Serum of sickle cell disease patients contains fetal hemoglobin silencing factors secreted from leukocytes

Figures & data

Figure 1 HbF response to HU is associated with hematological improvements in SCD patients.

Notes: Increases in HbF levels after HU therapy correlate with increases in total hemoglobin levels (A), MCV (B), and MCHC (C). Correlations were analyzed with 82 SCD patients who received HU therapy.
Abbreviations: HbF, fetal hemoglobin; HU, hydroxyurea; SCD, sickle cell disease.

Figure 2 There were no correlations between HU-induced HbF increases and reductions in the number of reticulocytes (A) and platelets (B) among the SCD patients who received HU therapy.

Abbreviations: HU, hydroxyurea; HbF, fetal hemoglobin; SCD, sickle cell disease.

Figure 3 Correlations between the levels of HbF increases upon HU therapy and pre-HU therapy leukocyte counts (A), post-HU therapy leukocyte counts (B), or pre-HU therapy HbF levels (C). Eighty-two SCD patients who received HU therapy were analyzed.

Abbreviations: HU, hydroxyurea; HbF, fetal hemoglobin; SCD, sickle cell disease.

Figure 4 Correlations between neutrophil (A) or lymphocyte/monocyte counts (B) and HU-induced HbF levels.

Note: Eighty-two SCD patients who received HU therapy were analyzed.
Abbreviations: HU, hydroxyurea; HbF, fetal hemoglobin; SCD, sickle cell disease.

Figure 5 Presence of HbF silencing factors in serum of SCD patients.

Notes: (A) Culture of CD34⁺-derived erythroblasts with 30% FBS (left panel) or 30% human AB serum (right panel). Cell photographs were taken on day 10 using EVOS FL Cell Imaging System. White bars indicate 400 µM. (B) Analysis of CD34⁺-derived erythroblasts that were harvested on day 14 by flow cytometry using anti-glycophorin A and anti-CD71 antibodies. (C) Immunoblotting analysis of γ-globin and GAPDH in whole cell lysates prepared from CD34⁺-derived erythroblasts. Lanes: 1, CD34⁺-derived erythroblasts cultured with 30% FBS; 2, CD34⁺-derived erythroblasts cultured with 30% human AB serum; 3 and 4, CD34⁺-derived erythroblasts cultured with 30% serum from steady-state SCD patients (Pt.1 and 2); 5, CD34⁺-derived erythroblasts cultured with 30% serum from SCD patients with HU therapy; 6, CD34⁺-derived erythroblasts cultured with 30% serum from SCD patients with HU therapy that was incubated with anti-GM-CSF antibody as stated in Materials and methods. GAPDH was used for internal control. (D) Quantitation of γ-globin mRNA levels in CD34⁺-derived erythroblasts cultured in (C) by RT-PCR. Lane designation is the same as that of (C). The γ-globin mRNA level of CD34⁺-derived erythroblasts cultured with 30% FBS was set to 100%. *P<0.05 compared to lane 1. **P<0.01 compared to lane 1.
Abbreviations: HbF, fetal hemoglobin; SCD, sickle cell disease; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HU, hydroxyurea; GM-CSF, granulocyte-macrophage colony-stimulating factor; RT-PCR, real-time PCR.

Figure 6 Possible model for the mechanisms that regulate HU-induced HbF expression.

Notes: HU has been shown to stimulate HbF expression in erythroid cells through the nitric oxide/sGC/cGMP pathway.^33–35 HU is expected to suppress the proliferation of leukocytes by inhibiting ribonucleotide reductase,³² which in turn inhibits the production of HbF silencing factors.
Abbreviations: HU, hydroxyurea; HbF, fetal hemoglobin; sGC, soluble guanylate cyclase.

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