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We present data on a patient of South Asian origin with recessive hereditary spherocytosis (HS) due to absence of protein 4.2 [4.2 (-) HS]. Protein 4.2 cDNA sequence analysis showed the presence of a novel 41-bp frameshift deletion that predicts a truncated peptide designated protein 4.2 Hammersmith. Quantitative reverse transcription-polymerase chain reaction indicated that the mutant mRNA was unstable. Sequencing of protein 4.2 genomic DNA revealed that the deletion stems from aberrant splicing. The proband was homozygous for a G>T substitution at position 1747 (cDNA numbering) that activates a cryptic acceptor splice site within exon 11 of the protein 4.2 gene (EPB42). The proband's mother was found to be heterozygous for this substitution. Unlike protein 4.2 null mice, the proband's red cells showed no evidence for abnormal cation permeability. Quantitation of red cell membrane proteins was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and flow cytometric measurement. CD47, a protein associated with the Rh complex, was markedly reduced to about 1% (in the proband) and 65% (in the mother) that found in healthy controls. The Rh-associated glycoprotein migrated with a higher than normal apparent molecular weight on SDS-PAGE. There was no obvious reduction in Rh polypeptides. These observations indicate that protein 4.2 and CD47 interact in the human red cell membrane. They provide further evidence for an association between the band 3 complex (band 3, ankyrin, protein 4.2, glycophorin A) and the Rh complex (Rh-associated glycoprotein, Rh polypeptides, glycophorin B, CD47, LW) and define a point of attachment between the Rh complex and the red cell cytoskeleton.

The glycoprotein CD47 (integrin-associated protein, IAP) is present on the surface of virtually all cells, including red blood cells (RBCs). CD47 acts like a marker of self by ligating the macrophage inhibitory receptor signal regulatory protein alpha (SIRPalpha). In this manner mild reactivity of wild-type RBCs with macrophage phagocytic receptors is tolerated, whereas otherwise identical CD47-deficient RBCs are rapidly eliminated. We show here that virtually all CD47-deficient nonobese diabetic (NOD) mice spontaneously develop severe lethal autoimmune hemolytic anemia (AIHA) at 180 to 280 days of age, whereas none of the control CD47(+) NOD mice develop lethal AIHA at least during the first year of life. This phenotype is at least partially due to a markedly increased rate of elimination of opsonized CD47(-/-) compared to CD47(+) RBCs. Similarly, CD47(-/-)C57BL/6 mice were much more sensitive than their wild-type counterparts to experimental passive AIHA induced by anti-RBC monoclonal antibodies. Thus, CD47-SIRPalpha signaling can have a profound influence on the severity of AIHA, making manipulation of this signaling pathway a theoretically appealing avenue in the treatment of the disease.

We investigated the impact of changes occurring during red blood cell (RBC) ageing on the RBC-binding activity of pathogenic anti-erythrocyte monoclonal antibodies derived from autoimmune-prone New Zealand black (NZB) mice. As assessed by flow cytometric analysis on in vivo biotinylated RBCs, all five NZB-derived anti-RBC mAb exhibited more efficient binding to aged RBCs than to young RBCs, and resulted in a selective elimination of more aged RBCs from the circulating blood. In addition, treatment of RBCs with proteases markedly enhanced the binding of all five anti-RBC mAb, raising the possibility that increased exposure of autoimmune epitopes on aged RBCs may be in part, a result of contacts with proteolytic enzymes during the lifetime of circulating RBCs. In marked contrast, the binding activity of mAb raised in non-autoimmune animals against antigens expressed on RBCs, such as CD44, CD47, CD147 and TER-119, was either decreased or unchanged with RBC ageing, and these epitopes, except for that recognized by anti-CD47 mAb, were highly sensitive to mild treatment with proteases. Our data unravel the unique molecular feature of RBC epitopes involved in autoimmune haemolytic anaemia, suggesting that membrane alterations in aged RBCs might play a significant role in the development of the autoantibody response to RBCs., (Copyright 2002 Elsevier Science Ltd.)

Integrin-associated protein (CD47) is a broadly expressed protein that costimulates T cells, facilitates leukocyte migration, and inhibits macrophage scavenger function. To determine the role of CD47 in regulating alloresponses, CD47(+/+) or CD47(-/-) T cells were infused into irradiated or nonconditioned major histocompatibility complex disparate recipients. Graft-versus-host disease lethality was markedly reduced with CD47(-/-) T cells. Donor CD47(-/-) T cells failed to engraft in immunodeficient allogeneic recipients. CD47(-/-) marrow was unable to reconstitute heavily irradiated allogeneic or congenic immune-deficient CD47(+/+) recipients. These data suggested that CD47(-/-) T cells and marrow cells were cleared by the innate immune system. To address this hypothesis, dye-labeled CD47(-/-) and CD47(+/+) lymphocytes or marrow cells were infused in vivo and clearance was followed. Dye-labeled CD47(-/-) cells were engulfed by splenic dendritic cells and macrophages resulting in the clearance of virtually all CD47(-/-) lymphohematopoietic cells within 1 day after infusion. Host phagocyte-depleted CD47(+/+) recipients partially accepted allogeneic CD47(-/-) T cells. Thus, dendritic cells and macrophages clear lymphohematopoietic cells that have downregulated CD47 density. CD47 expression may be a critical indicator for determining whether lymphohematopoietic cells will survive or be cleared.

