16alpha-bromoepiandrosterone, an antimalarial analogue of the hormone dehydroepiandrosterone, enhances phagocytosis of ring stage parasitized erythrocytes: a novel mechanism for antimalarial activity

Abstract

Dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S), which are the most abundant hormones secreted by the adrenal cortex and are present in plasma at approximately 6 micro M, as well as their analogue, 16alpha-bromoepiandrosterone (EPI), exerted antimalarial activities against two chloroquine-sensitive Plasmodium falciparum strains (Palo Alto, 50% inhibitory concentration [IC(50)] of EPI, 4.8 +/- 0.68 micro M; T996/86, IC(50) of EPI, 7.5 +/- 0.91 micro M, and IC(50) of DHEA-S, 19 +/- 2.6 micro M) and one mildly chloroquine-resistant strain (FCR-3, IC(50) of EPI, 6.5 +/- 1.01 micro M). Both EPI and DHEA/DHEA-S are potent inhibitors of glucose-6-phosphate dehydrogenase (G6PD), and G6PD deficiency is known to exert antimalaria protection via enhanced opsonization and phagocytosis of rings, the early forms of the parasite. Plasma-compatible antimalarial EPI concentrations did not inhibit G6PD activity and did not induce ring opsonization by immunoglobulin G and complement fragments, as observed in G6PD deficiency, but nevertheless remarkably stimulated ring phagocytosis. Plasma-compatible, low-micromolar concentrations of EPI induced exposure on the ring surface of phosphatidylserine, a signal for phagocytic removal independent of opsonization. We propose that enhanced ring phagocytosis due to exposure of negatively charged membrane phospholipids may explain the antimalarial activity of EPI.

Figures

FIG. 1.

Assay of basal and methylene blue-stimulated PPP flux in nonparasitized RBC treated or not with EPI (0.8 to 80 μM final concentration). After a 90-min preincubation at 37°C with and without EPI, PPP flux was measured (for details, see Materials and Methods). Methylene blue was addded at a final concentration of 150 μM. Data (mean values ± standard deviation [vertical bars] of four different experiments) are expressed as production of 14CO2 from d-[1-14C]glucose in counts per s (CPS)/10 μl of packed RBC. The significance of differences is relative to controls.For details, see Materials and Methods.

FIG. 2.

Assay of phagocytosis by human monocytes of rings (A), trophozoites (B), and nonparasitized RBC (A and B) treated or not with EPI (0.5 to 10 μM final concentration). Stage-separated parasitized (Palo Alto strain) and nonparasitized RBC were suspended in RPMI (pH 7.4) at a 10% hematocrit. After a 90-min preincubation at 37°C with and without EPI, phagocytosis by human monocytes was measured as detailed in Materials and Methods. Data (mean values ± standard deviation [vertical bars] of four different experiments per condition) are expressed as the number of ingested cells per monocyte. Data were corrected for nonparasitized RBC as detailed in Materials and Methods. (A) Significance of differences is relative to untreated rings. (B) None of the differences between EPI-treated and untreated trophozoites was statistically significant.

FIG. 3.

Binding of autologous IgG (A) and complement fragment C3c (B) to rings and nonparasitized RBC treated or not with EPI (1 to 2 μM final concentration). Ring stage parasitized (Palo Alto strain) and nonparasitized RBC were suspended in RPMI 1640 (pH 7.4) at a 10% hematocrit. After a 90-min preincubation at 37°C with and without EPI, binding of IgG (A) and complement fragment C3c (B) was measured as detailed in Materials and Methods. Data (mean values ± standard deviation [vertical bars] of three different experiments per each condition) are expressed as alkaline phosphatase activity of second antibodies given in milliunits of optical density at 405 nm (mOD405) per minute per milliliter of membranes. Data were corrected for nonparasitized RBC as detailed in Materials and Methods. None of the differences between treated and untreated rings was statistically significant.

FIG. 4.

Effect of EPI on cell-surface exposure of PS as measured by FACS. Shown are the results of FACS analysis of EPI (2 μM final concentration)-treated (open areas) or untreated (shaded areas) nonparasitized RBC (A), rings (B), and trophozoites (C) stained with FITC-annexin V. The data are representative of three independent experiments, all showing similar results.