Impairment of neutrophil oxidative burst in children with sickle cell disease is associated with heme oxygenase-1

Abstract

Sickle cell disease is a risk factor for invasive bacterial infections, and splenic dysfunction is believed to be the main underlying cause. We have previously shown that the liberation of heme in acute hemolysis can induce heme oxygenase-1 during granulopoiesis, impairing the ability of developing neutrophils to mount a bactericidal oxidative burst, and increasing susceptibility to bacterial infection. We hypothesized that this may also occur with the chronic hemolysis of sickle cell disease, potentially contributing to susceptibility to infections. We found that neutrophil oxidative burst activity was significantly lower in treatment-naïve children with sickle cell disease compared to age-, gender- and ethnicity-matched controls, whilst degranulation was similar. The defect in neutrophil oxidative burst was quantitatively related to both systemic heme oxygenase-1 activity (assessed by carboxyhemoglobin concentration) and neutrophil mobilization. A distinct population of heme oxygenase-1-expressing cells was present in the bone marrow of children with sickle cell disease, but not in healthy children, with a surface marker profile consistent with neutrophil progenitors (CD49d(Hi) CD24(Lo) CD15(Int) CD16(Int) CD11b(+/-)). Incubation of promyelocytic HL-60 cells with the heme oxygenase-1 substrate and inducer, hemin, demonstrated that heme oxygenase-1 induction during neutrophilic differentiation could reduce oxidative burst capacity. These findings indicate that impairment of neutrophil oxidative burst activity in sickle cell disease is associated with hemolysis and heme oxygenase-1 expression. Neutrophil dysfunction might contribute to risk of infection in sickle cell disease, and measurement of neutrophil oxidative burst might be used to identify patients at greatest risk of infection, who might benefit from enhanced prophylaxis.

Figure 1.

Neutrophil oxidative burst is impaired in children with sickle cell disease. (A) Representative histogram of rhodamine fluorescence intensity of unstimulated (dark fill) and PMA-stimulated (light fill) neutrophils from a child with sickle cell disease. (B–D) Comparison of unstimulated rhodamine fluorescence intensity (B), PMA-stimulated rhodamine fluorescence intensity (C), and PMA-stimulated upregulation of surface CD11b expression (D), in neutrophils of healthy control (n=25) and sickle cell disease subjects (n=26). The Mann-Witney test was used for all comparisons.

Figure 2.

Interacting effects of hemolysis, heme-oxygenase-1 (HO-1) induction, and neutrophil mobilization, on neutrophil oxidative burst. (A) Working model of the interactions between hemolysis, HO-1 induction, and neutrophil oxidative burst. Hemolysis has three interacting effects: stimulating the oxidative burst of mature neutrophils; inducing HO-1 expression in neutrophil progenitors in bone marrow (BM) which leads to impaired oxidative burst capacity during neutrophil maturation; and mobilization of neutrophils from BM to the blood. (B) Interaction between measured carboxyhemoglobin % (COHb, an indicator of hemolysis and HO-1 activity), neutrophil count in peripheral blood, and PMA-stimulated oxidative burst (rhodamine MFI) of neutrophils in patients with sickle cell disease. The fitted surface shows the predicted oxidative burst response (using a generalized linear model), and overlying black dots show individual patient data (n=20). (C) Predicted change in PMA-stimulated oxidative burst with neutrophil count at different levels of COHb [(representing the 25^th (dashed line), 50^th (dotted line), and 75^th centiles (dash-dot line) in subjects with sickle cell disease)]. The horizontal line represents the lowest value observed in control subjects.

Figure 3.

Heme oxygenase-1 (HO-1) is not induced in mature human neutrophils by hemin treatment or hemolysis (A) Illustrative flow cytometry analysis of the effect of incubation of human whole blood with varying concentrations of hemin for 18 h on expression of HO-1 in monocytes and neutrophils. Results shown from one healthy adult donor, representative of 5 independent experiments. (B) HO-1 expression in neutrophils of healthy control subjects (n=3) compared with subjects with sickle cell disease (n=5). The Mann-Witney test was used for comparison.

Figure 4.

Flow-cytometry analysis of heme oxygenase-1 (HO-1) expression in bone marrow (BM) from subjects with sickle cell disease and healthy donors. (A) Comparison of staining with anti-HO-1 antibody and control antibody in subjects with sickle cell disease (n=2) and healthy child BM donors (n=2, one HbAS, one HbAA), plotted against CD16 expression as a marker of neutrophil maturation. The gated population is CD16 intermediate, and is proposed to represent developing neutrophils. (B) Expression of surface markers which define the maturational stage of developing neutrophils in BM. For each subject with sickle cell disease, the expression of surface markers on the HO-1⁺CD16^intermediate population (gated in A) is presented in comparison with the expression of the same markers in total BM (upper panels). The HO-1⁺ cells express high levels of CD49d, low levels of CD24, intermediate-to-high levels of CD11b, and intermediate levels of CD15, suggesting they are around the point of transition from proliferating to non-proliferating neutrophil progenitors.

Figure 5.

Heme oxygenase-1 (HO-1) induction during maturation impairs the oxidative burst of neutrophil-like differentiated HL60 cells (A) Comparison of HO-1 expression in the promyelocytic HL-60 cell line incubated with or without the HO-1 substrate and inducer, hemin (50 μM), for five days in the presence or absence of 10⁻⁷ M all-trans retinoic acid (ATRA) (n=3 per group), which stimulates differentiation into neutrophil-like cells. (B) Representative morphology of hematoxylin and eosin stained HL-60 cells: (i) undifferentiated; (ii) differentiated cells following five days with 10⁻⁷ M ATRA; (iii) undifferentiated, hemin treated; (iv) differentiated, hemin treated. (C) Comparison of PMA-stimulated oxidative burst of HL-60 cells, undifferentiated or differentiated into neutrophil-like cells in the presence or absence of hemin (50 μM) (n=3 per group). Results representative of 3 independent experiments. Images were obtained at ×60 magnification using a Nikon Eclipse 50i microscope, all cells shown at the same scale. Statistical comparison was performed using unpaired t-test.