Figure 2: Effects of TSG-6 on Inflammatory Phenotypes, Cytokine Secretion, Foam Cell Formation, and Related Protein Expression in HMDMs

(A) Human monocytes were incubated for the indicated times in RPMI-1640 medium containing 10% human serum with or without TSG-6 (200 ng/ml). Cells were harvested for immunoblotting analysis for CD68 (differentiation marker), MARCO (M1 marker), or MRC1 (M2 marker). β-actin served as a loading control. The graph shows the expressions of MARCO on day 6 and MRC1 on day 3. n = 3; *p < 0.001 vs. 0 ng/ml of TSG-6. (B) HMDMs differentiated by 10% human serum without TSG-6 were incubated for 26 h in serum-free RPMI-1640 medium with or without TSG-6 (300 ng/ml). The concentrations of IL-6 and TNF-α in culture supernatants were measured by enzyme-linked immunosorbent assay. Baseline of control = 27.3 ± 11.9 pg/ml (IL-6) and 34.7 ± 3.1 pg/ml (TNF-α). n = 3; †p = 0.028 vs. control. (C) Human monocytes were incubated to differentiate into HMDMs for 7 days in RPMI-1640 with 10% human serum and the indicated concentrations of TSG-6, followed by a further 19 h with the same concentrations of TSG-6, 50 μg/ml oxLDL, and 0.1 mmol/l [3H]oleate. Foam cell formation was determined from the intracellular radioactivity of cholesterol-[3H]oleate. Baseline of control = 11.8 ± 4.0 nmol/mg cell protein. n = 4; ‡p = 0.042 vs. 0 ng/ml of TSG-6. (D to F) Before the addition of oxLDL, HMDMs were harvested for immunoblotting analysis of CD36, ACAT1, or ABCA1. β-actin served as a loading control. n = 6 to 7; #p = 0.021; §p = 0.014; ¶p < 0.01; and @p < 0.001 vs. 0 ng/ml of TSG-6. All values are presented as mean ± SEM. ACAT1 = acyl-CoA:cholesterol acyltransferase-1; oxLDL = oxidized low-density lipoprotein; other abbreviations as in Figure 1.