engleski

M2 POLARIZED SYNOVIAL MACROPHAGES FROM PATIENTS WITH OSTEOARTHRITIS KEEP PLASTICITY AND ARE ABLE TO INITIATE THE LPS-MEDIATED MATURATION PROGRAM IN VITRO

Introduction: Macrophages are cells present in synovial tissue in large number, and they potentiate the progression of osteoarthritis (OA). Aim: To analyse polarisation and maturation status, cytokine production, and endocytic capability of synovial macrophages under the influence of lipopolysaccharide (LPS). Material and methods: Mature synovial tissue samples were obtained during the knee alloarthroplasty due to OA. CD68 and arginase-1, IL-15 or TNF-α were labeled in paraffin-embedded tissue sections by double immunofluorescence. Maturation status and FITC-Dextran endocytosis were analyzed in the isolated and cultured synovial mononuclear cells in the medium only or with LPS (10 ng/mL). We developed a purification strategy for CD68+ cells from synovium. Results: CD68+ macrophages scattered in sublining synovial tissue were strongly arginase-1 positive, while rarely expressed TNF-α and IL-15, indicating their M2 polarisation. Back gating for CD68+ cells revealed ~45% of CD68+ cells in freshly isolated mononuclear cell suspensions, which remained viable after 18-hour culture. The frequency of HLA-DR (60-70%), CD80 and CD86 (50-60%) did not significantly differ between CD68+cells cultured in the medium only and with LPS. LPS decreased frequency of CD91 receptor and FITC Dextran uptake for about 20-30%, compared to the cells cultured in the medium only, suggesting a specific maturation process. CD68+ cells were enriched only up to 85% from the adherent mononuclear cell fraction using anti-CD68 mAb + goat anti-mouse antibody conjugated with nanobeads, due to cell clumping. Conclusion: The most of synovial macrophages are M2 polarized, but CD68+ cells keep plasticity and are able to initiate maturation program under the influence of LPS in vitro.

Macrophages ; Osteoarthritis ; Lipopolysaccharide ; Maturation ; Cytokine

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