Franco Turrini

Band 3/complement-mediated recognition and removal of normally senescent and pathological human erythrocytes.

Band 3 modifications that normally occur during physiological red blood cell (RBC) senescence in humans, and occasionally in pathological conditions are described in the context of their role in enhancing RBC recognition and phagocytic removal. Band 3 modifications are mostly due to oxidative insults that gradually accumulate during the RBC lifespan or impact massively in a shorter time period in pathological conditions. The oxidative insults that impact on the RBC, the protective mechanisms that counteract those damages and the phenotypic modifications that accumulate during the RBC lifespan are described. It is shown how specific oxidative as well as non-oxidative band 3 modifications enhance RBC membrane affinity for normally circulating anti-band 3 antibodies, and how membrane-bound anti-band 3 antibodies bring about a limited complement activation and membrane deposition of complement C3 fragments. The partially covalent complexes between anti-band 3 antibodies and complement C3 fragments are very powerful opsonins readily recognized by the CR1 complement receptor on the phagocyte. Band 3 modifications typically encountered in old RBCs have crystallized to a number of band 3-centered models of RBC senescence. One of those band 3-centered models, the so-called ‘band 3/complement RBC removal model’ first put up by Lutz et al. is discussed in more detail. Finally, it is shown how the genetic deficiency of glucose-6-phosphate dehydrogenase (G6PD) plus fava bean consumption, and a widespread RBC parasitic disease, P. falciparum malaria, may lead to massive and rapid destruction of RBCs by a mechanism comparable to a dramatic, time-compressed enhancement of normal RBC senescence.

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 μmol/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.

Naturally occurring anti-band 3 antibodies and red blood cell removal under physiological and pathological conditions

Naturally occurring antibodies (NAbs) directed to band 3 protein (major erythrocyte membrane protein) are involved in the clearance of red blood cell (RBC) at the end of their lifespan as well as in the removal of RBC in different hereditary haemolytic disorders and in malaria. In all cited situations RBC undergoes oxidative stress and hemichromes (haemoglobin degradation products) are formed. Hemichromes possess a strong affinity for band 3 cytoplasmic domain and, following their binding, lead to band 3 oxidation and clusterisation. Those band 3 clusters show increased affinity for NAbs which activate complement and finally trigger the phagocytosis of altered RBC. During intra-erythrocytic malaria parasite growth, NAbs begin to bind to RBC surface at early parasite development stages increasing their abundance in parallel with parasite development. Interestingly, a number of hereditary haemolytic disorders, known to exert a protective effect on malaria, tend to exacerbate this phenomenon leading to a more precocious and effective opsonization of diseased RBC infected by malaria parasites. The exact definition of band 3 neo-antigens and the mechanism of their surface exposure are still unclear. Also band 3 clusterisation is only superficially understood, new insights about band 3 phosphorylation by Src kinases suggest the presence of a complex regulatory pathway.

Metabolic indicators of oxidative stress correlate with haemichrome attachment to membrane, band 3 aggregation and erythrophagocytosis in β‐thalassaemia intermedia

Haematological data, genotype, transfusion requirements, metabolic indicators of oxidative stress (flux via hexose‐monophosphate shunt (HMPS); steady state level of GSH and GSSG, NADPH and NADP; activity of anti‐oxidant enzymes), parameters of membrane damage (aggregated band 3; membrane‐bound haemichromes, autologous immunoglobulins (Igs) and C3 complement fragments) and erythrophagocytosis were measured in erythrocytes (RBC) of 15 β‐thalassaemia intermedia patients (nine splenectomized) with low, if any, transfusion requirements. Patients presented increased aggregated band 3, bound haemichromes, Igs and C3 complement fragments, and increased erythrophagocytosis. Bound haemichromes strongly correlated with aggregated band 3. Anti‐band 3 Igs were predominantly associated with aggregated band 3. Erythrophagocytosis positively correlated with aggregated band 3, haemichromes and Igs, suggesting the involvement of haemichrome‐induced band 3 aggregation in phagocytic removal of β‐thalassaemic RBC. Splenectomized patients showed higher degrees of membrane damage and phagocytosis, significantly higher numbers of circulating RBC precursors, and tendentially higher numbers of reticulocytes. Basal flux via HMPS was increased twofold, but HMPS stimulation by methylene blue was decreased, as was the glucose flux via HMPS. GSH was remarkably decreased, whereas NADPH was increased. Except for unchanged catalase and glutathione reductase, anti‐oxidant enzymes had increased activity. Negative correlation between HMPS stimulation by methylene blue and bound haemichromes indicated that the ability to enhance HMPS may counteract haemichrome precipitation and limit consequent membrane damage leading to erythrophagocytosis.

