Prolonged storage of red blood cells affects aminophospholipid translocase activity.

BACKGROUND AND OBJECTIVES: Loss of phospholipid asymmetry in the membrane of red blood cells (RBC) results in exposure of phosphatidylserine (PS) and to subsequent removal from the circulation. In this study, we investigated the effect of long-term storage of RBCs on two activities affecting phospholipid asymmetry: the ATP-dependent aminophospholipid translocase (or flippase, transporting PS from the outer to the inner leaflet) and phospholipid scrambling (which will move PS from the inner to the outer leaflet). MATERIALS AND METHODS: Standard leukodepleted RBC concentrates were stored in saline-adenine-glucose-mannitol (SAGM) at 4 degrees C for up to 7 weeks. PS exposure was determined by measurement of AnnexinV-FITC binding to the cells, flippase activity by measurement of the inward translocation of NBD-labelled PS. Scrambling activity was determined by following the inward translocation of fluorescent NBD-phosphatidylcholine. In parallel, intracellular ATP levels were determined. RESULTS: PS exposure amounted to only 1.5 +/- 0.3% positive cells (n = 8) after 5 weeks of storage, which slightly increased to 3.5 +/- 0.7% (n = 8) after 7 weeks of storage. Flippase activity started to decrease after 21 days of storage and reached 81 +/- 5% of the control value after 5 weeks of storage (n = 6) and 59 +/- 6% (n = 6) after 7 weeks. Also in RBC obtained by apheresis, flippase activity decreased upon storage. Scrambling activity remained virtually absent during storage, explaining the low PS exposure despite the decrease in flippase activity. Rejuvenation of RBC after 7 weeks to increase ATP levels only partially restored flippase activity, but in combination with a correction of the intracellular pH to that of fresh cells, almost complete restoration was achieved. The decrease in flippase activity after prolonged storage did make the RBCs more prone to PS exposure after activation of phospholipid scrambling. CONCLUSION: This study shows that, although PS exposure remains low, prolonged storage does compromise the RBC membrane by affecting flippase activity. When the metabolic changes induced by storage are corrected, flippase activity can be restored.

Generation of singlet oxygen induces phospholipid scrambling in human erythrocytes.

Maintenance of phospholipid asymmetry of the plasma membrane is essential for cells to prevent phagocytic removal or acceleration of coagulation. Photodynamic treatment (PDT), which relies on the generation of reactive oxygen species to achieve inactivation of pathogens, might be a promising approach in the future for decontamination of red blood cell concentrates. To investigate whether PDT affects phospholipid asymmetry, erythrocytes were illuminated in the presence of 1,9-dimethyl-methylene blue (DMMB) as photosensitizer and subsequently labeled with FITC-labeled annexin V. This treatment resulted in about 10% annexin V positive cells, indicating exposure of phosphatidylserine (PS). Treatment of erythrocytes with N-ethylmaleimide (NEM) prior to illumination, to inhibit inward translocation of PS via the aminophospholipid translocase, resulted in enhanced PS exposure, while treatment with H(2)O(2) (previously shown to inhibit phospholipid scrambling) greatly diminished PS exposure, indicating the induction of phospholipid scrambling by PDT. Only erythrocytes illuminated in the presence of DMMB showed translocation of NBD-phosphatidylcholine (NBD-PC), confirming scrambling induction. Double label experiments indicated that PS exposure does not occur without concurrent scrambling activity. Induction of scrambling was only moderately affected by Ca(2+) depletion of the cells. In contrast, scavengers of singlet oxygen were found to prevent phospholipid scrambling induced by PDT. The results of this study show that phospholipid scrambling is induced in human erythrocytes by exposure to singlet oxygen.

Composition of the additive solution affects red blood cell integrity after photodynamic treatment.

