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

The Scripps Research Institute, Department of Immunology, La Jolla, California, USA

¹Correspondence: Scripps Research Institute, Department of Immunology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. E-mail: tobias{at}scrippsedu

SPECIFIC AIMS

Lipopolysaccharide (LPS) is a major component of the outer surface of Gram-negative bacteria and a potent activator of cells of the immune and inflammatory system, including endothelial cells (EC). The putative LPS signaling receptor, Toll-like receptor 4 (TLR4), is expressed in EC as in many other cells. Activation of the endothelial TLR4 by LPS plays a role in Gram-negative sepsis, where the body is overwhelmed with LPS, and in diseases linked to a low, but chronic, LPS burden. There is considerable evidence that endotoxin (LPS) contributes to the propagation of atherosclerosis, which shares many features with inflammatory disease. The aim of the present study was to investigate mechanisms of LPS-induced activation of human coronary artery endothelial cells (HCAEC).

PRINCIPAL FINDINGS

1. TLR4 is located and functions intracellularly in HCAEC
To begin a study of regulation and modification of TLR2 and TLR4 expression in HCAEC, we performed FACS analyses on those cells and on human peripheral blood monocytes. In contrast to monocytes, which showed clear surface staining for TLR2, TLR4, and CD14, we could not detect TLR4 or CD14 on the surface of HCAEC. However, TLR4 mRNA expression was the same in monocytes and HCAEC. Although a combination of LPS with IFN enhanced TLR4 mRNA expression in HCAEC, TLR4 was not detected on the surface. Upon transient transfection and overexpression of TLR4 or CD14 in HCAEC, some surface protein (1% of cells) was seen in FACS analyses, establishing the reliability of the detection system in HCAEC.

With the knowledge that TLR4 has been shown to reside in the Golgi apparatus in murine intestinal epithelial cell lines, we investigated HCAEC using confocal microscopy and found TLR4 located in a perinuclear region but not on the surface. As expected, VCAM-1 was seen intracellularly and on the surface after stimulation with LPS (Fig. 1 ). Since the number of TLR4 molecules on the HCAEC surface could be below the detection limit of confocal microscopy or FACS technology (although it has proved very sensitive in TLR2 detection), we performed functional assays.

View larger version (49K): [in this window] [in a new window]   Figure 1. 3-D confocal microscopy of HCAEC (A–D) and monocytes (E). 600x. A) TLR4, red; VE-cadherin (CD144) membrane staining, green; nucleus, blue. Note that CD144 as an exported protein can be found also intracellular. B) TLR4, red; CD144, green membrane staining; Golgi, green (Oregon Green® wheat germ agglutinin); nucleus, blue. C) TLR4, red; CD106, green; nucleus, blue. Cells were stimulated with LPS (10 ng/mL) for 4 h. The "cap" of the cell has been cut off optically and enables a view inside the cell. CD106 is found on the plasma membrane and inside the cytoplasm. TLR4 is seen only intracellularly. D) TLR4, red; F-actin staining, green; nucleus, blue. E) TLR4, red; mCD14, green; merge, yellow (colocalization of TLR4 and mCD14).

HCAEC activation was determined by measuring CD62E (E-selectin, ELAM) surface expression after stimulation with LPS or the TLR2 agonist, macrophage-activating lipopeptide-2 (MALP-2). We found that all TLR4 blocking antibodies used failed to inhibit LPS-induced HCAEC activation, although anti-TLR4 mAbs HTA405 and HTA125 blocked LPS-induced monocyte activation (Fig. 2 ). Given that monocytes express large amounts of TLR4 on the surface, as seen in FACS and microscopy, an equal amount of an anti-TLR4 antibody should have been able to block only a few surface TLR4 molecules on EC if TLR4 was there. In contrast to the anti-TLR4 antibodies, an anti-TLR2 antibody abolished MALP-2-induced EC activation, confirming that TLR2 is on the surface, where it can be reached by the antibody. Not surprisingly, the ability of LPS to stimulate cells was abolished by anti-LBP (mAb 2B5) or anti-CD14 (mAb 28C5) in EC and monocytes, because LBP is required to catalyze the formation of stimulatory LPS-sCD14 complexes that activate TLR4. We also found that Compound 406, a synthetic precursor of Escherichia coli lipid A that blocks the effects of LPS on human TLR4/MD-2, inhibited HCAEC activation. In contrast to antibodies, Compound 406 can enter cells like LPS.

View larger version (18K): [in this window] [in a new window]   Figure 2. Anti-TLR4 antibodies do not block LPS-induced HCAEC activation. TLR4 antibodies (20 µg/mL), anti-LBP (2B5; 20 µg/mL), anti-CD14 (28C5; 20 µg/mL), and anti-TLR2 (5 µg/mL; mAb 2392) were added 30 min before cell stimulation with 10 ng/mL LPS (E. coli O55:B5) or 100 ng/mL MALP-2 in HCAEC and with 1 ng/mL LPS (E. coli O55:B5) or 1 ng/mL MALP-2 in monocytes for another 4 h. Activation status was quantified in EC by measuring CD62E surface expression and in monocytes by measuring IL-8 secretion. Data are given as means ± SE from at least 5 independent experiments. **P < 0.01 (Mann Whitney U test).

Since there was still the possibility that EC use some receptor(s) other than TLR4 to detect LPS, lung microvascular and aortic EC from TLR4 knockout C57/BL6 mice were cultured and stimulated with various agonists. Even after 12 h incubation with 10 ng/mL of E. coli LPS (O55:B5), TLR4 –/– cells were completely unresponsive to this stimulus, whereas the response to murine TNF, MALP-2, or human IL-1 was independent of TLR4 genotype. Wild-type cells were activated by all stimuli.

