Hemolysis in the spleen drives erythrocyte turnover.

Red pulp macrophages (RPMs) of the spleen mediate turnover of billions of senescent erythrocytes per day. However, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition, and their subsequent degradation by RPMs remain unclear. In this study, we provide evidence that the splenic environment is of substantial importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPMs, we noted a substantial lack of macrophages that were in the process of phagocytosing intact erythrocytes. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By using in vivo imaging and transfusion experiments, we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis. In addition, we showed that erythrocyte adhesion molecules, which are specifically activated on aged erythrocytes, cause senescent erythrocytes to interact with extracellular matrix proteins that are exposed within the splenic architecture. Such adhesion molecule-driven retention of senescent erythrocytes under low shear conditions was found to result in steady shrinkage of the cell and ultimately resulted in hemolysis. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghost shells were prone to recognition and breakdown by RPMs. These data identify hemolysis as a key event in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated.

[Glucose-6-phosphate dehydrogenase deficiency: clinical presentation and eliciting factors]. / Glucose-6-fosfaatdehydrogenasedeficiëntie: klinische presentatie en uitlokkende factoren.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a hereditary X-linked disorder, is the most common enzymatic disorder of red blood cells in humans, affecting more than 200 million people worldwide. The prevalence is increasing in the Netherlands due to immigration of people from the Middle East and Africa. We present three different clinical manifestations of G6PD deficiency: neonatal jaundice, haemolysis provoked by infection and haemolysis caused by fava beans. The pathophysiology and treatment are discussed. Furthermore a recent update of chemicals which should be avoided in G6PD deficiency is provided.

From cooperative to uncorrelated clogging in cross-flow microfluidic membranes.

The operational lifetime of filtration membranes is reduced by the clogging of pores and subsequent build-up of a fouling or cake layer. Designing membrane operations in which clogging is delayed or even mitigated completely, requires in-depth insight into its origins. Due to the complexity of the clogging process, simplified model membranes fabricated in microfluidic chips have emerged as a powerful tool to study how clogs emerge and deteriorate membrane efficiency. However, to date, these have focussed solely on dead-end filtration, while cross-flow filtration is of greater practical relevance at the industrial scale. As such, the microscopic mechanisms of clogging in crossflow geometries have remained relatively ill-explored. Here we use a microfluidic filtration model to probe the kinetics and mechanisms of clogging in crossflow. Our study exposes two findings: (i) the primary clogging rate of individual pores depends only on the trans-membrane flux, whose strong effects are explained quantitatively by extending existing models with a term for flux-controlled flow-enhanced barrier crossing, (ii) cross-membrane flow affects the pore-pore communication, leading to a transition from correlated to uncorrelated clogging of the membrane, which we explain qualitatively by deriving a dimensionless number which captures two essential regimes of clogging at the microscale.

Glycophorin-C sialylation regulates Lu/BCAM adhesive capacity during erythrocyte aging.

Lutheran/basal cell adhesion molecule (Lu/BCAM) is a transmembrane adhesion molecule expressed by erythrocytes and endothelial cells that can interact with the extracellular matrix protein laminin-α5. In sickle cell disease, Lu/BCAM is thought to contribute to adhesion of sickle erythrocytes to the vascular wall, especially during vaso-occlusive crises. On healthy erythrocytes however, its function is unclear. Here we report that Lu/BCAM is activated during erythrocyte aging. We show that Lu/BCAM-mediated binding to laminin-α5 is restricted by interacting, in cis, with glycophorin-C-derived sialic acid residues. Following loss of sialic acid during erythrocyte aging, Lu/BCAM is released from glycophorin-C and allowed to interact with sialic acid residues on laminin-α5. Decreased glycophorin-C sialylation, as observed in individuals lacking exon 3 of glycophorin-C, the so-called Gerbich phenotype, was found to correlate with increased Lu/BCAM-dependent binding to laminin-α5. In addition, we identified the sialic acid-binding site within the third immunoglobulin-like domain within Lu/BCAM that accounts for the interaction with glycophorin-C and laminin-α5. Last, we present evidence that neuraminidase-expressing pathogens, such as Streptococcus pneumoniae, can similarly induce Lu/BCAM-mediated binding to laminin-α5, by cleaving terminal sialic acid residues from the erythrocyte membrane. These results shed new light on the mechanisms contributing to increased adhesiveness of erythrocytes at the end of their lifespan, possibly facilitating their clearance. Furthermore, this work may contribute to understanding the pathology induced by neuraminidase-positive bacteria, because they are especially harmful to patients suffering from sickle cell disease and are associated with the occurrence of vaso-occlusive crises.

