Suppression of Chronic Damage in Renal Allografts by Liver X Receptor (LXR) Activation
2011; Elsevier BV; Volume: 179; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2011.03.019
ISSN1525-2191
AutoresÉva Kiss, Zoran Popović, Jens Bedke, Shijun Wang, Mahnaz Bonrouhi, Norbert Gretz, Paula Stettner, Daniel Teupser, Joachim Thiery, Štefan Porubský, Judith E. Adams, Hermann-Josef Gröne,
Tópico(s)Nuclear Receptors and Signaling
ResumoLiver X receptors (LXR)-α,β regulate intracellular cholesterol homeostasis and inhibit inflammatory gene expression. We studied the effects of the LXRα,β-agonist GW3965 on acute and chronic organ damage in the F344-LEW rat kidney transplantation model. In addition, to gain LXR isoform and cell-specific insights BALB/c kidneys were transplanted into mice with macrophage overexpression of LXRα (mLXRα-tg) and evaluated 7 and 42 days after transplantation. After 56 days GW3965 improved significantly function and morphology of rat kidney allografts by substantial reduction of mononuclear cell infiltrate and fibrosis; in vitro GW3965 reduced inflammatory activity of bone marrow–derived macrophages (BMDMs) and alloreactivity of T cells. Kidneys transplanted into mLXRα-tg mice were also protected from development of chronic allograft dysfunction. Similarly to GW3965-activated BMDMs, mLXRα-tg macrophages secreted significantly less monocyte chemoattractant protein 1 and macrophage inflammatory protein 1β. Interestingly, 7 days after transplantation, when the total number of intragraft macrophages did not differ, evidently more arginase 1– and mannose receptor C type 1–positive cells were found in LXR rat and mice kidney allografts; in vitro both LXR activation by GW3965 and mLXRα overexpression accentuated the induction of alternative activation of BMDMs by IL-4/IL-13, suggesting an additional mechanism by LXRs to prevent graft damage. The results highlight the relevance of macrophage LXRα in allograft rejection and prevention of fibrosis. Liver X receptors (LXR)-α,β regulate intracellular cholesterol homeostasis and inhibit inflammatory gene expression. We studied the effects of the LXRα,β-agonist GW3965 on acute and chronic organ damage in the F344-LEW rat kidney transplantation model. In addition, to gain LXR isoform and cell-specific insights BALB/c kidneys were transplanted into mice with macrophage overexpression of LXRα (mLXRα-tg) and evaluated 7 and 42 days after transplantation. After 56 days GW3965 improved significantly function and morphology of rat kidney allografts by substantial reduction of mononuclear cell infiltrate and fibrosis; in vitro GW3965 reduced inflammatory activity of bone marrow–derived macrophages (BMDMs) and alloreactivity of T cells. Kidneys transplanted into mLXRα-tg mice were also protected from development of chronic allograft dysfunction. Similarly to GW3965-activated BMDMs, mLXRα-tg macrophages secreted significantly less monocyte chemoattractant protein 1 and macrophage inflammatory protein 1β. Interestingly, 7 days after transplantation, when the total number of intragraft macrophages did not differ, evidently more arginase 1– and mannose receptor C type 1–positive cells were found in LXR rat and mice kidney allografts; in vitro both LXR activation by GW3965 and mLXRα overexpression accentuated the induction of alternative activation of BMDMs by IL-4/IL-13, suggesting an additional mechanism by LXRs to prevent graft damage. The results highlight the relevance of macrophage LXRα in allograft rejection and prevention of fibrosis. Chronic renal allograft dysfunction, characterized by atrophic and fibrotic changes, is thought to be the result of complex interactions between innate and adaptive immune responses and parenchymal cells.1Yates P.J. Nicholson M.L. The aetiology and pathogenesis of chronic allograft nephropathy.Transpl Immunol. 