Carbon Monoxide Decreases Leukocytosis in Murine Sickle Cell Disease Models Via Decreased Granulopoiesis.

Abstract

Elevated white blood cell counts (WBC) in sickle cell disease (SCD) are risk factors for morbid vaso-occlusive events including acute chest syndrome and stroke. The mechanisms of leukocytosis in SCD include increased inflammation, oxidative stress, increased hematopoiesis, and hyposplenism. Heme oxygenase-1 (HO-1) plays critical roles in metabolizing the excess heme generated during hemolysis and in modulating vaso-occlusion in murine models of sickle cell disease (SCD). The products of HO-1 activity, carbon monoxide (CO), Fe2+/ferritin, and biliverdin/bilirubin have demonstrable anti-oxidant and anti-inflammatory effects. We have previously demonstrated that inhaled CO treatments decrease white blood cell counts (WBC), as well as liver redox-active iron and heme content in heterozygous BERK mice, a mouse model for sickle cell trait. To dissect the mechanism of this decrease in leukocytosis, we hypothesize that prolonged treatment with inhaled CO significantly decreases granulopoiesis in SCD mice. For this study, we exposed S+S-Antilles mice to 250 ppm inhaled CO for 1h 3X/week for 8–10 weeks. Upon completion of the treatment period animals were euthanized, blood was removed by cardiac puncture, and bone marrow and organs were harvested for analysis. Treatment for 10 weeks with 250 ppm CO significantly decreased total WBC (19.19±1.29 × 1000/ul (untreated) to 12.8±1.30 × 1000/ul (CO treated), p<0.05). The decrement in total WBC count was primarily due to a significant decrease in neutrophils (p<0.05) and lymphocytes (p<0.005), There was no significant change in the reticulocyte count, hematocrit, or bilirubin in CO-treated animals compared to controls. The spleens of the control and CO-treated animals had similar weights but no differences in their histopathology, ruling out the possibility of increased sequestration as a cause of the decrease in total WBC. Bone marrow staining reveals that CO-treated mice have a significant decrease in polymorphonuclear (PMN) precursors in bone marrow (p<0.05). Flow cytometry on bone marrow stained for hematopoietic markers CD117, CD45R/B220, SCA1, CD90.1, and CD135 demonstrates a significant (p<0.005) decrease in common lymphoid progenitor (CLP) and common myeloid progenitor (CMP) cells in CO-treated animals compared to controls. Flow cytometry for myeloperoxidase (MPO) and the granulocyte differentiation marker GR-1 (Ly-6G/6C) also demonstrates a significant (p<0.05) decrease in mature granulocytes cells in CO-treated mice. Colony-forming cell (CFC) assays verify the flow cytometry data, with a significant decrease (p<0.05) in CFU-GM in 250 ppm treated bone marrow compared to controls. Consistent with the lack of effect on hematocrit, CO had minimal effects on the erythroid marrow compartment since total CD71 positive erythroid cells were unchanged. In summary, we conclude that inhaled CO treatments decrease total WBC by decreasing granulopoiesis. 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.