Dynamics of blood electrolytes in repeated hyper- and/or hypoglycaemic events in patients with type 1 diabetes.

AIMS/HYPOTHESIS: Electrolyte disturbances are well-known consequences of the diabetic pathology. However, less is known about the cumulative effects of repeated changes in glycaemia, a characteristic of diabetes, on the electrolyte balance. We therefore investigated the ionic profiles of patients with type 1 diabetes during consecutive hyper- and/or hypoglycaemic events using the glucose clamp. METHODS: In protocol 1, two successive hyperglycaemic excursions to 18 mmol/l were induced; in protocol 2, a hypoglycaemic excursion (2.5 mmol/l) was followed by a hyperglycaemic excursion (12 mmol/l) and another hypoglycaemic episode (3.0 mmol/l). RESULTS: Blood osmolarity increased during hyperglycaemia and was unaffected by hypoglycaemia. Hyperglycaemia induced decreases in plasma Na(+) Cl(-) and Ca(2+) concentrations and increases in K(+) concentrations. These changes were faithfully reproduced during a second hyperglycaemia. Hypoglycaemia provoked rapid and rapidly reversible increases in Na(+), Cl(-) and Ca(2+). In sharp contrast, K(+) levels displayed a rapid and substantial fall from which they did not fully recover even 2 h after the re-establishment of euglycaemia. A second hypoglycaemia caused an additional fall. CONCLUSIONS/INTERPRETATION: Repeated hyperglycaemia events do not lead to any cumulative effects on blood electrolytes. However, repeated hypoglycaemias are cumulative with respect to K(+) levels due to a very slow recovery following hypoglycaemia. These results suggest that recurring hypoglycaemic events may lead to progressively lower K(+) levels despite rapid re-establishment of euglycaemia. This warrants close monitoring of plasma K(+) levels combined with continuous glucose monitoring particularly in patients under intensive insulin therapy who are subject to repeated hypoglycaemic episodes. TRIAL REGISTRATION: Clinicaltrial.gov NCT01060917.

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.

Isotype-specific detection of ABO blood group antibodies using a novel flow cytometric method.

Several methods to detect anti-A/B antibodies based on haemagglutination and haemolysis have been described. These methods measure predominantly anti-A/B immunoglobulin (Ig)M, whereas anti-A/B IgG and IgG subclasses are less well examined. We established a flow cytometry method (ABO-fluorescence-activated cell sorting; ABO-FACS) to quantify binding of anti-A/B IgM, IgG and IgG subclasses to human A or B red blood cells. Anti-A/B IgM were present in the majority of 120 blood donors, as expected from blood group typing. The sensitivity and specificity of anti-A/B IgM to predict the blood group was 93% and 96% respectively. Anti-A/B IgG was found in 34/38 blood group O samples (89%). Anti-B IgG in blood group A or anti-A IgG in blood group B was present in 4/28 (14%) and 1/28 (4%) samples, respectively, and absent in 26 AB sera. IgG2 was the predominant IgG subclass. The correlation of anti-A/B IgM and IgG in the ABO-FACS with haemagglutination titres was 0.870 and 0.783, respectively (n = 240; P < 0.001) whereas the comparison of ABO-FACS with ABO-enzyme-linked immunosorbent assay was less significant. In conclusion, ABO-FACS is a valid method to quantify anti-A/B IgM, IgG and IgG subclasses. It opens the possibility of isotype-specific monitoring of anti-A/B antibodies levels after ABO-incompatible solid organ and stem cell transplantation.

Innate immune and non-immune mediators of erythrocyte clearance.

Erythrocyte clearance is reviewed in the context of what is known in 2003 on clearance of apoptotic cells in vitro and in vivo. Thus, emphasis is put on the role of the innate immune system comprised of naturally occurring autoantibodies (NAbs) and complement. Oxidative damage, cellular senescence and diffusion-controlled exoplasmic cross-linking appear to generate oligomers of band 3 (anion transport protein) that are a prerequisite for anti-band 3 NAb binding to human red blood cells (RBC). Similar processes seem to be responsible for premature RBC clearance in hemoglobinopathies and membrane protein deficiencies. The review discusses why NAb binding alone is insufficient and how bound NAbs may enhance complement deposition. Clearance of RBC is not only the result of cell-bound opsonins, but is enhanced by the loss of RBC membrane constituents, such as CD47 and sialic acids. As long as these constituents are present on RBC in normal numbers and topologic arrangement, they bind to their respective receptors on macrophages, elicit a negative signal that appears to prevent the macrophage from engulfing bound RBC. Exposure of phosphatidylserine is not a primary signal for RBC removal and where exposed it initiates binding of CRP or of beta-2-glycoprotein I and NAbs.

