Nitric oxide synthase inhibition by haem oxygenase decreases macrophage nitric-oxide-dependent cytotoxicity: a negative feedback mechanism for the regulation of nitric oxide production

Nitric oxide (NO) production in macrophages by inducible nitric oxide synthase (NOS2) has multiple tissue damaging effects and is involved in the pathogenesis of inflammation and graft rejection. Haem oxygenase (HmOx) is the enzyme which degrades haem. Its inducible isoform, HmOx1, was recently shown to increase cellular resistance against oxidative stress and to decrease inflammation and graft rejection. Since haem is an essential cofactor for NOS2 activity, we investigated the effects of HmOx1-induction upon NO secretion in macrophages. We induced HmOx1 in BALB/c bone-marrow-derived macrophages by short-term exposure to haemin (20 mu mol/l, 30 min); then we incubated them for 24 h to allow maximal expression of HmOx1 activity. Next, we activated the macrophages with lipopolysaccharide (LPS) and measured their NO production and their NO-dependent cytotoxicity against P815 cells. We found that HmOx induction 24 h before LPS activation in mouse macrophages suppresses their production of NO, while HmOx inhibition (with zinc protoporphyrin) increases NO secretion. NOS2 inhibition is reflected by the decrease of macrophage NO-dependent cytotoxicity against the P815 targets. We therefore propose that HmOx1 is a physiological inhibitor of NOS2 in activated macrophages because it decreases haem availability for NOS2 synthesis. NOS2 inhibition may explain the antinflammatory effects of HmOx induction which could also be used therapeutically in situations when NO hyperproduction leads to cytotoxic effects such as inflammation or transplant rejection.