Toll-like receptor (TLR)-4 mediates anti-β2GPI/β2GPI-induced tissue factor expression in THP-1 cells

Abstract

Our previous study demonstrated that annexin A2 (ANX2) on cell surface could function as a mediator and stimulate tissue factor (TF) expression of monocytes by anti-β₂-glycoprotein I/β₂-glycoprotein I complex (anti-β₂GPI/β₂GPI). However, ANX2 is not a transmembrane protein and lacks the intracellular signal transduction pathway. Growing evidence suggests that Toll-like receptor 4 (TLR-4) might act as an 'adaptor' for intracellular signal transduction in anti-β₂GPI/β₂GPI-induced TF expressing cells. In the current study, we investigated the roles of TLR-4 and its related molecules, myeloid differentiation protein 2 (MD-2) and myeloid differentiation factor 88 (MyD88), in anti-β₂GPI/β₂GPI-induced TF expressing human monocytic-derived THP-1 (human acute monocytic leukaemia) cells. The relationship of TLR-4 and ANX2 in this process was also explored. Along with TF, expression of TLR-4, MD-2 and MyD88 in THP-1 cells increased significantly when treated by anti-β₂GPI (10 µg/ml)/β₂GPI (100 µg/ml) complex. The addition of paclitaxel, which competes with the MD-2 ligand, could inhibit the effects of anti-β₂GPI/β₂GPI on TLR-4, MD-2, MyD88 and TF expression. Both ANX2 and TLR-4 in THP-1 cell lysates could bind to β₂GPI that had been conjugated to a column (β₂GPI-Affi-Gel). Furthermore, TLR-4, MD-2, MyD88 and TF expression was remarkably diminished in THP-1 cells infected with ANX2-specific RNA interference (RNAi) lentivirus (LV-RNAi-ANX2), in spite of treatment with a similar concentration of anti-β₂GPI/β₂GPI complex. These results indicate that TLR-4 and its signal transduction pathway contribute to anti-β₂GPI/β₂GPI-induced TF expression in THP-1 cells, and the effects of TLR-4 with ANX2 are tightly co-operative.

Fig. 1

Tissue factor (TF) expression of THP-1 (human acute monocytic leukaemia) cells induced by anti-β₂-glycoprotein I/β₂-glycoprotein I (anti-β₂GPI/β₂GPI) complex. The THP-1 cells (2 × 10⁶) were treated with anti-β₂GPI (10 µg/ml)/β₂GPI (100 µg/ml) complex, isotype control rabbit immunoglobulin G (R-IgG) (10 µg/ml)/β₂GPI (100 µg/ml), lipopolysaccharide (LPS) (500 ng/ml) and anti-β₂GPI (10 µg/ml)/bovine serum albumin (BSA) (100 µg/ml) for 2 h (a) or 6 h (b). TF mRNA (a) and TF activity (b) were detected by real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) and TF activity kits, respectively, as described in Materials and methods. Data shown are from three independent experiments. *P < 0·05 versus control of untreated cells.

Fig. 2

Toll-like receptor (TLR)-4, myeloid differentiation factor 88 (MyD88) and myeloid differentiation protein 2 (MD-2) secretion in THP-1 (human acute monocytic leukaemia) cells stimulated with anti-β₂-glycoprotein I/β₂-glycoprotein I (anti-β₂GPI/β₂GPI) complex. The THP-1 cells (2 × 10⁶) were treated with anti-β₂GPI (10 µg/ml)/β₂GPI (100 µg/ml) complex, isotype control rabbit immunoglobulin G (R-IgG) (10 µg/ml)/β₂GPI (100 µg/ml) and lipopolysaccharide (LPS) (500 ng/ml) for 2 h (a) or 6 h (b). TLR-4, MyD88 and MD-2 mRNA levels (a) were detected by real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR), and their protein levels (b) were analysed by Western blotting. Data shown are from three independent experiments. *P < 0·05 versus control of untreated cells.