Cryoglobulin/albumin complexes in a patient with severe autoimmune syndrome.

We describe the case of a 30-year-old man with a severe autoimmune disease characterized by cryoglobulinaemia, pulmonary hypertension, Raynaud's phenomenon, lymphadenopathy, and glomerulonephritis. Despite initial remission following autologous stem cell transplantation, his disease relapsed and he died from pulmonary hypertension. At presentation the patient had hypergammaglobulinaemia and a number of autoantibodies, including rheumatoid factor (1:10240). The most striking feature was the extremely high level of cryoglobulins. The cryoprecipitate consisted of polyclonal IgM, IgG and albumin. Interestingly, the albumin in the cryoprecipitate was exclusively present in SS-bonded oligomeric forms, and contained an abnormal acidic component as judged by 2D gel electrophoresis. Oxidized albumin was also present in serum, and represented a small but significant fraction. None of the many known albumin variants have so far been associated with a particular disease; thus our results may represent the first description of an altered albumin associated with severe disease.

Hereditary spherocytosis and hemochromatosis.

A 37-year-old male, splenectomized at the age of 1 year, was admitted to the ward with severe chest pain and signs of cardiogenic shock. Clinical investigations revealed the presence of both hemochromatosis and hereditary spherocytosis (HS). HLA typing showed A3,B7 and A24,B57 haplotypes and genetic analysis revealed homozygosity for the C282Y mutation. A family study was performed. The parents and four brothers were heterozygous for the C282Y mutation. Two of the brothers also presented high levels of iron stores and they had been splenectomized because of HS, while two other siblings had neither spherocytosis nor hemochromatosis. The mother had a mild anemia with dehydrated red blood cells (RBC), while the father appeared to have low-density, but normal RBC; none of them presented with spherocytosis. All siblings with spherocytosis and elevated iron stores showed a RBC density distribution similar to the mother. We present the first case with genetically proven hemochromatosis in combination with spherocytosis, focusing on the various possibilities of iron accumulation in individuals with spherocytosis and heterozygosity for the C282Y mutation.

A C3 convertase assay for nephritic factor functional activity.

C3 nephritic factor (C3NeF) is an autoantibody against the C3 convertase which stabilizes this otherwise inherently labile neoenzyme and induces a continuous activation of the alternative pathway with C3 depletion. NeF is found in patients with membranoproliferative glomerulonephritis and/or partial lipodystrpohy. NeF activity is usually detected in plasma by hemolytic tests. In order to obtain reproducible data for the functional activity of purified C3NeF IgG a solid phase assay was developed. C3 convertase was generated on immobilized C3b by incubation with factors B and D in the presence of Ni(2+). Convertase sites were left to decay in the presence of normal IgG or NeF IgG. Residual convertase activity was measured by adding 125I-C3 and capturing nascent 125I-C3b on the plate surface via covalently coupled NH2-Glu-Tyr dipeptide. In the presence of factor H during C3 convertase decay, a dose dependent stabilizing activity was shown for NeF IgG including NeF IgG purified from urine. A second format of the assay was developed in which C3 convertase was assembled on C3b(2)-IgG complexes in the presence of Mg(2+). Since these complexes are more efficient as convertase precursors the signal was five-fold higher than with C3b. Convertase decay, on the other hand, was not influenced by the nature of the precursor and in both systems the stabilizing activity of NeF IgG was similar.

Band 3 protein clustering on human erythrocytes promotes binding of naturally occurring anti-band 3 and anti-spectrin antibodies.

