Innate immunity is the host's first line of defense against infectious pathogens. A number of innate receptors, including the Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns (PAMPs). TLR4 is the best characterized of the TLRs and serves as the receptor for lipopolysaccharide (LPS) as well as several other microbial and endogenous ligands. It is currently unknown how TLR4 recognizes such chemically diverse entities as LPS, carbohydrates, and oxidized lipids. TLR4 functions in association with two accessory proteins, CD14 and MD-2, and possibly other as yet unidentified proteins. We hypothesize that the combination of TLR4 and TLR4-associated proteins determines both specificity and the intracellular signal that is generated. TLR4 is thought to play a significant role in the development of auto immune and inflammatory diseases, including atherosclerotic plaques. There is evidence that atherosclerosis can be induced either by lipids or by infection. Although CD14 and MD-2 may play a significant role in infection induced atherosclerosis, thus far, CD14 and MD-2 do not appear to be necessary in lipid induced atherosclerosis. An understanding of the interaction between TLR4, CD14, and MD-2 is essential for a complete understanding of the cascades that can lead to atherosclerosis and other autoimmune and inflammatory diseases.

We extended previous observations that oxidized lipids (e.g. oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, oxPAPC) trigger TLR4 in macrovascular endothelial cells and demonstrated that oxPAPC activates TLR4 signaling in microvascular endothelial cells. In contrast, we could not confirm the widespread belief that low molecular weight fragments of hyaluronic acid (sHA) activate TLR4. Since all biological studies of sHA to date have used HA digested with hyaluronidases, we assayed chemically synthesized sHA for biological activity, eliminating concerns of DNA and endotoxin contamination and heterogeneity of samples. For the purpose of structural and biological studies, we expressed hCD14 in Pichia pastoris and hMD-2 in Escherichia coli, and refolded hMD-2 in vitro . This, together with TLR4 expressing cell lines, allowed assessment of combinatorial recognition of modified self by TLR4.