids represent an important adjunctive agent in the treatment of moderate-to-severe P. jiroveci pneumonia. However, problems of chemoprophylaxis and treatment failures, high rates of adverse drug reactions and drug intolerance to first-line antimicrobials, high rates of relapse or recurrence with second-line agents, and newer concerns about the development of P. jiroveci drug resistance represent formidable challenges to the management and treatment of AIDS-related P. jiroveci pneumonia. With the expanding global problem of HIV infection, the intolerance or unavailability of HAART to many individuals and limited access to healthcare for HIV-infected patients, P. jiroveci pneumonia will remain a major worldwide problem in the HIV-infected population. New drugs under development as anti-Pneumocystis agents such as echinocandins and pneumocandins, which inhibit beta-glucan synthesis, or sordarins, which inhibit fungal protein synthesis, show promise as effective agents. Continued basic research into the biology and genetics of P. jiroveci and host defense response to P. jiroveci will allow the development of newer antimicrobials and immunomodulatory therapeutic agents to more effectively treat life-threatening pneumonia caused by this organism.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/tratamento farmacológico , Síndrome da Imunodeficiência Adquirida/complicações , Anti-Infecciosos/uso terapêutico , Pneumocystis carinii , Pneumonia por Pneumocystis/tratamento farmacológico , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/etiologia , Corticosteroides/uso terapêutico , Adulto , Humanos , Pneumonia por Pneumocystis/diagnóstico , Pneumonia por Pneumocystis/etiologiaRESUMO
PURPOSE: To determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation. METHODS: Using sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFα by macrophages challenged with HIV miRNAs was measured by ELISA. RESULTS: HIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFα release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs released from infected macrophages as contributing to chronic immune activation in HIV-infected persons, and may represent a novel therapeutic target to limit AIDS pathogenesis. CONCLUSION: Novel HIV vmiR88 and vmiR99 are present in the systemic circulation of HIV+ persons and could exhibit biological function (independent of gene silencing) as ligands for TLR8 signaling that promote macrophage TNFα release, and may contribute to chronic immune activation. Targeting novel HIV-derived miRNAs may represent a therapeutic strategy to limit chronic immune activation and AIDS progression.