Elevated nitric oxide production in children with malarial anemia: hemozoin-induced nitric oxide synthase type 2 transcripts and nitric oxide in blood mononuclear cells

Abstract

Experiments outlined here investigate the role of nitric oxide (NO) in the pathogenesis of Plasmodium falciparum-induced malarial anemia (MA). The results show that ex vivo and in vitro NO synthase (NOS) activity in peripheral blood mononuclear cells (PBMCs) is significantly elevated in children with MA and inversely associated with hemoglobin levels. Additional experiments using PBMCs from non-malaria-exposed donors demonstrate that physiologic amounts of P. falciparum-derived hemozoin augment NOS type 2 (NOS2) transcripts and NO production. Results of these experiments illustrate that elevated NO production in children with MA is associated with decreased hemoglobin concentrations and that hemozoin can induce NOS2-derived NO formation in cultured blood mononuclear cells.

FIG. 1.

NOS enzyme activity in ex vivo PBMCs. Venous blood (3 ml) was obtained and PBMCs were collected from healthy Gabonese children (HC; n = 26) and children with mild malaria (MM; n = 19) or severe malaria (SM; n = 14). Cell lysates were prepared, and ex vivo NOS enzyme activity (in picomoles of citrulline per milligram of protein) was determined by measuring the conversion of l-[¹⁴C]arginine to l-[¹⁴C]citrulline. The graph shows the means ± the standard error of the means (SEM) of results from each of the groups. Statistical significance was determined by the Mann-Whitney U test. *, P value of <0.01 compared to results for HC.

FIG. 2.

Association of NOS enzyme activity with Hb. Venous blood (3 ml) was obtained and PBMCs were collected from children with malaria (n = 13), and cell lysates were prepared for ex vivo NOS enzyme activity determination (in picomoles of citrulline per milligram of protein). Hb was measured with a Hemocue. Regression analysis was used to examine the relationship between NOS enzyme activity and Hb levels.

FIG. 3.

NOS enzyme activity in cultured PBMCs. Venous blood (3 ml) was obtained and PBMCs were collected from healthy Gabonese children (HC; n = 26) and children with malaria (MAL; n = 14). PBMCs (10⁶ cells/ml) were cultured for 7 days with medium alone (control [Con]) or LPS (100 ng/ml) and IFN-γ (200 U/ml). Cell lysates were prepared from cultured PBMCs, and NOS enzyme activity (in picomoles of citrulline per milligram of protein) was determined. The graph shows means ± SEM of results for each of the groups. Statistical significance was determined by the Mann-Whitney U test. *, P value of <0.05 compared to results for HC; **, P value of <0.01 compared to results with unstimulated PBMCs from children with malaria.

FIG. 4.

Effect of hemozoin on NO_x production in cultured PBMCs. Venous blood (40 ml) was obtained from healthy, malaria-naïve U.S. donors, and PBMCs were isolated. Cultured PBMCs (10⁶ cells/ml) were stimulated with medium alone, LPS (100 ng/ml) and IFN-γ (200 U/ml), or LPS and IFN-γ in the presence of hemozoin (10, 1.0, or 0.1 μg/ml). The NO_x concentration in culture supernatants was determined at 48 h via the Griess reaction by previously defined methods (19). Values shown are the means ± SEM of results from 10 subjects. Statistical significance was determined by the Mann-Whitney U test. *, P value of <0.05 compared to results for LPS+IFN-γ stimulation conditions.

FIG. 5.

Effect of hemozoin on NOS2 mRNA expression. NOS2 mRNA was quantified for three individuals by real-time RT-PCR. PBMCs (10⁶ cells/ml) were obtained from healthy, malaria-naïve U.S. donors and cultured with medium alone, LPS (100 ng/ml) and IFN-γ (200 U/ml), or LPS and IFN-γ in the presence of hemozoin (10, 1.0, or 0.1 μg/ml). Cells were collected at 48 h for NOS2 mRNA determination by real-time RT-PCR. Data were compared using the ΔΔC_T method, where the endogenous control gene (β-actin) cycle threshold (C_T) value was subtracted from the experimental gene (NOS2) C_T for each sample. The change in C_T (ΔC_T) for each experimental sample was then subtracted from the ΔC_T of the unstimulated (medium alone) control sample. Change (n-fold) was expressed as 2^−ΔΔC_T relative to results from unstimulated conditions. Values are the means ± SEMs of results from three samples. Statistical significance was determined by the Mann-Whitney U test. *, P value of <0.05 compared to results for LPS+IFN-γ stimulation conditions.