Fig. production and degranulation by stimulated synovial tissue vs. normal blood NK cells was evaluated by intracellular cytokine staining. == Results == NK cells comprised nearly 30% of the AMG 548 CD45+ mononuclear cell infiltrate in synovial tissues from both primary and revision TJR. NK cells from both groups expressed CXCR3, CCR5, L-selectin, 4 integrins, and CLA. Synovial fluid (SF) from revision patients contained elevated concentrations of NK cell attractants CCL4, CCL5, CXCL9, CXCL10, and chemerin, all higher than in primary SF. Cytokine-stimulated IFN- production was significantly impaired in primary and revision NK cells compared to blood NK cells. == Conclusions == NK cells are a principal tissue infiltrating lymphocyte subset in OA and peri-prosthetic inflammation, and display a quiescent phenotype consistent with post-activation exhaustion. Patients with degenerative joint disease (most commonly OA) that is refractory to conservative management are candidates for TJR, which alleviates pain and restores limb function in >80% of cases (1,2). Aseptic loosening of joint prostheses is the most significant long-term C5AR1 complication, occurring in 718% of patients at 25-year follow up (1,2), and requires many to undergo revision surgery (3). The loss of bony fixation is due to inflammatory responses triggered by wear particles generated at the bearing surfaces and along the implant interface (4). Although NK cells are classically known for their role in killing virally-infected cells or tumor cells, NK cell infiltrates have been identified and characterized in a variety of other pathologic settings, such as psoriatic skin and RA SF (5,6). In one study, CD56+ CD16 NK cells comprised 16% of the infiltrating lymphocytes in RA SF (7), and most expressed the activation marker CD69 and the cytotoxic receptor NKp44, and lacked the inhibitory killer cell Ig-like receptors (KIRs) CD158a and CD158b (6,7), and 25% produced IFN- upon stimulationin vitro, suggesting that the cells are active participants in joint inflammation. The recruitment of NK cells to peripheral inflammatory sites is dependent on cell-expressed chemoattractant receptors and AMG 548 adhesion molecules, as well as chemoattractants present at the site. In peripheral blood, immunoregulatory CD16 NK cells express CXCR3, CCR5, and CCR7 and migrate to CXCL911, CCL35, CCL19, and CCL21in vitro(8). NK cells in inflamed joint SF were reported to express CCR5 and CXCR3, and chemokine ligands AMG 548 for these receptors have been detected in RA SF and synovial tissue (7,9), suggesting a role for CCR5 and AMG 548 CXCR3 in NK cell recruitment to the joint. Here we show that CD45+CD56+CD3 NK cells are a principal leukocyte infiltrate in synovial tissue from OA patients undergoing primary and revision TJR. We propose that the infiltrating CD56+ cells are recruited immunoregulatory NK cells, and that chronic activation in the joint environment leads to exhaustion of their capacity to secrete IFN-. == MATERIALS AND METHODS == == Human Sample Collection == Synovial tissues and fluid normally discarded during TJR surgery were obtained from 18 OA patients and 22 revision joint replacement patients (SeeSupplementary Table 1for patient demographics and additional information). PBMC were obtained from healthy blood donors (Stanford Blood Center). The Institutional Review Board at Stanford University School of Medicine (Stanford, CA) approved all human subject protocols. == Histological Analysis == Synovial tissue specimens from 4 patients undergoing primary joint replacement and 9 patients undergoing revision joint replacement were fixed, in 10% formalin, and paraffin blocks were prepared, sectioned, and stained with H&E by Histo-Tec laboratory (Hayward, CA). For immunofluorescence staining, 8-m cryostat sections were stained with directly conjugated antibodies as previously described (10). == Isolation and Stimulation of Untouched NK Cells == Minced synovial tissues were cultured in 60 U/mL rhIL-2 (eBioscience) for 4 days, and untouched NK cells from synovial tissues or freshly isolated PBMC were isolated via negative depletion with magnetic beads (Miltenyi Biotec), with a purity of >95% CD3CD56+. Purified NK cells were then cultured with 20 U/mL rhIL-2 for 48 hours prior to addition of one of the following cytokine combinations: IL-12 (10 ng/mL) and IL-15 (10 ng/mL), IL-12 and IL-18 (100 ng/mL), or IL-15 and IL-18 (eBioscience). After 18 hours, cells were incubated for 1 hour at 37C with LAMP-1-PE antibody (20uL/mL, BD Biosciences). Monensin (50 mM, eBioscience) and Brefeldin A (10 g/mL, Sigma) were added to cultures.