A pair of naturally occurring antibodies may dampen complement-dependent phagocytosis of red cells with a positive antiglobulin test in healthy blood donors.

BACKGROUND AND OBJECTIVE: It is known that red blood cells (RBC) from healthy blood donors with a positive direct antiglobulin test (DAT) for IgG continue to circulate despite carrying elevated numbers of IgG molecules. To unravel the properties of these RBC-bound IgG, we studied them not only on whole RBC populations, but also on density-fractionated RBCs. MATERIALS AND METHODS: The properties of acid-eluted RBC-bound IgG and plasma IgG were studied by ELISA for binding to RBC proteins and opsonins, and by blotting. In vitro phagocytosis was studied on density-separated RBCs. RESULTS: IgG-DAT-positive blood donors carried most IgG molecules on dense RBCs and had more RBCs of high density than DAT-negative controls. Their densest RBCs were older than the oldest RBCs of DAT-negative controls, based on the band 4.1a/b ratio. In vitro phagocytosis of senescent RBCs from IgG-DAT-positive donors was 1.5 to 2 fold higher than that of senescent control cells, but the same or less in the presence of physiological IgG concentrations, implying that RBC-bound IgGs impaired complement-dependent uptake. The IgG molecules on these DAT-positive RBCs comprised anti-band 3 naturally occurring antibodies (NAbs) and were two- to fivefold enriched in anti-C3 and framework-specific anti-idiotypic NAbs as compared to controls. Correspondingly, anti-C3 and framework-specific anti-idiotypic NAbs were proportionally elevated in the plasma of two-thirds of DAT+ donors. CONCLUSIONS: Extra-binding of anti-C3 together with anti-idiotypic NAbs to senescent RBC-associated C3 fragments may suppress complement-dependent RBC phagocytosis and may prolong the in vivo life span of RBCs.

Heavy transfusions and presence of an anti-protein 4.2 antibody in 4. 2(-) hereditary spherocytosis (949delG).

BACKGROUND AND OBJECTIVE: A patient with hereditary spherocytosis (HS) was found not to have red cell membrane protein 4.2. This rare form of HS, or 4.2 (-) HS, stems from mutations within the ELB42 or the EPB3 genes. The patient had long suffered from a gastric ulcer and impaired liver function. He had had several dramatic episodes of gastrointestinal tract bleeding and had received numerous transfusions. An antibody against a high frequency, undefined antigen was found, creating a transfusional deadlock. We elucidated the responsible mutation and searched for an anti-protein 4.2 antibody. DESIGN AND METHODS: Red cell membranes were analyzed by SDS-PAGE and by Western blotting. Nucleotide sequencing was performed after reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR. RESULTS: The not previously described mutation was a single base deletion: 949delG (CGCAECC, exon 7, codon 317) in the homozygous state. It was called protein 4.2 Nancy. The deletion placed a non-sense codon shortly downstream so that no viable polypeptide could be synthesized. The patient carried a strong antibody against protein 4.2 as shown by Western blotting. INTERPRETATION AND CONCLUSIONS: The manifestations resulting from the mutation described were compared with the picture of HS stemming from other ELB42 gene mutations. We discuss the mechanism through which the anti-protein 4.2 antibody developed. There was no way to establish or to rule out whether the antibody participated in the transfusional deadlock found in our patient.

High doses of immunoglobulin G attenuate immune aggregate-mediated complement activation by enhancing physiologic cleavage of C3b in C3bn-IgG complexes.

Intravenously applied human IgG has beneficial effects in treating inflammatory diseases, presumably because it has a complement attenuating role. This role of IgG was studied in vitro by following C3 activation and inactivation in sera that were supplemented with exogenous human IgG and incubated with immune aggregates. IgG added at 2 to 10 mg/mL stimulated the physiologic inactivation of C3b-containing complexes twofold to threefold in 20% sera. This, in turn, lowered the overall C3 activation by 28%, as new C3 convertases primarily assembled on C3b-containing complexes. Exogenous IgG (5 mg/mL) also stimulated inactivation of purified C3b2-IgG complexes, whereby their half-life dropped from 3-4 to 1.5 minutes in 20% serum. IgG appeared to act like a modulator of factor H and I because it did not stimulate inactivation of C3b-containing complexes in factor I-deficient serum. Thus, the known partial protection of C3bn-IgG complexes from inactivation by factor H and I was downregulated by high concentrations of IgG. The ability of high doses of IgG to stimulate complement inactivation is a novel regulatory role of IgG. This may be one of the molecular principles for its therapeutic efficacy in treating complement-mediated inflammations.

Naturally occurring anti-band 3 antibodies bind to protein rather than to carbohydrate on band 3.