Recognition of senescent and oxidatively stressed human erythrocytes appeared to be initiated by band 3 clustering, followed by bivalent binding of naturally occurring anti-band 3 autoantibodies (anti-band 3 NAbs), and complement deposition. The number of RBC-associated anti-band 3 NAbs was, however, low compared to the total amount of IgG that bound in vitro to RBC containing band 3 oligomers. This implied the involvement of yet other types of NAb, among which we focussed on anti-spectrin NAbs, since eluates from RBC of thalassemic patients contained these NAbs. Binding of affinity-purified anti-band 3 and anti-spectrin NAbs was studied to RBC on which band 3 oligomers were generated by exoplasmic cross-linking. This pretreatment increased binding not only of (125)I-iodinated anti-band 3, but also of anti-spectrin NAbs by 7-10-fold at 0 degrees C in the presence of nearly physiological IgG and HSA concentrations. Binding of anti-spectrin NAbs was not to spectrin as judged from surface-labeling of RBCs that were pretreated with cross-linker. Binding was dose and time dependent in both cases. Moreover, binding of anti-spectrin NAbs was not competed by high concentrations of anti-band 3 NAbs and anti-spectrin NAbs even stimulated binding of anti-band 3 F(ab')(2) by 30%. This suggests that anti-spectrin NAbs bound to band 3 or a protein associated with band 3 by virtue of their inherent polyreactivity.

Interaction of C3b(2)--IgG complexes with complement proteins properdin, factor B and factor H: implications for amplification.

Nascent C3b can form ester bonds with various target molecules on the cell surface and in the fluid phase. Previously, we showed that C3b(2)--IgG complexes represent the major covalent product of C3 activation in serum [Lutz, Stammler, Jelezarova, Nater and Späth (1996) Blood 88, 184--193]. In the present report, binding of alternative pathway proteins to purified C3b(2)--IgG complexes was studied in the fluid phase by using biotinylated IgG for C3b(2)--IgG generation and avidin-coated plates to capture complexes. Up to seven moles of properdin 'monomer' bound per mole of C3b(2)--IgG at physiological conditions in the absence of any other complement protein. At low properdin/C3b(2)--IgG ratios bivalent binding was preferred. Neither factor H nor factor B affected properdin binding. On the other hand, properdin strongly stimulated factor B binding. Interactions of all three proteins with C3b(2)--IgG exhibited pH optima. An ionic strength optimum was most pronounced for properdin, while factor B binding was largely independent of the salt concentration. C3b(2)--IgG complexes were powerful precursors of the alternative pathway C3 convertase. In the presence of properdin, C3 convertase generated from C3b(2)--IgG cleaved about sevenfold more C3 than the enzyme generated on C3b. C3b(2)--IgG complexes could therefore maintain the amplification loop of complement longer than free C3b.

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.

How pre-existing, germline-derived antibodies and complement may help induce a primary immune response to nonself.

In the hypothesis that pre-existing, germline-encoded antibodies (naturally occurring antibodies, NAb) bind to conserved epitopes on invading nonself antigens, bound NAbs may initiate complement deposition and become targets of nascent C3b, which generates C3b-C3b-NAb complexes that remain associated with the nonself antigen (C3b-C3b-NAb...antigen). The inactivated form of these complexes (C3dg-C3dg-NAb...nonself antigen) may bind bivalently and thus firmly to B cells via CR2, a process stimulating antigen presentation. In some cases, CR2-bound 'C3dg-C3dg-NAb...antigen complexes' may further be recognized by immunoglobulin (Ig) determinants on B cells, whereby an immune response is elicited. As conserved epitopes on the nonself antigen are already complexed to NAbs, only B cells carrying Ig determinants specific for nonself epitopes may be stimulated. This hypothesis can explain directed affinity maturation towards nonself, protection from a strong immune response to conserved epitopes, down-regulation of antibody formation and unresponsiveness to high-dose antigen.

Assembly and regulation of the complement amplification loop in blood: the role of C3b-C3b-IgG complexes.

