Microglial Activation by Components of Gram-Positive and -Negative Bacteria: Distinct and Common Routes to the Induction of Ion Channels and Cytokines

Abstract

Gram-positive Streptococcus pneumoniae is the major pathogen causing lethal meningitis in adults. We used pneumococcal cell walls (PCW) to investigate microglial consequences of a bacterial challenge and to determine the role of serum in the activation process. PCW caused the characteristic induction of an outwardly rectifying K+ channel (IK+(OR)), together with a concomitant suppression of the constitutively expressed inward rectifier K+ current, and evoked the release of tumor necrosis factor-alpha (TNF alpha), interleukin-6 (IL-6), IL-12, KC, macrophage inflammatory protein (MIP) 1alpha and MIP-2. Serum presence strongly facilitated the PCW effects, similarly as observed for lipopolysaccharide (LPS) from gram-negative Escherichia coli. The inflammatory cytokine, interferon-gamma (IFNgamma) induced the same electrophysiological changes, but independent of serum. Recombinant LPS binding protein (LBP) could partially replace serum activity in LPS stimulations. In contrast, neither LBP nor an antibody-mediated blockade of the LPS receptor, CD14 had significant influences on PCW-inducible changes. Cell surface interactions and cofactor involvement in microglial activation by gram-positive bacteria are thus distinct from the mechanisms employed by LPS. Moreover, tyrphostin AG126, a protein kinase inhibitor that prevents activation of the mitogen-activated protein kinase, p42MAPK (ERK2), potently blocked the PCW-stimulated cytokine release while having only a limited effect on LPS-inducible cytokines. In contrast, AG126 did not influence IK+(OR) inductions. This indicates that PCW recruits more than 1 intracellular signaling pathway to trigger the various responses and that different bacterial agents signal through both common and individual routes during microglial activation.

TLR2 controls random motility, while TLR7 regulates chemotaxis of microglial cells via distinct pathways

“…Primary microglia cultures were prepared from the cerebral cortex and midbrain of newborn male and female C57BL/6 and TLRKO mice (PO-P3), as described r.reviously (Prinz et al, 1999). In brief, the forebrain was carefully freed of blood vessels and meninges.…”

NTPDase1 activity attenuates microglial phagocytosis

“…Primary microglial and astrocyte cultures were prepared from cerebral cortex of newborn C57BL/6 mice as described previously [14].…”

The Microglia-activating Potential of Thrombin

“…Cultures routinely consisted of ϳ98% microglial cells as determined by staining with Griffonia simplicifolia isolectin B4 (Vector Laboratories, Burlingame, CA). Experiments were either carried out in FCS-containing DMEM or under serum-free conditions using macrophage serum-free medium supplemented with astrocyte-conditioned medium (30).…”

“…To exclude effects relating to FCS, experiments were also carried out under serum-free conditions (30). Thrombin still triggered a dose-dependent release (Fig.…”

“…LPS itself depends on serum factors (i.e. LPS-binding protein) (30), and microglia may behave differently under both culture conditions (25).…”