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• W2605762110 abstract "Purpose: Folate receptor (FR)-expressing macrophages have been shown to accumulate at sites of inflammation, where they promote the development of inflammatory symptoms. In our recently published cross-sectional pilot study (25 patients, 50 knees), we observed folate receptor positive macrophages in the synovium of the majority (76%) of osteoarthritic (OA) knees. The quantity of these cells was associated with radiographic severity of knee joint space narrowing and osteophyte. To better understand the identity and function of the cells expressing folate receptors, we characterized the cell populations from synovium and fluid and evaluated the association of functional folate receptor activity with concentrations of cytokines within synovial fluid. Methods: Under Duke Institutional Review Board approval, we collected synovial tissue (SV) and matched synovial fluid (SF) from patients with OA undergoing total knee arthroplasty (n = 18) at Duke University Hospital as waste surgical specimens. We developed an enzyme-free method to isolate cells from synovial tissue to prevent the loss of folate receptor function. Using polychromatic flow cytometry, we analyzed the cell populations from SV and SF for cell surface markers and functionality of folate receptor (FR). Functional folate receptor was quantified by uptake of a fluorescently labeled folate conjugated compound. We also measured SF cytokines (IL-1α, IL-1β, IL-6, IL-8, TNF-α, IL-1Ra and high-sensitivity C-reactive protein (HS-CRP)) and evaluated the correlations with cell populations. Results: Based on pilot studies using immortalized THP-1 (FR-β+) monocytes, we noticed that enzymes used to dissociate primary cells from tissue could adversely affect folate receptor ability to transport folate. We therefore developed an enzyme-free method of isolating cells from synovial tissue without compromising yields of haematopoietic cells (CD45+) including macrophages (CD14+). Comparing SF and SV cell profiles, lymphocytes (CD45+/CD3+) were of similar frequencies as a proportion of all cells (SF:20.0±4.6%; SV:12.7±1.9%). B cells (CD45+/CD19+) and mast cells (CD45+/CD117+) were detectable but in low abundance (<1%). Tissue fibroblasts (CD45-/CD90+) were detected in SF and SV (SF:0.19±0.03%; SV:4.44±0.7%). Macrophages (CD14+/CD11c+/HLADR+/CD11b+/CD16low) and neutrophils (CD14-/CD11c-/HLADR-/CD11b+/CD16high) were detected in SF and SV (see Table 1 for frequencies). SV Macrophages were significantly more abundant than neutrophils in SV. Neutrophils were significantly more abundant in SF than SV; macrophages were of similar abundance in SF and SV (Table 1). The percentage of cells expressing functional folate receptor was similar in SF (8.6±6.1%) and SV (7.7±6.2%) cells. There was little or no expression of functional folate receptors on lymphocytes, B cells, mast cells, or fibroblasts. Functional folate receptor was equally abundant on SF and SV macrophages and neutrophils (Table 1). However, SF and SV neutrophils took up significantly more folate conjugate demonstrated by internalization of higher amounts of the fluorescent signal (Table 1). Lastly, we observed a correlation of several SF inflammatory cytokines with the number of cells with functional folate receptor (Fig. 2). Conclusions: We have identified the inflammatory cell populations in synovial fluid and tissue of knee OA with functional folate receptor. Functional folate receptor is present on macrophages and neutrophils, but not lymphocytes. The correlation between secretory cytokines in SF and specific immune cells, especially the subpopulations with functional folate receptor, suggest that these cells could be potential therapeutic targets in OA.Acknowledgements: We would like to thank Endocyte, Inc. for supporting this study and for the folate-conjugate. We thank Duke Orthopaedic Surgery team for collecting all clinical specimens for this study.Table 1Phenotypic characterization of immune cells and subpopulations.% of live populationFR+(% of indicated cell)FR uptake intensitySynovial fluid Macrophage17.7 ± 3.420.3 ± 2.1153.4 ± 3.5 Neutrophil12.2 ± 4.021.0 ± 5.4239.6 ± 43.3 Lymphocyte20.0 ± 4.69.9 ± 1.5154.2 ± 8.6Synovium Macrophage13.2 ± 3.317.0 ± 2.9142.1 ± 2.6 Neutrophil2.0 ± 0.319.3 ± 2.7194.6 ± 19.7 Lymphocyte12.7 ± 1.97.9 ± 1.1141.0 ± 7.1 Open table in a new tab" @default.
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• W2605762110 date "2017-04-01" @default.
• W2605762110 modified "2023-09-23" @default.
• W2605762110 title "Functional folate receptor cells within synovium and fluid as therapeutic targets for osteoarthritis" @default.
• W2605762110 doi "https://doi.org/10.1016/j.joca.2017.02.082" @default.
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