Synovial tissue-infiltrating natural killer cells in osteoarthritis and periprosthetic inflammation

Abstract

Objective: Infiltrating immune cells play a central role in degenerative joint disease associated with osteoarthritis (OA) and particle-mediated periprosthetic osteolysis. The goal of this study was to characterize a newly identified population of synovial tissue-infiltrating natural killer (NK) cells obtained from patients with OA or patients with periprosthetic joint inflammation.

Methods: Synovial and interfacial tissue samples were collected from patients with OA who were undergoing primary or revision total joint replacement (TJR) surgery. The histologic features of OA synovium obtained from patients undergoing primary surgery and interfacial tissue obtained from patients undergoing revision surgery were determined by immunohistochemistry and immunofluorescence. Synovial tissue-infiltrating NK cells were evaluated for the expression of surface receptors, using flow cytometry. Chemoattractant and cytokine protein and RNA levels in synovial and interfacial tissue and fluid were assessed by Luminex assay and real-time quantitative polymerase chain reaction. Cytokine production and degranulation by stimulated synovial tissue versus normal blood NK cells were evaluated by intracellular cytokine staining.

Results: NK cells comprised nearly 30% of the CD45+ mononuclear cell infiltrate in synovial tissue obtained from patients undergoing primary TJR and from patients undergoing revision TJR. NK cells from both groups expressed CXCR3, CCR5, L-selectin, α4 integrins, and cutaneous lymphocyte antigen. Synovial fluid from patients undergoing revision surgery contained elevated concentrations of the NK cell attractants CCL4, CCL5, CXCL9, and CXCL10; all levels in synovial fluid obtained from patients undergoing revision surgery were higher than those in synovial fluid from patients undergoing primary surgery. Cytokine-stimulated interferon-γ production was significantly impaired in NK cells derived from primary and revision TJRs compared with blood NK cells.

Conclusion: NK cells are a principal tissue-infiltrating lymphocyte subset in patients with OA and patients with periprosthetic inflammation and display a quiescent phenotype that is consistent with postactivation exhaustion.

FIGURE 1

Histological features of primary OA synovium and revision interfacial tissues. Paraffin sections of synovial tissues from primary (A) or revision (B) TJR were stained with H&E to evaluate the synovial tissue architecture. (A) Lymphocytes in primary synovial tissues congregated in clusters among masses of largely acellular fibrous tissue. An enlargement of the boxed region is shown in the lower panel (white bar =100μm). (B) In contrast, lymphocytes in revision interfacial samples were scattered throughout the tissue (2 different patients shown). An enlargement of the boxed region from revision sample 2 is displayed in the bottom panel (white bar =100μm). White arrows indicate some of the infiltrating lymphocytes; black arrows indicate some of the macrophages. Cryostat sections (8 mm) of joint tissues from primary (C) or revision (D, E) patients were stained for CD45 (FITC), CD3 (PE), and CD94 (APC). (C) CD3+ lymphocytes were organized in lymphocyte clusters in primary synovial samples (3 different patients). (D) CD3+ T cells and CD3−CD94+ NK cells were scattered throughout revision interfacial tissues (2 different patients); white bar = 10μm. An enlargement of a stained revision section is shown in (E) (white bar = 20μm).

FIGURE 2

NK cells infiltrate primary and revision synovial joint tissue. (A) Freshly isolated synovial tissue leukocytes were harvested and analyzed by flow cytometry. A lymphocyte gate was established based on forward and side light scatter (FSC and SSC, respectively), and CD45 was used to identify leukocytes; CD56 co-staining is shown. (B) The percentage of the indicated CD-marker defined lymphocyte subset (of total CD45+ cells in the lymphocyte-light scatter gate) in primary (top panel) and revision patient (bottom panel) synovial samples is shown. The mean ± SD is displayed, n= 6 for each group. (C) Joint tissue-infiltrating NK cells (CD45+CD3−CD56+) were stained with antibodies for the indicated chemoattractant or homing receptors. Histograms are representative of n= 3 patients per group.

FIGURE 3

Synovial joint NK cells do not efficiently produce IFN-γ in response to stimulating cytokines. (A–C) Purified NK cells from blood and synovial tissues were stimulated as indicated. (A) Flow cytometric analysis of intracellular IFN-γ vs. surface expression of the degranulation marker, LAMP-1. Dot plots are representative of n=3 surgical patients or blood samples, except for primary synovial NK samples treated with IL-12/15 and IL-15/18, where n=2. (B and C) Percent of IFN-γ+ NK cells (B) or LAMP-1+ NK cells (C). Blood: peripheral blood NK Cells; Prim: primary synovial tissue NK cells; Rev: revision synovial tissue NK cells. For (B and C), error bars represent SEM where n=3 samples or more, and range where n=2. (D) The concentration of IL-12p40, IL-15, IL-18, and IFN-γ was assessed in primary SF (Prim, n=11) and revision SF (Rev, n=9) by Luminex. The circle symbols correspond to individual patient samples (mean of duplicate wells); the overall mean ± SEM within patient groups is shown. *p<0.05 by Student's t-test or Mann-Whitney rank sum test. For comparison, the reported ranges for IFN-γ in OA, RA, and spondyloarthropathy SF are 10–100 pg/ml, 20–900 pg/ml, and 1,000–5,000 pg/ml, respectively (15).