BACKGROUND AND OBJECTIVES: Photodynamic treatment is a promising technique for pathogen inactivation of red blood cell concentrates. For protocol optimization, the influence of the composition of the storage solution on the integrity of phototreated red cells was studied. MATERIALS AND METHODS: Red blood cells were resuspended in the storage solutions SAG-M or AS-3 to a haematocrit (Hct) of 30%. After addition of the photosensitizer, 1,9-dimethylmethylene blue (DMMB) (25 microm), the suspensions were illuminated with red light, and potassium leakage and delayed haemolysis were determined. In some experiments, the cells were washed after illumination and resuspended in modified storage solutions. RESULTS: Illumination of red cells in the presence of DMMB resulted in an immediate, light-dose-dependent increase in potassium leakage. The illumination conditions used induced no detectable haemolysis immediately after photodynamic treatment. Potassium leakage was higher when the illumination was performed in AS-3. In contrast, delayed haemolysis, measured after overnight storage, was considerably lower when cells were stored in AS-3. This protection was mainly a result of the presence of citrate in AS-3. In addition, other impermeant solutes protected against haemolysis. CONCLUSIONS: The additive solution strongly influences the integrity of red cells after photodynamic treatment. Whereas the solution in which the cells are illuminated has a small effect on red cell integrity, the main influence of the additive solution is during post-treatment storage. Red cell integrity is best maintained when illumination is performed in SAG-M followed by storage in AS-3. The presence of non-permeant solutes, such as citrate, in the solution used for storage, prevents haemolysis of the phototreated, cation-permeable cells by counterbalancing the osmotic activity of haemoglobin.

Additive effects of dipyridamole and Trolox in protecting human red cells during photodynamic treatment.

BACKGROUND AND OBJECTIVES: Photodynamic treatment (PDT) of red blood cell (RBC) suspensions has been reported to result in virus inactivation, but also in deterioration of cell quality. Recently, we have demonstrated the potential usefulness of the reactive oxygen species scavenger dipyridamole in selectively protecting RBCs against the harmful side-effects of PDT. Unfortunately, dipyridamole-conferred protection against long-term photohaemolysis was incomplete. In the present study, dipyridamole was applied in combination with Trolox (a hydrophilic vitamin E analogue) in order to augment RBC protection. MATERIALS AND METHODS: Leucodepleted RBC suspensions (30% haematocrit) were treated with 1,9-dimethylmethylene blue (DMMB) and red light, and the effect of inclusion of dipyridamole and Trolox was assessed on potassium leakage as well as on short-term and long-term photohaemolysis. Possible interference of the scavenger cocktail with virus inactivation was examined using extracellular pseudorabies virus (PRV). RESULTS: Treatment of RBC with DMMB and red light resulted in enhanced potassium leakage and both short- and long-term haemolysis. Dipyridamole and Trolox showed additive protective effects against induction of potassium leakage and photohaemolysis, suggesting different protection mechanisms for the two scavengers. Combined inclusion of dipyridamole and Trolox did not interfere with efficacy of PRV inactivation. CONCLUSIONS: Combined inclusion of dipyridamole and Trolox results in substantially improved selectivity of photodynamic treatment of RBC suspensions.

Four novel mutations in the gene encoding gp91-phox of human NADPH oxidase: consequences for oxidase assembly.

The superoxide-forming nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase of human phagocytes comprises membrane-bound and cytosolic proteins, which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients with chronic granulomatous disease (CGD) are defective in one of the phagocyte oxidase (phox) components, p47-phox or p67-phox, which reside in the cytosol of resting phagocytes, or gp91-phox or p22-phox, which constitute the membrane-bound cytochrome b(558). In four X-linked CGD patients we have identified novel missense mutations in CYBB, the gene encoding gp91-phox. These mutations were associated with normal amounts of nonfunctional cytochrome b(558) in the patients' neutrophils. In phorbol-myristate-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of p47-phox and p67-phox with the membrane fraction of the cells with Cys369-->Arg, Gly408-->Glu, and Glu568--> Lys substitutions was strongly disturbed. Only a Thr341-->Lys substitution, residing in a region of gp91-phox involved in flavin adenine dinucleotide (FAD) binding, supported a normal translocation. Thus, the introduction or reversal of charge at residues 369, 408, and 568 in gp91-phox destroys the correct binding of p47-phox and p67-phox to cytochrome b(558). Based on mutagenesis studies of structurally related flavin-dependent oxidoreductases, we propose that the Thr341-->Lys substitution results in impaired hydride transfer from NADPH to FAD. Because we found no electron transfer in solubilized neutrophil plasma membranes from any of the four patients, we conclude that all four amino acid replacements are critical for electron transfer. Apparently, an intimate relation exists between domains of gp91-phox involved in electron transfer and in p47/p67-phox binding. (Blood. 2000;95:666-673)

Disturbed interaction of p21-rac with mutated p67-phox causes chronic granulomatous disease.