In autoimmune hemolytic anemia (AIHA), circulating red blood cells (RBCs) opsonized with autoantibody are recognized by macrophage Fcgamma and complement receptors. This triggers phagocytosis and elimination of RBCs from the circulation by splenic macrophages. We recently found that CD47 on unopsonized RBCs binds macrophage signal regulatory protein alpha (SIRPalpha), generating a negative signal that prevents phagocytosis of the unopsonized RBCs. We show here that clearance and phagocytosis of opsonized RBCs is also regulated by CD47-SIRPalpha. The inhibition generated by CD47-SIRPalpha interaction is strongly attenuated but not absent in mice with only residual activity of the phosphatase Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1, suggesting that most SIRPalpha signaling in this system is mediated by SHP-1 phosphatase activity. The macrophage phagocytic response is controlled by an integration of the inhibitory SIRPalpha signal with prophagocytic signals such as from Fcgamma and complement receptor activation. Thus, augmentation of inhibitory CD47-SIRPalpha signaling may prevent or attenuate RBC clearance in AIHA.

Aims: The study evaluated the effects of glucose concentration on the extracellular and intracellular activation of the respiratory burst in fMet-Leu-Phe-stimulated normal human neutrophils., Methods: Specific extracellular respiratory burst activity was measured as superoxide dismutase (SOD)-inhibitable cytochrome c reduction. Intracellular respiratory burst activity was studied using luminol-enhanced chemiluminescence in the presence of SOD and catalase, to quench extracellular chemiluminescence activity. Myeloperoxidase (MPO) release from activated neutrophils was studied by using the guaiacol technique., Results: The extracellular respiratory burst following activation with 1 microM fMet-Leu-Phe was significantly reduced at 15 and 25 mM D-glucose (9.5 +/- 1.0 and 8.5 +/- 0.8 nmol/10(6) cells and 10 min; P < 0.01 and P < 0.001, respectively) as compared with that at 5 mM glucose (10.3 +/- 1.0 nmol/10(6) cells and 10 min). When specifically studying the intracellular respiratory burst, no difference was found between the responses at 5, 15 or 25 mM glucose. Increasing glucose concentrations also reduced the secretion of MPO from fMet-Leu-Phe-activated neutrophils., Conclusions: Elevated glucose concentrations inhibit the generation of extracellularly released reactive oxygen metabolites but have no effects on the intracellular respiratory burst in fMet-Leu-Phe-stimulated normal human neutrophils.

The immune system recognizes invaders as foreign because they express determinants that are absent on host cells or because they lack "markers of self" that are normally present. Here we show that CD47 (integrin-associated protein) functions as a marker of self on murine red blood cells. Red blood cells that lacked CD47 were rapidly cleared from the bloodstream by splenic red pulp macrophages. CD47 on normal red blood cells prevented this elimination by binding to the inhibitory receptor signal regulatory protein alpha (SIRPalpha). Thus, macrophages may use a number of nonspecific activating receptors and rely on the presence or absence of CD47 to distinguish self from foreign. CD47-SIRPalpha may represent a potential pathway for the control of hemolytic anemia.

The effects of glucose concentration on the chemokinetic effects of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) was evaluated for normal human neutrophils using a direct microscopic assay. fMet-Leu-Phe increased the rate of locomotion in the absence of glucose, but the chemokinetic effect of fMet-Leu-Phe was most potent at 5 mM glucose and not further changed at 15 mM glucose. The chemokinetic effects of fMet-Leu-Phe and glucose were essentially the same in blood clot-isolated and gradient-isolated neutrophils. However, in gradient-isolated neutrophils, the rate of locomotion under different experimental conditions was strictly negatively correlated to the fraction of non-locomoting cells and the degree of adhesion to the substratum. These results indicate that the chemokinetic effects of fMet-Leu-Phe are regulated by the glucose concentration by inducing locomotor activity in otherwise non-locomoting cells and by improving adhesion to the substratum.

