Mice and pristane treatment
Mice were bred and maintained under specific pathogen free (SPF) conditions at the University of Florida Animal Facility. IRF5−/− mice on a C57BL/6 (B6) background were provided by Dr. Katherine Fitzgerald (University of Massachusetts, MA) with permission from Dr. Tak Mak (University of Toronto, Canada) and were back-crossed to B6 for at least 10 generations. B6 MyD88−/− mice were provided by Dr. Lyle Moldawer (Department of Surgery, University of Florida). TLR7−/− mice on a BALB/c background were acquired from Oriental Bioservices (Kyoto, Japan) and IFNAR−/− mice backcrossed 9 generations onto a BALB/c background were provided by Dr. Joan Durbin (Nationwide Children’s Hospital, Ohio State University, Columbus OH), respectively. Wild type BALB/cJ, C57BL/6 and BALB/C X B6 F1 CB6F1/J mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice received a single intraperitoneal (I.P.) injection of 0.5 mL of pristane (2,6,10,14 tetramethylpentadecane, TMPD, Sigma, St. Louis, MO) filtered through a 0.25 µm filter or left untreated as controls These studies were approved by the Institutional Animal Care and Use Committee.
Real-time quantitative PCR (Q-PCR)
Q-PCR was performed as previously described (20
). In brief, total RNA was extracted from 106
peritoneal cells using TRIzol reagent (Invitrogen, Carlsbad, CA) and cDNA was synthesized using the Superscript II First-Strand Synthesis kit (Invitrogen) according to the manufacturer's protocol. SYBR green Q-PCR analysis was performed using an Opticon II thermocycler (Bio-Rad, Hercules, CA). Amplification conditions were as follows: 95°C for 10 min, followed by 45 cycles of 94°C for 15 s, 60°C for 25 s, and 72°C for 25 s. After the final extension (72°C for 10 min), a melting-curve analysis was performed to ensure specificity of the products. Primer sequences are listed as follows: ISG-15 Forward: GAGCTAGAGCCTGCAGCAAT, Reverse: TAAGACCGTCCTGGAGCACT; IRF7 Forward: ACAGCACAGGGCGTTTTATC, Reverse: GAGCCCAGCATTTTCTCTTG; Mx-1 Forward: GATCCGACTTCACTTCCAGATGG, Reverse: CATCTCAGTGGTAGTCCAACCC; CXCL5 Forward: CCCCTTCCTCAGTCATAGCC, Reverse: TGGATTCCGCTTAGCTTTCT; YM-2 Forward: CTGGGTAATGAGTGGGTTGG, Reverse: ACGTCCCTGGTGACAGAAAG; YM-1 Forward: TGAAGGAGCCACTGAGGTCT, Reverse: CACGGCACCTCCTAAATTGT; Fizz1 Forward: TGCTGGGATGACTGCTACTG, Reverse: AGCTGGGTTCTCCACCTCTT; IL23 P19 Forward: CATGGGGCTATCAGGGAGTA, Reverse: GACCCACAAGGACTCAAGGA.
Flow cytometry was performed as described previously (20
). Before surface staining, peritoneal or peripheral blood cells were incubated with anti-mouse CD16/32 (Fc Block, BD Bioscience, San Jose, CA) for 10 min. Cells then were stained with an optimized amount of primary antibody or the appropriate isotype control for 10 min at room temperature before washing and resuspending in PBS supplemented with 0.1% BSA. Ten thousand to 50,000 events per sample were acquired using a CYAN ADP flow cytometer (Beckman Coulter, Hialeah, FL) and analyzed with FCS Express 3 (De Novo Software, Ontario, Canada). The following conjugated antibodies were used: anti-CD8-APC, anti-Ly6G-PE, anti-B220-APC-CY7, anti-CD11c-APC, anti-B220-FITC, anti-Ly6C-FITC, CD69-FITC, CD69-PE-CY7, CD25-PE (BD Bioscience), anti-H2-Kb
-Biotin, avidin-PE-CY7, anti-CD11b-Brilliant Violet, anti-PDCA-1-biotin (Biolegend, San Diego, CA), anti-CD4-PE-CY7, CCR7-PE (eBioscience, San Diego, CA). For morphological analysis, 106
peritoneal cells were cytospun onto glass slides and stained using the Hema3 kit (modified Wright stain; Fisher Scientific, Pittsburgh, PA).
