Co-operation of TLR4 and raft proteins in LPS-induced pro-inflammatory signaling.

Toll-like receptor 4 (TLR4) is activated by lipopolysaccharide (LPS), a component of Gram-negative bacteria to induce production of pro-inflammatory mediators aiming at eradication of the bacteria. Dysregulation of the host responses to LPS can lead to a systemic inflammatory condition named sepsis. In a typical scenario, activation of TLR4 is preceded by binding of LPS to CD14 protein anchored in cholesterol- and sphingolipid-rich microdomains of the plasma membrane called rafts. CD14 then transfers the LPS to the TLR4/MD-2 complex which dimerizes and triggers MyD88- and TRIF-dependent production of pro-inflammatory cytokines and type I interferons. The TRIF-dependent signaling is linked with endocytosis of the activated TLR4, which is controlled by CD14. In addition to CD14, other raft proteins like Lyn tyrosine kinase of the Src family, acid sphingomyelinase, CD44, Hsp70, and CD36 participate in the TLR4 signaling triggered by LPS and non-microbial endogenous ligands. In this review, we summarize the current state of the knowledge on the involvement of rafts in TLR4 signaling, with an emphasis on how the raft proteins regulate the TLR4 signaling pathways. CD14-bearing rafts, and possibly CD36-rich rafts, are believed to be preferred sites of the assembly of a multimolecular complex which mediates the endocytosis of activated TLR4.

An interplay between scavenger receptor A and CD14 during activation of J774 cells by high concentrations of LPS.

Lipopolysaccharide (LPS) activates macrophages by binding to the TLR4/MD-2 complex and triggers two pro-inflammatory signaling pathways: one relies on MyD88 at the plasma membrane, and the other one depends on TRIF in endosomes. When present in high doses, LPS is internalized and undergoes detoxification. We found that the uptake of a high concentration of LPS (1000ng/ml) in macrophage-like J774 cells was upregulated upon inhibition of clathrin- and dynamin-mediated endocytosis which, on the other hand, strongly reduced the production of pro-inflammatory mediators TNF-α and RANTES. The binding and internalization of high amounts of LPS was mediated by scavenger receptor A (SR-A) with participation of CD14 without an engagement of TLR4. Occupation of SR-A by dextran sulfate or anti-SR-A antibodies enhanced LPS-induced production of TNF-α and RANTES by about 70%, with CD14 as a limiting factor. Dextran sulfate also elevated the cell surface levels of TLR4 and CD14, which could have contributed to the upregulation of the pro-inflammatory responses. Silencing of SR-A expression inhibited the LPS-triggered TNF-α production whereas RANTES release was unchanged. These data indicate that SR-A is required for maximal production of TNF-α in cells stimulated with LPS, possibly by modulating the cell surface levels of TLR4 and CD14.

CD14 mediates binding of high doses of LPS but is dispensable for TNF-α production.

Activation of macrophages with lipopolysaccharide (LPS) involves a sequential engagement of serum LPS-binding protein (LBP), plasma membrane CD14, and TLR4/MD-2 signaling complex. We analyzed participation of CD14 in TNF-α production stimulated with 1-1000 ng/mL of smooth or rough LPS (sLPS or rLPS) and in sLPS binding to RAW264 and J744 cells. CD14 was indispensable for TNF-α generation induced by a low concentration, 1 ng/mL, of sLPS and rLPS. At higher doses of both LPS forms (100-1000 ng/mL), TNF-α release required CD14 to much lower extent. Among the two forms of LPS, rLPS-induced TNF-α production was less CD14-dependent and could proceed in the absence of serum as an LBP source. On the other hand, the involvement of CD14 was crucial for the binding of 1000 ng/mL of sLPS judging from an inhibitory effect of the anti-CD14 antibody. The binding of sLPS was also strongly inhibited by dextran sulfate, a competitive ligand of scavenger receptors (SR). In the presence of dextran sulfate, sLPS-induced production of TNF-α was upregulated about 1.6-fold. The data indicate that CD14 together with SR participates in the binding of high doses of sLPS. However, CD14 contribution to TNF α production induced by high concentrations of sLPS and rLPS can be limited.

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

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.