Biomedical subjects
Publications and source records attributed to D Roos.
The NADPH oxidase of human eosinophils, measured in the cell-free system, shows the same characteristics as the enzyme from human neutrophils. All proteins required for activity of the enzyme are expressed in eosinophils at a higher level than in neutrophils. Eosinophils isolated from patients with chronic granulomatous disease show the same molecular defects as the neutrophils from these patients.
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The energy metabolism of human platelets was studied during storage of platelet concentrates. The platelets were prepared from buffy coats in PVC/DEHP bags and stored for 7 days at room temperature at a concentration of 1.0 x 10(9)/ml with horizontal agitation. The total amount of ATP and ADP decreased with 40% during this storage. This decrease correlated with the disc-to-sphere transformation associated with the loss of platelet viability. During storage, the ability to incorporate 3H-adenosine into metabolic ATP and ADP (45 min at 37 degrees C) decreased with 50%. Via measurement of the specific activity of actin-bound ADP and the amount of incorporated radioactivity into total ATP and ADP, we calculated the content of the metabolic and storage pools of ATP and ADP. The results indicate that the decrease in adenine nucleotide levels during storage was mainly caused by a depletion of ATP and ADP from the storage pool, whereas the metabolic pool remained nearly intact. After 7 days, the ATP:ADP ratio of the storage pool had decreased from 1.0 to 0.3, indicating hydrolysis of ATP. Diadenosine-triphosphate and diadenosine-tetraphosphate (present in the storage pool) decreased with only 30%, and the serotonin content remained nearly constant. Therefore, it is unlikely that the storage pool was completely secreted. Probably, the storage pool of nucleotides serves as an internal supply for maintaining the contents of the metabolic pool of ATP and ADP during storage of platelets.
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In this report, we present data on the activation of different neutrophil effector functions by two distinct Fc-gamma receptors, FcRII and FcRIII. We and others have shown previously that IgG-dependent activation of phagocytosis and superoxide generation is mediated via FcRII. IgG-dependent exocytosis of granule proteins was assessed with Staphylococcus aureus Oxford opsonized with human IgG or with IgG-coated latex. Both anti-FcRII mAb and anti-FcRIII-F(ab')2 mAb inhibited this release, whereas the combination of these mAb inhibited this process more strongly than either mAb alone. This indicates that both FcRII and FcRIII are involved in IgG-dependent release of granule proteins. Cross-linking of the receptors by anti-FcR mAb and F(ab')2 fragments of goat-anti-mouse-Ig showed again that both FcRII and FcRIII mediate lysozyme release, whereas cross-linking of a control antigen (CD67) did not. By measuring the release of elastase and lactoferrin, we found that cross-linking of either FcRII or FcRIII induced release of both azurophilic and specific granules. Under these conditions, we did not measure any activation of the respiratory burst. When FcRIII was removed by treatment of neutrophils with glycosylphosphatidylinositol-specific phospholipase C, the lysozyme release induced by cross-linking of FcRIII was lower than the release from control neutrophils, whereas the release induced by cross-linking of FcRII was similar. Therefore, we conclude that IgG-dependent activation of neutrophils follows two distinct pathways: one via transmembrane FcRII, activating both the NADPH oxidase and the release of granule proteins (as was demonstrated previously by us and by others), and the other via phosphatidylinositol-linked FcRIII, activating exocytosis of granule proteins.
Phagocytic leukocytes contain an activatable NADPH:O2 oxidoreductase. Components of this enzyme system include cytochrome b558, and three soluble oxidase components (SOC I, SOC II, and SOC III) found in the cytosol of resting cells. Previously, we found that SOC II copurifies with, and is probably identical to, a 47-kDa substrate of protein kinase C. In the present study we investigated the change in location of several of these oxidase components after activation of intact neutrophils with phorbol myristate acetate (PMA) and separation of subcellular fraction on sucrose density gradients. On Western blots with fractions of resting cells, the alpha subunit of cytochrome b558 was detected with a monoclonal antibody as a doublet of Mr 22,000 and 24,000 in the specific granules and as a single band of Mr 24,000 in the plasma membrane. PMA induced an increase of cytochrome b558 in the plasma membrane, including the Mr 22,000 band. PMA also induced translocation of the 47-kDa protein from the cytosol to the membrane fraction, as revealed by in vitro phosphorylation experiments. When NADPH oxidase activity was determined in a cell-free system in the presence of sodium dodecyl sulfate and GTP with plasma membranes from resting cells, cytosol from PMA-treated cells was deficient compared with cytosol from resting cells. This deficiency could be partially restored by the addition of SOC I. Concomitantly, SOC I activity appeared in the plasma membranes of PMA-treated cells. These studies support the hypothesis that PMA stimulation of neutrophils results in assembly of oxidase components from the cytosol and the specific granules in the plasma membrane with subsequent expression of NADPH oxidase activity.
