Toll‐like receptor 4 functions intracellularly in human coronary artery endothelial cells: roles of LBP and sCD14 in mediating LPS‐responses

Abstract

Endothelial cells are activated by microbial agonists through Toll-like receptors (TLRs) to express inflammatory mediators; this is of significance in acute as well as chronic inflammatory states such as septic shock and atherosclerosis, respectively. We investigated mechanisms of lipopolysaccharide (LPS)-induced cell activation in human coronary artery endothelial cells (HCAEC) using a combination of FACS, confocal microscopy, RT-PCR, and functional assays. We found that TLR4, in contrast to TLR2, is not only located intracellularly but also functions intracellularly. That being the case, internalization of LPS is required for activation. We further characterized the HCAEC LPS uptake system and found that HCAEC exhibit an effective LPS uptake only in the presence of LPS binding protein (LBP). In addition to its function as a catalyst for LPS-CD14 complex formation, LBP enables HCAEC activation at low LPS concentrations by facilitating the uptake, and therefore delivery, of LPS-CD14 complexes to intracellular TLR4-MD-2. LBP-dependent uptake involves a scavenger receptor pathway. Our findings may be of pathophysiological relevance in the initial response of the organism to infection. Results further suggest that LBP levels, which vary as LBP is an acute phase reactant, could be relevant to initiating inflammatory responses in the vasculature in response to chronic or recurring low LPS.

Heat shock up‐regulates expression of Toll‐like receptor‐2 and Toll‐like receptor‐4 in human monocytes via p38 kinase signal pathway

“…TLR4 was reported to reside and mediate LPS response intracellularly in epithelial and endothelial cells. [22][23][24] In human peripheral monocytes, TLR4 could be expressed either on cell surface or in the cytoplasm, and rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and plasma membrane could be observed. 25 These results suggested that both cell surface and intracellular TLR4 might contribute to cell response to LPS.…”

Immune Regulation by Microvascular Endothelial Cells: Directing Innate and Adaptive Immunity, Coagulation, and Inflammation

“…TLR4 expression has been demonstrated on various ECs and significantly increases under inflammatory conditions. TLR4 is expressed in coronary ECs (29) and is overexpressed and colocalizes with the p65 subunit of NF-B in coronary atherosclerotic plaques, suggesting activation of TLR4 at this site (21). This possibility is supported by the demonstration that LPS activates NF-B in dermal microvascular ECs and that LPS, IFN-␥, and TNF-␣ up-regulate TLR4 mRNA and protein (23).…”

Lipopolysaccharide signaling in endothelial cells

“…Studies using affinity chromatography, and later confirmed by fluorescence resonance energy transfer (FRET), revealed that LPS associates with the heat shock proteins, Hsp70 and Hsp90, chemokine receptor 4 (CXCR4) and growth differentiation factor 5 (GDF5). 49,50 In human coronary artery endothelial cells, TLR4 functions intracellularly, 51 suggesting that LPS uptake may be necessary for optimal signal transduction. Similarly, TLR4 colocalizes with LPS within the Golgi of intestinal epithelial cells 52 and functions within this intracellular compartment to recognize internalized LPS.…”