Fig. 5

NOS activity, nitrite/nitrate, and NOS2 antigen in mononuclear cells from control subjects and RA patients. (A) NOS activity in freshly isolated and cultured mononuclear cells from control subjects and RA patients. Blood mononuclear cells were prepared and extracts from freshly isolated cells (“fresh”) or from cells cultured 5 days with no additions, with 1 µg/ml LPS alone, with 500 U/ml IFN-γ alone, or with 500 U/ml IFN-γ and 1 µg/ml LPS were assayed for NOS activity (ability to convert L-arginine to L-citrulline). Assays were done as six replicates for each individual subject. Results are shown as medians (horizontal bar), means (circle), the interquartile range (box), and the 10th to 90th percentile range (vertical lines). There were 20 control subjects and 25 RA patients. Using the Wilcoxon Rank Sum test, RA patients’ NOS activities differed significantly from control subjects’ NOS activities in the categories Fresh (p < 0.003), No additions (p < 0.005), LPS (p < 0.002), IFN-γ (p < 0.002), and LPS/IFN-γ (p < 0.002). In analyses of cultured cells, the within-group comparison was significant for RA patients (p < 0.001), but not for control subjects. Pairwise comparisons for cells from RA patients revealed significant differences for treatments which included IFN-γ [No additions vs. IFN-γ (p < 0.003), and No additions vs. LPS/IFN-γ (p < 0.003)]. (B) Nitrite/nitrate production by cultured mononuclear cells from normal subjects and RA patients. Blood mononuclear cells were prepared and cultured 5 days with no additions, 1 µg/ml LPS alone, 500 U/ml IFN-γ alone, or 500 U/ml IFN-γ and 1 µg/ml LPS. Supernatant media were then measured for nitrite/nitrate. Assays were done as six replicates for each individual subject. Results are shown as medians (horizontal bar), means (circle), the interquartile range (box), and the 10th to 90th percentile range (vertical lines). There were 20 control subjects and 25 RA patients. Using the Wilcoxon Rank Sum test, RA patients’ nitrite/nitrate levels differed significantly from control subjects’ nitrite/nitrate levels in the categories LPS (p < 0.01) and LPS + IFN-γ (p < 0.02). Within-group comparison was significant for control subjects (p < 0.02), but not for RA patients. Pairwise comparisons for cells from control subjects revealed significant differences for No additions vs. LPS (p < 0.02) and “LPS” vs. IFN-γ (p < 0.003). (C) Immunoblot analysis of mononuclear cells from normal controls and RA patients for NOS2 expression. Blood mononuclear cells were isolated and extracts were analyzed for NOS2 antigen content using an NOS2-specific mouse monoclonal anti-NOS2 antibody. The NOS2 antigen has a molecular weight of approximately 130 kD. Extracts from the murine macrophage cell line cells J774 and RAW 264 were used as negative and positive controls. Forty micrograms of protein from the extracts were used in each lane. Parallel gels and blots done using isotype-specific control immunoglobulin showed no reactivity. J = J774 control; J+ = J774 cells cultured with LPS + IFN-γ; r+ = RAW 264 cells cultured with LPS + IFN-γ; R1−5 = samples from 5 separate RA patients; N1−5 = samples from 5 separate normal (control) subjects. [Reproduced with permission (67).]