2006; 16: 148-157Crossref PubMed Scopus (78) Google Scholar Acting synergistically with lymphocytes, macrophages contribute to both innate and acquired immunity.2Joosten S.A. Sijpkens Y.W. van Kooten C. Paul L.C. Chronic renal allograft rejection: pathophysiologic considerations.Kidney Int. 2005; 68: 1-13Crossref PubMed Scopus (166) Google Scholar, 3Wyburn K.R. Jose M.D. Wu H. Atkins R.C. Chadban S.J. The role of macrophages in allograft rejection.Transplantation. 2005; 80: 1641-1647Crossref PubMed Scopus (123) Google Scholar The contribution of macrophages to the pathogenesis of renal fibrosis is well documented, although, depending on the inflammatory microenvironment, macrophages may change their phenotype and take on an anti-inflammatory, reparative, and matrix remodeling role.3Wyburn K.R. Jose M.D. Wu H. Atkins R.C. Chadban S.J. The role of macrophages in allograft rejection.Transplantation. 2005; 80: 1641-1647Crossref PubMed Scopus (123) Google Scholar, 4Martinez F.O. Helming L. Gordon S. Alternative activation of macrophages: an immunologic functional perspective.Annu Rev Immunol. 2009; 27: 451-483Crossref PubMed Scopus (2051) Google Scholar, 5Wang Y. Wang Y.P. Zheng G. Lee V.W. Ouyang L. Chang D.H. Mahajan D. Coombs J. Wang Y.M. Alexander S.I. Harris D.C. Ex vivo programmed macrophages ameliorate experimental chronic inflammatory renal disease.Kidney Int. 2007; 72: 290-299Crossref PubMed Scopus (293) Google Scholar Recently, macrophages expressing arginase 1 (Arg1) have been found to reduce T-helper cell 2 (Th2)–mediated liver inflammation and fibrosis.6Pesce J.T. Ramalingam T.R. Mentink-Kane M.M. Wilson M.S. El Kasmi K.C. Smith A.M. Thompson R.W. Cheever A.W. Murray P.J. Wynn T.A. Arginase-1-expressing macrophages suppress Th2 cytokine-driven inflammation and fibrosis.PLoS Pathog. 2009; 5: e1000371Crossref PubMed Scopus (590) Google Scholar We have shown that Ccr5 deficiency could induce the alternative activation (M2) pathway in macrophages; the resulting M2 phenotype apparently had an important role in the attenuation of chronic renal allograft rejection.7Dehmel S. Wang S. Schmidt C. Kiss E. Loewe R.P. Chilla S. Schlöndorff D. Gröne H.J. Luckow B. Chemokine receptor Ccr5 deficiency induces alternative macrophage activation and improves long-term renal allograft outcome.Eur J Immunol. 2010; 40: 267-278Crossref PubMed Scopus (32) Google Scholar Liver X receptors (LXRs) are lipid-ligand–activated nuclear receptors. The two LXR isotypes, α and β, have high-sequence homology and are activated by oxidized cholesterols (oxysterols) or synthetic agonists such as T0901317 or GW3965.8Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. 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Zelcer N. Janssen E.M. Hausner M.A. Shih R. Parks J.S. Edwards P.A. Jamieson B.D. Tontonoz P. LXR signaling couples sterol metabolism to proliferation in the acquired immune response.Cell. 2008; 134: 97-111Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar After ligand binding LXRs form permissive heterodimers with the retinoid X receptor. These dimers control transcriptional programs involved in lipid metabolism and inflammation.10Repa J.J. Mangelsdorf D.J. The liver X receptor gene team: potential new