LPS (Escherichia coli: Strain 55:B5), CPB from porcine pancreas, 3-morpholinylsydneimine hydrochloride (SIN-1), the xanthine oxidase inhibitor allopurinol and the iNOS inhibitor 1400W [N-(3-(amino methyl)benzyl)acetamide 2HCl] were from Sigma Chemical Co. (St Louis, MO, USA). The spin trap DMPO (5, 5-dimethyl-1-pyrroline N-oxide) was obtained from Alexis Biochemicals, (San Diego, CA, USA). The eNOS inhibitors L-NIO (1-imino-3-butenyl)-L-ornithine, and L-NAME (L-ω-nitroarginine methyl ester) were from Cayman Chemicals, Ann Arbor, MI, USA. (Sigma Chemical Co, USA).
Adult male, pathogen-free, 8–10 week old C57BL6/J mice (Jackson Laboratories) weighing 23–27 g on arrival were housed for 1 week, one to a cage, before any experimental use. Experiments using mice that contained the disrupted iNOS (iNOS−/−), gp91phox (gp91phox−/−) and eNOS (eNOS−/−) genes were treated identically. Age-matched mice of C57BL6/J origin that had normal iNOS, NADPH oxidase, and eNOS activity served as the control animals for knockout experiments. Mice had ad libitum access to food and water and were housed in a temperature-controlled room at 23–24 °C with a 12-hour light/dark schedule. All animals were treated in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals, and the experiments were approved by the institutional review board.
LPS-induced systemic inflammation model and spin trapping of protein-centered radicals
Mice received a bolus infusion of 6 or 12 mg kg−1LPS at 0 h. Mice screened for protein radical adduct formation also received DMPO, a total of 2 g/kg in two divided doses at +4 and +5 hours post-LPS administration. The sham-treated group received saline in place of LPS and/or DMPO. LPS and DMPO were dissolved in pyrogen-free saline and were administered through the intraperitoneal (i.p.) route. At +6 h, all mice were sacrificed and the spleen and other organs collected and immediately snap-frozen in liquid nitrogen. Tissues were homogenized in phosphate buffer containing 100 μM DTPA and centrifuged at 3,000 RPM at 4 °C for 20 minutes. The samples were stored at −80 °C until further use.
Administration of allopurinol and NOS inhibitors
Allopurinol, a specific inhibitor of xanthine oxidase, the relatively specific eNOS inhibitors (1-imino-3-butenyl)-L-ornithine (L-NIO), 20mg/kg, and L-ω-nitroarginine methyl ester (L-NAME) (Cayman Chemicals, Ann Arbor, MI, USA) and the iNOS inhibitor N-(3-(amino methyl)benzyl) acetamide 2HCl (1400W, Sigma Chemical Co, USA) were all administered i.p. in a single bolus dose of 20 mg/kg 30 minutes prior to LPS treatment.
Mass spectometric analysis for protein identifications
Protein bands manually excised from gels, cut into small pieces, and transferred into a 96-well microtiter plate were subjected to automatic tryptic digestion and analyzed by MALDI/MS and LC/ESI/MS17
. For all MS analyses, data-dependent acquisitions were acquired in a fully automated mode such that a mass spectrum was acquired followed by MS/MS of the most abundant ions in the spectrum as described in the Supplemental Data. Searches were performed against the NCBI nonredundant protein database using both the MS and the MS/MS data as described in the Supplemental Data.
Immunoprecipitation of DMPO nitrone-adduct and CPB1
Immunoprecipitation of DMPO nitrone adducts was carried out with the Seize X Mammalian immunoprecipitation Kit (Pierce, Rockford, IL, USA) with some modifications. Solubilized spleen cell homogenates were collected and protein concentrations measured with a Micro BCA Protein Assay Kit (Pierce, Rockford, IL, USA) and adjusted to a concentration of 200 μg/sample. The samples were pre-cleared (1 h at room temperature) with 200 μl of Protein G slurry (50%). The homogenate was incubated overnight with 25 μg of monoclonal antibody to DMPO, and this antigen-antibody mixture was then incubated overnight with the Protein G slurry. Immune complexes were eluted with elution buffer according to the manufacturer’s instructions and collected in tubes with 20 μl Tris buffer (pH 9.6). 30 μl of the elution fractions were then resuspended in NuPAGE LDS sample loading buffer with 5 μl of 140 mmol/L 2-mercaptoethanol, heated at 85 °C for 10 minutes and immediately resolved by reducing SDS-PAGE in 4–12% Bis Tris gels (Invitrogen, Carlsbad, CA, USA). CPB1 was immunoprecipitated with polyclonal antibody to CPB1 (R & D Systems, Minneapolis, MN, USA) using the Protein A Immunoprecipitation kit (Pierce, Rockford, IL, USA.) following manufacturer’s instructions.
