Objective: We recently reported that activation of neutrophils obtained from pregnant women resulted in production of tumor necrosis factor-alpha by a thromboxane- and cyclooxygenase-2- dependent mechanism. Activated neutrophils also generate reactive oxygen species such as the superoxide anion, which can lead to oxidative damage of biomolecules. In this study, we tested the possibility that thromboxane plays a role in neutrophil superoxide generation in pregnancy via cyclooxygenase-2 by inhibiting key enzymes in the pathway leading to its synthesis.
Study design: Neutrophils were isolated from normal pregnant women and incubated for 2 hours in phosphate-buffered saline with glucose alone or with treatments. Experiment 1 treatments were: (1) indomethacin at a dose sufficient to inhibit phospholipase A2 (100 microM); (2) aspirin (100 microM), a cyclooxygenase-1 and cyclooxygenase-2 inhibitor; (3) NS-398 (10 microM), a specific cyclooxygenase-2 inhibitor; (4) nordihydroguaiaretic acid (10 microM), a lipoxygenase inhibitor; and (5) pinane thromboxane, a thromboxane synthase inhibitor. Experiment 2 treatments were arachidonic acid (50 microM and 100 microM) and arachidonic acid (100 microM) alone or in combination with phorbol myristic acid plus varying doses of pinane thromboxane (5 microM to 100 microM).
Results: Indomethacin inhibited superoxide production to one fourth of control. Aspirin, NS-398, and pinane thromboxane also significantly decreased neutrophil superoxide production. Nordihydroguaiaretic acid had no significant affect on neutrophil superoxide generation. Arachidonic acid stimulated superoxide generation by neutrophils, and this was inhibited by pinane thromboxane in a dose-dependent manner. Pinane thromboxane also significantly inhibited superoxide production by neutrophils exposed to arachidonic acid plus phorbol myristic acid, a known activator of neutrophils.
Conclusion: These data demonstrate that thromboxane is involved in the production of superoxide by neutrophils obtained from pregnant women. Neutrophil superoxide generation is significantly decreased by inhibition of thromboxane synthase, cyclooxygenase-2, or phospholipase A2. NS-398 inhibits neutrophil superoxide generation as effectively as aspirin, suggesting that cyclooxygenase-2 plays an important role in neutrophil superoxide production mediated by thromboxane.