Increased IkappaB expression and diminished nuclear NF-kappaB in human mononuclear cells following hydrocortisone...

We have recently demonstrated that hydrocortisone and other glucocorticoids inhibit reactive oxygen species (ROS) generation by mononuclear (MNC) and polymorphonuclear leucocytes (PMNL). Since NF-kappaB/IkappaB system regulates the transcription of proinflammatory genes, including those responsible for ROS generation, we tested the hypothesis that hydrocortisone may stimulate IkappaB production thus inhibiting NF-kappaB translocation from the cytosol into the nucleus in MNC, in vivo. One hundred milligram of hydrocortisone was injected intravenously into 4 normal subjects. Blood samples were obtained prior to the injection and at 1, 2, 4, 8 and 24 hr after the injection. Nuclear extracts and total cell lysates were prepared from MNC by standard techniques. IkappaB levels in MNC homogenates increased at 1 hr, peaked at 2-4 hr, started to decrease at 8 hr, and returned to baseline levels at 24 hr. NF-kappaB in MNC nuclear extracts decreased at 1 hr, reached a nadir at 4 hr, gradually increased at 8 hr and returned back to baseline levels at 24 hr. The total protein content of NF-kappaB subunit (P65) in MNC lysates also showed a decrease following hydrocortisone injection. This decrease was observed at 2 hr, reached a nadir at 4 hr, and returned to baseline levels at 24 hr. ROS generation inhibition paralleled NF-kappaB levels in the nucleus. It was inhibited at 1 hr, reached a nadir at 2-4 hr, started to increase at 8 hr, and returned to basal levels at 24 hr. Our data demonstrate that hydrocortisone induces IkappaB and suppresses NF-kappaB expression in MNC in parallel. IkappaB further reduces the translocation of NF-kappaB into the nucleus thus preventing the expression of proinflammatory genes.