CirculationAHA/2006/659912-R1 Zirlik et al. Online Supplement: -1- Online Supplement Methods: Atherosclerotic mice LDLR-deficient and CD40-deficient mice on a C57/BL6 background were obtained from Jackson Laboratories (Bar Harbor, ME) and cross-bred to generate CD40-/-/LDLR-/- mice. Genotyping of each mouse used polymerase chain reaction (PCR) and employed the following primers: LDLR, 5’-CCA TAT gCA TCC CCA gTC TT-3’ (Common primer), 5’-gCg ATg gAT ACA CTC ACT gC-3’ (WT primer), 5’-AAT CCA TCT TgT TCA ATg gCC gAT C-3’ (MUT primer); CD40, 5’-ggC AgT AAg ACg ATg TgA cAA Cag Ag-3’ (forward), 5’-gAg ATg AgA Agg AAg AAT ggg AAA AC-3’ (reverse). Six to eight week old LDLR-/- and CD40-/-/LDLR-/- mice (N≥12 animals per group) consumed a high cholesterol diet (Research Diets, New Brunswick, NJ) for 8 and 16 weeks. Similarly, in the Mac-1 in vivo study, 6-8 week old LDLR-/- mice consumed a high cholesterol diet for 10 weeks, receiving 3 times a week intraperitoneal injections of 75µg antimurine Mac-1 antibody (Pharmingen, San Jose, CA) or respective carrier only. Finally, mice were killed, the hearts and aortas removed, and analyzed as described. Aortic roots and arches were embedded in OCT (Sakura Tissue-Tek, Torrance, CA) and snap-frozen in liquid nitrogen while thoraco-abdominal aortas were fixed in 10% buffered formalin, as described previously 1-3 . Total cholesterol and triglyceride levels were assayed in EDTA-plasma from blood obtained by retroorbital bleeding before feeding and drawn from the right ventricle upon harvest. All mice were housed under specific pathogen-free conditions and procedures were approved by the Animal Care Committee of the University of Freiburg and Harvard Medical School CirculationAHA/2006/659912-R1 Zirlik et al. -2- Online Supplement Tissue preparation and histological analysis Serial longitudinal sections (6µm) of mouse aortic arches and serial transverse sections of mouse aortic roots (Leica Cryostat, Germany) were fixed in acetone (-20°C, 5 min), air dried, and stained by the avidin-biotin-peroxidase method, as described previously 1-3 . Antibodies used included rat anti-mouse Mac-3 (Pharmingen, San Diego, CA) and monoclonal anti-human smooth muscle anti-actin (Dako, Glostrup, Denmark). En face analysis of abdominal aortas determined lipid deposition by Oil-Red-O staining. Thoraco-abdominal aortas were opened longitudinally, pinned on the surface of black silicone, stained with Oil-Red-O solution (Sigma, St. Louis, MO), and washed in 85% propylene glycol solution. Formalin-fixed sections of the aortic arch and root were stained similarly after dehydration in 85% propylene glycol, as described previously 1-3 . Quantification of the extent and composition of the aortic lesions used methods described previously. The total wall area (=intima + media), intimal lesion area (intima), and medial area (media) as well as the percentage of positively stained area for macrophages, lipids, and smooth muscle cells was quantified employing computer-assisted image analysis software (Image Pro, Media Cybernetics, Silver Springs, MD). Flow cytometry Human blood (100µl) obtained from healthy donors or monocytes isolated by Ficoll-density gradient centrifugation from human blood were stimulated with and without PMA (200 ng/ml) for 10 min, incubated with FLAG-tagged human recombinant sCD40L (5 µg/ml, Alexis, Lausen, Switzerland) for 10 min at room temperature in the presence or absence of anti-Mac-1 (100 µg/ml) antibody (Dako, Hamburg, Germany); subsequently, a FITC-labeled mouse anti-flag CirculationAHA/2006/659912-R1 Zirlik et al. -3- Online Supplement antibody (1:100, Alexis) was added for another 10 min. Cells were washed with 2% bovine serum albumin/PBS, erythrocytes were lysed, and cells were fixed (BD lysing and fixing solution, Beckton Dickinson, BD, Franklin Lakes, NJ) and analyzed with a fluorescence-activated cell sorter (FACS Calibur, BD). The mean fluorescence indices (MFI) were analyzed employing the BD software. Binding of 125I-labelled sCD40L to Mac-1 Recombinant sCD40L protein (Leinco Technologies, St. Louis, Missouri, USA) was labelled with 125 I (GE Healthcare, NJ, USA) for 10 min and then labelled protein was separated from the free 125I through iodo-beads column (Pierce, Rockford, IL, USA). Radioiodine incorporation into sCD40L was monitored with a γ-counter (Packard RIASTAR, GMI, MI, USA). CHO cells expressing non-activated, wild-type (CHO-Wt-Mac-1) as well as GFFKR-deleted, permanently