Extracellular proton release by stimulated neutrophils.

We have tried to elucidate the mechanism of phagosome acidification in human neutrophils. Assuming that phenomena occurring at the plasma membrane reflect reactions in the phagocytic vacuoles, we have stimulated human neutrophils with agents that induce a "respiratory burst," and we have measured the release of protons into the extracellular medium. Phorbol myristate acetate, N-formyl-methionyl-leucyl-phenylalanine and serum-opsonized zymosan particles each caused a rapid release of protons, concomitant with the increase in oxygen consumption. The stimulated release of protons was strictly coupled to the increase respiration of the cells, because inhibition of the respiration of either anaerobiosis, chlorpromazine, or glycolytic inhibitors also inhibited the release of protons. Also, in the presence of the above-mentioned stimulating agents, neutrophils from three patients with chronic granulomatous disease enhanced neither respiration not proton release. In normal cells, the ratio of deltaH+/-deltaO2 was 1.04 +/- 0.19 (mean +/ SD, n = 13). The mechanism of this proton release is not clear. The amount of lactic and carbonic acid produced by stimulated neutrophils was inadequate to explain the amount of protons released. Perhydroxyl radicals were also ruled out as the source of the protons. Because the cells did not release measurable amounts of phosphate ions, a phosphate-hydroxyl-ion antiport was also excluded. Finally, the lack of any effect of uncouplers renders it unlikely that a respiration-driven proton gradient is built up across the plasma membrane.

A phosphoprotein of Mr 47,000, defective in autosomal chronic granulomatous disease, copurifies with one of two soluble components required for NADPH:O2 oxidoreductase activity in human neutrophils.

The NADPH:O2 oxidoreductase (NADPH oxidase) of human neutrophils is converted from a dormant to an active state upon stimulation of the cells. We have studied the soluble fraction that is required for NADPH oxidase activation in a cell-free system. Human neutrophils were separated in a membrane-containing and a soluble fraction. The soluble fraction was separated on carboxymethyl (CM) Sepharose in 10 mM 4-morpholino-ethanesulfonic acid buffer of pH 6.8. Reconstitution of the NADPH oxidase activity, measured as O2 consumption, was only found when the membrane fraction was combined with the flowthrough of the CM Sepharose column as well as with a fraction that eluted at 125 mM NaCl. This result indicates that at least two soluble components are necessary for reconstitution of the NADPH oxidase activity: one that does not bind to CM Sepharose and one that does bind. These components were designated soluble oxidase component (SOC) I and SOC II, respectively. Boiling destroyed the activity in both fractions. In the soluble fraction of human lymphocytes and thrombocytes neither SOC I nor SOC II activity was found. SOC II copurified with a 47-kD phosphoprotein, previously found defective in patients with the autosomal form of chronic granulomatous disease (CGD). Inactive soluble fractions of cells from autosomal CGD patients were reconstituted with a SOC II fraction from control cells. The result of this experiment indicates that autosomal CGD patients are normal in SOC I but defective in SOC II.

Cytochrome b deficiency in an autosomal form of chronic granulomatous disease. A third form of chronic granulomatous disease recognized by monocyte hybridization.

