Plasma from rheumatoid arthritis patients promotes pro-atherogenic cholesterol transport gene expression in THP-1 human macrophages

Abstract

Immunologic derangements in rheumatoid arthritis (RA) patients likely contribute to premature atherosclerotic cardiovascular disease (CVD). Traditional CVD risk factors do not reliably identify at-risk RA patients, probably because disease-associated mechanisms are not taken into account. The purpose of this study was to determine whether plasma from subjects with RA exhibits atheroma-promoting properties leading to disruption of cholesterol homeostasis in human monocytes/macrophages. Twenty-one healthy controls (HC) and 22 RA patients were enrolled in an IRB approved study at Winthrop University Hospital. Naïve THP-1 macrophages were exposed to plasma from each HC and RA patient. Following incubation, RNA and protein were isolated. QRT-PCR and Western blotting techniques were then used to measure expression of proteins responsible for cholesterol efflux (ATP binding cassette transporter (ABC)A1, ABCG1, 27-hydroxylase) and cholesterol uptake (CD36, ScR-A1, lectin oxidized low density lipoprotein receptor (LOX)-1, CXCL16). To confirm the pro-atherogenic effects of RA plasma on macrophages, foam cell formation was quantified. Results showed that RA plasma downregulates cholesterol efflux proteins and upregulates scavenger receptors CD36, LOX1 and CXCL16. These pro-atherogenic changes in gene expression in the presence of RA plasma are associated with augmented lipid accumulation and foam cell formation by THP-1 macrophages. RA plasma induces macrophage cholesterol overload. Demonstration of disrupted cholesterol homeostasis mediated by RA plasma provides further evidence of the involvement of the immune system in atherogenesis. Our data suggest that chronic exposure to RA plasma adversely affects the capacity of monocytes/macrophages in the arterial wall to metabolize cholesterol and maintain lipid homeostasis, thereby contributing to the development of premature atherosclerosis.

Keywords: Rheumatoid arthritis; atherosclerosis; cholesterol transport; macrophage.

Figure 1

Effect of RA plasma on the expression of scavenger receptors in human THP-1 macrophages. THP-1 macrophages were incubated in the presence of 10% plasma from each of 22 RA or each of 21 HC subjects for 18 h and 24 h, as indicated in methods. Following incubation, total RNA isolated from cells of each condition was reverse transcribed and amplified by QRT-PCR with GAPDH message as an internal standard. Gene expression levels were graphed as relative mRNA expression with mean of HC set at 100%. The data represent the mean for 22 RA or 21 HC±SEM of three independent experiments. Protein expression is presented as relative comparison between protein abundance of the sample and loading control (β-actin). *P < 0.05; ***P < 0.001 versus THP-1 macrophages exposed to HC plasma

Figure 2

Effect of RA plasma on the expression of cholesterol efflux proteins in human THP-1 macrophages. THP-1 macrophages were incubated in the presence of 10% plasma from each of 22 RA or each of 21 HC subjects for 18 h and 24 h, as indicated in Materials and methods section. Following incubation, total RNA isolated from cells of each condition was reverse transcribed and amplified by QRT-PCR with GAPDH message as an internal standard. Gene expression levels were graphed as relative mRNA expression with mean of HC set at 100%. The data represent the mean for 22 RA or 21 HC±SEM of three independent experiments. Protein expression is presented as relative comparison between protein abundance of the sample and loading control (β-actin). 27-OH=27-hydroxylase. **P < 0.01; ***P < 0.001 versus THP-1 macrophages exposed to HC plasma

Figure 3

Foam cell formation (FCF) in THP-1 macrophages exposed to plasma from RA patients and HC. Cultured THP-1 macrophages were exposed to RA or HC plasma (3 h for oxLDL uptake assay and 24 h for FCF analysis) as indicated in methods. (a) FCF was calculated as a percentage of oil-red-O stained cells. All results are expressed as mean for 22 RA or 21 HC±SEM of three independent experiments. *P < 0.05 versus % FCF in THP-1 macrophages exposed to HC plasma. (b) Accumulation of Dil-oxLDL in cells was determined by fluorescent intensity (FU) of accumulated Dil-oxLDL. Fluorescent intensity was quantified and graphed. ##P < 0.01 versus FU of THP-1 macrophages exposed to HC plasma