Naturally occurring anti-band 3 antibodies were affinity purified from pooled human IgG (Sandoglobulin) (Lutz, H. U., Flepp, R., and Stringaro-Wipf, G. (1984) J. Immunol. 133, 2610-2618). They bound to the major integral membrane protein of human red blood cells and its 55-kDa NH2-terminal chymotryptic fragment but not to the carbohydrate-rich 38-kDa fragment on blots. Likewise, neither an endo-beta-galactosidase nor a neuraminidase treatment of band 3 on intact red cells reduced their binding to the blotted antigen. Lactoferrin (10 micrograms/ml) had no significant effect on their binding to band 3 and to its 55-kDa chymotryptic fragment. Even in the presence of 20 micrograms/ml lactoferrin anti-band 3 antibodies bound specifically to chymotrypsin-pretreated and oxidatively stressed red cells. Thus, naturally occurring anti-band 3 antibodies bind to protein rather than carbohydrate within band 3 protein, irrespectively of whether the antibodies were depleted of anti-idiotypic and other IgG-reactive antibodies or not.

Naturally occurring anti-band 3 antibodies have a unique affinity for C3.

Naturally occurring anti-band 3 antibodies appear to mediate opsonization of oxidatively stressed and in vivo aged red cells. Their low concentration in plasma (< 100 ng/ml) and weak affinity (estimated association constant, 5-7 x 10(6) l/mol) contrasted with their biological efficiency. In compensating for their inadequate properties they have an affinity for C3 at a site independent of the antigen binding domain, with an estimated association constant of 2-3 x 10(5) l/mol. Though weak, their binding to C3 was about 100 times higher than that of whole IgG, which is known to have an affinity for C3. The affinity for C3 may render these antibodies preferred targets of the short-lived nascent C3b and result in a preferential C3b-anti-band 3 complex formation. C3b-IgG complexes represent the best opsonins and can nucleate alternative complement pathway C3 convertases by which opsonization is further enhanced.

Preferential formation of C3b-IgG complexes in vitro and in vivo from nascent C3b and naturally occurring anti-band 3 antibodies.

Naturally occurring anti-band 3 antibodies appear to have tissue homeostatic functions in the clearance of senescent red cells and in eliciting selective phagocytosis of oxidatively stressed red cells by mediating C3b deposition under conditions that favor the alternative complement pathway (Lutz, H. U., Bussolino, F., Flepp, R., Fasler, S., Stammler, P., Kazatchkine, M. D., and Arese, P. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7368-7372). They overcome the notoriously low affinities of naturally occurring antibodies by having affinity for C3 which renders these antibodies preferred targets of nascent C3b. Anti-band 3 antibodies preferentially formed covalently linked C3b-IgG complexes, when C3 was activated randomly by trypsin. IgG depleted of anti-band 3 antibodies had almost lost the ability to form C3b-IgG complexes. Likewise, anti-band 3 antibodies, but not anti-spectrin antibodies, preferentially formed C3b-IgG complexes on oxidatively stressed red cells in the presence of a 10(3)-fold excess of other serum IgG, when complement deposition was initiated by antibody binding in diluted serum. Moreover, anti-band 3 antibodies preferentially formed C3b-IgG complexes at a 10(5)-fold excess of other IgG on in vivo aging red cells, since C3b-IgG complexes from senescent red cells contained exclusively anti-band 3 antibodies with an affinity for C3. Thus, the low titer, low affinity naturally occurring antibody became functionally relevant by preferred generation of C3b-IgG complexes that can nucleate alternative complement pathway C3 convertases and represent the most effective opsonins (Fries, L. F., Siwik, S. A., Malbran, A., and Frank, M. M. (1987) Immunology 62, 45-51).

Release of vesicles enriched in complement receptor 1 from human erythrocytes.

Vesicles released from human E by Ca(2+)-loading, ATP-depletion, or storage are enriched in several glycosylphosphatidylinositol-anchored proteins such as acetylcholinesterase (AchE) and decay-accelerating factor (DAF). As a result of this, the remaining E are depleted of these proteins. We analyzed whether vesiculation induced by ATP-depletion in vitro was also responsible for a loss of C receptor 1 (CR1), which is a transmembrane protein arranged predominantly in small clusters. ATP-depleted E had lost 15.4% to 33.9% of their CR1. This loss was similar to that of AchE and DAF. The released vesicles contained CR1. The number of CR1 per band 3 protein was 1.7 to 2.7 that in the original E, indicating an enrichment of CR1 in vesicles. This enrichment was similar to that observed for AchE and DAF (1.83- and 2.6-fold, respectively). The capacity of the vesicles and the ATP-depleted E to bind C3b-coated immune complexes correlated with the CR1 number, suggesting that there was no preferential loss of CR1 clusters. Vesicles released from human E during C attack also contained CR1. In conclusion, in vitro aging induced by ATP-depletion is responsible not only for a loss of glycosylphosphatidylinositol-anchored proteins, but also of CR1. Whether vesiculation explains the loss of CR1 from aging E in vivo and from E of patients with SLE or AIDS remains to be studied.

