Bacterial endotoxin induces [Ca2+]i transients and changes the organization of actin in microglia

Abstract

We have employed amoeboid microglia purified from primary cultures of neonatal rat brain to examine the effect of bacterial lipopolysaccharide (LPS), a potent activator of immune cells, on intracellular calcium concentration ([Ca²⁺]i) in brain macrophages. In single brain macrophages loaded with indo 1, pulse administration of LPS elicited a rapid and transient increase in [Ca²⁺]i From a total of 70 cells examined, all responded to LPS with a similar [Ca²⁺]i transient, indicating a good homogeneity of the cell population with regard to the LPS response. It was concluded that the rise of cytosolic [Ca²⁺]i originated from intracellular stores because the response to LPS occured similarly in the presence or in the absence of extracellular Ca²⁺. A second administration of LPS to the same cells resulted in a second but reduced [Ca²⁺]i transient. In contrast to the first response to LPS, this second response was totally dependent on the presence of Ca²⁺ in the extracellular medium. The first response to LPS was strongly inhibited by ruthenium red and could be suppressed in a reversible manner by pre incubating the cells with caffeine in the absence of Ca²⁺ in the extracellular medium. These results indicate that caffeinesensitive intracellular Ca²⁺ stores may be the major source of Ca²⁺ in the response of brain macrophages to LPS. The possible release of Ca²⁺ from phosphatidylinositol(3,4,5)‐trisphosphate (IP₃)‐sensitive stores in brain macrophages was also evaluated by stimulating cells with the IP₃‐mobilizing agonist histamine. Brain macrophages were heterogeneous with regard to the histamine response since histamine induced a [Ca²⁺]i rise in only 30% of cells examined.The increase in [Ca²⁺]i triggered by LPS may in turn activate several intracellular events involved in the transition from one microglial functional state to another. We examined the effect of LPS on the state of organization of actin, which is an essential component of chemoattractant signal transduction. Immunofluorescence staining with anti‐actin antibody which recognizes actin in both filamentous and nonfilamentous configurations, indicated that LPS produced drastic changes in the actin organization. LPS‐treated cells appeared more intensely fluorescent than untreated cells due to the concentration of diffuse fluorescence near the center of the cell. In addition, prominent fluorescent dots were present in the subplasmalemmal region in cells stimulated with LPS. This specific LPS‐induced reorganization of actin was also observed in cells preincubated in the external medium without Ca²⁺. Thus, it is likely that the LPS‐induced mobilization of Ca²⁺ from intracellular stores may be responsible for the changes observed in the actin organization.