Cytoadherence of Plasmodium falciparum-infected erythrocytes is mediated by a redox-dependent conformational fraction of CD36.

The adherence of Plasmodium falciparum-infected RBC (IRBC) to postcapillary venular endothelium is an important determinant of the pathogenesis of severe malaria complications. Cytoadherence of IRBC to endothelial cells involves specific receptor/ligand interactions. The glycoprotein CD36 expressed on endothelial cells is the major receptor involved in this interaction. Treatment of CD36-expressing cells with reducing agents, such as DTT and N-acetylcysteine, was followed by CD36 conformational change monitorable by the appearance of the Mo91 mAb epitope. Only a fraction of the surface expressed CD36 molecules became Mo91 positive, suggesting the presence of two subpopulations of molecules with different sensitivities to reduction. The Mo91 epitope has been localized on a peptide (residues 260-279) of the C-terminal, cysteine-rich region of CD36. Treatment with reducing agents inhibited the CD36-dependent cytoadherence of IRBC to CD36-expressing cells and dissolved pre-existent CD36-mediated IRBC/CD36-expressing cell aggregates. CD36 reduction did not impair the functionality of CD36, since the reactivity of other anti-CD36 mAbs as well as the binding of oxidized low density lipoprotein, a CD36 ligand, were maintained. The modifications induced by reduction were reversible. After 14 h CD36 was reoxidized, the cells did not express the Mo91 epitope, and cytoadherence to IRBC was restored. The results indicate that IRBCs bind only to a redox-modulated fraction of CD36 molecules expressed on the cell surface. The present data indicate the therapeutic potential of reducing agents, such as the nontoxic drug N-acetylcysteine, to prevent or treat malaria complications due to IRBC cytoadhesion.

Phagocytosis of malarial pigment haemozoin by human monocytes: a confocal microscopy study.

Haem from host erythrocyte (RBC) haemoglobin is polymerized in the digestive organelle of Plasmodium falciparum to haemozoin (HZ), a crystaLline, insoluble substance. Human monocytes avidly ingest HZ that persists undigested for long periods of time, and generates potent bioactive lipid peroxide derivatives. Protein kinase C, an effector of signal transduction, phagolysosome formation and acidification, is inhibited in HZ-fed monocytes. Inability to digest HZ might derive from impairment in phagolysosome formation or acidification. Time-course and extent of HZ phagocytosis and acidification of phagolysosomes were studied by quantitative confocal microscopy. From 180 min until 72 h after the start of phagocytosis approximately 75-79% of the monocytes contained massive amounts of HZ. Coincidence between red (HZ) and green (acidic organelles) fluorescent compartments was very high. Confocal images showed that at 30-60 min after the start of phagocytosis, HZ was preferentially present as separated particles, with full co-localization of red and green fluorescence. Later on HZ-laden phagolysosomes tended to fuse together. In conclusion, phagolysosome formation and acidification were normal in HZ-fed monocytes during the 72-h observation time. The presence of HZ in the phagolysosome, the site of antigen processing, may offer a physical link with immunodepression in malaria.

Growth of Plasmodium falciparum induces stage-dependent haemichrome formation, oxidative aggregation of band 3, membrane deposition of complement and antibodies, and phagocytosis of parasitized erythrocytes.

Plasmodium falciparum-parasitized erythrocytes (RBCs) are progressively transformed into non-self cells, phagocytosed by human monocytes. Haemichromes, aggregated band 3 (Bd3) and membrane-bound complement fragment C3c and IgG were assayed in serum-opsonized stage-separated parasitized RBCs. All parameters progressed from control to rings to trophozoites to schizonts: haemichromes, nil; 0.64 +/- 0.12; 5.6 +/- 1.91; 8.4 +/- 2.8 (nmol/ml membrane); Bd3, 1 +/- 0.1; 4.3 +/- 1.5; 23 +/- 5; 25 +/- 6 (percentage aggregated); C3c, 31 +/- 11; 223 +/- 86; 446 +/- 157; 620 +/- 120 (mOD405/min/ml membrane); IgG, 35 +/- 12; 65 +/- 23; 436 +/- 127; 590 +/- 196 (mOD405/min/ml membrane). All increments in rings versus controls and in trophozoites versus rings were highly significant. Parasite development in the presence of 100 micromol/l beta-mercaptoethanol largely reverted haemichrome formation, Bd3 aggregation, C3c and IgG deposition and phagocytosis. Membrane proteins extracted by detergent C12E8 were separated on Sepharose CL-6B. Haemichromes, C3c and IgG were present exclusively in the high-molecular-weight fractions together with approximately 30% of Bd3, indicating the oxidative formation of immunogenic Bd3 aggregates. Immunoblots of separated membrane proteins with anti-Bd3 antibodies confirmed Bd3 aggregates that, in part, did not enter the gel. Immunoprecipitated antibodies eluted from trophozoites reacted preferentially with aggregated Bd3. Changes in parasitized RBC membranes and induction of phagocytosis were similar to oxidatively damaged, senescent or thalassaemic RBC, indicating that parasite-induced oxidative modifications of Bd3 were per se sufficient to induce and enhance phagocytosis of malaria-parasitized RBC.