Chronic granulomatous disease (CGD) is characterized by the failure of phagocytic leukocytes to generate superoxide, needed for the intracellular killing of microorganisms. This is caused by mutations in any one of the four subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In a rare, autosomal recessive form of CGD, a 67-kD cytosolic component of this enzyme (p67-phox) is missing. We here report on a patient with a mutation in the p67-phox gene that leads to expression of a nonfunctional p67-phox protein. The purified granulocytes of this patient failed to produce superoxide and contained about half of the normal amount of p67-phox. Analysis of the cDNA and genomic DNA of this patient showed that the patient is a compound heterozygote for a triplet nucleotide deletion in the p67-phox gene, predicting an in-frame deletion of lysine 58 in the p67-phox protein and a larger deletion of 11-13 kb in the other allele. Interestingly, the 58Lys deletion in p67-phox disrupts the interaction with p21-rac1, a ras-related protein involved in the activation of the NADPH oxidase. In contrast to normal neutrophils, in which p47-phox and p67-phox translocate to the plasma membrane upon cell activation, the cells of the patient did not show this translocation, indicating that an interaction between p67-phox and p21-rac1 is essential for translocation of these cytosolic proteins and activation of the NADPH oxidase. Moreover, this CGD patient represents the first case of disease caused by a disturbed binding of a ras-related protein to its target protein.

Interactions between the cytosolic components p47phox and p67phox of the human neutrophil NADPH oxidase that are not required for activation in the cell-free system.

Activation of the human NADPH oxidase requires the interaction of at least four cytosolic proteins and one membrane-bound heterodimeric protein. Src homology 3 (SH3) domains and their proline-rich counterstructures have been shown to play an important role in protein-protein interactions. Because it was found that the cytosolic oxidase components p67phox, p47phox, and p40phox reside in a complex in resting neutrophils, we studied the role of SH3 domains in their interaction by use of an overlay technique. Wild-type and mutated 35S-labeled p67phox and p47phox were used to detect immobilized cytosolic proteins on a protein blot. A specific association of native p67phox to blotted p47phox and blotted p40phox was found. These interactions were not disturbed by deleting the only proline-rich region (amino acids 227-231) in p67phox. We also found a specific association of native p47phox with blotted p67phox. Deletions in a putative SH3-binding region of p47phox completely abrogated the interaction with p67phox. Other results suggest that the C terminus of p47phox exposes this SH3-binding domain for interaction with p67phox. Similar results were obtained when the binding of cytosolic p67phox to wild-type or mutated p47phox were studied in solution. Interestingly, mutants of p47phox unable to bind to p67phox were fully capable of supporting superoxide production under cell-free activation conditions. We conclude that an interaction between the C-terminal proline-rich region of p47phox and the second SH3 domain of p67phox is not required for oxidase activity in the cell-free assay.

156Pro-->Gln substitution in the light chain of cytochrome b558 of the human NADPH oxidase (p22-phox) leads to defective translocation of the cytosolic proteins p47-phox and p67-phox.

Src homology 3 (SH3) domains have been suggested to play an important role in the assembly of the superoxide-forming nicotinamide adenine dinucleotide phosphate (NADPH) oxidase upon activation of phagocytes, which involves the association of membrane-bound and cytosolic components. We studied the translocation of the cytosolic proteins to the plasma membrane in neutrophils of a patient with a point mutation in the gene encoding the light chain of cytochrome b558. This mutation leads to a substitution at residue 156 of a proline into a glutamine in a putative SH3 binding domain of p22-phox (Dinauer, M., E. A. Pierce, R. W. Erickson, T. Muhlebach, H. Messner, R. A. Seger, S. H. Orkin, and J. T. Curnutte. 1991. Proc. Natl. Acad. Sci. 88:11231). In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient's neutrophils was virtually absent. In contrast, when solubilized membranes of the patient's neutrophils were activated with phospholipids in the absence of cytosol (Koshkin, V., and E. Pick. 1993. FEBS [Fed. Eur. Biochem. Soc.] Lett. 327:57), the rate of NADPH-dependent oxygen uptake was observed at a rate similar to that of control membranes. We suggest that the binding of an SH3 domain of p47-phox to p22-phox, and thus activation of the oxidase, does not occur in the neutrophils of this patient, although under artificial conditions, electron flow from NADPH to oxygen in cytochrome b558 is possible.