players in atherosclerosis.Nat Med. 2002; 8: 1243-1248Crossref PubMed Scopus (339) Google Scholar, 12Hong C. Tontonoz P. Coordination of inflammation and metabolism by PPAR and LXR nuclear receptors.Curr Opin Genet Dev. 2008; 18: 461-467Crossref PubMed Scopus (180) Google Scholar In murine macrophages ligand-activated LXRs have been shown to blunt the expression of inflammatory genes, such as iNOS, COX-2, and MMP-9, and various chemokines [eg, monocyte chemoattractant protein (MCP)-1] in response to lipopolysaccharide, tumor necrosis factor-α, and IL-1β.13Ogawa S. Lozach J. Benner C. Pascual G. Tangirala R.K. Westin S. Hoffmann A. Subramaniam S. David M. Rosenfeld M.G. Glass C.K. Molecular determinants of crosstalk between nuclear receptors and toll-like receptors.Cell. 2005; 122: 707-721Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar, 14Castrillo A. Joseph S.B. Marathe C. Mangelsdorf D.J. Tontonoz P. Liver X receptor-dependent repression of matrix metalloproteinase-9 expression in macrophages.J Biol Chem. 2003; 278: 10443-10449Crossref PubMed Scopus (266) Google Scholar, 15Joseph S.B. Castrillo A. Laffitte B.A. Mangelsdorf D.J. Tontonoz P. Reciprocal regulation of inflammation and lipid metabolism by liver X receptors.Nat Med. 2003; 9: 213-219Crossref PubMed Scopus (1006) Google Scholar In RAW cells and primary mouse macrophages LXRα has been documented to positively regulate arginase II, a gene that may have anti-inflammatory effects through antagonism of nitric oxide signaling.16Marathe C. Bradley M.N. Hong C. Lopez F. Ruiz de Galarreta C.M. Tontonoz P. Castrillo A. The arginase II gene is an anti-inflammatory target of liver X receptor in macrophages.J Biol Chem. 2006; 281: 32197-32206Crossref PubMed Scopus (77) Google Scholar It has been reported that the transcriptional regulation of intracellular cholesterol homeostasis by LXRβ influences lymphocyte proliferation and acquired immune responses.11Bensinger S.J. Bradley M.N. Joseph S.B. Zelcer N. Janssen E.M. Hausner M.A. Shih R. Parks J.S. Edwards P.A. Jamieson B.D. Tontonoz P. LXR signaling couples sterol metabolism to proliferation in the acquired immune response.Cell. 2008; 134: 97-111Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar Collectively, these data have led us to expect a relevant immunomodulatory activity of LXRs in chronic fibrosing inflammation, which we exemplarily analyzed in renal allografts with chronic damage. We have studied the effects of ligand activation of LXRs by GW3965 on acute and chronic rejection phenomena of F344-to Lew rat renal allografts. In addition, we have investigated whether in a fully major histocompatibility complex–mismatched model of mouse renal transplantation the cell-specific transgenic expression of LXRα in recipient macrophages might affect renal allograft rejection. Our results have shown that the LXR agonist GW3965 could prevent the development of chronic lesions in rat renal allografts by a reduction of the mononuclear cell infiltrate and by reduced intragraft pro-inflammatory/profibrotic gene [MCP-1/CCL2, macrophage inflammatory protein (MIP)-1β/CCL4] expression. Macrophage LXRα had an important effect on chronic organ damage because kidneys transplanted into mice with selective overexpression of LXRα in macrophages showed considerably better function and morphology 42 days after transplantation. In addition to the quantitative reduction of the pro-inflammatory macrophage phenotype, we could detect the induction of anti-inflammatory alternatively activated macrophages by LXRs