Inhaled Carbon Monoxide: An Anti-Inflammatory Modulator in Transgenic Sickle Mice.

Abstract

Inflammation plays a prominent role in vaso-occlusion and organ pathology in sickle cell disease (SCD). We have shown that heme oxygenase-1 (HO-1) plays a critical role in metabolizing the excess heme generated in this hemolytic disease, thereby modulating hypoxia/reoxygenation-induced vaso-occlusion in murine models of SCD. The products of HO-1 activity, carbon monoxide (CO), Fe2+ (ultimately incorporated into ferritin), and biliverdin/bilirubin have demonstrable anti-oxidant and anti-inflammatory effects. We have observed that brief (1 h/d × 3 d) inhalation of CO in SCD mice modulates vaso-occlusion (JCI , 116: 808–816; 2006). We hypothesize that prolonged treatment with inhaled CO will significantly decrease the inflammatory phenotype of SCD mice. Starting at three weeks of age, C57BL-6J and heterozygous BERK mice (mβ/hβS) were treated with inhaled CO at 0, 25, or 250 ppm (1 h/d × 3 d/wk × 10 wks). Over the duration of the ten week study no animals died as result of the CO treatments. Upon completion of the ten week treatment period animals were euthanized, blood was removed by cardiac puncture, and organs were harvested and homogenized. Carboxyhemoglobin levels immediately after 1h of CO treatment, (0, 25, and 250 ppm) were <1%,<1%, and 12%, respectively, but decreased to normal 24 h later. Untreated heterozygous BERK mice had a higher mean white blood cell count (WBC)(14,400/μL) than C57BL-6J mice (9,469/ μL, p<0.005). Treatment for 10 weeks with either 25 or 250 ppm CO significantly decreased heterozygous BERK WBC (8827 and 9222/μl, p<0.02), but had no effect on C57BL-6J WBC. The decrement in heterozygous BERK WBC was primarily due to a significant decrease in neutrophils (p<0.05). There was no significant difference in either the mean hematocrits or reticulocyte counts in CO-treated mice. Since redox-active iron (Fe2+) promotes oxidative stress and inflammation, we measured redox-active iron with Ferene-S in liver homogenates. In untreated heterozygous BERK mice, redox-active iron was two-fold greater than C57BL-6J mice (p<0.05) and treatment with 25 or 250 ppm CO significantly (p<0.01, p<0.02 respectively) decreased the redox-active iron (Fe2+) to levels comparable to untreated C57BL-6J. Since there is red cell congestion in heterozygous BERK livers, it was not surprising that the heme-content of untreated heterozygous BERK mice was three-fold (p<0.05) higher than untreated C57BL-6J. Treatment with 25 or 250 ppm CO significantly (p<0.05) decreased the heme-content of the livers to levels comparable to untreated C57BL-6J. The cytoprotective proteins HO-1 and ferritin heavy-chain were significantly increased in the livers of untreated heterozygous BERK mice compared to C57BL-6J (p<0.02). However, CO treatments had no significant effects on expression of these proteins on Western blots. We conclude that inhaled CO treatments decrease WBC, neutrophils, as well as liver redox-active iron and heme content in heterozygous BERK mice. Further studies are evaluating the effects of CO on inflammation, vaso-occlusion, and organ pathology in the homozygous BERK mouse model. We speculate that inhaled CO treatments may be a potential therapy for patients with SCD by acting as a modulator of oxidative stress and inflammation.