Abstract
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.
Highlights
Mechanisms of the innate immunity assure a rapid response directed against microbes which have successfully overcome physical barriers protecting the body
We found that this blocking of function of CD14 nearly abolished tumor necrosis factor-α (TNF-α) and RANTES production induced by 1 ng/mL of smooth LPS (sLPS)
The blocking of the LPS/CD14 interaction by the 4C1 antibody inhibited the production of TNF-α by 25–27% only (Figure 1(a)) while RANTES generation was reduced by 58–76% (Figure 1(b))
Summary
Mechanisms of the innate immunity assure a rapid response directed against microbes which have successfully overcome physical barriers protecting the body. These reactions are triggered upon recognition of evolutionarily conserved constituents of microorganisms named pathogen-associated molecular patterns (PAMPs) by distinct cellular receptors among which Toll-like receptors (TLR) are of great importance [1]. The prototypical PAMP is lipopolysaccharide (LPS, endotoxin), a major constituent of the outer membrane of Gram-negative bacteria. LPS activates TLR4 of leukocytes and initiates signalling cascades leading to production of proinflammatory mediators exemplified by tumor necrosis factor-α (TNF-α), chemokines like MIP-2 and RANTES, and type I interferons [2, 3]. In certain species or mutants of Gram-negative bacteria, or in distinct growth conditions, the O-specific chain may be absent giving rise to a so-called rLPS
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