Taking the data from FACS experiments, RT-PCR, confocal microscopy, and functional assays together, we conclude that TLR4 is not only located but also functions intracellularly in HCAEC. Intracellular function is further supported by the finding that the TLR4 signaling adaptor molecule MyD88 was rapidly recruited to a perinuclear region upon LPS stimulation of HCAEC.

2. LBP but not CD14 or TLR4 facilitates the uptake of LPS and LPS-CD14 complexes in EC
Cognizant that LPS needs to be internalized to activate TLR4 in HCAEC, we investigated requirements for LPS internalization in these cells. We found that LBP can dramatically enhance the uptake of LPS and of LPS-sCD14 complexes. The cellular uptake of LPS when bound to LBP alone was enhanced 10-fold, whereas uptake of in vitro preformed LPS-sCD14 complexes was enhanced by LBP 5-fold. However, LBP is not mandatory for the uptake of LPS at high concentrations and a "basal" uptake exists that is insensitive to anti-TLR4, anti-LBP, or anti-CD14. In cells expressing membrane CD14, LPS is internalized via CD14-dependent mechanisms. In contrast, HCAEC LPS uptake enhanced by LBP was CD14 independent but activation was CD14 dependent. Uptake mediated by LBP in the absence of CD14 did not lead to cell activation.

When TLR4-deficient murine endothelial cells were incubated with tritiated LPS (³H-LPS) for 2 h with various concentrations of LBP and ³H-LPS uptake was measured, no difference was found compared with wild-type cells, suggesting that uptake occurs independent of TLR4 phenotype.

LBP-facilitated uptake was blocked by scavenger receptor blocking agents (polyanions), suggesting that LBP-bound LPS is taken up by scavenger receptors in preference to LPS alone. Data obtained with ³²P-labeled LBP showed that LBP is internalized with LPS and that uptake follows the same pattern observed when LPS uptake was measured. Confocal microscopy showed clear colocalization of FITC-LPS and LBP, FITC-LPS and intracellular TLR4, sCD14 and TLR4 in the presence of LBP, and finally LBP and TLR4 in the presence of sCD14.

3. LBP enables a response of HCAEC to low endotoxin
Because LBP enhanced the uptake of stimulatory LPS-sCD14 complexes, it seemed likely that LBP would also enhance HCAEC activation. This was observed to be the case, but only at low LPS (0.1 ng/mL) concentrations. At higher LPS-sCD14 concentrations (10 ng/mL), LBP-independent uptake apparently is sufficient to allow activation of the cells. Low concentrations of preformed LPS-sCD14 complexes (0.1 ng/mL) did not stimulate cells without additional LBP; thus LBP is needed for cell activation at low LPS concentrations.

CONCLUSIONS AND SIGNIFICANCE

The present study provides for the first time experimental evidence that TLR4 functions intracellularly in primary EC. FACS data and confocal microscopy clearly demonstrated that this receptor, in contrast to TLR2, is not on the surface of HCAEC but is intracellular. In these cells, LPS signaling is initiated intracellularly since blocking anti-TLR4 antibodies could not block LPS activation. Experiments with TLR4 –/– EC confirmed that TLR4 is required for LPS activation of EC. The mechanisms that keep TLR4 intracellular in HCAEC and differentiate TLR2 from TLR4 are unclear.

We show a role for LBP in LPS uptake and on cellular activation. The effect of LBP on cell stimulation has been thought to be limited to its role in forming LPS-sCD14 complexes. In this work, the role of LBP in enhancing activation is also dependant on its novel role in enhancing uptake. By facilitating the uptake of LPS-sCD14 complexes at low concentrations and therefore delivery to intracellular TLR4, LBP participates in HCAEC activation. Since HCAEC exhibit an effective LPS uptake only in the presence of the acute phase reactant LBP, our data may provide a mechanism that enables HCAEC to respond to LPS in the early stage of infection or to a chronic low endotoxin burden.

Atherosclerosis is a chronic inflammatory disease of the arteries (including EC) and low LPS has been suggested to be an etiologic agent. Low levels of circulating LPS are found even in healthy human subjects. One is tempted to speculate that TLR4 is not surface expressed in HCAEC to protect the cells from continuous activation by removing TLR4 from exposure to every circulating LPS molecule. Endothelial cells are generally less sensitive to LPS than monocytes, and the intracellular location of TLR4 may be responsible for this. It is our contention that CRP is unlikely to play an etiologic role in atherosclerosis, although its clinical utility as a risk indicator seems clear. On the other hand, LBP is an acute phase reactant and, according to these results, likely to have an important role in initiating inflammatory responses in the vasculature in response to low, chronic LPS. Perhaps plasma LBP levels should be monitored as well as CRP.

View larger version (27K): [in this window] [in a new window]   Figure 3. Scheme of mechanisms of LPS-induced HCAEC activation. TLR4 is located and functions intracellularly while TLR2 functions at the surface. In contrast to anti-TLR2, which blocks MALP-2-induced cell activation, anti-TLR4 antibodies are inactive because they cannot reach their target. LBP facilitates the uptake of LPS and LPS-sCD14 complexes. Only the latter results in cellular activation, illustrated by expression of E-selectin (CD62E). Scavenger receptors mediate LPS uptake by HCAEC.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1263fje; doi: 10.1096/fj.03-1263fje

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