Density separation of human red blood cells on self forming Percoll gradients: correlation with cell age.

Human red blood cells were density separated on self-forming Percoll gradients. Redistribution of density fractionated red blood cells was studied by recentrifugation on self-forming Percoll gradients. A protocol that avoids centrifugation of red cells prior to removal of white cells and introduces EDTA before red cell pelleting completely avoided redistribution. Dense red cells separated according to this method were senescent on the basis of a biochemical and a physical criterion: the increase in the band 4.1a:4.1b ratio (Mueller, T., Jackson, C.W., Dockter, M.E. and Morrison, M. (1987) J. Clin. Invest. 79, 492-499) and the loss of maximum deformability. Characterization also included the relative content of two surface proteins (complement receptor 1, CR1 (Ripoche, J. and Sim, R.B. (1986) Biochem. J. 235, 815-821); decay accelerating factor, DAF) on density fractionated red cells. Unlike cytoplasmic proteins, these proteins face similar conditions, whether located on circulating reticulocytes or aging red cells. Both components were lost linearly within experimental errors with cell density and were lower by 60 and 40% in dense than light cells, respectively.

Covalent binding of detergent-solubilized membrane glycoproteins to 'Chemobond' plates for ELISA.

An ELISA method is presented which is based on covalent binding of detergent-solubilized membrane proteins to surface-modified polystyrene plates (Chemobond plates). These plates carried 0.52-0.65 nmol of aldehyde groups per well (150 microliters) and allowed coupling of protein by Schiff base formation either at high pH and subsequent reduction with NaBH4 or by trapping reduced imines at pH 6-6.8 with cyanoborohydride. They bound 15 times the amount of normal plates. Sodium chloride (0.5 M) increased binding 2-3-fold. Binding was essentially resistant to elution by 1% sodium dodecyl sulfate. Reduction of uncoated plates with NaBH4 eliminated the high extent of binding. ELISA tests on Chemobond plates with a rabbit anti-band 3 antibody gave a ten-fold higher signal than plates to which band 3 protein was merely adsorbed. The use of an antigen-enzyme conjugate to detect bound antibody allowed to perform antibody binding and detection of bound antibody simultaneously in the presence of 0.05% Triton X-100. A competitive, one step ELISA system allowed determination of rabbit anti-band 3 antibodies in diluted serum with a sensitivity range of 0.02-0.4 microgram/ml.

How naturally occurring anti-band 3 antibodies stimulate C3b deposition to senescent and oxidatively stressed red blood cells.

Several naturally occurring antibodies stimulate alternative complement pathway C3b deposition. One type of these antibodies, human anti-band 3 antibody, is involved in opsonizing senescent and oxidatively stressed red cells with C3b (Proc. Natl. Acad. Sci. USA 84[1987]7368). The mechanism was investigated, by which it stimulates alternative complement pathway. Anti-band 3 antibodies are preferred targets for nascent C3b which forms ester bonds with IgG and generates C3b-IgG complexes. Since C3b-IgG and free C3b can equally well nucleate alternative convertases and since C3b in C3b-IgG is protected from degradation (J. Exp. Med. 160[1984]1640), generation of C3b-IgG complexes means stimulation of alternative complement pathway. Anti-band 3 antibodies are preferred targets for the shortlived, nascent C3b since they bind to native C3 by a site distant from the antigen binding site. In vitro generation of C3b-IgG complexes confirmed a preferential formation of C3b-anti-band 3 complexes. Thus, an affinity for C3 may be all what is required to make an antibody a stimulator of the alternative complement pathway and thereby a potent opsonin.

An affinity for complement C3 as a possible reason for the potency of naturally occurring antibodies in mediating tissue homeostasis.

Certain naturally occurring antibodies stimulate alternative complement pathway C3b deposition. We propose that only those IgG antibodies stimulate alternative complement pathway which have an affinity for C3. Their weak binding to C3 in plasma increases the probability that covalently linked C3b-IgG complexes are formed during C3 activation. Such complexes are known to be much more efficient than C3b in mediating positive feedback of C3 activation, since they are more stable against inactivation by factor I and H. The hypothesis is supported by functional properties of naturally occurring antibodies to erythrocyte band 3 protein. Their ability to stimulate alternative complement pathway C3b deposition increases the potency of these low titer antibodies as opsonins.