Neutrophils express two distinct types of receptor for the Fc region of IgG, FcRII and FcRIII, in amounts of 10,000 to 20,000 FcRII (40 Kd) and 100,000 to 200,000 FcRIII (50 to 80 Kd) per neutrophil. We showed that the FcRIII exhibits genetically determined heterogeneity, detectable by differences in electrophoretic mobility with sodium dodecyl sulfate (SDS) as well as by reaction with antibodies against the biallelic neutrophil-specific antigen system NA. FcRIII was precipitated with an FcRIII-specific monoclonal antibody (MoAb) from the neutrophils of 35 donors. NA1NA1 donors expressed an FcRIII with a molecular weight (mol wt) of 50 to 65 Kd, NA1NA2 donors expressed an FcRIII with a mol wt of 50 to 80 Kd, and NA2NA2 donors expressed an FcRIII with a mol wt of 65 to 80 Kd. Statistical analysis showed that the electrophoretic heterogeneity corresponds with the NA polymorphism (k = 1). Sequential immunoprecipitation with a MoAb against NA1 and a MoAb against anti-FcRIII, followed by SDS-polyacrylamide gel electrophoresis (PAGE), showed that NA1-FcRIII is distinct from NA2-FcRIII. Moreover, immunoprecipitation with a MoAb against NA1 yielded a protein of 50 to 65 Kd, and immunoprecipitation with human anti-NA2 sera or an MoAb against NA2 yielded a protein of 65 to 80 Kd. Preincubation of NA1NA2 neutrophils with F(ab')2 fragments of an MoAb against anti-NA1 reduced binding of IgG dimers to these cells with about 50%, whereas it completely prevented binding of the dimers to NA1NA1 neutrophils. Inhibition experiments with the MoAb against NA2 yielded the same results for NA1NA2 cells, whereas binding of IgG dimers to NA2NA2 cells was completely prevented. Thus, the products of both NA alleles bind IgG. Immunoprecipitation from the medium of neutrophils either stimulated with formyl- methionyl-leucyl-phenylalanine (FMLP) or treated with glycosyl-phosphatidyl-inositol-specific phospholipase C (GPI- PLC) showed that both the NA1-FcRIII and the NA2-FcRIII are released from the cell surface, indicating that both forms of FcRIII have some structural features in common. Deglycosylation of FcRIII from homozygous donors yielded material that showed several bands on SDS-PAGE. GPI-PLC treatment of neutrophils indicated that all of this material is phosphatidyl-inositol linked.
Human neutrophils aggregate after stimulation with various stimuli; this response is completely absent in neutrophils from patients with leukocyte adhesion deficiency (LAD). To investigate the cellular requirements of this process a method was used in which neutrophils are separately loaded with hydroethidine (HE) and sulfofluorescein (SFDA), to give them either red fluorescence or green fluorescence. After mixing HE- and SFDA-labeled cells in a ratio of 1:1, the number of double-colored aggregates formed after activation was determined by analysis on a fluorescence-activated cell sorter. In this way, essential information is obtained when cells of different origin are used. The formation of aggregates between neutrophils of an LAD patient and control neutrophils was thus quantified. Because neutrophil aggregation is dependent mostly on the presence of complement receptor type 3 (CR3), which is not present on LAD neutrophils, this result revealed the presence of a counter-structure for CR3 on LAD neutrophils (and hence on normal human neutrophils). In addition to the presence of these proteins on the cell surface, aggregation required continuous cell triggering as indicated by the transient aggregation induced by short-term activation of protein kinase C. This phenomenon was substantiated by the fact that energy depletion caused profound disaggregation. The present study reveals that neutrophil aggregation is a well-controlled process, which needs constant activation of CR3 for a stable interaction with a constitutively expressed counter-structure.
