Differential induction of BLT receptor expression on human endothelial cells by lipopolysaccharide, cytokines, and leukotriene B4

Abstract

Leukotriene (LT) B4 is a powerful chemotactic and immune modulating agent that signals via two receptors denoted BLT1 and BLT2. Here we report that BLT1 and BLT2 are expressed at low levels in an apparently silent state in human umbilical vein endothelial cells (HUVEC). However, treatment with LPS leads to a >10 fold increase in the levels of BLT1 mRNA without any significant effects on BLT2 mRNA. In parallel, LPS also increases the amounts of BLT1 protein. Tumor necrosis factor-alpha (TNF-alpha) increases the expression of BLT2 mRNA approximately 6 times above basal levels with only a modest increase in BLT1 mRNA. Interleukin-1beta causes variable and parallel increases of both BLT1 and BLT2 mRNA. The natural ligand LTB4 also increases BLT1, but not BLT2, mRNA and protein expression. Along with the induction of BLT1 and/or BLT2, HUVEC acquire the capacity to respond to LTB4 with increased levels of intracellular calcium and these signals can be blocked by isotype selective BLT antagonists, CP-105696 and LY-255283. In addition, treatment of HUVEC with LTB4 causes increased release of both nitrite, presumably reflecting nitric oxide (NO), and monocyte chemoattractant protein-1. Our data indicate that expression of functional BLT receptors may occur at the surface of endothelial cells in response to LPS, cytokines, and ligand, which in turn may have functional consequences during the early vascular responses to inflammation. Moreover, the results point to BLT receptors as potential targets for pharmacological intervention in LT-dependent inflammatory diseases such as asthma, rheumatoid arthritis, and arteriosclerosis.

Fig. 1.

Increase in BLT₁ mRNA and protein in HUVEC stimulated with LPS. HUVEC were incubated with LPS (100 ng/ml) and harvested at different time points to prepare total RNA and protein extracts. mRNA and protein levels were analyzed by semiquantitative RT-PCR and Western blot, as described in Methods. (Upper) The rapid (within 60 min), almost 12-fold, increase in the levels of BLT₁ mRNA (filled circles) induced by treatment of HUVEC with LPS. In contrast, only a weak effect on the levels of BLT₂ mRNA is observed (open circles). (Lower) The increased levels in BLT₁ mRNA corresponded to elevated amounts of immunoreactive BLT₁ protein.

Fig. 2.

Increase in BLT₂ mRNA in HUVEC stimulated with TNF-α. HUVEC were incubated with TNF-α (100 ng/ml) and harvested at different time points to prepare total RNA. mRNA levels were analyzed by semiquantitative RT-PCR, as described in Methods. The time course for the increase in BLT₁ (filled circles) and BLT₂ (open circles) mRNA levels in HUVEC upon treatment with TNF-α is shown.

Fig. 3.

Increase in BLT₁ mRNA and protein expression in HUVEC stimulated with LTB₄. HUVEC were incubated with LTB₄ (100 nM) and harvested at different time points to prepare total RNA and protein extracts. mRNA and protein levels were analyzed by semiquantitative RT-PCR and Western blot as described in Methods. (Upper) Time course for the increase in BLT₁ mRNA (filled circles) and decrease in BLT₂ mRNA (open circles) in HUVEC treated with the natural agonist LTB₄. (Lower) Levels of immunoreactive BLT₁ protein in HUVEC treated with LTB₄.

Fig. 4.

LTB₄-induced Ca²⁺ mobilization in HUVEC after treatment with LPS or TNF-α. LTB₄ induced Ca²⁺ responses when HUVEC had been treated with LPS (Upper) or TNF-α (Lower) for 3 h but only marginally when treated with HBSS. The kinetics of the TNF-α responses were consistently more rapidly emerging and transient, whereas LPS responses were slower in onset but persistent. (Inset) The Ca²⁺ response of neutrophils to LTB₄ alone.

Fig. 5.

Modulation of Ca²⁺ responses, elicited by LTB₄ in cytokine-treated HUVEC, by the antagonist LY-255283. When HUVEC had been treated with LPS for 3 h, followed by LY-255283 (1 μM) for 20 min, most of the Ca²⁺ response elicited by addition of LTB₄ remained (Upper). When the same procedure was repeated and LPS was substituted by TNF-α, no LTB₄-induced Ca²⁺ responses could be observed (Lower).

Fig. 6.

Ca²⁺ responses elicited by LTB₄ in LTB₄-treated HUVEC. When HUVEC had been treated with LTB₄ for 4 h, followed by activation by LTB₄, a major response that was 8.3-fold higher than that of HUVEC pretreated with HBSS alone was noted. When LTB₄ was substituted by LY-255283 (control) as pretreatment, no Ca²⁺ response was observed.

Fig. 7.

LTB₄ increases the generation of nitrite and release of MCP-1 from HUVEC. Cultured HUVEC were incubated as described in Methods. Stimulation of cultured HUVEC with LTB₄ for 15 min leads to generation of nitrite (Left), as assessed by the Griess reaction for nitrite. Pretreatment of HUVEC with LPS for 3 h followed by stimulation with LTB₄ increases the nitrite release further, indicating up-regulation of BLT₁. Results are expressed as percentage of nitrite generation by control cells (stimulated with HBSS alone); mean value was 2.3 ± 0.4 μM, n = 28. Preincubation of HUVEC with LTB₄ (250 nM) for 3 h increases the response of a second 15-min challenge with LTB₄ (Right). Addition of the BLT₁ antagonist CP-105696 (CP) during the preincubation period reduces most of the response. Results are expressed as percentage of untreated control HUVEC; mean value was 656 ± 221 pg/ml, n = 18.