Amplification of complement activation in blood and serum starts on multi-protein complexes that act as precursors of an alternative C3 convertase. Among these covalently linked C4b-, C3b-, and IgG-containing complexes C3b-C3b-IgG complexes represent the major species containing C3b and IgG. Recent work on their purification and characterization is discussed. Special emphasis is placed on the arrangement of ester bonds in these complexes and their dual type of partial protection from inactivation. Partial protection from inactivation is mediated by properdin which binds to these complexes in the complete absence of any other complement protein. High dose IgG, known to stimulate inactivation of these complexes, appears to lower properdin binding in a process that also involves factor H. Properdin stimulates factor B binding to these complexes and renders them far better precursors of a C3 convertase than C3b. The available information allows a suggestion for a new scheme on how the amplification loop is assembled and regulated in blood and serum.

Rabbit IgG antibodies against cord red blood cell membranes bind to complement receptor 1 (CD35).

We have previously shown that a subpopulation of cord/fetal red blood cells (RBC) binds rabbit IgG antibodies raised against cord RBC and absorbed on adult RBC (F-IgG), while control IgG, raised against and absorbed on adult RBC (A-IgG), fails to do so. In the present study, F-IgG maintained its binding to cord RBC surface antigens following absorption on spectrin but not after absorption on skeleton-stripped RBC membranes. Spectrin-absorbed F-IgG- but not A-IgG-affinity-purified material from cord RBC contained polypeptides with apparent MW of complement receptor 1 (CR1) allotypes. Moreover, on immunoblotting these polypeptides reacted with 125I-F-IgG as well as with 125I-anti-CR1 mAb, and binding of 125I-anti-CR1 mAb was inhibited by unlabelled F-IgG. In addition, cord RBC incubated with F-IgG prior to reaction with anti-CR1 showed decreased fluorescence intensity on flow cytometry. Taken together the results suggest that F-IgG binds to CR1 which shows increased expression/accessibility on a subpopulation of cord/fetal RBC.

CR1, CD35 in synovial fluid from patients with inflammatory joint diseases.

OBJECTIVE: To investigate synovial fluid (SF) for the presence of CR1 and to study its relationship to SF leukocytes and to serum levels of soluble CR1 (sCR1) in patients with rheumatic diseases. METHODS: Synovial fluids were collected from 35 patients with rheumatoid arthritis (RA) and 26 patients with other inflammatory joint diseases. Total CR1 in the SF and serum were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) that recognized both soluble and transmembrane forms of CR1. The characteristics of CR1 in SF were analyzed by ultracentrifugation and by a second ELISA specific for transmembrane CR1. RESULTS: CR1 was found in all SF samples tested (range 5-281 ng/ml). SF CR1 was higher in patients with RA (mean +/- SD 81 +/- 66 ng/ml) than in those with other inflammatory joint diseases (31.8 +/- 23.8 ng/ml) (P < 0.001). Serum sCR1 was not significantly increased in the patients compared with the normal subjects. There was no correlation between serum sCR1 and SF CR1. In 44% of the patients, the SF CR1 level was higher than the serum sCR1 level. A fraction (30-80%) of SF CR1 was pelleted by ultracentrifugation and, unlike serum sCR1, it reacted in an ELISA specific for transmembrane CR1. Thus, SF contained 2 forms of CR1: a membrane-associated and a soluble form, which was confirmed by sucrose density-gradient ultracentrifugation. SF CR1 levels correlated directly with the number of SF total leukocytes and polymorphonuclear leukocytes (PMN). These 2 forms of CR1 were also found in the supernatant of in vitro-activated PMN from normal subjects. SF CR1 exhibited the capacity to act as a cofactor for the factor I degradation of C3b. CONCLUSION: CR1 is found in the SF of patients with joint inflammation. The data suggest that SF CR1 originates from the infiltrating leukocytes, which shed both a soluble and a membrane-associated form. Whether SF CR1 participates in the local regulation of complement activation remains to be examined.

Tissue homeostatic role of naturally occurring anti-band 3 antibodies.