during the acute phase of rejection which may have contributed to prevention of late graft damage. Male inbred Lewis (LEW, RT11) and Fisher (F344, RT11v1) rats as well as BALB/c mice were purchased from Charles River GmbH, Sulzfeld, Germany. Mice with macrophage overexpression of LXRα (C57BL/6 background) were provided by Daniel Teupser (University of Leipzig, Germany)17Teupser D. Kretzschmar D. Tennert C. Burkhardt R. Wilfert W. Fengler D. Naumann R. Sippel A.E. Thiery J. Effect of macrophage overexpression of murine liver X receptor-alpha (LXR-alpha) on atherosclerosis in LDL-receptor deficient mice.Arterioscler Thromb Vasc Biol. 2008; 28: 2009-2015Crossref PubMed Scopus (60) Google Scholar and bred in our animal facility. These mice were designated as mLXRα-tg. Animal experiments were performed according to German laws on animal protection. Transplantation was performed under ether drop anesthesia.18Adams J. Kiss E. Arroyo A.B. Bonrouhi M. Sun Q. Li Z. Gretz N. Schnitger A. Zouboulis C.C. Wiesel M. Wagner J. Nelson P.J. Gröne H.J. 13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar The left kidney of the donor rat (F344) was isolated, perfused with ice-cold isotonic sodium chloride solution, excised, and transplanted orthotopically into a weight-matched (200 to 220 g) Lewis recipient. In the recipient, the left renal vein and artery were mobilized and clamped, the ureter was cut, and the left kidney was excised. End-to-end anastomoses of renal vessels and of ureter, without ureteral stenting, were performed with 10-0 nonabsorbable nylon sutures. Total ischemic time of the donor kidney varied between 30 and 45 minutes. The right kidney was left in place to enhance rejection and damage to the transplant by avoidance of potential endogenous immunosuppressive effects of renal insufficiency and by a reduction of the work load of the transplanted kidney18Adams J. Kiss E. Arroyo A.B. Bonrouhi M. Sun Q. Li Z. Gretz N. Schnitger A. Zouboulis C.C. Wiesel M. Wagner J. Nelson P.J. Gröne H.J. 13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 19Bedke J. Kiss E. Schaefer L. Behnes C.L. Bonrouhi M. Gretz N. Horuk R. Diedrichs-Moehring M. Wildner G. Nelson P.J. Gröne H.J. Beneficial effects of CCR1 blockade on the progression of chronic renal allograft damage.Am J Transplant. 2007; 7: 527-537Crossref PubMed Scopus (48) Google Scholar; to obtain functional parameters of the graft at the end of the experiments, the right kidney was removed 48 hours before sacrifice. The animals were fed a standard rat chow without or with the synthetic LXR agonist GW3965 (20 mg/kg of body weight/day)12Hong C. Tontonoz P. Coordination of inflammation and metabolism by PPAR and LXR nuclear receptors.Curr Opin Genet Dev. 2008; 18: 461-467Crossref PubMed Scopus (180) Google Scholar and sacrificed 7 or 56 days after transplantation. The dose of 20 mg/kg of body weight/day GW3965 corresponds to a dose by which anti-inflammatory effects of the substance have been found.15Joseph S.B. Castrillo A. Laffitte B.A. Mangelsdorf D.J. Tontonoz P. Reciprocal regulation of inflammation and lipid metabolism by liver X receptors.Nat Med. 2003; 9: 213-219Crossref PubMed Scopus (1006) Google Scholar, 20Li N. Rivéra-Bermúdez M.A. Zhang M. Tejada J. Glasson S.S. Collins-Racie L.A. Lavallie E.R. Wang Y. Chang K.C. Nagpal S. Morris E.A. Flannery C.R. Yang Z. LXR modulation blocks prostaglandin E2 production and matrix degradation in cartilage and alleviates pain in a rat osteoarthritis model.Proc Natl Acad Sci U S A. 2010; 107: 3734-3739Crossref