CD14 and TLR4 Mediate Cytokine Release Promoted by Electronegative LDL in Monocytes

Aims: Electronegative LDL (LDL(-)), a minor modified LDL present in the circulation, induces cytokine release in monocytes. We aimed to determine the role of the receptor CD14 and toll-like receptors 2 and 4 (TLR2, TLR4) in the inflammatory action promoted by LDL(-) in human monocytes.

Methods And Results: Monocytes were preincubated with antibodies to neutralize CD14, TLR2 and TLR4. The release of monocyte chemoattractant protein 1 (MCP1), and interleukin 6 and 10 (IL6 and IL10) promoted by LDL(-) was inhibited 70-80% by antiCD14 and antiTLR4, and 15-25% by antiTLR2. The involvement of CD14 and TLR4 was confirmed by gene silencing experiments. The human monocytic THP1 cell line overexpressing CD14 released more cytokines in response to LDL(-) than the same THP1 cell line without expressing CD14. VIPER, a specific inhibitor of the TLR4 signaling pathway, blocked 75-90% the cytokine release promoted by LDL(-). Cell binding experiments showed that monocytes preincubated with neutralizing antibodies presented lesser LDL(-) binding than non-preincubated monocytes The inhibitory capacity was antiCD14>antiTLR4>>antiTLR2. Cell-free experiments performed in CD14-coated microtiter wells confirmed that CD14 was involved in LDL(-) binding. When LDL(-) and lipopolysaccharide (LPS) were added simultaneously to monocytes, cytokine release was similar to that promoted by LDL(-) alone. Binding experiments showed that LDL(-) and LPS competed for binding to monocytes and to CD14 coated-wells.

Conclusions: CD14 and TLR4 mediate cytokine release induced by LDL(-) in human monocytes. The cross-competition between LPS and LDL(-) for the same receptors could be a counteracting action of LDL(-) in inflammatory situations.