Western Blot Analysis
Following separation by SDS-PAGE, proteins were transferred electrophoretically (40 volts for 40 minutes) to nitrocellulose membranes (Invitrogen, Carlsbad, CA, USA) which were blocked (overnight, 4°C) with 2% Immunoblot blocking reagent (non-fat dry milk) (Upstate Technologies, Temecula, CA, USA) in 0.1M bicarbonate buffer (pH 9.6). For detection of DMPO-nitrone adducts, blots were incubated with rabbit polyclonal antibody to DMPO (1:1000). CPB1 was detected using goat polyclonal antibody to CPB1 (1:1000) (R&D systems, Minneapolis, MN USA). Antibodies specific for xanthine oxidase was purchased from Abcam (Cambridge, MA, USA). After four 10 minute washes in phosphate buffered saline-Tween (PBS-T), the above immunocomplexed membranes were probed (1h at RT) with goat anti-rabbit (1:5000, Upstate Biotechnologies, Temecula, CA, USA), donkey anti-goat (1:3000, R&D Systems, Minneapolis, MN, USA), goat-anti-mouse (1:5000) horseradish peroxidase conjugated secondary antibodies respectively. Probed membranes were washed (10 minutes, PBS-T) four times and immunoreactive proteins were detected using enhanced chemiluminescence (LumiGLO Chemiluminescence Substrate, Upstate, Temecula, CA, USA). The images were subjected to densitometry analysis using LabImage 2006 Professional™ 1D gel analysis software from KAPLEAN Bioimaging Solutions, Germany.
Confocal microscopy of spleen tissue
Mice were administered LPS, Enos inhibitor L-NIO and xanthine oxidase inhibitor allopurinol. DMPO was injected in two divided doses of 1 g/kg at 2h and 1h prior to sacrifice. Spleens were fixed in 10% neutral buffered formalin and soaked in 30% sucrose for 24h. The frozen sections (10 micron) were cryocut using a frozen tissue processor (Leica Instruments, USA) at the immunohistochemistry core facility at NIEHS. Tissue slices were then permeabilised, and blocked (2% nonfat dry milk, Pierce Biomedical, USA). Antibody specific to DMPO nitrone adducts and Alexafluor 488 goat anti-rabbit antibody (Molecular Probes, USA) were used as primary and secondary antibodies, respectively. Confocal images were taken on a Zeiss LSM510-UV meta (Carl Zeiss Inc, Oberkochen, Germany) using a Plan-NeoFluar 40X/1.3 Oil DIC objective. The 488 nm line from an Argon laser was used for producing polarized light for a DIC image as well as fluorescence excitation of the Alexa488 secondary antibody. A longpass 505 emission filter was used to collect the fluorescence images with a pinhole setting of 81 microns. All images were acquired with equal excitation power (5%) and identical detection gain (532 volts).
MetaMorph Offline 184.108.40.206 (Molecular Devices, Downingtown, PA, USA) was used for the calculation of the average fluorescence pixel intensity of the entire image. This data was then entered into Microcal Origin software for statistical analysis
Carboxypeptidase B-like activity assay of CPB1
The mouse carboxypeptidase B1 chromogenic activity assay was carried out using an Actichrome TAFI activity kit (American Diagnostica Inc., Stamford, CT, USA) as per the manufacturer’s instructions with some modifications.
Nitric oxide synthase activity assay
Nitric oxide synthase activity in spleen cell homogenates was measured using the colorimetric assay kit from Calbiochem (La Jolla, CA, USA) following the manufacturer’s instructions.
All in vivo experiments were repeated three times with 3 mice per group. Quantitative data from Western blots as depicted from relative light intensity of the bands were analyzed by performing a one-tailed Student’s t test. P<0.05 was considered statistically significant.