Inducible nitric oxide synthase is expressed in synovial fluid granulocytes

Abstract

The objective of the study was to evaluate the NO-producing potential of synovial fluid (SF) cells. SF from 15 patients with arthritis was compared with blood from the same individuals and with blood from 10 healthy controls. Cellular expression of inducible nitric oxide synthase (iNOS) was analysed by flow cytometry. High-performance liquid chromatography was used to measure l-arginine and l-citrulline. Nitrite and nitrate were measured colourimetrically utilizing the Griess' reaction. Compared to whole blood granulocytes in patients with chronic arthritis, a prominent iNOS expression was observed in SF granulocytes (P < 0.001). A slight, but statistically significant, increase in iNOS expression was also recorded in lymphocytes and monocytes from SF. l-arginine was elevated in SF compared to serum (257 +/- 78 versus 176 +/- 65 micro mol/l, P = 0.008), whereas a slight increase in l-citrulline (33 +/- 11 versus 26 +/- 9 micro mol/l), did not reach statistical significance. Great variations but no significant differences were observed comparing serum and SF levels of nitrite and nitrate, respectively, although the sum of nitrite and nitrate tended to be elevated in SF (19.2 +/- 20.7 versus 8.6 +/- 6.5 micro mol/l, P = 0.054). Synovial fluid leucocytes, in particular granulocytes, express iNOS and may thus contribute to intra-articular NO production in arthritis.

Fig. 1

Fluorescence after labelling of exudated polymorhonuclear leucocytes with different concentrations of antibodies/Ig. (a) Fixation and permeabilization with Intrastain®. (b) Samples are fixed and permeabilized with PFA 4% and saponin 0·1%, respectively. ♦, Polyclonal rabbit IgG; ▪, polyclonal rabbit IgG anti-iNOS; ▴, polyclonal mouse IgG; ×, monoclonal mouse IgG anti-iNOS.

Fig. 2

A representative example of flow cytometric iNOS expression in different cell populations in synovial fluid (patient number 9). (a) Forward/side scatter plot providing cell size versus granularity. The cell populations were gated and the resulting fluorescence profiles are shown in (b). The granulocyte population is clearly iNOS⁺ while mononuclear cells show lower iNOS expression. R1 represents granulocytes, R2 monocytes and R3 lymphocytes. C is FITC-labelled rabbit IgG in an R1-gating (granulocyte population).

Fig. 3

iNOS expression in different cell populations from control blood (n =10), patient blood (n =15) and synovial fluid (n =15). All synovial fluid leucocyte populations show statistically significant iNOS expression compared to the corresponding cell population in blood. *** =P < 0.001; ** =P < 0.01. Mean values ± standard deviations are given.