LPS induces phosphorylation of actin‐regulatory proteins leading to actin reassembly and macrophage motility

Abstract

Upon bacterial infection lipopolysaccharide (LPS) induces migration of monocytes/macrophages to the invaded region and production of pro-inflammatory mediators. We examined mechanisms of LPS-stimulated motility and found that LPS at 100 ng/ml induced rapid elongation and ruffling of macrophage-like J774 cells. A wound-healing assay revealed that LPS also activated directed cell movement that was followed by TNF-α production. The CD14 and TLR4 receptors of LPS translocated to the leading lamella of polarized cells, where they transiently colocalized triggering local accumulation of actin filaments and phosphatidylinositol 4,5-bisphosphate. Fractionation of Triton X-100 cell lysates revealed that LPS induced polymerization of cytoskeletal actin filaments by 50%, which coincided with the peak of cell motility. This microfilament population appeared at the expense of short filaments composing the plasma membrane skeleton of unstimulated cells and actin monomers consisting prior to the LPS stimulation about 60% of cellular actin. Simultaneously with actin polymerization, LPS stimulated phosphorylation of two actin-regulatory proteins, paxillin on tyrosine 118 by 80% and N-WASP on serine 484/485 by 20%, and these events preceded activation of NF-κB. LPS-induced protein phosphorylation and reorganization of the actin cytoskeleton were inhibited by PP2, a drug affecting activity of tyrosine kinases of the Src family. The data indicate that paxillin and N-WASP are involved in the reorganization of actin cytoskeleton driving motility of LPS-stimulated cells. Disturbances of actin organization induced by cytochalasin D did not inhibit TNF-α production suggesting that LPS-induced cell motility is not required for TNF-α release.

c-Abl mediated tyrosine phosphorylation of paxillin regulates LPS-induced endothelial dysfunction and lung injury

“…Earlier studies have demonstrated that LPS enhanced paxillin tyrosine phosphorylation in macrophages and bovine pulmonary arterial endothelial cells (7,19); however, the role of tyrosine phosphorylation of paxillin on LPS-induced endothelial barrier function is not well defined. We determined whether LPS modulates paxillin tyrosine phosphorylation in HLMVECs.…”

“…Paxillin has been shown to differentially regulate endothelial barrier function by growth factors via modulation of Rac-Rho signaling (4). Recent studies showed that LPS treatment is known to induce phosphorylation of paxillin in macrophages (19) and bovine pulmonary arterial ECs (7); however, little is known about the potential role of tyrosine phosphorylation of paxillin in regulating LPS-induced endothelial barrier dysfunction.…”

A rapid screening method to evaluate the impact of nanoparticles on macrophages

“…Lipopolysaccharide (LPS), which is a bacterially derived endotoxin used as a positive proinflammatory control, promoted adhesion and spreading of cells, inducing the typical morphology of immune-activated macrophages (arrows). 30 In contrast, exposure to citrate AuNP, the NH 2 PVA polymer or NH 2 PVA-AuNP did not induce any change in morphology, with cells remaining small and round. Some material aggregates (arrows) were seen with NH 2 PVA polymer exposure.…”

Relationship between macrophage differentiation and the chemotactic activity toward damaged myoblast cells

“…It is considered to be possible that glucocorticoid contributes to the macrophage migration during the process of skeletal muscle repair after acute exercise. On the other hand, lipopolysaccharide (LPS), which strongly induces the differentiation into inflammatory macrophages, also affects the macrophage motility (Kleveta et al, 2012). A recent study indicated that a phenotype switch was not observed in CCR2 deficient mice (Lumeng et al, 2007), suggesting that the MCP1/CCR2 pathway likely contributes to macrophage polarization.…”

“…Nevertheless, the exact cellular and biological events that cause this macrophage migration toward injured muscle are not completely understood. The evaluations of macrophage infiltration have mainly consisted of two strategies: the immunohistochemical evaluations (examining macrophage accumulation in tissue) (Shireman et al, 2006;Kleveta et al, 2012), and the evaluation of chemotaxis by the Boyden chamber assay (Boyden, 1962) or a modified assay. Both of these made it difficult or impossible to evaluate the speed and direction of horizontal movement of the macrophages toward damaged muscle.…”