Three patients (two sisters and a brother) in one family are described with chronic granulomatous disease. The granulocytes of these patients did not respond with a metabolic burst to various stimuli and failed to kill catalase-positive microorganisms. The magnitude of the cytochrome b signal in the optical spectrum of the patients' granulocytes was less than 4% of the normal value, whereas the amount of noncovalently bound flavin in these cells was normal. The mode of inheritance of the genetic defect in this family is autosomal because the granulocytes of both parents (first cousins) and a nonaffected sister of the patients expressed 70-80% of the normal cytochrome b signal, showed low-normal or subnormal oxidative reactions during stimulation, and did not display mosaicism in the stimulated nitroblue-tetrazolium slide test. Somatic cell hybridization was performed between the monocytes from the affected boy in this family with monocytes from either a cytochrome b-negative male patient with X-linked chronic granulomatous disease or a cytochrome b-positive male patient with the classic autosomal form of this disease. In both combinations, monocyte hybrids were observed with nitroblue tetrazolium reductase activity after stimulation with phorbol myristate acetate. This complementation of the oxidase activity required protein synthesis. Our results prove that the defect in this family is genetically distinct from that in the other two forms of chronic granulomatous disease. Moreover, our results also indicate that the expression of cytochrome b in human phagocytes is coded by at least two loci, one on the X chromosome and one on an autosome.

A family with complement factor D deficiency.

A complement factor D deficiency was found in a young woman who had experienced a serious Neisseria meningitidis infection, in a deceased family member with a history of meningitis, and in three relatives without a history of serious infections. The patient and these three relatives showed a normal activity of the classical complement pathway, but a very low activity of the alternative complement pathway and a very low capacity to opsonize Escherichia coli and N. meningitidis (isolated from the patient) for phagocytosis by normal human neutrophils. The alternative pathway-dependent hemolytic activity and the opsonizing capacity of these sera were restored by addition of purified factor D. The family had a high degree of consanguinity, and several other family members exhibited decreased levels of factor D. The gene encoding factor D was found to contain a point mutation that changed the TCG codon for serine 42 into a TAG stop codon. This mutation was found in both alleles of the five completely factor D-deficient family members and in one allele of 21 other members of the same family who had decreased or low-normal factor D levels in their serum. The gene sequence of the signal peptide of human factor D was also identified. Our report is the first, to our knowledge, to document a Factor D gene mutation. The mode of inheritance of factor D deficiency is autosomal recessive, in accordance with the localization of the Factor D gene on chromosome 19. Increased susceptibility for infections in individuals with a partial factor D deficiency is unlikely.

[Acute anaemia in a Vietnamese patient with alpha-thalassaemia and a parvovirus infection]. / Acute anemie bij een Vietnamese patiënt met alpha-thalassemie en een parvovirus-infectie.

A 14-year-old girl of Vietnamese descent with an unremarkable medical history presented with haemodynamic shock due to severe anaemia. This was caused by an aplastic crisis resulting from the combined effects of a Parvovirus infection and HbH disease. The HbH disease was a result of compound heterozygosity for the South East Asia (SEA) deletion and the Constant Spring mutation in the genes coding for alpha-globin chains (HbH/Hb Bart's). The girl had multiple blood transfusions and recovered. Family investigation revealed that, in addition to these 2 mutations in the alpha-globin gene, some family members also carried the 3.7-kb deletion of the alpha-globin gene, a mutation in the beta-globin gene resulting in HbE, and a novel mutation of unknown clinical significance in the beta-globin gene. This case demonstrates that essentially asymptomatic carriership of thalassaemia can have serious consequences when coupled with a concurrent infection.

Limitations on the use of dihydrorhodamine 123 for flow cytometric analysis of the neutrophil respiratory burst.

Intracellular oxidation of dihydrorhodamine 123 (DHR) to the fluorescent compound rhodamine 123 (Rho123) was used to detect the production of oxygen metabolites in activated neutrophils. Total leukocyte preparations can be used in this assay, which is a great advantage when priming of the respiratory burst is studied. We have defined the conditions that should be taken into account when priming is studied with this assay. We found that neither the extent nor the kinetics of DHR oxidation match those of NADPH oxidase activity. In addition, DHR oxidation is influenced by the absolute and relative number of neutrophils in the leukocyte suspension, by the DHR concentration and by myeloperoxidase availability. The results presented in this study emphasize the need for carefully designed experiments when DHR is used to study the respiratory burst in neutrophils.

