From captured images, each glomerular lesion was scored based on the involved area as 0 (no staining), 1 (<25%), 2 (25C50%), 3 (50C75%), and 4 (>75%). inflammatory monocytes in the development of SLE. These monocytes migrate to the peritoneal cavity in WT and IRF7-deficient mice but not in IRF8-deficient mice, and there they produce both type I IFN and proinflammatory cytokines in WT mice, while in IRF7-deficient mice they only produce proinflammatory cytokines. Upon migration to the spleen, Ly6Chigh inflammatory monocytes differentiate into dendritic cells (DCs) which are capable of generating proinflammatory cytokines in response to dsDNA autoantigen. Collectively, type I IFN produced from inflammatory monocytes/monocyte-derived DCs might be essential for autoantibody production whereas proinflammatory cytokines produced from them might mediate tissue damages in this model. Our study reveals PLA2G3 a specialized role for monocyte-derived antigen presenting cells in autoimmunity. Plasticity of monocyte might play an important role not only in the pathogenesis of the disease but also in flare-ups of the disease. mice [13] were obtained from Dr. Keiko Ozato (National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA). CD45.1+mice were given a single i.p. injection of 0.5??ml of TMPD (pristane) (Funakoshi, Tokyo) or PBS (vehicle). Ten months later, urine, blood, and kidneys were harvested. In some experiments, blood, kidneys and peritoneal cells were harvested two weeks after injection. 2.3. Measurement of urine protein Proteinuria was assessed by a urinary test strip (Wako, Osaka) and graded as 0 (none), 1+ (trace; 10C20??mg/dl), GSK-923295 2+ (30??mg/dl), 3+ (100??mg/dl), 4+ (300??mg/dl), and 5+ (>1000??mg/dl). 2.4. Direct immunofluorescence Kidneys from WT, mice treated with TMPD or PBS were harvested 10 months after the disease induction, frozen in OCT medium, and stored at ?80??C. Cryosections were prepared at 6??m thickness and incubated with FITC-anti-mouse IgG Ab GSK-923295 (SouthernBiotech, Birmingham, AL), or Alexa Fluor 488-anti-mouse C3 Ab (Novus Biologicals, Littleton, CO). Nuclei were stained with Hoechst 33,258 (ThermoFisher Scientific, Waltham, MA) and examined by fluorescence microscopy (Keyence, Osaka). For the evaluation of glomerular lesions, images of 5 glomeruli per mouse were captured with a constant exposure time on fluorescence microscopy. From captured images, each glomerular lesion was scored based on the involved area as 0 (no staining), 1 (<25%), 2 (25C50%), 3 (50C75%), and 4 (>75%). The average severity grade was calculated and defined as the renal score of the mouse. For CD11c and CD45.1 double immunofluorescence staining, 6??m frozen tissue sections of the spleen were fixed with chilly acetone, incubated with FITC-CD45.1 (clone A20) (eBiosciences, Tokyo) and PE-CD11c Ab (HL3) (BD pharmingen, Tokyo). In some experiments, double immunofluorescence staining was performed with FITC-CD45.2??Ab (clone 104) (Biolegend, San Diego, CA) and PE-CD11c Ab. The sections were observed by fluorescence microscopy (Keyence). 2.5. Indirect immunofluorescence Hep2 cells were cultured in 8-well CultureSlide (BD Falcon, Tokyo), fixed with chilly acetone, and blocked with 3% BSA and 1% FCS in PBS for 1??h. Sera from your mice 10 months after TMPD or PBS injection GSK-923295 were diluted at 1:100 and slides were incubated with diluted sera overnight. Slides were then incubated with FITC-conjugated anti-IgG Ab for 30??min, mounted and examined by fluorescence microscopy (Olympus, Tokyo). 2.6. ELISA The sera were obtained 10 months after TMPD or PBS injection. Serum concentrations of anti-nuclear antibody (ANA), anti-nRNP Ab (Alpha diagnostic international, San Antonio, TX), and anti-dsDNA Ab (FUJIFILM Wako Shibayagi, Gunma, Japan) were assayed by ELISA. In some experiments, the sera were obtained 2 weeks after injection and the serum levels of TNF- and IL-6 were determined by ELISA (R&D systems, Minneapolis, MN). 2.7. Harvesting of peritoneal cells The peritoneal cavity was lavaged with 2??ml of complete RPMI plus 10 U/ml heparin. Cells were collected by centrifugation, depleted of RBC by ACK lysing buffer and then resuspended in total RPMI. 2.8. Culture of peritoneal cells, lymph node cells, and spleen cells Freshly isolated peritoneal cells from WT, mice were produced in total RPMI in the presence or absence of TMPD. Due to its insolubility in aqueous medium, TMPD was added as the inclusion complexes with -cyclodextrin.