Naturally occurring anti-band 3 antibodies and complement in phagocytosis of oxidatively-stressed and in clearance of senescent red cells.

Treatment of human red blood cells with diamide and opsonization with whole serum enhanced their phagocytosis by mononuclear phagocytes. Opsonization of diamide-treated red cells with whole serum containing 20-100 times the physiologic concentration of naturally occurring anti-band 3 antibodies further increased the extent of phagocytosis. Enhanced phagocytosis was due to an anti-band 3 mediated binding of C3b to red cells via the alternative pathway. Red cell-bound anti-band 3 was slightly elevated on diamide-treated cells and elicited a C3 binding that exceeded the amount of bound antibody by two orders of magnitude. Pretreatment of red cells with a monoclonal anti-CR1 did not significantly inhibit opsonization and phagocytosis if cells were opsonized at elevated anti-band 3 concentrations. On the other hand, phagocytosis of mildly oxidized (20 microM diamide) red cells was completely inhibited by blocking CR1 if cells were opsonized with serum containing physiologic concentrations of anti-band 3. The results suggest that two types of opsonization mediate in vitro phagocytosis: one operating at physiologic anti-band 3 concentrations with mildly oxidized red cells (IC-like mechanism) and one that operates with either heavily oxidized (greater than 200 microM diamide) red cells at physiologic anti-band 3 concentrations, or with mildly oxidized cells opsonized at elevated concentration of anti-band 3. The latter mechanism is relevant in vivo. It is most likely that it starts by Fab-dependent binding of anti-band 3 to diamide-induced band 3 protein oligomers. Complement activation may occur by assembly of an alternative convertase on C3b covalently bound to red cell-associated anti-band 3. This mechanism is also likely to mediate clearance of senescent red cells, as it was primarily from senescent red cells that we could isolate complexes containing IgG covalently bound to C3b.

[Alternative complement pathway activation by anti-band 3 antibodies]. / "Anti-Bande-3"-Antikörper aktivieren Komplement über den alternativen Weg.

Naturally occurring antibodies against the anion exchange protein of red cells (band 3 protein) can elicit in whole serum a strong C3b deposition to red cells under conditions which favor alternative complement pathway activation. Such a mode of opsonization calls for generation of an alternative C3 convertase nucleated by C3b covalently bound to anti-band 3. Senescent, but not young, red cells should also carry "C3b-anti-band 3" complexes, if clearance of in vivo aged red cells occurred by the same mechanism. We succeeded in isolating covalently linked complexes of C3b and IgG primarily from membranes of senescent red cells.

Naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes.

Treatment of erythrocytes with the thiol-specific oxidant azodicarboxylic acid bis(dimethylamide) (diamide) enhances their phagocytosis by adherent monocytes. Phagocytosis of diamide-treated erythrocytes required that the cells were opsonized with whole serum, since complement inactivation abolished phagocytosis. Opsonization with whole serum containing 20-100 times the physiological concentration of naturally occurring anti-band-3 antibodies enhanced phagocytosis of diamide-treated erythrocytes. High inputs of anti-band-3 also restored phagocytosis of erythrocytes that had been incubated with complement-inactivated serum. Elevated concentrations of anti-spectrin antibodies were ineffective in whole and complement-inactivated serum. Specific recognition of diamide-treated erythrocytes by anti-band-3 antibodies may be due to generation of anti-band-3 reactive protein oligomers on intact diamide-treated erythrocytes. Generation of such oligomers was dose-dependent with respect to diamide. Bound anti-band-3 alone was not sufficient to mediate phagocytosis. It resulted in deposition of complement component C3b on the cells through activation of the alternative complement pathway in amounts exceeding that of bound antibodies by two orders of magnitude. Thus, anti-band-3 and complement together mediate phagocytosis of oxidatively stressed erythrocytes, which stimulate senescent erythrocytes with respect to bound antibody and complement.

Diamide enhances phagocytosis of human red cells in a complement- and anti band 3 antibody-dependent process.

Human red cells treated with 20-200 microM diamide were opsonized by serum and phagocytized by adherent monocytes. Phagocytosis was dependent on complement. It was enhanced by supplementing whole serum and was restored by supplementing complement-inactivated serum with naturally occurring antibodies which bind to the major integral protein of the human red cell, band 3 protein. Diamide induced oligomers of anti band 3 reactive oligomers, and enhanced anti band 3 binding to red cells. Complement C3 binding paralleled that of anti band 3 and exceeded it by two orders of magnitude. Thus, naturally occurring antibodies have functional properties which were not abolished by other serum Ig and may be involved together with complement in the clearance of red cells subjected to oxidant stress.