Nacife V P, Soeiro M D, Araújo-Jorge T C, Castro-Faria Neto H C, Meirelles M D
Lab. Ultra-estrutura e Celular, Dept. de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brasil.
Cell Struct Funct. 2000 Dec;25(6):337-50. doi: 10.1247/csf.25.337.
In the present paper we performed a morphological characterization of mouse peritoneal cells stimulated in vivo for 24 h with carrageenan (CAR) and lipopolysaccharide (LPS) by ultrastructural and flow cytometry analysis. In all samples, the flow cytometry studies showed the presence of three major populations consisting of monocytes, macrophages and lymphocytes. A special recruitment of monocytes was detected in CAR-injected mice. Macrophages and monocytes from CAR-treated mice displayed a characteristic phenotype, with a larger number of cytoplasmic vacuoles and numerous membrane projections, as compared to the cells collected from LPS- and PBS-injected mice. The induction of vacuolization was also confirmed upon in vitro treatment with CAR for 15 min to 24 h. The in vivo CAR-induced vacuoles were not related to lipid storage as judged by the lack of lipidic labeling after imidazole treatment at the ultrastructural level. In order to investigate the acidic nature of the vacuoles we used acidothropic probes, Lysotracker Yellow (LY) and Acridine Orange (AO). CAR injection activated the ability of peritoneal cells to incorporate LY around 2-5 times higher than control cells. However, the AO incorporation was 10-fold lower in CAR-stimulated cells than in LPS-stimulated ones. It is possible that the increase in intracellular vacuolization observed in CAR-stimulated cells could be related to exocytosis, since in most vacuoles the inflammatory protein MRP-14 was immunolocalized. The presence of MRP-14 in the culture supernatant of adherent peritoneal cells from CAR-injected mice was further comfirmed by ELISA, suggesting the discharge of MRP-14 enriched vacuole contents in the extracellular medium. We concluded that the morphological characteristics of activated monocytes and macrophages may depend on the nature of the triggering stimuli. Our observations reflect different functional phenotypes of monocytes/macrophages after in vivo stimulation with inflammatory agents such as CAR and LPS.
在本论文中，我们通过超微结构和流式细胞术分析，对用角叉菜胶（CAR）和脂多糖（LPS）在体内刺激24小时的小鼠腹膜细胞进行了形态学表征。在所有样本中，流式细胞术研究显示存在由单核细胞、巨噬细胞和淋巴细胞组成的三个主要群体。在注射CAR的小鼠中检测到单核细胞的特殊募集。与从注射LPS和PBS的小鼠收集的细胞相比，来自CAR处理小鼠的巨噬细胞和单核细胞表现出特征性表型，具有更多的细胞质空泡和大量膜突起。在用CAR体外处理15分钟至24小时后，空泡化的诱导也得到证实。通过超微结构水平的咪唑处理后缺乏脂质标记判断，体内CAR诱导的空泡与脂质储存无关。为了研究空泡的酸性性质，我们使用了酸otropic探针，溶酶体示踪剂黄（LY）和吖啶橙（AO）。注射CAR激活了腹膜细胞摄取LY的能力，比对照细胞高约2 - 5倍。然而，CAR刺激细胞中AO的摄取比LPS刺激细胞低10倍。CAR刺激细胞中观察到的细胞内空泡化增加可能与胞吐作用有关，因为在大多数空泡中炎症蛋白MRP - 14被免疫定位。通过ELISA进一步证实了注射CAR的小鼠贴壁腹膜细胞培养上清液中存在MRP - 14，表明富含MRP - 14的空泡内容物释放到细胞外培养基中。我们得出结论，活化的单核细胞和巨噬细胞的形态特征可能取决于触发刺激的性质。我们的观察结果反映了用CAR和LPS等炎症剂在体内刺激后单核细胞/巨噬细胞的不同功能表型。