Oxidized and poorly glycosylated band 3 is selectively phosphorylated by Syk kinase to form large membrane clusters in normal and G6PD-deficient red blood cells

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 BS(3) (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.

Acute haemolysis in childhood falciparum malaria

Acute haemolysis associated with clinical episodes of high-level Plasmodium falciparum parasitaemia was studied in 20 children from an holoendemic area (coastal Tanzania). The change in blood haemoglobin (Hb) concentration ranged from -46 to g/L during the 72-h observation period and was linearly related to maximum parasitaemia. Balance studies between loss of blood Hb, increase in plasma Hb and appearance of Hb in the urine indicated that extravascular clearance of red cells was the predominant mode of erythrocyte clearance. Most subjects, however, showed minor signs of intravascular haemolysis. The plasma Hb was << 1% of blood Hb and haemoglobinuria was detected in 14/20 children but the excretion of Hb in urine was < 0.5% of total Hb loss. Haemoglobinuria was, however, a marker of severe haemolysis, since the maximum blood Hb loss in children without haemoglobinuria was 10 g/L. Erythrocyte-bound opsonins known to induce erythrophagocytosis, i.e., complement C3c fragments and autologous IgG, were increased in all patients. In the patients with major haemolysis, the changes correlated to the haemolysis over time. Hence, a similar mechanism for predominantly extravascular erythrocyte clearance may be operative in acute malarial anaemia, normal erythrocyte senescence and other forms of acute haemolysis.

Phagocytosis of P. falciparum malarial pigment hemozoin by human monocytes inactivates monocyte protein kinase C

Hemozoin (malarial pigment) is a ferriprotoporphyrin IX-rich hemoglobin degradation product present in parasitized RBC. Avidly phagocytosed hemozoin abolishes phagocyte TPA-induced oxidative burst. Membrane-associated PKC increased transiently in hemozoin-fed monocytes by 50% after 30 min and decreased irreversibly to 20% of initial value within 5 h after phagocytosis. Control RBC-fed monocytes showed transient decay of membrane-associated PKC followed by complete recovery 12 h after phagocytosis. Cytosolic PKC was not impaired within 12 h and diminished drastically 24 h after phagocytosis of hemozoin. Results are compatible with increased degradation of membrane-translocated PKC, possibly by iron/H2O2-mediated damage of cysteine-rich regulatory domains of PKC.

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.

Current knowledge about the functional roles of phosphorylative changes of membrane proteins in normal and diseased red cells

With the advent of proteomic techniques the number of known post-translational modifications (PTMs) affecting red cell membrane proteins is rapidly growing but the understanding of their role under physiological and pathological conditions is incompletely established. The wide range of hereditary diseases affecting different red cell membrane functions and the membrane modifications induced by malaria parasite intracellular growth represent a unique opportunity to study PTMs in response to variable cellular stresses. In the present review, some of the major areas of interest in red cell membrane research have been considered as modifications of erythrocyte deformability and maintenance of the surface area, membrane transport alterations, and removal of diseased and senescent red cells. In all mentioned research areas the functional roles of PTMs are prevalently restricted to the phosphorylative changes of the more abundant membrane proteins. The insufficient information about the PTMs occurring in a large majority of the red membrane proteins and the general lack of mass spectrometry data evidence the need of new comprehensive, proteomic approaches to improve the understanding of the red cell membrane physiology.

Inhibition of heat shock proteins (HSP) expression by quercetin and differential doxorubicin sensitization in neuroblastoma and Ewing's sarcoma cell lines

Neuroblastoma (NB) and Ewing’s sarcoma (ES) represent the most common extracranial solid tumors of childhood. Heat shock proteins (HSP) are elevated in cancer cells and their over‐expression was correlated to drug‐resistance. In this work we identified the HSP by a sensitive proteomic analysis of NB and ES cell lines, then, we studied the HSP response to doxorubicin. Some identified HSP were constitutively more expressed in NB than in ES cells. Doxorubicin‐stimulated HSP response only in NB cells. Quercetin was found to inhibit HSP expression depleting heat shock factor 1 (HSF1) cellular stores. Quercetin caused a higher anti‐proliferative effect in NB (IC50: 6.9 ± 5.8 μmol/L) than in ES cells (IC50: 85.5 ± 53.1 μmol/L). Moreover, quercetin caused a very pronounced doxorubicin sensitizing effect in NB cells (241 fold IC50 decrease) and a moderate effect in ES cells. HSP involvement in NB cells sensitization was confirmed by the silencing of HSF1. Quercetin treatment and HSF1 silencing increased the pro‐apoptotic effect of doxorubicin. In conclusion, the higher HSP levels, observed in NB cells, did not confer increased resistance to doxorubicin; on the contrary, HSP inhibition by quercetin or gene silencing caused higher sensitization to doxorubicin. These results may have a potential application in the treatment of NB.