Self antigens exposed to the immune system constitutively or in the process of tissue homeostasis may stimulate a TH2 type immunity giving rise to low titer, low affinity naturally occurring antibodies which are involved in actively maintaining peripheral tolerance to self and in tissue homeostatic clearance processes. In reviewing the tissue homeostatic aspect of naturally occurring antibodies to band 3 protein of the human erythrocyte membrane, we address crucial issues of how these and other types of naturally occurring antibodies (NAb) (eg. anti-spectrin NAb) gain functionality and how this can induce opsonization by complement C3b under physiological conditions. Exoplasmic, chemical cross-linking of band 3 protein is sufficient to increase specific anti-band 3 binding under physiological conditions. Formation of oligomers following this non-oxidative cross-linking protocol disfavors a recognition mechanism involving exposure of a neoantigen. New data on NAb binding to erythrocytes further demonstrates that specific binding of any low affinity NAb can only be determined in the presence of whole human IgG and physiological ionic strength, where competition of the predominantly positively charged NAb for binding to the negatively charged cell surface is high. Hence, specific and physiologically relevant binding of low affinity NAb is gained by bivalent binding and suppression of exclusive charge-charge interactions by other NAb sharing the range of pl values. Therefore, many investigations on NAb/cell interactions which have been carried out in the absence of whole IgG have yielded controversial data.

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 human anti-band 3 autoantibodies accelerate clearance of erythrocytes in guinea pigs.

A variety of naturally occurring autoantibodies (NOAs) have been found in sera of animals and humans. Although their specific homeostatic role in the clearance of altered or senescent cells has been proposed and in vitro studies support such functions, in vivo evidence has been lacking. We studied the effect of affinity-purified human anti-band 3 NOA on the survival of untreated and diamide-treated erythrocytes in normal and complement C3-deficient guinea pigs. In vitro exposure to diamide, an oxidative agent, severely reduced the erythrocyte deformability and increased the amount of high-molecular-weight forms of band 3 protein and band 3-hemoglobin adducts in erythrocyte membranes, thereby markedly shortening the survival of these cells in vivo. Human anti-band 3 NOA bound in a dose-dependent manner to erythrocytes, and binding increased with exposure to diamide. In normal guinea pigs anti-band 3 NOA significantly accelerated the clearance of erythrocytes that were mildly damaged by iodine surface labeling and of those that were further oxidized by diamide. However, the anti-band 3 effect was transient and small. In contrast, anti-band 3 NOA did not significantly alter erythrocyte survival in functionally C3-deficient guinea pigs, thereby supporting the C3b requirement for anti-band 3 NOA activity. On the other hand, a pretreatment of animals with purified human band 3 protein slowed down the clearance of erythrocytes incubated with IgG depleted of anti-band 3 NOA. These results provide the first in vivo evidence of a role for anti-band 3 NOA in the clearance of erythrocytes.

An indirect effect of an antibody on complement deposition and lysis of differently sensitized surrounding cells.

Lysis of papain-treated group A and B erythrocytes by human complement was studied by an anti-A (BRIC. 131) and an anti-B (BRIC. 30) IgM monoclonal antibody in 51Cr release assays. The indirect effect of membrane-bound antibody, i.e. its influence on complement binding to sensitized surrounding cells, was examined in a cold target competition test in which sensitized, non-labelled cells are present along with sensitized labelled cells and complement. The mode by which anti-A antibodies indirectly suppressed lysis of sensitized B cells up to 20-fold was studied by following C1q and C3b binding. C1q binding to both types of erythrocytes was not altered in mixed populations of erythrocytes in the presence of both antibodies. Binding of C3b to a mixture of both cell types was, however, suppressed, when both antibodies were present. C3b deposition in mixed cell populations did not reach a significantly higher extent than deposited to one type of erythrocyte alone. This was consistent with the results from competitive lysis and suggests that the anti-A captured most C3b at high anti-A concentrations and deprived the similarly sensitized B erythrocytes of complement. We think that this phenomenon is not due to an uneven removal of complement regulatory proteins from A and B erythrocytes by papain. Instead, the phenomenon might be due to an inherent property of anti-A mAb to better produce nucleation sites for C3 convertases which, upon binding factor B, better compete for the limiting factor D. A mathematical analysis of cold target competition experiment (containing 2430 individual measurements) also shows that the distribution of complement between the competing A and B erythrocyte population is uneven, since it predicts that in any given antibody combination the majority of complement is bound to A erythrocytes. This is consistent with the measured average percentage of lysis.

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.