Leukocyte adhesion to endothelial cells and migration into the subendothelial matrix was studied with a three-dimensional model system, consisting of human endothelial cells cultured on a loose collagen matrix. We developed a new method to separate the endothelial cell monolayer and adhering leukocytes, from the subendothelial matrix, allowing simultaneous analysis of leukocyte adhesion and transendothelial migration. Monocytes adhered more avidly to untreated endothelial cells than did neutrophils (2.5 +/- 0.3 vs. 1.0 +/- 0.2 leukocytes per endothelial cell). Only a small fraction (10%-20%) of these leukocytes migrated into the subendothelium. Pretreatment of endothelial cells with interleukin 1 (IL 1) enhanced adhesion (20%), but not migration of monocytes. In contrast, neutrophil adhesion was markedly and in a time-dependent manner increased by IL 1 treatment (i.e. 200% after 6 h and 110% after 24 h of IL 1 treatment). Moreover, IL 1 pretreatment enhanced neutrophil migration twofold. Activation of leukocytes with formyl-methionyl-leucyl-phenylalanine (fMLP) enhanced both monocyte and neutrophil adhesion, but did not affect leukocyte migration. Under all conditions, monocyte adhesion was only partly (30%-40%) inhibited by monoclonal antibodies (mAb) against the common beta subunit of the leukocyte-cell adhesion molecules (LeuCAM: CD18) and 25%-30% by mAb against the alpha subunit of LFA-1 (CD11a). In contrast, mAb against the alpha subunits of Mac-1 (CD11b) and p150.95 (CD11c) were hardly effective. fMLP-mediated neutrophil adhesion was reduced to below baseline levels by anti-LeuCAM (CD18) mAb, whereas the LeuCAM contribution in IL 1-mediated neutrophil adhesion was less pronounced and varied in time. IL 1-mediated neutrophil migration, however, was completely blocked by anti-LeuCAM mAb. fMLP-mediated neutrophil adhesion was inhibited by mAb against the alpha subunits of Mac, while mAb against the alpha subunits of LFA-1 and Mac-1 both reduced IL 1-mediated adherence. In summary, we describe a novel leukocyte adhesion/migration method and demonstrate that the contribution of the LeuCAM complex in leukocyte-endothelium interaction varies depending on cell type and stimulus used.
Upon activation, basophilic granulocytes release inflammatory mediators, such as histamine. We studied histamine release (HR) of purified (64 +/- 10%) human basophils after cross-linking of membrane-bound IgE via anti-IgE or after binding of the chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP). A variability in the extent of histamine release upon stimulation by either anti-IgE or FMLP was found between donors. Non-responders for FMLP showed high histamine release for anti-IgE, and vice versa. Inhibition of protein kinase C (PKC) by staurosporine (STSP) resulted in partial inhibition of the anti-IgE-induced HR, whereas inhibition of a PKC-independent pathway by wortmannin (WTM) totally blocked the anti-IgE induced histamine release. The HR induced by FMLP was not affected by either of these inhibitors. We conclude that major differences exist in the signal-response coupling between the anti-IgE and FMLP-induced HR in human basophils. The so-called releasability of human basophils may be due to the availability of different cell activation pathways.
The complement fragments C3b and iC3b were purified from human serum by affinity chromatography with Sepharose-coupled monoclonal antibody against the C3d region of C3. The resulting preparations were more than 95% pure and contained less than 0.1% native IgG. Purified C3b and iC3b were coupled to latex beads (0.8 micron diameter) by means of F(ab')2 fragments of monoclonal antibodies against the beta chain or the C3d region of C3, thus orienting the C3b and the iC3b on the latex with the C3b- and iC3b-specific regions outwards. These particles were found to activate the respiratory burst of freshly isolated human neutrophils to 20-30% of the maximal capacity. Latex particles randomly coated with C3b or iC3b were about 3 times less stimulatory. C3b, iC3b and IgG coupled to latex in an oriented fashion were about equally effective in stimulating the respiratory burst. Neutrophils from a patient with a total deficiency of CR3 responded normally to C3b-coated latex but did not respond to iC3b-coated latex. A monoclonal antibody against the alpha chain of CR3 inhibited the activation by iC3b-coated latex and a polyclonal antibody against CR1 partially inhibited the activation by C3b-coated latex. We found an additive effect between IgG-coated latex and C3b-coated latex, regardless of the presence of IgG and C3b on the same particle or on different particles. Thus, binding of ligands to either CR1 or CR3 per se is sufficient to induce an activating signal to the NADPH oxidase in human neutrophils.
