RESUMEN
Murine models of Mycobacterium tuberculosis (Mtb) infection demonstrate progression of M1-like (proinflammatory) and M2-like (anti-inflammatory) macrophage morphology following primary granuloma formation. The Mtb cell wall cording factor, trehalose 6,6'-dimycolate (TDM), is a physiologically relevant and useful molecule for modeling early macrophage-mediated events during establishment of the tuberculosis-induced granuloma pathogenesis. Here, it is shown that TDM is a major driver of the early M1-like macrophage response as seen during initiation of the granulomas of primary pathology. Proinflammatory cytokines tumor necrosis factor-α, IL-1ß, IL-6, and IL-12p40 are produced in lung tissue after administration of TDM to mice. Furthermore, CD11b+CD45+ macrophages with a high surface expression of the M1-like markers CD38 and CD86 were found present in regions of pathology in lungs of mice at 7 days post-TDM introduction. Conversely, only low phenotypic marker expression of M2-like markers CD206 and EGR-2 were present on macrophages. These findings suggest that TDM plays a role in establishment of the M1-like shift in the microenvironment during primary tuberculosis.
Asunto(s)
Adyuvantes Inmunológicos/toxicidad , Factores Cordón/toxicidad , Granuloma/patología , Mediadores de Inflamación/metabolismo , Macrófagos/patología , Mycobacterium/metabolismo , Neumonía/patología , Animales , Femenino , Granuloma/inducido químicamente , Granuloma/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Neumonía/inducido químicamente , Neumonía/metabolismoRESUMEN
The movement of neutrophils between blood and tissues appears to be regulated by chemoattractants and chemorepellents. Compared with neutrophil chemoattractants, relatively little is known about neutrophil chemorepellents. Slit proteins are endogenously cleaved into a variety of N- and C-terminal fragments, and these fragments are neuronal chemorepellents and inhibit chemoattraction of many cell types, including neutrophils. In this report, we show that the â¼140-kDa N-terminal Slit2 fragment (Slit2-N) is a chemoattractant and the â¼110-kDa N-terminal Slit2 fragment (Slit2-S) is a chemorepellent for human neutrophils. The effects of both Slit2 fragments were blocked by Abs to the Slit2 receptor Roundabout homolog 1 or the Slit2 coreceptor Syndecan-4. Slit2-N did not appear to activate Ras but increased phosphatidylinositol 3,4,5-triphosphate levels. Slit2-N-induced chemoattraction was unaffected by Ras inhibitors, reversed by PI3K inhibitors, and blocked by Cdc42 and Rac inhibitors. In contrast, Slit2-S activated Ras but did not increase phosphatidylinositol 3,4,5-triphosphate levels. Slit2-S-induced chemorepulsion was blocked by Ras and Rac inhibitors, not affected by PI3K inhibitors, and reversed by Cdc42 inhibitors. Slit2-N, but not Slit2-S, increased neutrophil adhesion, myosin L chain 2 phosphorylation, and polarized actin formation and single pseudopods at the leading edge of cells. Slit2-S induced multiple pseudopods. These data suggest that Slit2 isoforms use similar receptors but different intracellular signaling pathways and have different effects on the cytoskeleton and pseudopods to induce neutrophil chemoattraction or chemorepulsion.
Asunto(s)
Citoesqueleto de Actina/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neutrófilos/fisiología , Isoformas de Proteínas/metabolismo , Seudópodos/metabolismo , Anticuerpos Bloqueadores/metabolismo , Orientación del Axón , Adhesión Celular , Células Cultivadas , Quimiotaxis/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/inmunología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Seudópodos/ultraestructura , Transducción de Señal , Sindecano-4/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas ras/metabolismoRESUMEN
Fibrocytes are monocyte-derived fibroblast like cells that participate in wound healing, but little is known about what initiates fibrocyte differentiation. Blood platelets contain 60-100-mer polymers of phosphate groups called polyphosphate, and when activated, platelets induce blood clotting (the first step in wound healing) in part by the release of polyphosphate. We find that activated platelets release a factor that promotes fibrocyte differentiation. The factor is abolished by treating the crude platelet factor with the polyphosphate-degrading enzyme polyphosphatase, and polyphosphate promotes fibrocyte differentiation. Macrophages and recruited neutrophils also potentiate wound healing, and polyphosphate also promotes macrophage differentiation and induces chemoattraction of neutrophils. In support of the hypothesis that polyphosphate is a signal that affects leukocytes, we observe saturable binding of polyphosphate to these cells. Polyphosphate also inhibits leukocyte proliferation and proteasome activity. These results suggest new roles for extracellular polyphosphate as a mediator of wound healing and inflammation and also provide a potential link between platelet activation and the progression of fibrosing diseases.
Asunto(s)
Diferenciación Celular/fisiología , Proliferación Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Leucocitos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Polifosfatos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Coagulación Sanguínea/fisiología , Fibroblastos/metabolismo , Humanos , Leucocitos/metabolismo , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Neutrófilos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
Compared to neutrophil chemoattractants, relatively little is known about the mechanism neutrophils use to respond to chemorepellents. We previously found that the soluble extracellular protein dipeptidyl peptidase IV (DPPIV) is a neutrophil chemorepellent. In this report, we show that an inhibitor of the protease activated receptor 2 (PAR2) blocks DPPIV-induced human neutrophil chemorepulsion, and that PAR2 agonists such as trypsin, tryptase, 2f-LIGRL, SLIGKV, and AC55541 induce human neutrophil chemorepulsion. Several PAR2 agonists in turn block the ability of the chemoattractant fMLP to attract neutrophils. Compared to neutrophils from male and female C57BL/6 mice, neutrophils from male and female mice lacking PAR2 are insensitive to the chemorepulsive effects of DPPIV or PAR2 agonists. Acute respiratory distress syndrome (ARDS) involves an insult-mediated influx of neutrophils into the lungs. In a mouse model of ARDS, aspiration of PAR2 agonists starting 24 h after an insult reduce neutrophil numbers in the bronchoalveolar lavage (BAL) fluid, as well as the post-BAL lung tissue. Together, these results indicate that the PAR2 receptor mediates DPPIV-induced chemorepulsion, and that PAR2 agonists might be useful to induce neutrophil chemorepulsion.