Atheroprotective effects of methotrexate on reverse cholesterol transport proteins and foam cell transformation in human THP-1 monocyte/macrophages

Abstract

Objective: To determine whether methotrexate (MTX) can overcome the atherogenic effects of cyclooxygenase 2 (COX-2) inhibitors and interferon-gamma (IFNgamma), both of which suppress cholesterol efflux protein and promote foam cell transformation in human THP-1 monocyte/macrophages.

Methods: Message and protein levels of the reverse cholesterol transport proteins cholesterol 27-hydroxylase and ATP-binding cassette transporter A1 (ABCA1) in THP-1 cells were evaluated by real-time polymerase chain reaction and immunoblot, respectively. Expression was evaluated in cells incubated in the presence or absence of the COX-2 inhibitor NS398 or IFNgamma, with and without MTX. Foam cell transformation of lipid-laden THP-1 macrophages was detected with oil red O staining and light microscopy.

Results: MTX increased 27-hydroxylase message and completely blocked NS398-induced down-regulation of 27-hydroxylase (mean +/- SEM 112.8 +/- 13.1% for NS398 plus MTX versus 71.1 +/- 4.3% for NS398 alone; P < 0.01). MTX also negated COX-2 inhibitor-mediated down-regulation of ABCA1. The ability of MTX to reverse inhibitory effects on 27-hydroxylase and ABCA1 was blocked by the adenosine A2A receptor-specific antagonist ZM241385. MTX also prevented NS398 and IFNgamma from increasing transformation of lipid-laden THP-1 macrophages into foam cells.

Conclusion: This study provides evidence supporting the notion of an atheroprotective effect of MTX. Through adenosine A2A receptor activation, MTX promotes reverse cholesterol transport and limits foam cell formation in THP-1 macrophages. This is the first reported evidence that any commonly used medication can increase expression of antiatherogenic reverse cholesterol transport proteins and can counteract the effects of COX-2 inhibition. Our results suggest that one mechanism by which MTX protects against cardiovascular disease in rheumatoid arthritis patients is through facilitation of cholesterol outflow from cells of the artery wall.

Figure 1. Effect of MTX on 27-hydroxylase message in THP-1 monocytes and human PBMC

(a) Quantitation of 27-hydroxylase message in NS-398-treated THP-1 cells exposed to MTX. COX-2 inhibitor-mediated decrease in 27-hydroxylase mRNA is prevented by MTX. THP-1 human monocytes were exposed to the following conditions represented by the four bars (from left to right): (1) Control RPMI 1640, (2) MTX (5 μM, 18 hr), (3) NS398 (50μM, 18 hr), (4) MTX (5 μM, 18 hr) and NS398 (50μM, 18hr). Cells were extracted for total RNA, and evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. *p<0.05, control vs. NS398. #p<0.01, NS398 + MTX vs. NS398. (b) Quantitation of 27-hydroxylase message in IFN-γ-treated THP-1 cells exposed to MTX. IFN-γ-mediated decrease in 27-hydroxylase mRNA is prevented by MTX. THP-1 human monocytes were exposed to the following conditions represented by the three bars (from left to right): (1) Control RPMI 1640, (2) IFN-γ (500 U/ml, 18 hr), (3) IFN-γ (500 U/ml, 18 hr) and MTX (5 μM, 18 hr). Cells were extracted for total RNA, and evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. ***p=0.02 IFN-γ + MTX vs IFN-γ. (c) Effect of MTX on 27-hydroxylase message in healthy donor PBMC. Isolated PBMCs were incubated in RPMI 1640 with and without MTX (5 μM, 18 hr). Cells were extracted for total RNA, and evaluated for 27-hydroxylase mRNA expression by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. ****p=0.004 MTX vs control untreated.

Figure 2. Detection and quantitation of cholesterol 27-hydroxylase in NS398-treated THP-1 cells exposed to increasing doses of MTX

(a) Decrease in 27-hydroxylase protein in THP-1 monocytes treated with the COX-2 inhibitor NS398 is corrected with increasing concentrations of MTX. Cultured THP-1 monocytic cells were untreated or exposed to NS398 (50μM, 18hr) then untreated or exposed to increasing doses of MTX for 24 hr. Total cell protein was isolated and 27-hydroxylase detected with specific rabbit polyclonal anti-human 27-hydroxylase antibody. Western blotting was also performed with an anti-beta actin antibody to confirm equal protein loading. (b) COX-2-inhibitor-mediated suppression of 27-hydroxylase mRNA expression in THP-1 monocytes is overcome with MTX. Cultured THP-1 monocytic cells were incubated in NS398 (50μM, 48hr) then untreated or exposed to increasing doses of MTX for 24 hr. Following isolation of total RNA, the RNA was reverse transcribed and the cDNA amplified by QRT-PCR as described. Signals obtained from the amplification of GAPDH message were used as internal controls. *p<0.05, **p<0.01, MTX vs Control (C). #p<0.01, NS+MTX vs NS398 (NS).

