Human monocyte-derived macrophages ingest diamide-treated red blood cells (RBC), anti-D immunoglobulin (Ig)G-opsonized RBC, or Plasmodium falciparum ring-stage parasitized RBC (RPRBC), degrade ingested hemoglobin rapidly, and can repeat the phagocytic cycle. Monocytes fed with trophozoite-parasitized RBC (TPRBC), which contain malarial pigment, or fed with isolated pigment are virtually unable to degrade the ingested material and to repeat the phagocytic cycle. Monocytes fed with pigment display a long-lasting oxidative burst that does not occur when they phagocytose diamide-treated RBC or RPRBC. The phorbol myristate acetate-elicited oxidative burst is irreversibly suppressed in monocytes fed with TPRBC or pigment, but not in monocytes fed with diamide-treated or IgG-opsonized RBC. This pattern of inhibition of phagocytosis and oxidative burst suggests that malarial pigment is responsible for the toxic effects. Pigment iron released in the monocyte phagolysosome may be the responsible element. 3% of total pigment iron is labile and easily detached under conditions simulating the internal environment of the phagolysosome, i.e., pH 5.5 and 10 microM H2O2. Iron liberated from pigment could account for the lipid peroxidation and increased production of malondialdehyde observed in monocytes fed with pigment or in RBC ghosts and liposomes incubated at pH 6.5 in presence of pigment and low amounts of H2O2. Removal of the labile iron fraction from pigment by repeated treatments with 0.1 mM H2O2 at pH 5.5 reduces pigment toxicity. It is suggested that iron released from ingested pigment is responsible for the intoxication of monocytes. In acute and chronic falciparum infections, circulating and tissue-resident phagocytes are seen filled with TPRBC and pigment particles over long periods of time. Moreover, human monocytes previously fed with TPRBC are unable to neutralize pathogenic bacteria, fungi, and tumor cells, and macrophage responses decline during the course of human and animal malaria. The present results may offer a mechanistic explanation for depression of cellular immunity in malaria.

Oxidative events involving band 3 (Anion Exchanger 1) have been associated with RBC (red blood cell) removal through binding of NAbs (naturally occurring antibodies); however, the underlying mechanism has been only partially characterized. In addition to inducing direct membrane protein oxidative modification, oxidative treatment specifically triggers the phosphorylation of band 3 tyrosine residues. The present study reports that diamide, a thiol group oxidant, induces disulfide cross-linking of poorly glycosylated band 3 and that the oligomerized band 3 fraction is selectively tyrosine phosphorylated both in G6PD (glucose-6-phosphate dehydrogenase)-deficient and control RBCs. This phenomenon is irreversible in G6PD-deficient RBCs, whereas it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. Diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size-exclusion separation of Triton X-100-extracted membrane proteins, Syk was found only in the high-molecular-mass fraction containing oligomerized/phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 tyrosine phosphorylation, band 3 clustering and NAbs binding to the RBC surface, suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS³ (bis-sulfosuccinimidyl-suberate) showed that band 3 tyrosine phosphorylation enhances its capability to form large aggregates. The results of the present study suggest that selective tyrosine phosphorylation of oxidized band 3 by Syk may play a role in the recruitment of oxidized band 3 in large membrane aggregates that show a high affinity to NAbs, leading to RBC removal from the circulation.

The British Journal of Haematology publishes original research papers in clinical, laboratory and experimental haematology. The Journal also features annotations, reviews, short reports, images in haematology and Letters to the Editor.

Nitric oxide (NO), a highly diffusible cellular mediator involved in a wide range of biological effects, has been indicated as one of the cytotoxic agents released by leukocytes to counteract malaria infection. On the other hand, NO has been implicated as a mediator of the neuropathological symptoms of cerebral malaria. In such circumstances NO production has been thought to be induced in host tissues by host-derived cytokines. Here we provide evidence for the first time that human red blood cells infected by Plasmodium falciparum (IRBC) synthesize NO. The synthesis of NO (measured as citrulline and nitrate production) appeared to be very high in comparison with human endothelial cells; no citrulline and nitrate production was detectable in noninfected red blood cells. The NO synthase (NOS) activity was very high in the lysate of IRBC (while not measurable in that of normal red blood cells) and was inhibited in a dose-dependent way by three different NOS inhibitors (L-canavanine, NG-amino-L-arginine, and NG-nitro-L-arginine). NOS activity in P. falciparum IRBC is Ca++ independent, and the enzyme shows an apparent molecular mass < 100 kD, suggesting that the parasite expresses an isoform different from those found in mammalian cells. IRBC release a soluble factor able to induce NOS in human endothelial cells. Such NOS-inducing activity is not tissue specific, is time and dose dependent, requires de novo protein synthesis, and is probably associated with a thermolabile protein having a molecular mass > 100 kD. Our data suggest that an increased NO synthesis in P. falciparum malaria can be directly elicited by soluble factor(s) by the blood stages of the parasite, without necessarily requiring the intervention of host cytokines.