To investigate whether changes in platelet condition during platelet storage correlate with an altered expression of platelet membrane proteins, the binding of monoclonal antibodies (MoAbs) to fresh platelets was compared with MoAbs' binding to thrombin-activated platelets and to platelets stored as platelet concentrates. The MoAbs included antibodies against the platelet glycoprotein (GP) IIb/IIIa complex and against two activation-dependent antigens, one of which was a component of the internal platelet alpha-granule membrane (GMP 140) and the other of which was a 53-kD protein derived from platelet lysosomes. The binding of MoAbs to platelets fixed with 1 percent paraformaldehyde was measured by flow cytometry. In thrombin-activated platelets, a threefold increase was found in the expression of GP IIb/IIIa over that in fresh platelets. The binding of the activation-dependent MoAbs increased from 2 to 3 percent to 70 to 80 percent of the platelets. Storage of platelet concentrates for 5 days resulted in a 60 percent increase in GP IIb/IIIa expression compared to Day 0 and increased binding of the MoAbs directed against GMP-140 from 3 to 16 percent and against the 53-kD protein from 2 to 8 percent of the platelets, respectively. These changes correlated with modifications in platelet morphology (decrease in swirling), leakage of lactate dehydrogenase, and release of beta-thromboglobulin. These data indicate that platelets become activated and are damaged during the storage of platelet concentrates.(ABSTRACT TRUNCATED AT 250 WORDS)
The activation of platelets during the preparation of platelet concentrates (PCs) by two methods was compared. To eliminate interdonor differences, 2 units of whole blood were pooled and subsequently divided into two batches. From one batch, the platelets were harvested as pelleted platelets from platelet-rich plasma (PRP) and from the other as nonpelleted platelets from the buffy coat (BC). The activation of platelets in these PCs was studied immediately after preparation and during storage for up to 9 days at 22 degrees C with gentle agitation. The binding of monoclonal antibodies (MoAbs) against the GP IIb/IIIa complex and against activation-dependent antigens (GMP 140 from the alpha granules and a 53-kDa glycoprotein from the lysosomal granules) was measured. Beta-thromboglobulin (beta-TG) release was also determined. Disc-to-sphere transformation was quantitated by measuring on an aggregometer the difference in light transmission during stirring at different rates and also by light microscopy. Immediately after preparation, platelets derived from PRP had a more spheric morphology (p less than 0.01), had a higher beta-TG release (p less than 0.01), bound more MoAbs against GP IIb/IIIa (p less than 0.01), and expressed more GMP 140 and 53-kDa glycoprotein (p less than 0.01) than did BC-derived platelets. However, these differences had disappeared after 2 days of storage. It was concluded that, immediately after preparation, PRP-derived platelets are more activated than BC-derived platelets. This is most likely a result of the pelleting that follows the second high-speed centrifugation of the PRP.