Discrepancies in the oxygen balance of whole human neutrophils and neutrophil homogenates.

The oxygen consumption, superoxide production and hydrogen peroxide generation was studied in human neutrophils phagocytosing zymosan particles. Application of sodium azide, as an inhibitor of catalase, and/or 1,3-bis(chloroethyl)-1-nitrosourea (BCNU), as an inhibitor of glutathione reductase, led to the conclusion that neutrophils convert about half of the oxygen consumed in the respiratory burst to hydrogen peroxide; the other half is used for formation of organic peroxides, disulfide bridges, etc. These products are rapidly degraded to water by catalase and/or the glutathione redox cycle. Reduction of exogenous cytochrome C accounted for only about 15% of the consumed oxygen. Neutrophil homogenates contain a badly damaged oxidase system, because oxygen consumption and hydrogen peroxide formation were only about one-tenth of that observed with whole cells. In contrast, cytochrome-C reduction was about three times as high as that found with intact cells. Probably, cytochrome C partly reconstitutes damaged oxidase systems, thus artificially increasing the oxidase activity. We conclude that cytochrome-C reduction is not a good parameter to characterize cell-free oxidase preparations.

[Molecular-genetic studies of carrier stage of glucose-6-phosphate dehydrogenase deficiency]. / Moleculair-genetisch onderzoek naar draagsterschap van glucose-6-fosfaat-dehydrogenase-deficiëntie.

OBJECTIVE: Determining the reliability of a new DNA analysis in the detection of carriers of 6 mutations that cause glucose-6-phosphate dehydrogenase (G6PD) deficiency. DESIGN: Validation of a diagnostic test. SETTING: Central Laboratory of the Netherlands Red Cross Blood Transfusion Service in Amsterdam, the Netherlands. METHOD: With polymerase chain reactions (PCR) and restriction enzyme analyses, the DNA of 78 proven patients or carriers was compared with the DNA of 51 patients suffering from haemolytic anaemia (possibly due to G6PD deficiency) and of 50 healthy blood donors. RESULTS: In 60 of the 78 patients, 1 or 2 of the 6 mutations were found that lead--according to the literature--to G6PD deficiency. In 2 of the 51 anaemic patients a clinically relevant mutation was found, while such a mutation was revealed in 3 of the 50 blood donors. All 3 had been born in Curaçao or Surinam, areas with a higher incidence of G6PD deficiency than the Netherlands. CONCLUSION: In comparison with G6PD activity tests, which leave 50% of carriers undetected, the described PCR method is a reliable test. Because G6PD activity measurement is independent of mutation analysis, we conclude that a combination of these tests will detect carriers of G6PD deficiency with a higher sensitivity than either of these tests separately.

[Determination of spectrin in erythrocytes: an important aid in the diagnosis of hereditary spherocytosis]. / Bepaling van spectrine in erytrocyten: een belangrijk hulpmiddel bij de diagnostiek van hereditaire sferocytose.

OBJECTIVE: Assay of spectrin in erythrocytes as a diagnostic test in hereditary spherocytosis (HS). DESIGN: Validation of a diagnostic test. SETTING: Central Laboratory of the Netherlands Red Cross Blood Transfusion Service in Amsterdam, the Netherlands. METHOD: A radiolabelled rabbit antiserum against human spectrin was used to determine the amount of spectrin in erythrocytes from 64 patients with proven or supposed HS, suffering from inborn, sometimes familial anaemia and a decreased osmotic resistance of the erythrocytes. These amounts of spectrin were compared with those of 12 patients with decreased osmotic resistance suffering from haemolytic anaemia of unknown cause, 16 patients with various other erythrocyte disorders and 30 healthy blood donors. RESULTS: The intradonor and interdonor variations in the amount of spectrin in erythrocytes from healthy blood donors were found to be less than 7%. In 56 of the 64 patients with HS (88%), the erythrocytes contained less than 86% of the normal amount of spectrin. A similar result was found in 4 of the 12 patients suffering from non-characterised haemolytic anaemia (33%). In contrast, a normal amount of spectrin was found in the erythrocytes of patients with other erythrocytic disorders. CONCLUSION: The radio-immunoassay of spectrin in erythrocytes is more specific for the diagnosis of HS than the osmotic fragility test of the erythrocytes. The normal amount of spectrin found in 8 of the 64 patients possibly suffering from HS may be due to a rare molecular origin of HS not leading to a decreased spectrin level or may be related to other causes of anaemia than HS.