Figure 3. Detection and quantitation of cholesterol 27-hydroxylase and ABCA1 mRNA in NS398-treated THP-1 cells exposed to MTX in the presence and absence of A2A receptor antagonism with ZM-241385

(a) Suppression of 27-hydroxylase message in THP-1 cells by NS398 is reversed by MTX and this reversal is blocked by ZM-241385. THP-1 monocytes were exposed to the following conditions represented by the four bars from left to right: (1) Control RPMI 1640, (2) NS398 (50μM, 24 hr), (3) NS398 (50μM, 24 hr) then add MTX (5 μM, 24 hr), (4) NS398 (50μM) and ZM-241385 (10μM) for 24 hr, then add MTX (5μM) for 24 hr. Cells were extracted for total RNA, and evaluated for 27-hydroxylase mRNA by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) Suppression of ABCA1 message in THP-1 cells by NS398 is reversed by MTX and this reversal is blocked by ZM-241385. THP-1 monocytes were exposed to the following conditions represented by the four bars from left to right: (1) Control RPMI 1640, (2) NS398 (50μM, 24 hr), (3) NS398 (50μM, 24 hr) then add MTX (5 μM, 24 hr), (4) NS398 (50μM) and ZM-241385 (10μM) for 24 hr, then add MTX (5μM) for 24 hr. Cells were extracted for total RNA, and evaluated for 27-hydroxylase mRNA by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. *p<0.05, **p<0.01, MTX vs control (C). #p<0.01, NS398+MTX vs NS398.

Figure 4. Detection and quantitation of cholesterol 27-hydroxylase mRNA and protein and ABCA1 mRNA in IFN-γ-stimulated THP-1 macrophages exposed to MTX in the presence and absence of A2A receptor antagonism with ZM-241385

(a) Suppression of 27-hydroxylase message in THP-1 macrophages by IFN-γ is reversed by MTX and this reversal is blocked by ZM-241385. THP-1 macrophages were exposed to the following conditions represented by the eight bars from left to right: (1) Control RPMI 1640, (2) ZM-241385 (10μM, 24 hr), (3) MTX (5 μM, 24 hr), (4) IFN-γ (500 U/ml, 24 hr), (5) IFN-γ (500 U/ml, 24 hr), then add MTX (5μM, 24 hr), (6) ZM-241385 (10 μM, 24 hr), then add MTX (5μM, 24 hr), (7) ZM-241385 (10 μM) and IFN-γ (500 U/ml) for 24 hr, then add MTX (5μM, 24 hr), (8) ZM-241385 (10 μM) and NS398 (50μM) for 24 hr, then add MTX (5μM, 24 hr). Cells were extracted for total RNA, and evaluated for 27-hydroxylase mRNA by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. (b) Suppression of 27-hydroxylase protein in THP-1 cells by IFN-γ is reversed by MTX and this reversal is blocked by ZM-241385. THP-1 macrophages were exposed to identical conditions 1-8 as in part (a) of this figure represented by the eight lanes of the immunoblot from left to right. Total cell protein was isolated and 27-hydroxylase detected with specific rabbit polyclonal anti-human 27-hydroxylase antibody. Western blotting was also performed with an anti-beta actin antibody to confirm equal protein loading. (c) Suppression of ABCA1 message in THP-1 macrophages by IFN-γ is reversed by MTX and this reversal is blocked by ZM-241385. THP-1 macrophages were exposed to identical conditions 1-8 as in part (a) of this figure represented by the eight bars from left to right. Cells were extracted for total RNA, and evaluated for 27-hydroxylase mRNA by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. **p<0.01, IFN-γ+ MTX vs IFN-γ. #p<0.01, ZM+MTX vs MTX.

Figure 5. Effect of the A2AR agonist CGS0-21680 on NS398-induced suppression of 27-hydroxylase expression in THP-1 monocytes

a) 27-hydroxylase message level is decreased by the COX-2 inhibitor, NS398 (50μM), and this decrease is reversed by the addition of the adenosine A2AR agonist, CGS-21680 (10 μM). THP-1 monocytes were exposed to the following conditions represented by the four bars from left to right: (1) Control RPMI 1640, (2) CGS-21680 (10μM, 18 hr), (3) NS398 (50 μM, 18 hr), (4) NS398 (50μM, 18 hr) and CGS-21680 (10 μM, 18 hr). Cells were extracted for total RNA, and evaluated for 27-hydroxylase mRNA by QRT-PCR. Signals obtained from the amplification of GAPDH message were used as internal controls. b) 27-hydroxylase protein level is decreased by the COX-2 inhibitor, NS398, and this decrease is reversed by the addition of the adenosine A2AR agonist, CGS-21680. THP-1 monocytes were exposed to the following conditions represented by the four bands from left to right: (1) Control RPMI 1640, (2) CGS-21680 (10μM, 18 hr), (3) NS398 (50 μM, 18 hr), (4) NS398 (50μM, 18 hr) and CGS-21680 (10 μM, 18 hr). *p<0.01, control vs. NS398. #p<0.01, NS398 + CGS-21680 vs. NS398.

Figure 6. Effect of MTX on NS398 and IFN-γ-induced foam cell transformation in lipid loaded THP-1 macrophages

Representative photomicrographs at 40 X magnification of lipid laden macrophages stained with oil red-O. A) Acetylated LDL-treated THP-1 macrophages showed a significant decrease in foam cell transformation in the presence of MTX compared to control B) MTX prevented the NS398-induced increase in foam cell formation in THP-1 macrophages. C) MTX prevented the IFN-γ-induced increase in foam cell formation in THP-1 macrophages. D) Effectiveness of MTX in decreasing foam cell formation is abolished by A2AR antagonism with ZM-241385