FcRIII (the CD16-antigen), a low affinity receptor for IgG, is expressed by neutrophils, natural killer lymphocytes, and macrophages. We have developed a sensitive radioimmunoassay to quantify FcRIII. A soluble form of FcRIII was identified in human plasma. Immunoprecipitation of FcRIII from plasma showed that the plasma form of FcRIII has an identical electrophoretic mobility as the FcRIII expressed by neutrophils. Moreover, the plasma form of FcRIII exhibited the same polymorphism as does the neutrophil FcRIII. The neutrophil expresses the phosphatidylinositol-linked form of FcRIII, encoded by the gene FcRIII-1. Because it is not known whether this gene is also active in nonhematopoietic cells, we analyzed patients with an acquired clonal disorder of their hematopoietic cells, paroxysmal nocturnal hemoglobinuria (PNH). PNH patients appeared to have a strongly reduced expression of FcRIII on their neutrophils. The concentration of FcRIII in the plasma of these patients was also reduced, indicating that plasma FcRIII originates from neutrophils. A patient deficient in FcRIII-1 but with a normal expression of FcRIII-2 had no soluble FcRIII in her plasma, also indicating that plasma FcRIII originates from neutrophils. The electrophoretic mobility of the protein backbone of plasma FcRIII and FcRIII released by activated neutrophils was identical, whereas deglycosylated FcRIII obtained from a lysate of neutrophils migrated slower. This indicates that plasma FcRIII originates from activation-induced release by neutrophils. Stimulation of neutrophils or neutrophil cytoplasts (closed membrane vesicles filled with cytoplasm) with low concentrations of FMLP (10(-9)-10(-8) M) or phorbol myristate acetate (1-10 ng/ml) induced a dose-dependent release of FcRIII. The plasma concentration of FcRIII was relatively constant (range 40-280% of the mean). Soluble FcRIII was also detected in inflamed joint fluids of arthritis patients, suggesting that FcRIII is also released by activated neutrophils in vivo.
To determine the efficacy of an autologous predeposit blood program in a 2000-bed university hospital, we studied a total of 38,500 homologous transfusions administered to 8,806 patients in 1988. Only 4,170 patients were transfused; 18% of those received greater than 12 units and required 61% of all blood products, while patients with 1 to 6 units (65%) used only 22%. Patients were categorized according to similar diagnoses and therapy. For patients with elective surgery and possible predeposit blood we calculated a) the fraction of patients which presumably would not require homologous blood and b) how many of 1 to 10 predonated autologous units would not be outdated. The estimates of utilization are presented as "benefit-disadvantage-diagrams". With increasing numbers of predonations, the fraction of patients with additional needs of homologous blood decreases and the wastage rate of autologous blood increases: Favourable autologous/homologous proportions were found in orthopedic and cardiovascular surgery patients and some women with reduction mammaplasty. The "break-even" point for eligible diagnosis is set on 70 to 90% utilisation rate in patients without prospective need of homologous blood. The optimal number of predonations ranges from 3 to 8 units; depending on diagnosis, surgical patients should predeposit only in selected cases. An estimated maximum of 18 percent of all transfused patients in our hospital may thus be efficiently enrolled in a preoperative autologous blood program.
We started our preoperative autologous blood donation program in January 1987, comprising orthopedic, cardiological and gynecological patients with prospective perioperative homologous blood requirements. The safety and practicability of our program could be improved by a precise definition of organizational procedures and the development of a special EDP program. The number of patients/units of autologous blood increased from 35/85 in 1987 to 68/260 in 1988 and will reach approximately 120/500 in 1989. Severe side effects did not occur during that period, 72% of the autologous units were transfused, 80% of the patients did not need additional homologous blood transfusions. Considering unsolved problems of homologous blood transfusions, further development of preoperative autologous blood programs is necessary.
Up to six units of autologous blood can be provided for patients with heart surgery, hip joint replacement or scoliosis. This study was undertaken to evaluate hematological parameters in juvenile and elderly patients and the tolerance of 6 weeks preoperative autologous blood donations. We furthermore investigated the approximate "net blood gain" of the autologous procedure. For an optimal stimulation of erythropoiesis, under vigorous substitution of ferrous sulfate, the autologous donations should start as early as 4 to 6 weeks instead of 2-3 weeks prior to the scheduled surgery, even if only 3 units are required prospectively. The net Hb gain of the autologous procedure in 12-68 years old patients reached a mean of 141 g and 231 g Hb at 4 and 6 donations, respectively. This is equivalent to 2.5 and 4.1 homologous units of RBC (approximately 56g Hb each). Up to 6 units of autologous blood can easily be provided by employing "additive solutions" (PAGGS mannitol), avoiding tedious alternatives like "leap frog-techniques" or freezing of blood.
Exchange of blood units between transfusion services and blood depots, using EDP systems requires standardisation of unit identification and bar-codes. The ISBT Working Party on Data Processing defined recommendations for blood-unit identification by Codabar. Our group (German Society of Transfusion Medicine and Immunohematology, Section Data Processing and Standardization) extended and adapted these standards to the German blood bank settings. This paper describes the basic definitions and standards. A complete outline will be published in the next future.