[An opsonin-independent strain of Staphylococcus epidermidis]. / Opsoninnezavisimyi shtamm Staphylococcus epidermidis.

Human neutrophils, subjected to stimulation under different conditions (phorbol myristate acetate, opsonized zymosan, formylmethionyl-leucinephenylalanine, nonopsonized staphylococci), produced a factor (denoted as clumping factor, or CF) with a capacity for highly selective clumping and opsonization of staphylococci. Out of 68 strains of different species of staphylococci, only a single strains (S.epidermidis) was sensitive of CF. CF negative staphylococci were capable of inducing the release of CF by neutrophils, but were not bound by this factor. Extracts, obtained by the mechanical destruction of neutrophils (sonication, repeated freezing and thawing), had no clumping activity. CF had a mol. wt. exceeding 100 kD, was positively charged and disintegrated at 100 degrees C. The capacity of S.epidermidis 178 M for binding CF completely disappeared after the treatment of bacteria with pronase and partially disappeared after boiling and treatment with trypsin and periodate. Neuraminidase and heating at 80 degrees C produced no effect. These data are the first demonstration of highly selective (strain-specific) interaction between secretory products of neutrophils and bacteria.

Cytochrome b, flavins, and ubiquinone-50 in enucleated human neutrophils (polymorphonuclear leukocyte cytoplasts).

Neutrophilic granulocytes contain an oxidase system in their plasma membrane that can be activated to generate superoxide radicals and hydrogen peroxide. Cytochrome b, flavoprotein, and ubiquinone-50 have been proposed as components of this oxidase system. These components have been quantitated, but the results are obscured by different isolation procedures for plasma membranes from resting and activated neutrophils. This problem has now been avoided by the use of enucleated neutrophils (polymorphonuclear leukocyte cytoplasts), which are almost completely devoid of intracellular structures but contain an intact, activatable oxidase system (Roos, D., Voetman, A.A., and Meerhof, L.J. (1983) J. Cell Biol. 97, 368-377). Membranes of resting and phorbol myristate acetate-stimulated cytoplasts contain equal amounts of cytochrome b (4 pmol/milliunit of alkaline phosphatase) and also equal amounts of noncovalently bound FAD (2 pmol/milliunit of alkaline phosphatase). These findings refute the hypothesis that incorporation of cytochrome b and/or a flavoprotein into the plasma membrane constitutes the mechanism of activation of the oxidase system. Ubiquinone-50 is present neither in intact neutrophils nor in cytoplasts, excluding a role for this compound in the generation of bactericidal oxygen species by neutrophils.

Immunocytochemical discovery of the 22- to 23-Kd subunit of cytochrome b558 at the surface of human peripheral phagocytes.

A monoclonal antibody raised against cytochrome b558 reacted specifically with the 22- to 23-Kd protein, the small subunit of this cytochrome. Cytochemical studies showed that the epitope was located on the surfaces of human neutrophils and monocytes. The small subunit of cytochrome b558, therefore, was expressed at least in part on the outer surface of these cells.

A convenient diagnostic function test of peripheral blood neutrophils in glycogen storage disease type Ib.

