[PubMed] [Google Scholar] 62. lacked this adaptor. We used the NOD. B10 strain because SS pathogenesis in this model closely recapitulates the human disease, as these animals have a female disease predilection and develop both local and systemic disease spontaneously by 26 wk of age [26, C, 28]. We found that NOD.B10females had attenuated disease, as lymphocytic infiltration was reduced in exocrine tissue, as well as in the lung and kidney. In addition, NOD.B10mice maintained salivary flow with disease progression and had diminished total and ANA\specific autoantibody titers. Therefore, Myd88 is usually a critical mediator of SS pathology, both in exocrine glands and in other C-178 peripheral tissues. MATERIALS AND METHODS Mice NOD.B10, BL/10, and B6.129P2(SJL)\animals by crossing NOD.B10 mice with mice [B6.129P2(SJL)\mice. We confirmed that these mice were congenic with the parental NOD.B10 strain using a panel of microsatellite markers (The Jackson Laboratory). Animals were bred and managed at the University or college at Buffalo, The State University or college of New York (Buffalo, NY, USA), and housed in identical conditions. Females were used in all experiments. This study was carried out in accordance with the recommendations of the animal research guidelines, IACUC. The protocol was approved by the University or college at Buffalo, The State University or college of New York, IACUC. Histologic processing and analyses SMG and lacrimal gland tissues were collected. In addition, lung and kidney were harvested. All tissue was formalin fixed, paraffin embedded, and stained with H&E. Slides were scanned using Aperio software (Leica Biosystems, Buffalo Grove, IL, USA), and ImageJ (NIH, Bethesda, MD, USA) was used to measure the lymphocytic infiltration present in the tissue [29]. This was quantified by the division of the area of infiltration by the total tissue area examined, as described previously [30]. Splenocyte harvest and culture Spleens were harvested and mechanically disrupted. Cells (5 106) were cultured in RPMI 1640 made up of 5% warmth\inactivated FBS, 2 mM l\glutamine, 50 M 2\ME, 100 U/ml penicillin, and 100 g/ml streptomycin in 12\well plates. Splenocytes were stimulated C-178 with LPS (25 g/ml; Sigma\Aldrich, St. Louis, MO, USA) or anti\IgM (10 g/ml; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and IL\4 (20 ng/ml; PeproTech, Rocky Hill, NJ, USA) for 24 h before assay, as described previously [31]. Cells and supernatants were harvested and assayed by circulation cytometry and ELISA, respectively. Circulation cytometry Splenocytes were stained by immunofluorescence for B220 (clone RA3\6B2; BD Biosciences, Franklin Lakes, NJ, USA), CD23 (clone B3B4; BD Biosciences), CD21/35 (clone 7G6; BD Biosciences), CD4 (clone GK1.5; BD Biosciences), CD86 (clone GL1; BD Biosciences), and CD69 (clone H1.2F3; BioLegend, San Diego, CA, USA). All incubations were performed on ice. In brief, cells were incubated C-178 with CD16/32 (BD Biosciences) in staining buffer (2% FBS in PBS) for 30 min, followed by addition of fluorescently labeled antibodies at a 1:1000 dilution for 30 min. Cells were washed once in staining buffer before data acquisition. Data analysis was performed using FlowJo (TreeStar, Ashland, OR, USA). Sera and saliva collection Sera and saliva were harvested from female NOD.B10, NOD.B10mice and age\ and gender\matched BL/10 controls [32]. Sera were harvested by cardiac puncture immediately following euthanasia and stored at ?20C until Rabbit Polyclonal to OGFR use. Saliva was collected for 10 min following intraperitoneal injection with pilocarpine HCl (0.3 mg/100 l; Sigma\Aldrich). Saliva was centrifuged briefly and measured by pipette, as explained previously [30]. ELISAs Serum IgM, IgG, IgG1, and IgG2a ELISAs were performed in accordance with the manufacturers instructions (Bethyl Laboratories, Montgomery, TX, USA). Murine anti\ANA IgM and IgG ELISAs C-178 (Alpha Diagnostic International, San Antonio, TX, USA) were also carried out in accordance with the manufacturers instructions with the following modifications: HRP conjugated to anti\IgG or \IgM, respectively, was used to detect isotype\specific ANA (SouthernBiotech, Birmingham, AL, USA), as explained.