activated Mac-1 (CHO-Del-Mac-1: deletion of a highly conserved GFFKR motif in the cytoplasmic domain of αM, as described previously 4, 5) were grown in DMEM, 10% FCS, 1% penicillin/streptomycin, 1% L-glutamine, 1% non-essential amino acids, 700 μg/ml geneticin and 250 μg/ml zeocin, were split at a ratio of 1:6 and 24 hours later washed with pre-warmed PBS and trypsinized for 5 min. Human monocytes were isolated and activated as described above. CHO cells expressing recombinant Mac-1 and monocytes were resuspended in modified Tyrode’s buffer at 1x106 cells/ml. 2 g of labelled sCD40L were added to 1ml of resuspended cells and incubated at room temperature for 40 min. Following incubation, cells were washed 3 times in modified Tyrode’s buffer and binding of labelled sCD40L was measured with a γcounter. For the evaluation of Mac-1-specific binding, cells were pre-incubated with the blocking anti-Mac-1 mAb (Dako) for 30 min at room temperature. CirculationAHA/2006/659912-R1 Zirlik et al. Immunoprecipitation and immunoblotting -4- Online Supplement CHO cells expressing wild-type Mac-1 (CHO-Wt-Mac-1) and GFFKR-deleted Mac-1 (CHODel-Mac-1) were cultured as described above. After 24hrs, cells were lysed in PBS lysis buffer containing 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% Triton X-100, protease inhibitor cocktail (Roche, Basel, Switzerland), 10mM NaF, 1mM Na3VO4 and 10mM NaP2O7 and sonicated for 10 sec. Sonicated samples were centrifuged at 13,000 rpm for 10 min at 4C. The lysates were precleared by incubation with 30 l of protein A-sepharose beads (Santa Cruz Biotechnologies, CA, USA) for 1 hour at 4C. Beads were pelleted and supernatant transferred to a fresh tube containing recombinant CD40L proteins (5 g) and anti-CD40L (1-2 g) antibody (Santa Cruz Biotechnologies). After incubation for 2 hours at 4C, 30 l of fresh protein A-sepharose beads were added to the lysate/protein/antibody mixture and incubated overnight at 4C. The immune complexes were washed with lysis buffer a minimum of 5 times and eluted in 40 l of SDS sample loading buffer and heated to 95C for 10 min. The samples were separated on 12% SDSPAGE and transferred onto a Hybond-C nitrocellulose membrane (Amersham, Buckinghamshire, England). Immunoblots were probed with anti-Mac-1 (1:1000) (R&D Systems, Minneapolis, MN, USA) and anti-CD40L (1:1000) (Santa Cruz Biotechnologies). Horseradish peroxidaseconjugated secondary antibodies against mouse and rabbit (Jackson ImmunoResearch, West Grove, PA, USA) were used at their recommended dilutions and protein bands were detected with enhanced chemiluminescent substrate (Supersignal West Pico, Pierce, Rockford, IL, USA). Static and dynamic adhesion assays Monocytes: Static adhesion assays were performed as described previously 5, 6 . In brief, 96-well plates (Nunc ImmunoPlate, MaxiSorp®) were coated with sCD40L (10 µg/ml, R&D) in PBS at CirculationAHA/2006/659912-R1 Zirlik et al. -5- Online Supplement 4°C overnight, blocked with 0.1% agarose for 1 hour at room temperature, washed with PBS twice, and incubated with monocytes (0.7 x 106/ml) isolated by Ficoll density centrifugation from human blood for 50 min at 37°C in the presence or absence of anti-Mac-1 antibody (100 µg/ml). After 5x washing with PBS (Sigma), permeabilization buffer (6 mg/ml phosphatase substrate (Sigma), 1% Triton X-100, 50mM sodium acetate, pH 5.5) was added for 1 hour at 37°C, and absorbance was read at 405 nm in a plate reader (Spectramax, Molecular Devices, Sunnyvale, CA). Plates coated with 0.1% agarose served as negative control, plates coated with fibrinogen (30µg/ml) served as positive control. CHO cells expressing recombinant Mac-1: Plates with immobilized sCD40L were prepared as described above for monocytes. Non-expressing CHO cells (CHO), CHO cells transfected with wild-type Mac-1 (CHO-Wt-Mac-1), and with CHO cells transfected with permanently activated Mac-1 (CHO-Del-Mac-1) at a density of 0.6 x 106/ml cells per well were allowed to adhere for 50 min and adhesion was analyzed under static conditions in the presence or absence of antiMac-1 antibody (100 µg/ml). For dynamic adhesion assays, 35 mm dishes (Costar, Bethesda, MD) coated with 1% BSA (negative control), sCD40L (20 µg/ml), or fibrinogen (positive control, 30 µg/ml) were subjected to the flow chamber, as described previously 7 . In brief, the Glycotech flow chamber (Gaithersburg, MD) was assembled with the dish as the bottom of the resulting parallel flow