Human macrophages limit oxidation products in low density lipoprotein

Abstract

This study tested the hypothesis that human macrophages have the ability to modify oxidation products in LDL and oxidized LDL (oxLDL) via a cellular antioxidant defence system. While many studies have focused on macrophage LDL oxidation in atherosclerosis development, less attention has been given to the cellular antioxidant capacity of these cells. Compared to cell-free controls (6.2 +/- 0.7 nmol/mg LDL), macrophages reduced TBARS to 4.42 +/- 0.4 nmol/mg LDL after 24 h incubation with LDL (P = 0.022). After 2 h incubation with oxLDL, TBARS were 3.69 +/- 0.5 nmol/mg LDL in cell-free media, and 2.48 +/- 0.9 nmol/mg LDL in the presence of macrophages (P = 0.034). A reduction of lipid peroxides in LDL (33.7 +/- 6.6 nmol/mg LDL) was found in the presence of cells after 24 h compared to cell-free incubation (105.0 +/- 14.1 nmol/mg LDL) (P = 0.005). The levels of lipid peroxides in oxLDL were 137.9 +/- 59.9 nmol/mg LDL and in cell-free media 242 +/- 60.0 nmol/mg LDL (P = 0.012). Similar results were obtained for hydrogen peroxide. Reactive oxygen species were detected in LDL, acetylated LDL, and oxLDL by isoluminol-enhanced chemiluminescence (CL). Interestingly, oxLDL alone gives a high CL signal. Macrophages reduced the CL response in oxLDL by 45% (P = 0.0016). The increased levels of glutathione in oxLDL-treated macrophages were accompanied by enhanced catalase and glutathione peroxidase activities. Our results suggest that macrophages respond to oxidative stress by endogenous antioxidant activity, which is sufficient to decrease reactive oxygen species both in LDL and oxLDL. This may suggest that the antioxidant activity is insufficient during atherosclerosis development. Thus, macrophages may play a dual role in atherogenesis, i.e. both by promoting and limiting LDL-oxidation.

Figure 1

Effect of macrophages on oxidation products in LDL and oxLDL. RPMI 1640 containing 100 μg/mL of LDL or oxLDL (oxidized for 2 h) was incubated in cell culture wells at 37°C with and without macrophages (HMDM) for 2 h and 24 h. Values are expressed as nmol/mg LDL, TBARS (n = 5) (A), lipid peroxides (n = 6) (B), H₂O₂ (n = 6), (C). Values of Apo B are expressed as % change compared to cell free control incubations (n = 4) (D).

Figure 2

Production of reactive oxygen species from LDL, oxLDL and macrophages as measured by isoluminol-enhanced chemiluminescence. The incubation mixture of 1.0 mL KRG contained 5 × 10⁵ cells (HMDM), 10 μg isoluminol, 4 U horseradish peroxidase, and 100 μg of LDL, oxLDL (oxidized for 2 h), or acLDL. The chemiluminescence was measured every 2 minutes for a total of 100 minutes at 37°C (n = 5).

Figure 3

Production of reactive oxygen species from oxLDL and macrophages as measured by isoluminol-enhanced chemiluminescence. The incubation mixture of 1.0 mL KRG, containing 10 μg isoluminol, 4 U horseradish peroxidase, and 100 μg of oxLDL (oxidized for either 2 h, 8 h or 20 h), was used alone or in combination with 5 × 10⁵ macrophages (HMDM) (n = 3).

Figure 4

Dose-response effect of oxLDL on production of reactive oxygen species as measured by isoluminol-enhanced chemiluminescence. The incubation mixture of 1.0 ml KRG contained 10 μg isoluminol, and different concentrations of oxLDL oxidized for 2 h, alone and in combination with HMDM (5 × 10⁵ cells). Data are shown as means of maximal peak values in mV ± SE for triplicate determinations within a single experiment and are representative of two independent experiments. Results of oxLDL in cell-free incubations versus oxLDL in the presence of macrophages were analyzed by ANOVA.

Figure 5

Production of reactive oxygen species from oxLDL and non-viable macrophages as measured by isoluminol-enhanced chemiluminescence. The incubation mixture of 1.0 mL KRG, containing 10 μg isoluminol, 4 U horseradish peroxidase, and 100 μg of oxLDL (oxidized for 2 h), was used alone or in combination with 5 × 10⁵ non-viable macrophages. Data are shown from a typical experiment with 2 different cell donors.

Figure 6

Effect of LDL and oxLDL on the intracellular antioxidant defenses in macrophages. The intracellular activity of catalase (A), glutathione peroxidase (B), and the levels of glutathione (C) were measured in crude extracts from macrophages (n = 6) incubated with LDL or oxLDL (oxidized for 2 h). Control cells were incubated in the absence of LDL. Results were analyzed by ANOVA.