Spleen cell proliferation assay
Spleen cells were collected by squeezing the spleen through a 70 μm cell strainer, erythrocytes were removed by an erythrocyte lysis buffer (0.15 M NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA, pH 7.3). Splenocytes were cultured at 2×105 cells/well in 96-wells plates in RPMI 1640 (2 mM L-Glutamine, 10% FCS) for 24-48 hours. Concanavalin A (Con A; Sigma-Diagnostics, MO) was used as a positive control. Cultures were pulsed for the final 16 hours with [6-3H]-thymidine (1 μCi/well, Amersham Biosciences, The Netherlands). Subsequently, cells were washed, lysed and [3H]-thymidine incorporation was measured. Responses are expressed as stimulation index (SI): ratio of mean counts per minute of triplicate cultures with antigen to triplicate cultures without antigen.
Flow cytometric analysis
Splenocytes were incubated with oxLDL (1-10 mg/ml) in the presence of anti-CD3 (5 mg/ml) and anti-CD28 (5 mg/ml). 48 hours later cells were harvested, incubated with 1% normal mouse serum and stained with PerCP-conjugated anti-CD3 and FITC-conjugated anti-F4/80 (0.5 mg Ab/200,000 cells). After washing, cells were analyzed by flow cytometry on a FACSCalibur. To detect CD4+CD25+Foxp3+ T cells, a three color flow cytometry was performed. Mononuclear cells were isolated from spleen, mesenteric lymph nodes, Peyer’s patches, and blood using Lympholyte (Cedarlane, Canada). Cells were stained with FITC-conjugated anti-CD4 (0.125 mg/sample) and APC-conjugated anti-CD25 (0.06 mg/sample) mAb, cells were washed, fixed and permeabilized. Subsequently cells were stained with PE-conjugated anti-Foxp3 (0.2 mg/sample). Cells were washed and analyzed by FACS. Data were analyzed with CELLQuest software (BD Biosciences, The Netherlands) and antibodies were from eBioscience, Belgium.
Real-time PCR assays
Carotid arteries from control and oxLDL-treated mice were isolated and mRNA was extracted using the guanidium isothiocyanate (GTC) method and reverse transcribed (RevertAid M-MuLV reverse transcriptase). Quantitative gene expression analysis was performed on an ABI PRISM 7700 sequence detector (Applied Biosystems, CA) using SYBR green technology. The following primer pairs were used: 5’-GGAGCCGCAAGCTAAAAGC-3’ and 5’-TGCCTTCGTGCCCACTGT-3’ for Foxp3; 5’-CTTATATTGCAAATGTGGCACAATC-3’ and 5’-ATCAATCATCAGTGGGACAATCTG-3’ for CD25; 5’-CGAGGTCCTGCACCAACTG-3’ and 5’-TCCATCACCATCGGTTTATGC-3’ for CTLA4. Acidic ribosomal phosphoprotein PO (36B4) was used as the endogenous reference gene and detected using the primers 5’-GGACCCGAGAAGACCTCCTT-3’ and 5’-GCACATCACTCAGAATTTCAATGG-3’.
Plaque analysis
Six weeks after collar placement the mice were anaesthetized with ketamine-hypnorm and perfused with FormalFixx. Common carotid arteries and both carotid bifurcations were removed for analysis.27 Arteries were embedded in OCT compound (TissueTek; Sakura Finetek, The Netherlands) and 5 mm sections were made on a Leica Cryostat proximal to the collar. Cryosections were stained with hematoxylin (Sigma Diagnostics, MO) and eosin (Merck Diagnostica, Germany). For analysis of the aortic root 10mm thick sections were made of the aortic root containing the aortic valves. Sections were stained with Oil-Red-O and hematoxylin. Plaque sizes were measured using a Leica DM-RE microscope and LeicaQwin software. Sections were also stained immunohistochemically using antibodies against a macrophage-specific antigen (MOMA-2, Research Diagnostics Inc) and α-smooth muscle cell actin (monoclonal mouse IgG2a, Sigma) exactly as described.28