Previous reports indicate that the effects of phytosterols on LXR and SREBP-2 target genes are cell type dependent. To determine their effects in macrophages, we evaluated ABCA1 and LDLR protein abundance in elicited MPMs following treatment with individual non-cholesterol sterols differing at the 22 and 24 carbon positions within the cholesterol side chain (). Cells were harvested, cultured for 24 hr and incubated in the presence of serum free medium (Control) supplemented with the indicated sterol for 48 hr. Consistent with previous reports in Y1 adrenal cells, stigmasterol and 22(R)-dehydrocholesterol increased ABCA1 expression and decreased LDLR abundance whereas the remaining sterols had no effect. One potential explanation for the differences in responses among the sterols is their entry and accumulation in macrophages. We extracted lipids from control and sterol treated cells and analyzed cholesterol and phytosterol content by GC-MS. Although there were substantial differences in the mass of sterols extracted from cultured macrophages, no correlation between cell-associated sterols and the expression of either ABCA1 or LDLR could be established (not shown).
Figure 1 Effect of 22 and 24 substitution of the cholesterol side-chain on expression of ABCA1 and LDLR and sterol accumulation in MPMs. A) MPMs were isolated and cultured as described in Methods. On day 2, the medium was removed and replaced with medium containing (more ...)
The concentrations of phytosterols used in this and previous studies are substantially greater than what are typically observed in plasma of individuals consuming phytosterol supplements. To determine if phytosterols affected the expression of ABCA1 and LDLR at concentrations that are observed in vivo
(4–20 μg/ml [15
]), we conducted a time-course experiment using 10 μg/ml phytosterol (). Among the commonly consumed phytosterols only stigmasterol increased expression of ABCA1 and decreased expression of LDLR. Neither campesterol nor sitosterol altered immunoreactive ABCA1 and LDLR during over the 48 hour period (not shown). Densitometric analysis indicated that the effects of stigmasterol on ABCA1 and LDLR were discernable by 4 hours and persisted up to 48 hours (Figure S1). However, LDLR expression decreased in control cells after 24 hours, suggesting that reductions over this period were unrelated to the presence of stigmasterol. This result also implies that the cellular content of cholesterol is dynamic over the 72 hr culture period and that the effects of stigmasterol may be dependent upon, or secondary to changes in endogenous cholesterol synthesis, a known source of LXR ligands [17
]. To address this concern and to determine the minimal concentration of stigmasterol required to elicit changes in ABCA1 and LDLR abundance, a dose-response experiment was conducted in the presence of the HMG-CoA Reductase (HMGCR) inhibitor, compactin (). The presence of compactin suppresses ABCA1 and upregulates LDLR. Therefore, 25-hydroxycholesterol was used as a positive control since it is known to be both an LXR agonist and a suppressor of SREBP processing. Stigmasterol treatment resulted in an increase in ABCA1 that was detectible at 1 μg/ml, increased substantially at 10 μg/ml, and continued to increase, albeit to a lesser extent from 10 to 50 μg/ml (Figure S1). For LDLR, a modest suppression was observed at 0.5 μg/ml and further increases in stigmasterol resulted in complete suppression of immunoreactive LDL receptor. These results demonstrate that the effects of stigmasterol on LDLR and ABCA1 in macrophages are dose-dependent and not due to accumulation of cholesterol biosynthetic intermediates.
Figure 2 Effect of time and concentration of stigmasterol on immunoreactive ABCA1 and LDLR in elicited MPMs. A) MPMs were isolated and cultured as in . On day 2, the medium was removed and replaced with Medium B containing stigmasterol (10 μg/ml) (more ...)
Next we determined if the effect of stigmasterol on ABCA1 abundance was associated with changes in mRNA levels for this and other LXR target genes (). Each of the LXR target genes was increased by stigmasterol, but not sitosterol. Similarly, campesterol did not alter LXR target gene expression. We also evaluated expression of SREBPs and selected targets. Not surprisingly, SREBP-1c was also upregulated by stigmasterol as well as its downstream targets fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC1). However, stigmasterol had no effect on SREBP2 or its target genes, suggesting the mechanism by which stigmasterol suppresses LDLR protein is post-transcriptional, distinct from that of 25-OH-C, and independent of interference with SREBP processing.
Figure 3 Effect of stigmasterol on the expression of LXR (A) and SREBP1 (B), SREBP2 (C) and selected target genes in mouse peritoneal macrophages. Macrophages were elicited and cultured as in . On day 2, the medium was removed and replaced with Medium (more ...)
