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1.
Immunity ; 39(3): 599-610, 2013 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-24012416

RESUMO

It is thought that monocytes rapidly differentiate to macrophages or dendritic cells (DCs) upon leaving blood. Here we have shown that Ly-6C⁺ monocytes constitutively trafficked into skin, lung, and lymph nodes (LNs). Entry was unaffected in gnotobiotic mice. Monocytes in resting lung and LN had similar gene expression profiles to blood monocytes but elevated transcripts of a limited number of genes including cyclo-oxygenase-2 (COX-2) and major histocompatibility complex class II (MHCII), induced by monocyte interaction with endothelium. Parabiosis, bromodoxyuridine (BrdU) pulse-chase analysis, and intranasal instillation of tracers indicated that instead of contributing to resident macrophages in the lung, recruited endogenous monocytes acquired antigen for carriage to draining LNs, a function redundant with DCs though differentiation to DCs did not occur. Thus, monocytes can enter steady-state nonlymphoid organs and recirculate to LNs without differentiation to macrophages or DCs, revising a long-held view that monocytes become tissue-resident macrophages by default.


Assuntos
Diferenciação Celular , Células Dendríticas/metabolismo , Linfonodos/citologia , Macrófagos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Animais , Antígenos Ly/metabolismo , Movimento Celular , Ciclo-Oxigenase 2/genética , Células Dendríticas/citologia , Células Dendríticas/imunologia , Endotélio/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Pulmão/citologia , Linfonodos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Pele/citologia
2.
J Vis Exp ; (45)2010 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-21113122

RESUMO

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection. In vitro and in vivo studies have also observed prion retention by dendritic cells, although their role in trafficking remains unclear. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection. These prior studies also suffer from the problem of differentiating between endogenous PrP(C) and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.


Assuntos
Encefalopatias/imunologia , Sistema Imunitário/fisiologia , Monitorização Imunológica/métodos , Proteínas PrPC/imunologia , Doenças Priônicas/imunologia , Animais , Linfócitos B/imunologia , Células Dendríticas/imunologia , Citometria de Fluxo/métodos , Imunidade Celular , Macrófagos/imunologia , Camundongos , Linfócitos T/imunologia
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