PubMed Scopus (66) Google Scholar Mice (male, 8 to 10 weeks old, 20 to 25 g) were anesthetized with Tribromoethanol (Avertin) intraperitoneally. Kidneys from BALB/c(H-2d) donor mice were orthotopically transplanted into C57BL/6 (H-2b) wild-type (WT) or mLXRα-tg recipients as described.21Wang S. Schmaderer C. Kiss E. Schmidt C. Bonrouhi M. Porubsky S. Gretz N. Schaefer L. Kirschning C.J. Popovic Z.V. Gröne H.J. Recipient Toll-like receptors contribute to chronic graft dysfunction by both MyD88- and TRIF-dependent signaling.Dis Model Mech. 2010; 3: 92-103Crossref PubMed Scopus (79) Google Scholar Briefly, the abdomen of the donor was opened through a midline incision, the left kidney with its vessels was attached to a segment of the aorta, and the vena cava along with ureter was removed en bloc. The donor aorta and inferior vena cava were then anastomosed end-to-side to the recipient abdominal aorta and inferior vena cava below the level of the native renal vessels, respectively. The native left kidney was removed before revascularization. Donor and recipient ureters were anastomosed at the ureteropelvic junction end to end. The native right kidney was removed after grafting. Mice were sacrificed on day 7 or day 42 after transplantation. All transplanted kidneys with hydronephrosis, which was evaluated both macroscopically and by light microscopy, were excluded from the experimental groups. For measurements of serum creatinine and albuminuria animals were kept in metabolic cages 24 hours before the end of the experiment. Serum creatinine (enzymatic determination), urea, cholesterol, triglycerides, glutamate-oxalate-transaminase, glutamate-pyruvate-transferase, alkaline phosphatase, and albumin in urine were analyzed by a Hitachi 9-17-E autoanalyzer (Hitachi, Frankfurt, Germany).22Keppler A. Gretz N. Schmidt R. Kloetzer H.M. Groene H.J. Lelongt B. Meyer M. Sadick M. Pill J. Plasma creatinine determination in mice and rats: an enzymatic method compares favorably with a high-performance liquid chromatography assay.Kidney Int. 2007; 71: 74-78Crossref PubMed Scopus (102) Google Scholar Renal allografts were removed in deep anesthesia, quickly blotted free of blood, weighed, and processed as required for histology, immunohistology, and molecular analysis. For histology and immunohistology the kidneys were cut into 1-mm coronal slices and immersion fixed in 4% formaldehyde in PBS, Methacarn (rat kidney samples), or zinc solution (mice kidney samples) and then embedded in paraffin. In addition tissue slices were snap frozen in liquid nitrogen and stored at −80°C. Light microscopy was performed on 3-μm sections stained by PAS. Kidneys were evaluated for evidence of acute and chronic vascular, glomerular, and tubulointerstitial damage as previously described.17Teupser D. Kretzschmar D. Tennert C. Burkhardt R. Wilfert W. Fengler D. Naumann R. Sippel A.E. Thiery J. Effect of macrophage overexpression of murine liver X receptor-alpha (LXR-alpha) on atherosclerosis in LDL-receptor deficient mice.Arterioscler Thromb Vasc Biol. 2008; 28: 2009-2015Crossref PubMed Scopus (60) Google Scholar, 23Kiss E. Adams J. Grone H.J. Wagner J. Isotretinoin ameliorates renal damage in experimental acute renal allograft rejection.Transplantation. 