A point mutation in gp91-phox of cytochrome b558 of the human NADPH oxidase leading to defective translocation of the cytosolic proteins p47-phox and p67-phox.

The superoxide-forming NADPH oxidase of human phagocytes is composed of membrane-bound and cytosolic proteins which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients suffering from chronic granulomatous disease (CGD) are defective in one of the following components: p47-phox and p67-phox, residing in the cytosol of resting phagocytes, and gp91-phox and p22-phox, constituting the membrane-bound cytochrome b558. In an X-linked CGD patient we identified a novel missense mutation predicting an Asp-->Gly substitution at residue 500 of gp91-phox, associated with normal amounts of nonfunctional cytochrome b558 in the patient's neutrophils. In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient was strongly disturbed. Furthermore, a synthetic peptide mimicking domain 491-504 of gp91-phox inhibited NADPH oxidase activity in the cell-free assay (IC50 about 10 microM), and the translocation of p47-phox and p67-phox in the cell-free translocation assay. We conclude that residue 500 of gp91-phox resides in a region critical for stable binding of p47-phox and p67-phox.

Inhibition of neutrophil NADPH oxidase assembly by a myristoylated pseudosubstrate of protein kinase C.

To further define the role played by protein kinase C (PKC) in the activation of the neutrophil NADPH oxidase, we have utilized a pseudosubstrate of PKC which was myristoylated at the N terminus. In electropermeabilized neutrophils, the myristoylated pseudosubstrate Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln (myr-psi PKC) inhibited PMA-induced protein phosphorylations and activation of the NADPH oxidase, induced either by PMA or by the receptor agonist formyl-methionyl-leucyl-phenylalanine. Both the pseudosubstrate lacking the N-terminal myristate (psi PKC) and a myristoylated control peptide (Phe-Ala-Glu-Asp-Gly-Ala-Leu-Glu-Gln, myr-CP) were without effect on these responses. The myristoylated pseudosubstrate was also tested in a cell-free system, in which NADPH oxidase activation can be achieved by addition of SDS and guanosine 5'-3-O-(thio)triphosphate in a staurosporine-insensitive manner. Myr-psi PKC, but not psi PKC or myr-CP, proved to be a potent inhibitor of NADPH oxidase activity in the cell-free system, indicating that the inhibition observed in permeabilized neutrophils may have been caused by an effect other than PKC inhibition. In the presence of myr-psi PKC, translocation in the cell-free system of the cytosolic oxidase components p47-phox and p67-phox to the plasma membrane was inhibited. From these results we conclude that myristoylation profoundly increases the ability of pseudosubstrates of PKC to inhibit not only PKC-mediated phosphorylations, but also NADPH oxidase activation. The latter effect, however, is most probably not related to PKC inhibition but may indicate a critical role of the membrane surface charge in the translocation of the cytosolic oxidase components p47-phox and p67-phox.

Absence of both subunits of cytochrome b558 in the UM384 cell line relative to the inability to generate superoxide anions.

Phagocytic cells are characterized by their ability to generate superoxide anions upon activation by appropriate stimuli. UM384, a myelomonocytic cell line, was shown to be defective in this oxidase activity as measured by nitroblue tetrazolium or cytochrome c reduction. Cytochrome b558, a unique pigment present in phagocytes and implicated in electron transfer from NADPH to O2, was absent in the differentiated UM384 cells. Both subunits of the cytochrome b558 appeared to be absent or present in strongly reduced amounts compared to the mother cell line U937, as indicated by immunocytochemistry or Western blot analysis using monoclonal antibodies (MABs). On the other hand, cytosolic factors also involved in NADPH oxidase activity were shown to be present, either immunologically or by using the capacity of the cytosol to activate the oxidase in a membrane fraction from bovine neutrophils. At the molecular level, the mRNA that encodes the gp91-phox was shown to be absent in the differentiated UM384 cells, whereas the mRNA that encodes the p22-phox was normally expressed. These results suggest that the defect in superoxide production by the UM384 cells is related to the absence of cytochrome b558, a situation mimicking that observed in phagocytes from patients with X-linked chronic granulomatous disease (X-CGD).