Although plant sterols may affect gene expression and cholesterol trafficking when added directly to the culture medium, macrophage foam cells acquire plant sterols from modified lipoproteins in vivo
. Aggregation of LDL by vortexing substantially increased the incorporation of phytosterols into LDL particles compared to oxidation and acetlyation (not shown). However, LDL aggregates are poorly processed by mouse peritoneal macrophages [25
]. Therefore we selected THP-1 cells since these cells are an established model of macrophage foam cells that readily internalize and process agLDL in lysosomes [21
]. First, we confirmed that the effects of individual phytosterols on ABC transporter expression would persist in lipid loaded cells and that they were not unique to MPMs. Following differentiation, THP-1 macrophages were cultured with medium supplemented with agLDL (Control) or agLDL prepared in the presence of the indicated sterol. As a positive control, cells were incubated in medium containing both agLDL and an LXR agonist (TO901317). As an additional control, agLDL was prepared in the presence of cholesterol to maintain equality of total added sterols. Following 48 hr of treatment, membrane proteins were prepared and analyzed by SDS-PAGE and immunoblotting (). Incubation of THP-1 macrophages with agLDL in the absence of additional sterols increases ABCA1 and suppresses LDLR below the limits of detection (not shown). The addition of the LXR agonist further increased ABCA1 protein in agLDL loaded macrophages. ABCG1 was also increased in THP-1 loaded macrophages, the measurements of which proved difficult in mouse macrophages using commercially available antibodies. The incorporation of cholesterol, sitosterol and campesterol in agLDL had no effect on ABCA1 or ABCG1 abundance. Consistent with mRNA data in MPMs, the inclusion of stigmasterol in agLDL increased both transporters in human macrophages, whereas other phytosterols had no effect.
Figure 4 Effect of major phytosterols on expression of ABCA1 and ABCG1 and cholesterol efflux to ApoAI and HDL in agLDL loaded THP-1 macrophages. Native human LDL (1 mg protein) was aggregated in the presence of carrier (Control), TO901317 (10 μM), cholesterol, (more ...)
Cellular sterol content was determined before and after incubation with agLDL (). Lipids were extracted and analyzed by GC-MS. Incubation of THP-1 macrophages with agLDL resulted in a 5–6 fold increase in total cellular sterol content. The addition of phytosterols collectively and individually had no effect on the extent of cholesterol accumulation or total cellular sterol content when compared to the cholesterol control, indicating that changes in ABC transporter expression are not merely a function of total cholesterol or sterol content of THP-1 macrophages.
Table 1 Cellular sterol content (μg/mg total cell protein) following incubation with agLDL prepared in the presence (+) of the indicated sterol. Values represent means ±standard deviations. Empty cells indicate that levels were below the limits (more ...)
Next we determined if phytosterols altered efflux of cholesterol from agLDL loaded THP-1 macrophages to Apo AI and HDL (). THP-1 monocytes were differentiated into macrophages and incubated with agLDL prepared in the presence of [3H]-cholesterol and the indicated sterol for 48 hours (100 μg/ml LDL, 10 μg/ml sterol, 1 μCi/ml [3H]-cholesterol). Following the loading phase, the cells were washed and allowed to equilibrate in serum free medium for 2 hr. The equilibration medium was removed, the cells were washed and the medium replaced in Medium B containing Apo AI (30 μg/ml) or HDL (100 μg/ml) for 4hr. Relative to control cells, in which no additional sterols were added to the LDL aggregates, TO901317 enhanced efflux of [3H]-cholesterol to both Apo AI and HDL (p<0.05). Sitosterol had no effect on efflux to either acceptor, although there was a tendency for a decrease to HDL. The presence of stigmasterol increased efflux to Apo AI by 25% (p<0.05) and tended to increase efflux to HDL. Campesterol resulted in a modest, but significant decrease in efflux to HDL (p<0.05), but did not alter efflux to Apo AI. Similar stimulatory effects of stigmasterol were observed in MPMs (Figure S2). These results indicate that the effects of individual phytosterols on ABC transporter expression and cholesterol efflux are largely consistent among cultured macrophages of both human and mouse origin.
Beyond the accumulation of lipid, macrophages contribute to the inflammatory state of the atherosclerotic lesion. To determine if phytosterols alter the inflammatory response to agLDL loading, we evaluated the secretion of inflammatory cytokines in the culture medium using a commercially available cytometric bead assay (CBA) inflammation panel (). First, we used pretreatment with interferon (IFN) -γ followed by lipopolysaccharide (LPS) as a control for classical activation of macrophages. Following pretreatment with IFN-γ, LPS dramatically increased the secretion of TNFα, IL-6 and IL-1β. Compared to untreated cells, incubation with agLDL increased the expression of each of these proinflammatory cytokines. The effect of phytosterols on the response to agLDL loading was assessed by comparing the levels of secreted cytokines to cells treated with agLDL prepared in the presence of the carrier (ethanol). The presence of stigmasterol decreased agLDL-induced secretion of TNFα, IL-6 and IL-1β. The presence of sitosterol increased the secretion of TNFα and IL-1β, but not IL-6. Campesterol had no effect on the inflammatory response to agLDL. Levels of IL-8 were unaffected by treatments. IL-10 and IL12p70 were below the limits of detection in our assay (not shown).
Figure 5 Sterol loading of THP-1 cells induces the synthesis and secretion of TNFα, IL-6 and IL-1β. THP-1 macrophages were incubated in medium (control), agLDL (100 μg/ml) prepared in the presence of carrier or the indicated sterol (10 (more ...)