Activation of liver X receptor induces macrophage interleukin-5 expression

Abstract

IL-5 stimulates production of T15/EO6 IgM antibodies that can block the uptake of oxidized low density lipoprotein by macrophages, whereas a deficiency in macrophage IL-5 expression accelerates development of atherosclerosis. Liver X receptors (LXRs) are ligand-activated transcription factors that can induce macrophage ABCA1 expression and cholesterol efflux, thereby inhibiting the development of atherosclerosis. However, it remains unknown whether additional mechanisms, such as the regulation of macrophage IL-5 expression, are related to the anti-atherogenic properties of LXR. We initially defined IL-5 expression in macrophages where the LXR ligand (T0901317) induced macrophage IL-5 protein expression and secretion. The overexpression of LXR increased, whereas its knockdown inhibited IL-5 expression. Furthermore, we found that LXR activation increased IL-5 transcripts, promoter activity, formation of an LXR·LXR-responsive element complex, and IL-5 protein stability. In vivo, we found that T0901317 increased IL-5 and total IgM levels in plasma and IL-5 expression in multiple tissues in wild type mice. In LDL receptor knock-out (LDLR(-/-)) mice, T0901317 increased IL-5 expression in the aortic root area. Taken together, our studies demonstrate that macrophage IL-5 is a target gene for LXR activation, and the induction of macrophage IL-5 expression can be related to LXR-inhibited atherosclerosis.

FIGURE 1.

Expression pattern of IL-5 in mouse tissues and macrophages. Total cellular protein or RNA was extracted from mouse tissues or macrophages or EL4 cells. Expression of IL-5 protein (A) and mRNA (B) was determined by Western blot and RT-PCR, respectively. Sp, spleen; Lv, liver; Ln, lymph node; pMφ, peritoneal macrophages. The results of statistical analysis of blots are presented at the bottom of each panel.

FIGURE 2.

Regulation of macrophage IL-5 expression by LXR activation. A and B, RAW macrophages (A) or mouse peritoneal macrophages (B) were treated with T0901317 at the indicated concentrations for 8 h or treated with 200 nm T0901317 for the indicated times. C, both RAW and peritoneal macrophages in 12-well plates were treated with T0901317 at the indicated concentrations for 16 h. The treatment medium was used to determine the secreted IL-5 protein by ELISA. *, significantly different from the corresponding controls at p < 0.01 using Student's t test (n = 3). D, RAW cells in 12-well plate were transfected with DNA for the pEGFP-C2 vector, pEGFP-LXRα, or pEGFP-LXRβ expression vector at the indicated concentrations for 24 h. E, primary macrophages in 6-well plates were transfected with shRNA against LXRα or LXRβ or control shRNA at the indicated concentrations for 24 h. Ctrl, control.

FIGURE 3.

Induction of IL-5 expression is LXRE-dependent. A, mouse peritoneal macrophages were treated with T0901317 at the indicated concentrations for 6 h. Expression of IL-5 mRNA was determined by real time RT-PCR. *, p < 0.01 versus control (n = 3). B, EL4 cells in 24-well plates were transfected with DNA for p1218IL-5 and Renilla luciferase. After 4 h of transfection, the cells were treated with 32 nm phorbol 12-myristate 13-acetate (PMA) plus 1 mm N₆,O₂′-dibutyryladenosine 3′:5′-cAMP (Bt2cAMP) or T0901317 at the indicated concentrations overnight followed by determination of activities of firefly and Renilla luciferases. *, p < 0.01 versus control (open bars; n = 3). C, EL4 cells were transfected with DNA for the indicated IL-5 promoters and treated with 200 nm T0901317 overnight. *, p < 0.01 versus controls (n = 3). D, left panel, nuclear proteins were extracted from T0901317-treated RAW cells and used to react with the ³²P-labeled IL-5 LXRE probe in the presence or absence of the indicated competitor. Right panel, RAW cells were treated with T0901317 at different concentrations overnight. The binding of nuclear proteins with the ³²P-labeled IL-5 LXRE probe was determined using EMSA.

FIGURE 4.

Activation of LXR increases IL-5 protein stability and induces IL-5 expression in EL4 cells. A, RAW macrophages were treated with 2 μg/ml cycloheximide (CHX) or cycloheximide plus 200 nm T0901317 (T0) for the indicated times. B, macrophages were treated with T0901317 at different concentrations overnight and 5 μm MG132 for 5 h before the end of the experiment. After treatment, 200 μg of total cellular protein were used to conduct immunoprecipitation (IP) with anti-IL-5 antibody and protein A-conjugated agarose. The ubiquitinated IL-5 (IL-5-Ub) and total IL-5 protein and the loading in precipitates were determined by Western blot with anti-ubiquitin (Ub) and anti-rabbit IgG antibodies, respectively. The input was determined by Western blot with 20 μg of cellular proteins with anti-IL-5, ubiquitin, and GAPDH antibodies. IB, immunoblot. C, EL4 cells were treated with T0901317 at the indicated concentrations overnight followed by determination of IL-5 protein expression.

FIGURE 5.

Activation of LXR induces IL-5 production in vivo. C57BL/6 wild type mice (10-week-old females; n = 5) were fed normal chow (control) or chow containing T0901317 (5 mg/100 g of food) for 10 days. After treatment, the following assays were completed. A, serum IL-5 concentrations were determined using an ELISA kit. *, p < 0.001 versus control (n = 10; each serum sample was analyzed in duplicate). B, IL-5 protein expression in mouse tissues was determined by Western blot. C, T cells isolated from spleen or lymph nodes were sorted with anti-TCRβ plus anti-CD3 antibodies. After lysis, IL-5 protein expression in the T cells was determined by Western blot. D, serum total IgG, IgM, and anti-oxLDL antibodies levels were determined using ELISA kits. *, p < 0.01 versus control (n = 5). E, left panel, immunohistochemical analysis of IL-5 protein in the liver; right panel, IL-5 protein in T cells or Kupffer cells in the liver was determined by immunofluorescent staining. Bars, 100 μm.

FIGURE 6.

Activation of LXR induces IL-5 expression in LDLR^−/− mouse aortic lesions. LDLR^−/− mice were fed a high fat diet or the diet containing T0901317 (1 mg/100 g of food) for 20 weeks. Mouse aortic root was collected and used to determine the presence of IL-5 protein and macrophages by immunohistochemical staining (A) or immunofluorescent staining (B), respectively. MOMA-2, monocyte/macrophage marker. a or b is the part in arterial wall of control or T0-treated mouse with greater magnification to demonstrates the location of IL-5 protein within macrophages. Bars, 50 μm.