Neutrophils from patients suffering from glycogen storage disease type Ib (GSD-Ib) show several defects. one of which is a decreased rate of glucose utilization. In this study, we established experimental conditions to show the stimulation of the neutrophil respiratory burst by extracellular glucose. With phorbol-myristate-acetate as stimulus of the burst, the activity of the NADPH oxidase in GSD-Ib neutrophils hardly increased on addition of glucose. In control and GSD-type Ia neutrophils, a clear increase was observed. The lack of response to extracellular glucose in GSD-Ib neutrophils is correlated with the inability to raise intracellular glucose-6-P levels on glucose addition, thereby limiting the activity of the generation of NADPH in the hexose-monophosphate shunt. Our study shows the usefulness of this test for the diagnosis of neutrophil function abnormality in GSD-Ib patients.

Purification and partial characterization of the b-type cytochrome from human polymorphonuclear leukocytes.

Polymorphonuclear leukocytes contain an oxidase system that can be activated to produce superoxide radicals and hydrogen peroxide. A nonmitochondrial b cytochrome, functioning in the generation of these oxygen species, has been purified to apparent homogeneity from human polymorphonuclear phagocytes. After solubilization of the cytochrome with Triton X-100, the cell extract was subsequently chromatographed on Blue Sepharose and Sephacryl S-300. The final preparation was maximally purified 170-fold with a specific content of 5.33 +/- 2.03 nmol mg-1 of protein (mean +/- S.D.; n = 7) and a yield of 21 +/- 13% (n = 5). The apparent molecular mass of the nondenatured cytochrome was estimated by gel filtration to be 235 kDa. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single polypeptide was found with a molecular mass of 127 kDa. From the pyridine hemochrome spectrum 1 protoheme IX/polypeptide was calculated. The light absorbance bands of the dithionite-reduced cytochrome were found to be at 558.5 (alpha), 529 (beta), and 426 nm (Soret), and that of the oxidized cytochrome at 413.5 nm. The difference absorbance coefficients are delta epsilon (426.5 - 440 nm) = 160.6 +/- 11 mM-1 cm-1 and delta epsilon (558.5 - 542 nm) = 29.3 +/- 2 mM-1 cm-1 (mean +/- S.D.; n = 5). Carbon monoxide binds to the cytochrome in a time-dependent fashion (maximum binding after 50-60 min). The midpoint potential of the solubilized nonpurified cytochrome is identical to the cytochrome in situ (Em7.0 = -218 +/- 7 mV (mean +/- S.D.; n = 5)). However, purified cytochrome b shows a significantly decreased midpoint potential, estimated at -407 +/- 18 mV (n = 4). The protein does not contain noncovalently bound FAD or FMN, and no spectral evidence was obtained for the presence of covalently bound flavin. Preliminary amino acid analysis of the cytochrome shows a high content of hydrophilic residues.

Neutrophil Fc gamma RIIIb deficiency, nature, and clinical consequences: a study of 21 individuals from 14 families.

Several individuals have been described whose neutrophils lack the normally abundantly expressed IgG Fc gamma receptor IIIb (Fc gamma RIIIb). We now studied the responsible genomic defect and analyzed the medical history in detail of 21 Fc gamma RIIIb-negative donors identified in 14 unrelated families. We developed a polymerase chain reaction allele-specific-primer annealing assay to genotype for the NA polymorphism of the Fc gamma RIIIB gene. All Fc gamma RIIIb-deficient individuals were negative for both the NA1 and the NA2 allele. In all cases the complete absence of the Fc gamma RIIIB alleles was confirmed using a Southern blot-based restriction fragment length polymorphism assay. Furthermore, an additional deletion of the next more telomeric located Fc gamma RIIC gene was found. Family studies showed that at least one Fc gamma RIIIB allele was absent in both parents in 6 families, whereas in 2 families the father had a normal phenotype. Two individuals suffered from an autoimmune thyroiditis. Four individuals had had multiple episodes of infection, 3 had only incidental infections, and 14 never had any serious infection. Genotyping showed a normal Fc gamma RIIa phenotype distribution among the Fc gamma RIIIb-negative individuals, thus excluding the possibility that the presence of the favorable IgG2-binding low-responder isoform of Fc gamma RIIa (131-H) contributed to the overall absence of recurrent bacterial infections.