Methionine 156 in the immunodominant domain of CD36 contributes to define the epitope recognized by the NL07 MoAb.

CD36 is a membrane glycoprotein expressed by several cell types, and play a role as a receptor for different physiological and pathological ligands. An immunodominant domain of CD36 has been described in the amino acidic region 155-183, where many ligands and monoclonal antibodies (MoAbs) react. MoAbs against CD36 have proved useful in structural as well as functional studies. One of these antibodies, MoAb NL07, recognizes a conformational epitope that is acquired in the late steps of the CD36 maturation. The NL07 epitope appears to be functionally relevant and blocks CD36-mediated binding to red blood cells infected with the malaria parasite Plasmodium falciparum (IRBC). In this work a mutant COS-7 clone expressing NL07-negative CD36 molecules on the cell surface was investigated. In the mutant, the methionine in position 156 of the wild type CD36 sequence was replaced by a valine. It was determined that methionine 156 was essential for NL07 reactivity, mapping the NL07 epitope to the vicinity of the functionally important immunodominant domain (aa 155-183) of CD36. Although methionine 156 is located in this region, the CD36V156 mutated molecule was apparently functional and able to bind IRBC and oxidized LDL.

Hemozoin stability and dormant induction of heme oxygenase in hemozoin-fed human monocytes.

Human monocytes avidly ingest malarial pigment, hemozoin. Phagocytosed hemozoin persists in the monocyte for a long time and modifies important monocyte functions. Stability of phagocytosed hemozoin may depend on modifications of the hemozoin heme moiety or reduced ability to express heme-inducible heme oxygenase. We show here that the spectral characteristics of alkali-solubilized hemozoin were identical to those of authentic heme, although hemozoin was solubilized by alkali much more slowly than authentic heme. Alkali-solubilized hemozoin was a substrate of microsomal rat heme oxygenase and bilirubin reductase, with bilirubin as the main final product. Hemozoin feeding to human monocytes did not induce heme oxygenase, but authentic heme and alkali-solubilized hemozoin supplemented to hemozoin-fed monocytes induced heme oxygenase and were degraded normally. Lysosomes isolated from hemozoin-fed monocytes released only traces of heme while lysosomes from erythrocyte-fed monocytes liberated considerable quantities of heme. Lack of heme release from hemozoin did not depend on proteolysis-resistant, heme-binding proteins, since lysosomal proteases fully degraded hemozoin-associated proteins but did not solubilize hemozoin. In conclusion, our data indicate that lack of induction of HO1 is due to the intrinsic structural characteristics of hemozoin and not to hemozoin-mediated impairment of the mechanism of HO1 induction.

Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum may explain malaria protection in G6PD deficiency.

