Synergistic Interactions between Pro-Inflammatory Cytokines in the Induction of Inos in Human Vascular Smooth Muscle Cells
Inducible nitric oxide synthase (iNOS) is thought to play an important role in atherosclerosis. However, the precise role of LPS and individual cytokines in the induction of iNOS in humans remains undetermined. We have used human aortic vascular or internal mammary artery segments to assay the efficacy of LPS (1 g/ml), IFN-γ (400 units/ml), TNF-α (1000 units/ml) and IL-16 (100 units/ml), either alone or in combination to induce the expression of iNOS and subsequent accumulation of NO2 detected by chemiluminescence. Combination of all 3 cytokines induced a significant increase in NO2 accumulation after 36 (210%) and 48 (893%) hours incubation, which could be inhibited by L-NMMA (10−4M) and cycloheximide (10−6M). Medial smooth muscle of sections of vessel segments stained positive with an antibody raised against human iNOS. Incubation of IFN-γ and IL-16 alone both caused a small but significant increase in the level of NO2 above control over a 48 hour period, by 50% and 75% respectively (p<0.05). TNF-α was unable to induce a significant rise in NO2 over the same time period. Experiments with different combinations of the cytokines revealed a synergistic interaction between IFN-γ and IL-16, with the increase in NO2 levels of 869%. A combination of IFN-γ and TNF-α, or IL-16 and TNF-α yielded no enhanced accumulation of NO2 compared to the action of each cytokine alone. Incubation of LPS alone was unable to cause an increase in NO2 accumulation after 48 hours. Combination of LPS with IFN-γ or IFN-γ and IL-16, gave no significant enhancement of NO2 accumulation. However, LPS was capable of increasing the level of NO2 accumulation in response to a combination of IFN-γ and TNF-α from 158% to 295%. These results highlight a central role for IFN-γ in the induction of iNOS and reveal a complex series of interactions between IFN-γ and other mediators.