chamber. The chamber and tubes were filled with PBS without serum prior to the experiment. Subsequently, shear stress was applied with a syringe pump (Harvard apparatus PHD2000, Holliston, MA) with increasing flow rates of 0.5 dyne/cm² (venous flow; a total of 5 min), and 15 dyne/cm² (arterial flow, 1 min), respectively. Adherent cells were quantified under the microscope. Five time points per minute were pooled. Data from at least six different experiments were analyzed. CirculationAHA/2006/659912-R1 Zirlik et al. -6- Online Supplement Migration assays Migration assays were performed in a modified Boyden chamber, as described previously 8. Briefly, the lower chamber was filled with sCD40L (30 µg/ml) in RPMI, the upper chamber with monocytes (0.5 x 106/ml). Cells migrated for 90 min in the presence or absence of anti-Mac-1 antibody (100 µg/ml). Cells were quantified under the microscope by blinded investigators (CM and IA). RPMI without serum and FLMP (100 nM) served as negative and positive control, respectively. Quantification of myeloperoxidase (MPO) release from monocytes MPO secretion of monocytes was quantified in the supernatants from the static adhesion assays with a commercially available ELISA according to the manufacturer’s instructions (Oxis Research, Portland, OR). Mouse peritonitis LDLR-/-, CD40L-/-/LDLR-/-, and CD40-/-/LDLR-/- mice were treated intraperitoneally with PBS or 100 µg of mAb against mouse Mac-1 (Pharmingen, San Jose, CA) 30 min before injection of 4% thioglycollate broth (Sigma). After 4 h, mice were euthanized with CO2, the peritoneal cavity was flushed with 6 ml of RPMI for 2 min. Leukocytes were quantified in a CASY counter and manually under the microscope in a Neubauer chamber. In parallel, leukocyte counts from blood, obtained from the right ventricle, were analyzed. Similarly, mice were intraperitoneally injected with sCD40L (75 µg/mouse), PBS, wild-type murine fibroblasts (5x106), and murine fibroblasts (5x106), which overexpress membrane-bound CD40L. The resulting leukocyte migration was measured after 4 and 72 h. CirculationAHA/2006/659912-R1 Zirlik et al. -7- Online Supplement Statistical analysis Data are expressed as means ± SEM of absolute or normalized values. Groups were compared employing the Student’s t-test. A value of P<0.05 was considered significant. Data points from the dynamic adhesion assays in the flow chamber were compared by repeated measurements of multivariance analysis using the GLM procedure in collaboration with the Institute of Statistics of the University of Freiburg. Online Figure Legend: Fig. 1: Monocytes isolated from a pool of 16 mice were allowed to adhere to sCD40L-coated plates in the presence or absence of anti-Mac-1 antibody. Adherent cells were permeabilized, incubated with substrate, and analyzed colorimetrically in triplicates. Results are given in generic units normalized to values of adhesion to agarose-coated control plates. * indicates a P-value of <0.05. CirculationAHA/2006/659912-R1 Zirlik et al. Online References: 1. -8- Online Supplement Bavendiek U, Zirlik A, LaClair S, et al. Atherogenesis in mice does not require CD40 ligand from bone marrow-derived cells. Arterioscler Thromb Vasc Biol. Jun 2005;25(6):1244-1249. 2. Mach F, Schonbeck U, Sukhova GK, et al. Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature. Jul 9 1998;394(6689):200-203. 3. Schonbeck U, Sukhova GK, Shimizu K, et al. Inhibition of CD40 signaling limits evolution of established atherosclerosis in mice. Proc Natl Acad Sci U S A. Jun 20 2000;97(13):7458-7463. 4. O'Toole TE, Katagiri Y, Faull RJ, et al. Integrin cytoplasmic domains mediate inside-out signal transduction. J Cell Biol. Mar 1994;124(6):1047-1059. 5. Peter K, O'Toole TE. Modulation of cell adhesion by changes in alpha L beta 2 (LFA-1, CD11a/CD18) cytoplasmic domain/cytoskeleton interaction. J Exp Med. Jan 1 1995;181(1):315-326. 6. Peter K, Schwarz M, Conradt C, et al. Heparin inhibits ligand binding to the leukocyte integrin Mac-1 (CD11b/CD18). Circulation. Oct 5 1999;100(14):1533-1539. 7. Ahrens IG, Moran N, Aylward K, et al. Evidence for a differential functional regulation of the two beta(3)-integrins alpha(V)beta(3) and alpha(IIb)beta(3). Exp Cell Res. Jan 20 2006. 8. Marx N, Walcher D, Raichle C, et al. C-peptide colocalizes with macrophages in early arteriosclerotic lesions of diabetic subjects and induces monocyte chemotaxis in vitro. Arterioscler Thromb Vasc Biol. Mar 2004;24(3):540-545.