Objective and Design: To further understand the mechanisms behind defective neutrophil function in diabetes mellitus, the ability of insulin to affect the production and metabolism of reactive oxygen metabolites in normal human neutrophils was studied., Materials and Methods: Neutrophil granulocytes from healthy adults were studied for their N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated production of reactive oxygen metabolites and release of myeloperoxidase or elastase following treatment with insulin., Results: Preincubation of the neutrophils for 30 min with insulin (40-320 microU/ml), before activation with 1 microM fMet-Leu-Phe, revealed that the hormone at 80 and 160 microU/ml reduced the luminol-enhanced chemiluminescence without affecting the superoxide secretion. The insulin-induced reduction of the chemiluminescence response was reversed by the addition of exogenous peroxidase and was also paralleled by a reduced myeloperoxidase activity, with no effect on the elastase activity, in cell-free supernatants from fMet-Leu-Phe-stimulated neutrophils. Superoxide dismutase removed the inhibitory effect of insulin on myeloperoxidase activity., Conclusions: These results suggest that elevated levels of insulin do not affect the NADPH-oxidase activity but, together with superoxide anions, interfere with myeloperoxidase availability and a subsequent myeloperoxidase-dependent generation of reactive oxygen metabolites in fMet-Leu-Phe-stimulated normal human neutrophils.

This study was performed to test whether the inhibitory effect of elevated D-glucose concentrations on insulin-stimulated chemokinesis in normal human neutrophils is mediated by increase in protein kinase C (PKC) activity. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) at 0-100 nM dose-dependently inhibited neutrophil random locomotion in the absence of insulin. Sub-optimal concentrations of PMA (0.1-0.5 nM) inhibited the chemokinetic effect of 160 microU/mL insulin in a dose-dependent way. The specific PKC inhibitor bisindolylmaleimide (GF 109203x) did not affect the insulin-stimulated chemokinesis at 5 mM glucose but restored the chemokinetic effect of insulin at 15 mM glucose. These results therefore suggest that glucose-induced PKC activation may mediate the inhibitory effects of high glucose levels on insulin-stimulated chemokinesis in normal human neutrophils.

This study shows that D-glucose and insulin, alone and in certain combinations, have chemokinetic effects on neutrophil granulocytes from healthy humans. A stimulatory chemokinetic effect of insulin was present at 40-160 microU/mL insulin but only in the presence of 5 mM, and not at 15 mM, D-glucose. D-Glucose alone dose-dependently increased the neutrophil locomotion, showing a maximum effect at 5-15 mM and a return toward the basal locomotion at 25 mM glucose. Because the stimulatory chemokinetic effects of 5 and 15 mM D-glucose alone were the same, it seems that the high glucose level does not inhibit the basic motile properties of the cells but may generate intracellular signals interfering with the signal transduction underlying the insulin effect. The tyrosine kinase inhibitor, genistein, or the phosphatidylinositol 3-kinase inhibitor, wortmannin, abolished the chemokinetic effect of 160 microU/mL insulin in the presence of 5 mM D-glucose. This indicates that the insulin effect on locomotion is indeed mediated by receptor-associated tyrosine kinase activation and involving phosphatidylinositol 3-kinase.

The response to D-glucose (0-21 mM) was studied in neutrophil granulocytes from obese, hyperglycemic and hyperinsulinemic Umeå ob/ob mice and their lean, littermate controls in order to further elucidate the effects of in vivo and in vitro hyperglycemia on neutrophil function. Neutrophil random locomotion on glass and neutrophil resting luminol-enhanced chemiluminescence in cell suspension were studied. Random locomotion was stimulated by D-glucose in neutrophils from both Umeå ob/ob and control mice but the locomotive activity in Umeå ob/ob mouse neutrophils was significantly higher than that found in the controls at 4-21 mM glucose. In both types of mice, the stimulatory effect of D-glucose on random locomotion was diminished at 21 mM glucose (not significantly different from that at 0 mM glucose). Resting chemiluminescence from mouse neutrophils was also stimulated by glucose but here the magnitude of response was similar in neutrophils from both types of mice. These results indicate that chronic hyperglycemia and hyperinsulinemia in the Umeå ob/ob mouse may be associated with an increased neutrophil random locomotive activity but a similar resting production of reactive oxygen species, as compared with neutrophils from control mice at physiological and hyperglycemic glucose concentrations in vitro.

The effects of glucose (5-25 mM) and insulin concentration (40-320 microU/ml) on the cell shape of neutrophil granulocytes from healthy humans were studied. Both non-activated and N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-activated neutrophils in suspension were used as a model for initial chemotactic activation of neutrophil locomotion. D-glucose, but not the non-metabolizable analogue 3-O-methyl-D-glucose, dose-dependently reduced the fMet-Leu-Phe-induced (10(-8)M) neutrophil elongation. Insulin, either alone or in combination with 25 mM D-glucose, was without effect on the fMet-Leu-Phe-induced neutrophil elongation. Furthermore, the inhibitory effect of D-glucose was observed already after 1 min of exposure to D-glucose and fMet-Leu-Phe. D-glucose diminished the fraction of neutrophils with elongated locomotor shape by changing it into an irregular cell shape, suggesting that at least part of the D-glucose effect could be associated with mechanisms determining the typical locomotor shape. The present results suggest that D-glucose through its metabolism, but without the involvement of insulin, reduces chemotactically induced elongation to a locomotor neutrophil shape, and thus neutrophil motility, and that this effect of glucose appears prior to adhesion. This glucose-induced inhibition of the neutrophil chemotactic response may be involved in the neutrophil deficiency seen in diabetes mellitus.