Departamento de Fisiología Médica y Biofísica, Facultad de Medicina, Universidad de Sevilla, Sevilla, Spain.
Department of Neuroscience, Yale School of Medicine, New Haven, CT, USA.
Cell Physiol Biochem. 2021 Jan 15;55(1):17-32. doi: 10.33594/000000324.
BACKGROUND/AIMS: Microglia are the dynamic motile phagocytes of the brain considered the first line of defense against threats or disturbances to the Central Nervous System (CNS). Microglia help orchestrate the immunological response by interacting with others immune cells. Mast cells (MCs) are effector cells of the innate immune system distributed in all organs and vascularized tissues, brain included. Several molecular mechanisms for potential interactions between MCs and microglia have been determined. However, the effect of MCs on regulated exocytosis and phagocytic clearance in microglia has not been explored.
Cocktails of MCs mediators (MCM) obtained at 37°C and 53°C were used to induce microglia activation. Changes in intracellular calcium [Ca] and ATP release were studied by calcium and quinacrine fluorescence imaging. Fluorescent latex beads were used to assay phagocytosis in microglia after MCM treatment and compared to that measured in the presence of histamine, ATP and lipopolysaccharide (LPS). Iba-1 expression and area were quantified by immunofluorescence and histamine levels evaluated by ELISA techniques.
Local application onto microglia of the MC mediator cocktail elicited Ca transients and exocytotic release associated with quinacrine dye de-staining. Ca signals were mimicked by histamine and blocked by the H1 receptor (H1R) antagonist, cetirizine. Hydrolysis of ATP by apyrase also affected Ca transients to a lesser extent. Iba-1 fluorescence, cell area and phagocytosis were enhanced by histamine through H1R. However, ATP prevented iba-1 expression and microglial phagocytosis. MCM showed combined effects of histamine and ATP, increasing the number of internalized microbeads per cell and area without raising iba1 expression.
Our results highlight the relevance of MC-derived histamine and ATP in the modulation of secretory and phagocytic activities that would explain the heterogeneity of microglial responses in different pathological contexts.
背景/目的：小胶质细胞是大脑中具有动态运动能力的吞噬细胞，被认为是抵抗中枢神经系统（CNS）威胁或干扰的第一道防线。小胶质细胞通过与其他免疫细胞相互作用来帮助协调免疫反应。肥大细胞（MCs）是先天免疫系统的效应细胞，分布于所有器官和血管组织，包括大脑。已经确定了 MCs 和小胶质细胞之间潜在相互作用的几种分子机制。然而，MCs 对小胶质细胞调节性胞吐和吞噬清除的影响尚未被探索。
在 37°C 和 53°C 下获得 MC 介质（MCM）混合物，用于诱导小胶质细胞激活。通过钙和吖啶橙荧光成像研究细胞内钙 [Ca] 和三磷酸腺苷（ATP）释放的变化。在用 MCM 处理后，使用荧光乳胶珠检测小胶质细胞的吞噬作用，并与在组胺、ATP 和脂多糖（LPS）存在下测量的吞噬作用进行比较。通过免疫荧光定量 Iba-1 表达和面积，通过 ELISA 技术评估组胺水平。
将 MC 介质混合物局部应用于小胶质细胞，引发与吖啶橙染料褪色相关的 Ca 瞬变和胞吐释放。组胺模拟 Ca 信号，并被 H1 受体（H1R）拮抗剂西替利嗪阻断。三磷酸腺苷的水解通过 apyrase 也会产生较小程度的 Ca 瞬变。组胺通过 H1R 增强 Iba-1 荧光、细胞面积和吞噬作用。然而，ATP 会阻止 iba-1 表达和小胶质细胞吞噬作用。MCM 显示组胺和 ATP 的联合作用，增加每个细胞内化的微生物珠的数量和面积，而不会增加 iba1 表达。
我们的研究结果强调了 MC 衍生的组胺和 ATP 在调节分泌和吞噬活性方面的相关性，这可以解释不同病理情况下小胶质细胞反应的异质性。
