Ex vivo Nitrite Exposure and Response of Peripheral Blood Mononuclear Cells from Osteoarthritis Cases and Controls: Proliferation and Nitric Oxide Generation

Author(s): Junu Shrestha, Lisa Beltz, Catherine Zeman

This ex vivo pilot study examined the effect of low dose nitrite exposure on peripheral blood mononuclear cells (PBMC) proliferation and nitric oxide (NO) generation among people with osteoarthritis (cases) and without osteoarthritis (controls). The hypothesis was that nitrite exposure produced different effects on proliferation and NO generation between the cases and controls. Human PBMC, composed of lymphocytes and monocytes, were isolated from the whole blood of 31 participants (15 cases and 16 controls) using density gradient centrifugation, then exposed to five different nitrite concentrations: 0 (negative controls), 1 mg/l (0.02 mM), 5 mg/l (0.10 mM), 10 mg/l (0.21 mM), and 15 mg/l (0.32 mM). PBMC proliferation and NO generation were determined after 96-hours and 48-hours, respectively, using a Muse Unit (Cell Analyzer/Flow Cytometer). Increasing nitrite exposure significantly suppressed the non-stimulated PBMC proliferation (F = 7.07, p<.0001). The study also showed PBMC proliferation was significantly suppressed among all participants when exposed to 5 mg/l nitrites, but that the degree of suppression was higher among participants with osteoarthritis (F=4.23, p<0.05). Increasing nitrite exposure together with stressful life events, use of multiple drugs (prescribed and supplements), and chronic disease also significantly impacted the NO generating cells and PBMC proliferation.