SHIP deficiency causes Crohn's disease-like ileitis

Kerr, William G; Park, Mi-Young; Maubert, Monique; Engelman, Robert W

The development and production of SHIP À/À mice has been described previously. In brief SHIP À/À mice were generated by deletion of the promoter and first exon of SHIP via a Cre-LoxP strategy and then backcrossed to the C57BL6/J background. SHIP DIP/DIP mice were a kind gift of Dr Jeffery Ravetch (Rockefeller University, New York). 47 MxCreSHIP flox/flox mice were described previously. 33 SHIP expression is ablated in these mice following three intraperitoneal injections of polyI/C at 625m/ dose. MxCreSHIP flox/flox mice were sacrificed for tissue harvest 4e5 weeks after the last polyI/C injection. The PI3K haploinsufficient SHIP À/À mice analysed in this study possess a mutation in the p85 regulatory subunit of PI3K 48 and a SHIP mutation created by Helgason et al. 41 All mice were between 6 and 13 weeks of age at time of sacrifice. All mice were maintained in an accredited, barrier facility confirmed free of a comprehensive list of potential parasites and microbial pathogens including Citrobacter rodentium, Pseudomonas aeruginosa, Salmonella spp., and Clostridium perfringens, Adoptive transfer experiments BM cells were flushed from intact femur and tibia and collected in tissue media (TM) consisting of RPMI, 3% fetal bovine serum (FBS) and 10 mM HEPES (Invitrogen, Carlsbad, California, USA). Spleens were crushed with a 10 ml syringe plunger. The single cell suspension was then filtered through a 70 mm strainer (BD Bioscience, San Jose, CA) and red blood cell (RBC) lysis performed at room temperature for 5 min in 13 RBC lysis buffer (eBioscience, San Diego, California, USA). Cells were centrifuged and resuspended in 13 Dulbecco phosphate-buffered saline (D-PBS). C57BL/6 recipients were given antibiotic water prior to receiving a split dose of 1100 Rads (600+500) from an x-ray irradiator. Irradiated recipients were then transplanted with 5310 5 BM cells or splenocytes as indicated via retro-orbital injection. For adoptive transfer of T and NK cells, CD3 + NKp46 À T and NKp46 + CD3 À NK cells were simultaneously sorted from spleens of SHIP À/À mice and adoptively transferred into irradiated C57BL6 hosts (550 Rads) via retro-orbital injection. Each C57BL/6 host received 2.4310 5 SHIP À/À T cells and 7.5310 3 SHIP À/À NK cells.