2003; 76: 480-489Crossref PubMed Scopus (36) Google Scholar In short, acute vascular injury was assessed as 0, indicating no injury; 0.5, sticking of mononuclear cells to the endothelium; 1, subendothelial location of mononuclear cells; 2, inflammation of the media, including transmural infiltration; or 3, fibrinoid necrosis of the vessel wall or thrombosis of the vessel or both in addition to the inflammatory reaction. Chronic vascular injury was evaluated as negative (−) or positive (+), determined as narrowing of the luminal area by fibrous thickening of the subendothelial space with or without the presence of foam cells and given as percentage of positive vessels. Acute glomerular injury was defined as 0, indicating no injury; 0.5, sticking of mononuclear cells to the capillary endothelium in 50% of the convolute; 2, mesangiolysis with or without sticking of mononuclear cells; or 3, aneurysm, thrombosis, or necrosis of the capillary loops. Chronic glomerular injury was defined as 0, indicating no sclerosis; 0.5, sclerosis of 75% of the capillary loops. The acute and chronic glomerular injury was evaluated in ≥50 glomeruli per sections. Chronic tubulointerstitial damage was defined as broadening of the basement membrane of the tubuli with flattened epithelium, tubular atrophy, and interstitial matrix increase; it was judged as 0.5, indicating focal chronic damage, and 1, diffuse chronic damage. Tubulointerstitial inflammation was judged as 0, indicating no mononuclear cells in the interstitium; 0.5, focal mononuclear cell infiltration in the interstitium; 1, focal mononuclear infiltration in the interstitium with tubulitis; 2, diffuse mononuclear cell infiltration of the interstitium; or 3, diffuse mononuclear cell infiltration of the interstitium with tubulitis. Tubulitis was defined as ≥1 mononuclear cells/tubular cross section. A tubulointerstitial inflammation index was defined as the percentage of fields with respective degree of the injury encountered in 10 fields (objective 20×) of cortex and outer stripe of outer medulla. The final tubulointerstitial inflammation score was calculated as the sum of all specific indices, whereby the index of fields with degree 0.5 was multiplied by 0.5, that of degree 1 × 1, that of degree 2 × 2, and that of degree 3 × 3. Vascular and glomerular injury were scored in an analogous pattern.17Teupser D. Kretzschmar D. Tennert C. Burkhardt R. Wilfert W. Fengler D. Naumann R. Sippel A.E. Thiery J. Effect of macrophage overexpression of murine liver X receptor-alpha (LXR-alpha) on atherosclerosis in LDL-receptor deficient mice.Arterioscler Thromb Vasc Biol. 2008; 28: 2009-2015Crossref PubMed Scopus (60) Google Scholar, 23Kiss E. Adams J. Grone H.J. Wagner J. Isotretinoin ameliorates renal damage in experimental acute renal allograft rejection.Transplantation. 2003; 76: 480-489Crossref PubMed Scopus (36) Google Scholar Morphometric analysis was performed with the use of a semiautomatic image analyzing system (Leica Q600 Qwin, Cambridge, UK) on sections stained with Goldner-Masson-Trichrom to quantify scared areas in tubulointerstitium: 10 randomly selected fields (objective 10×) of cortex and outer stripe of outer medulla were evaluated. Results were expressed as a percentage of the total tubulointerstitial area, obtained after exclusion of glomeruli.18Adams J. Kiss E. Arroyo A.B. Bonrouhi M. Sun Q. Li Z. Gretz N. Schnitger A. Zouboulis C.C. Wiesel M. Wagner J. Nelson P.J. Gröne H.J. 13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Immunohistochemical (IHC) staining was done on sections of frozen or paraffin-embedded kidney samples. The antibodies used for rat tissues included mouse anti-rat monoclonal antibodies against ED1, CD4, CD8 (Serotec, Oxford, UK), Ki-67 (clone MIB-5; Dianova, Hamburg, Germany), rat anti-rat FoxP3 (NatuTec, Frankfurt, Germany), rabbit anti-rat polyclonal antibodies against collagen I (Biogenesis, Poole, UK), collagen III (Chemicon, Temecula, CA), C4d (Hycult Biotech, Beutelsbach, Germany), and antibodies to α-smooth muscle actin (α-SMA) (mouse ascites fluid; Sigma, Schnelldorf, Germany). Mouse kidneys were stained with rat anti-mouse monoclonal antibodies against F4/80 (Serotec), CD3 (Santa Cruz Biotechnology, Santa Cruz, CA), CD4 (BD Biosciences Pharmingen, San Diego, CA), FoxP3 (NatuTec), α-SMA (Sigma), and rabbit anti-mouse against collagen I/III (Biogenesis). Anti-Arg1 (BD Biosciences Pharmingen), anti-mannose receptor-1 (CD206; Abcam) monoclonal antibodies were used to detect markers of alternatively activated macrophages. Positive glomerular cells were counted in ≥50 glomerular cross sections and given as the mean per glomerular section; interstitial positive cells were counted in 20 high-power fields (×40) of cortex and outer medulla and recorded as mean per high-power field. The intensity of the staining for collagen was evaluated as not detectable (degree 0), faint (degree 1), moderate (degree 2), and intense staining (degree 3). Scores were calculated as described.18Adams J. Kiss E. Arroyo A.B. Bonrouhi M. Sun Q. Li Z. Gretz N. Schnitger A. Zouboulis C.C. Wiesel M. Wagner J. Nelson P.J. Gröne H.J. 13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Generation of murine bone marrow–derived macrophages (BMDMs) of WT and mLXRα-tg mice was performed according to standard protocols.24Weischenfeldt J. Porse B. Bone marrow-derived macrophages (BMM): isolation and applications.CSH Protoc. 2008; https://doi.org/10.1101/pdb.prot5080Crossref PubMed Scopus (619) Google Scholar, 25Gersuk G.M. Razai L.W. Marr K.A. Methods of in vitro macrophage maturation confer variable inflammatory responses in association with altered expression of cell surface dectin-1.J Immunol Methods. 2008; 329: 157-166Crossref PubMed Scopus (35) Google Scholar In brief, mouse femurs were dissected, and each bone was flushed with 10 mL of PBS. A bone marrow cell suspension was collected and centrifuged. Pellets were resuspended in RPMI 1640 medium supplemented by 20% macrophage colony-stimulating factor–containing L929 medium. The cells were plated on non-TC–treated 10-cm petri dishes and incubated at 37°C/5% CO2. Fresh medium was provided at days 3 and 5, and experiments were performed at day 7. After pre-incubation with dimethyl sulfoxide (DMSO; 0.05%) (WT and mLXRα-tg) or 3 μmol/L GW3965 dissolved in DMSO (WT) for 16 hours, macrophages were stimulated with murine IL-4 and IL-13 (10 ng/mL; PrepoTech) overnight. Cells and supernatant fluids were collected for further analyses. Splenic T cells and T cell–depleted splenocytes were prepared as described.26Porubsky S. Wang S. Kiss E. Dehmel S. Bonrouhi M. Dorn T. Luckow B. Brakebusch C. Gröne H.J. Rhoh deficiency reduces peripheral T-cell function and attenuates allogenic transplant rejection.Eur J Immunol. 2011; 41: 76-88Crossref PubMed Scopus (10) Google Scholar Briefly, spleens from BALB/c (n = 4) and C57BL/6 mice (n = 4) were mechanically disrupted in 6-well plates with 5 mL of digestion solution [RPMI 1640, 10 mmol/L HEPES, 0.1% bovine serum albumin (BSA), 0.5 mg/mL collagenase IA, and 4.5 kU/mL DNase I; all from Sigma-Aldrich] and incubated for 15 minutes at 37°C and 5% CO2. After repetitive pipetting, cell suspensions were sieved through filters with 100-μm and 30-μm pore sizes (BD, Heidelberg, Germany). Purified splenic T cells and T cell–depleted splenocytes were