In population-based studies it has been established that inherited deficiency of erythrocyte (E) glucose-6-phosphate dehydrogenase (G6PD) confers protection against severe Plasmodium falciparum (P falciparum) malaria. Impaired growth of parasites in G6PD-deficient E in vitro has been reported in some studies, but not in others. In a systematic analysis, we have found that with five different strains of P falciparum (FCR-3, KI, C10, HB3B, and T9/96), there was no significant difference in either invasion or maturation when the parasites were grown in either normal or G6PD-deficient (Mediterranean variant) E. With all of these strains and at different maturation stages, we were unable to detect any difference in the amount of P falciparum-specific G6PD mRNA in normal versus deficient parasitized E. The rate of 14C-CO2 production from D-[1-14C] glucose (which closely reflects intracellular activity of G6PD) contributed by the parasite was very similar in intact normal and deficient E. By contrast, in studies of phagocytosis of parasitized E by human adherent monocytes, we found that when the parasites were at the ring stage (ring-stage parasitized E [RPE]), deficient RPE were phagocytosed 2.3 times more intensely than normal RPE (P = .001), whereas there was no difference when the parasites were at the more mature trophozoite stage (trophozoite-stage parasitized E [TPE]). Phagocytic removal markers (autologous IgG and complement C3 fragments) were significantly higher in deficient RPE than in normal RPE, while they were very similar in normal and deficient TPE. The level of reduced glutathione was remarkably lower in deficient RPE compared with normal RPE. We conclude that impaired antioxidant defense in deficient RPE may be responsible for membrane damage followed by phagocytosis. Because RPE, unlike TPE, are nontoxic to phagocytes, the increased removal by phagocytosis of RPE would reduce maturation to TPE and to schizonts and may be a highly efficient mechanism of malaria resistance in deficient subjects.

Phagocytosis of the malarial pigment, hemozoin, impairs expression of major histocompatibility complex class II antigen, CD54, and CD11c in human monocytes.

In Plasmodium falciparum malaria, large proportions of resident macrophages and circulating monocytes and leukocytes contain massive amounts of the malarial pigment, hemozoin. Previous studies have shown that important functions (e.g., the generation of the oxidative burst, the ability to repeat phagocytosis, and protein kinase C activity) were severely impaired in hemozoin-loaded monocytes. Expression of membrane antigens directly involved in the immune response and in the phagocytic process, and/or under protein kinase C control, in hemozoin-loaded human monocytes was studied. Expression of major histocompatibility complex (MHC) class II after gamma interferon stimulation was blocked in hemozoin-loaded monocytes at the protein expression and gene transcription levels but was preserved in control monocytes loaded with opsonized latex beads or anti-D(Rho)-immunoglobulin G (IgG)-opsonized human erythrocytes. Expression of CD54 (intracellular adhesion molecule 1) and CD11c (p150,95 integrin) was also decreased in hemozoin-loaded monocytes. Expression of MHC class I, CD16 (low-affinity Fc receptor for aggregated IgG), CD32 (low-affinity Fc receptor for aggregated IgG), CD64 (high-affinity receptor for IgG), CD11b (receptor for complement component iC3b [CR3]), CD35 (receptor for complement components C3b and C4b [CR1]), and CD36 (non-class-A scavenger receptor) was not specifically affected by hemozoin loading. These results suggest that hemozoin loading may contribute to the impairment of the immune response and the derangement of antigen presentation reported in previous studies of P. falciparum malaria.

Erythrocyte stages of Plasmodium falciparum exhibit a high nitric oxide synthase (NOS) activity and release an NOS-inducing soluble factor.

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.

Binding of naturally occurring antibodies to oxidatively and nonoxidatively modified erythrocyte band 3.

Both oxidative clustering (elicited by diamide treatment) and nonoxidative clustering (elicited by zinc/BS3 (bis[sulfosuccinimidyl]suberate) treatment) of erythrocyte integral membrane proteins induce binding of autologous antibodies with anti-band 3 specificity, followed by complement deposition and phagocytosis. Autologous antibodies eluted from nonoxidatively clustered erythrocytes bind to and stimulate phagocytosis of oxidatively damaged erythrocytes. Those eluted antibodies bind specifically to disulfide-crosslinked band 3 dimers generated by diamide treatment. Band 3 dimerization and antibody binding are abrogated by cleavage of band 3 cytoplasmic domain. Thus, disulfide-crosslinked band 3 dimers are the minimal band 3 aggregate with enhanced affinity for anti-band 3 antibodies. The eluted antibodies do not bind to band 3 dimers generated nonoxidatively by BS3 treatment but bind avidly to larger band 3 clusters generated nonoxidatively by zinc/BS3 treatment. Possibly, disulfide crosslinking of cytoplasmic domain cysteines induces reorientation of intramembrane domains as to expose putative anti-band 3 epitopes and allow bivalent binding of anti-band 3 antibodies. Extensive nonoxidative band 3 clustering appears to disrupt the native band 3 conformation and generate reoriented dimers which expose putative anti-band 3 epitopes in the proper distance and orientation as to allow bivalent antibody binding.

Impairment of macrophage functions after ingestion of Plasmodium falciparum-infected erythrocytes or isolated malarial pigment.

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.