Intracellular cytokine staining
Intracellular IFN-γ, IL-4 and IL-17 were detected after culturing peritoneal cells or splenocytes at a concentration of 2.5 × 106 cells/ml in RPMI medium with 10% FBS, PMA (50 ng/mL), and ionomycin (1 µg/ml), and GolgiStop (BD; 5 µg/ml) for 6 h at 37°C in a 5% CO2 incubator. After stimulation, cells were surface stained, fixed in Fixation/Permeabilization buffer (eBioscience), washed in Perm/Wash buffer (eBioscience), and then stained with PE-labeled anti–IFNγ, APC-labeled anti-IL-4 (BD Biosciences), APC-labeled anti-IL-17 (eBioscience), or isotype controls, according to the manufacturer’s protocol. Cells were washed twice in Perm/Wash buffer and resuspended in PBS and analyzed by flow cytometry. Data were analyzed with FCS Express 3 software (De Novo Software).
Immunoglobulin and autoantibody ELISAs
Total immunoglobulin levels were measured as described (20
). Briefly, microtiter plates (Nunc, Immobilizer Amino) were coated with goat anti-mouse L-chain (κ and λ specific) antibodies (Southern Biotechnology, Birmingham, AL). Sera were diluted 1:200,000 in 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 2mM EDTA, 0.5% BSA, 0.3% NP40, 0.05% NaN3
(NET/NP40). Alkaline phosphatase-conjugated goat anti-mouse polyclonal antibodies against μ, γ1, γ2c, γ2b, or γ3 H-chain (Southern Biotechnology, 1:1000 dilutions) were used as secondary antibodies. Antigen capture ELISAs for anti-nRNP/Sm antibodies were performed as described (20
), using mouse sera at a dilution of 1:400 and goat anti-mouse IgG second antibodies (Southern Biotechnology). Levels of anti-dsDNA antibodies were tested (1:400 serum dilution) by ELISA using S1 nuclease-treated calf thymus DNA as antigen as previously described (22
). Anti-U1A (subset of anti-RNP) antibodies were tested by ELISA as described (23
). Briefly, recombinant U1A antigen-coated wells were incubated with mouse sera (1:400 dilutions) followed by alkaline phosphatase-conjugated goat anti-mouse μ or γ1, γ2c, γ2b, or γ3 H-chain-specific antibodies. ELISAs were developed with p
-nitrophenyl phosphate substrate (Sigma-Aldrich) and optical density at 405 nm (OD405
) was read using a VersaMax microplate reader (Molecular Devices Corporation, Sunnyvale, CA).
CCL2 (MCP-1) levels in peritoneal lavage fluid were measured using the Mouse MCP-1 OptEIA Set (BD Bioscience) following the manufacturer’s instructions. IL-12 p40/p70 in peritoneal lavage fluid was measured as described previously (23
) using rat monoclonal antibody pairs for IL-12 (BD Biosciences). After incubation with biotinylated IL-12-specific antibodies, streptavidin-conjugated alkaline phosphatase (1:1000 dilution, Southern Biotechnology) was added for 30 minutes at 22°C and the reaction was developed with p
-nitrophenyl phosphate substrate. OD405
of each sample was converted into cytokine concentrations based on the standard curves of recombinant cytokines using the Softmax Pro 4.3 program (Molecular Devices) with a four-parameter logistic equation.
Autoantibody analysis by immunoprecipitation
Immunoprecipitation of radiolabeled cell extracts to detect serum autoantibodies against nRNP/Sm was performed as previously described (20
Assessment of glomerulonephritis
Glomerular cellularity was evaluated by counting the number of nuclei per glomerular cross-section after staining with hematoxylin and eosin (H&E) as described (24
). For assessing renal immune complex deposition, 4-µm frozen sections were stained with FITC rat anti-mouse complement component C3 monoclonal antibody (Cedarlane) and examined by fluorescence microscopy (24
Bone marrow chimeras
Bone marrow chimeras were generated as described (25
). Briefly, lethally irradiated (1000 Rad) BALB/c X B6 F1 (CB6F1/J) mice were reconstituted with bone marrow from wild type CB6F1/J mice (H2-Kb
positive) mice mixed 1:1 with bone marrow from either wild type BALB/c or BALB/c IFNAR−/− mice (H2-Dd
single positive). After 6 weeks, the reconstituted mice were treated with 0.5 ml of pristane i.p. and 3 weeks later the presence of activated (CD4+
) T cells was determined in the peritoneum and spleen.
Statistical analyses were performed using Prism 4.0 software (GraphPad Software, Inc, La Jolla, CA). Differences between groups were analyzed by the unpaired Student's t test or the Mann-Whitney U-test. The data are shown as mean +/− SD for normally distributed data sets, and as median and interquartile range for non-normally distributed data sets. Student's t-test was used for normally distributed data, and the Mann-Whitney U test for non-normally distributed data. Normality was determined by D’agostino and Person omnibus normality test using Prism 4.0. All tests were two-sided, and P < 0.05 was considered significant.