prepared with magnetic bead–coupled anti-CD90.2 antibodies (clone 30-H12; Miltenyi Biotec, Galdbach, Germany) and with magnetically activated cell sorter mass spectrometric columns and separators (both Miltenyi Biotec) according to the manufacturer's instructions. After pretreatment with DMSO 0.05% or 3 μmol/L GW3965 dissolved in DMSO WT for 16 hours 105 T cells purified from C57BL/6 mice were incubated with 105 T cell–depleted splenocytes from BALB/c mice. Culture supernatant fluids were harvested after 24 and 72 hours, and concentrations of IL-2 and interferon-γ (IFN-γ) were analyzed as described below. Supernatant fluids of BMDM and splenocyte/T cell co-cultures were measured for MCP-1, MIP-1β and IL-2, and IFN-γ, respectively, by FACSCalibur with BD CBA Flex-Set bead assays (BD). Sample files were analyzed by FCAP Array 1.0.1 software. Total RNA was extracted from the kidney allografts and macrophages (BMDMs) with the method of Chomczynski and Sacchi27Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63149) Google Scholar (n = four to six animals/group). RNA quality was checked by a RNA6000 Nanochip (Agilent Technologies, Waldbronn, Germany). Total RNA (10 μg) was digested with DNase I according to standard protocol. Total RNA (3 μg; DNA free) was used for the first-strand cDNA synthesis with the use of Superscript II Reverse Transcriptase and oligo d(T)12–18 as primer (LifeTechnologies, Karlsruhe, Germany). Real-time PCR was performed by LightCycler with the use of LightCyler-FastStart DNA MasterSYBR Green I kit (Roche Diagnostics, Mannheim, Germany) as described.18Adams J. Kiss E. Arroyo A.B. Bonrouhi M. Sun Q. Li Z. Gretz N. Schnitger A. Zouboulis C.C. Wiesel M. Wagner J. Nelson P.J. Gröne H.J. 13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy.Am J Pathol. 2005; 167: 285-298Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar The primer sequences for target genes are shown in Table 1.Table 1Sequences of Primers Used for Real-Time RT-PCR AnalysisGeneSenseAntisenseRat Cyclophilin5′-AGGTGAAAGAAGGCATAGC-3′5′-TTACAGGGTATTGCGAGCAG-3′ MCP-1/CCL25′-GCTGACCCCAATAAGGAATG-3′5′-GTTGTGGAAAAGAGAGTGGATG-3′ MIP-1β5′-GCTCTGACCCTCCCACTTC-3′5′-ACTCATTGACCCAGGGCTC-3′ IL-45′-ACGGCAACAAGGAACACCAC-3′5′-TTCAGACCGCTGACACCTCTAC-3′ IL-105′-CATGGGTCTTGGGAAGAGAA-3′5′-GCTTTCGAGACTGGAAGTGG-3′ IL-135′-CATGGTATGGAGCGTGGAC-3′5′-GAGGCCTTTTGGTTACAGAGG-3′ MRC15′-AGTGGTCATCGTGGTCCTTC-3′5′-AATGACCGCGATGCTCATTCT-3′Mouse GAPDH5′-ACTCCCACTCTTCCACCTTC-3′5′-GGTCCAGGGTTTCTTTACTCC-3′ Tubulin5′-TCTCTCACCCTCGCCTTCTA-3′5′-GGGTCCCAGGTCTACGAACA-3′ MCP-1/CCL25′-ACCAAGCTCAAGAGAGAGG-3′5′-ACATTCAAAGGTGCTGAAGAC-3′ MIP-1β5′-GCTGTTTCTCTTACACCTCC-3′5′-ACTCATGTACTCAGTGACCC-3′ IL-45′-TCCACGGATGCGACAAAAAT-3′5′-TTCTTCTTCAAGCATGGAGT-3′ IL-105′-ACCTGGCAAACAAAATGAGG-3′5′-CTCTGACCTGCTGTCATGGA-3′ IL-135′-CTCACTGGCTCTGGGCTTCA-3′5′-CTCATTAGAAGGGGCCGTGG-3′ Arg-15′-ACCACGGCAGTGGCTTTAACC-3′5′-GGTTTTCATGTGGCGCATTC-3′ MRC15′-GCGTTGCACATACCTCAAGA-3′5′-GCTAAATGATCGCATGCTCA-3′ Chi3l3/Ym15′-GAAGGAGCCACTGAGGTCTG-3′5′-CACGGCACCTCCTAAATTGT-3′ Fizz15′-TCCCAGTGAATACTGATGAGA-3′5′-CCACTCTGGATCTCCCAAGA-3′ Pdcd1lg25′-GCCACACGTGAGTTAT-3′5′-TTGAACATGCCAAGCT-3′ Open table in a new tab All data were presented as mean ± SEM. Data were analyzed by the non-parametric Mann-Whitney U-test or unpaired t-test as appropriate. A P value <0.05 was considered to show a significant difference between two groups. At day 7 after transplantation treatment with GW3965 led to a moderate increase of triglyceride levels (data not sh
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