Beyond this initial β2 integrin binding, myeloid cells also encounter β2 integrin ligands within the extracellular matrix while en route to their intended

targets. Here these ligands would be modified DNA Damage inhibitor by local inflammatory mediators [46], suggesting that distinct β2 integrin ligands may differentially regulate TLR responses in a manner that targets inflammatory cytokine production to the infected tissue and therefore minimizes damage to the host. C57BL/6 mice were purchased from Charles River Laboratories. CD18-deficient (Itgb2−/−) mice [22] were backcrossed six generations against C57BL/6 mice and were provided by Dr. Clifford Lowell (University of California, San Francisco). CD11a-deficient (Itgal−/−) and CD11b-deficient (Itgam−/−) animals were purchased from Jackson Laboratories [23, 47]. Cbl-b-deficient (Cblb−/−) Bortezomib in vitro mice were backcrossed 12 generations against C57BL/6 and were provided by Dr. Phil Greenberg (University of Washington)

[48]. All animals were housed in specific-pathogen-free facilities and all experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at the Benaroya Research Institute. BM cells were flushed from femurs and tibias, followed by erythrocyte lysis in ACK buffer (Lonza). For macrophages, BM cells were plated onto a 10 cm petri dish (Fisher Scientific) using 10 mL of BM macrophage growth medium, which consisted of DMEM supplemented with 10% FBS (Sigma), 2 mM L-glutamine (Gibco), 1 mM sodium pyruvate (Gibco), 10 mM HEPES (Lonza), penicillin/streptomycin (Gibco) and 10% CMG14–12 cell conditioned media as a source of CSF-1 [49]. BM-derived DCs were grown in DC medium, which consisted of RPMI 1640 supplemented with 10%

FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, penicillin/streptomycin and 10 ng/mL GM-CSF (Peprotech). For both macrophages and DCs, an additional 10 mL of growth medium was added after 3 days of culture. Day 6 DCs were isolated from culture by magnetic bead enrichment heptaminol for MHCII+ cells. Cells were treated with anti-FcγRII/III (2.4G2) followed by staining with anti-MHC II-biotin (M5/114.15.2/eBioscience), antibiotin microbeads (Miltenyi biotech) and sorting with MACS columns according to the manufacturer’s instructions. The purity of CD11c+ cells was >90% in WT cultures. BM-derived macrophages and DCs were used at day 6 of culture. Mice were injected i.p. with 3% thioglycollate broth and peritoneal cells were isolated by lavage with Cell Dissociation Buffer (Invitrogen) 5 days after injection. Macrophages were purified by magnetic bead enrichment using anti-F4/80-biotin (BM8/eBioscience) followed by incubation with antibiotin microbeads and then sorted by MACS according to the manufacturer’s instructions. F4/80+ macrophages were cultured in DMEM supplemented with 10% FBS (Sigma).