Fig. 3

Analysis of β₂-glycoprotein I (β₂GPI) binding with annexin A2 (ANX2) and Toll-like receptor (TLR)-4 on THP-1 (human acute monocytic leukaemia) cell membrane. The cell lysates were extracted from 1 × 10⁷ of THP-1 cells and subjected to affinity chromatography on β₂GPI-Affi-Gel column, then the column was washed and eluted as described in Materials and methods. The proteins in each solution were analysed by Western blotting using anti-ANX2 or anti-TLR-4 antibodies. The protein bands of approximately 36 kD (ANX2) (a) and 100 kD (TLR-4) (b) were found only in Tris/350 mM NaCl-eluted solution.

Fig. 4

Paclitaxel decreased anti-β₂-glycoprotein I/β₂-glycoprotein I (anti-β₂GPI/β₂GPI)-induced Toll-like receptor (TLR)-4, myeloid differentiation factor 88 (MyD88) and myeloid differentiation protein 2 (MD-2) expression in THP-1 (human acute monocytic leukaemia) cells. The THP-1 cells were pretreated with paclitaxel (1 µM) for 1 h, then stimulated by anti-β₂GPI (10 µg/ml)/β₂GPI (100 µg/ml) complex, isotype control rabbit immunoglobulin G (R-IgG) (10 µg/ml)/β₂GPI (100 µg/ml) and lipopolysaccharide (LPS) (500 ng/ml) for 2 h (a) or 6 h (b). The mRNA levels and the protein expression of TLR-4 (a1, a2), MyD88 (b1, b2) and MD-2 (c1, c2) were detected by real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) and Western blotting. Data shown are from three separate experiments. *P < 0·05 versus no paclitaxel-treated cells.

Fig. 5

Paclitaxel inhibited anti-β₂-glycoprotein I/β₂-glycoprotein I (anti-β₂GPI/β₂GPI)-stimulated tissue factor (TF) expression on THP-1 (human acute monocytic leukaemia) cells. The THP-1 cells were pretreated with paclitaxel (1 µM) for 1 h, then stimulated by anti-β₂GPI (10 µg/ml)/β₂GPI (100 µg/ml) complex, isotype control rabbit immunoglobulin G (R-IgG) (10 µg/ml)/β₂GPI (100 µg/ml) and lipopolysaccharide (LPS) (500 ng/ml) for 2 h (a) or 6 h (b). The TF mRNA (a) and TF activity (b) were measured by real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) and TF activity kits, respectively. Data shown are from three separate experiments. **P < 0·01 versus no paclitaxel-treated cells.

Fig. 6

Annexin A2 (ANX2) expression in lentiviral-infected THP-1 (human acute monocytic leukaemia) cells. The empty lentivirus (LV-GFP) and ANX2 siRNA (LV-RNAi-ANX2) were transferred into target THP-1 cells at multiplicity of infection (MOI) equal to 100 with enhanced infection solution (ENi.S) and 5 µg/ml polybrene. After 72 h, the ANX2 mRNA (a) and its protein (b) levels on the cells were detected by real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) or Western blot. **P < 0·01 versus no lentivirus cells. Data shown are from three separate experiments.

Fig. 7

Toll-like receptor (TLR)-4, myeloid differentiation factor 88 (MyD88) and myeloid differentiation protein 2 (MD-2) expression in lentiviral-infected THP-1 (human acute monocytic leukaemia) cells. The THP-1 cells infected with LV-GFP or LV-RNAi-annexin A2 (ANX2) were incubated with anti-β₂-glycoprotein I (β₂GPI) (10 µg/ml)/β₂GPI (100 µg/ml) for 2 h or 6 h. The mRNA levels and the protein expression of TLR-4 (a1, a2), MyD88 (b1, b2) and MD-2 (c1, c2) were detected by real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) and Western blotting. Data shown are from three separate experiments. *P < 0·05 versus no lentivirus cells.

Fig. 8

Tissue factor (TF) expression in lentiviral-infected THP-1 (human acute monocytic leukaemia) cells. The THP-1 cells infected with LV-GFP or LV-RNAi-annexin A2 (ANX2) were incubated with anti-β₂-glycoprotein I (β₂GPI) (10 µg/ml)/β₂GPI (100 µg/ml), lipopolysaccharide (LPS) (500 ng/ml) for 2 h or 6 h. TF mRNA (a) and TF activity (b) were examined by real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) and and TF activity kits, respectively. Data shown are from three separate experiments. *P < 0·05 versus no lentivirus cells.