Complement receptor 1 inhibitors for prevention of immune-mediated red cell destruction: potential use in transfusion therapy

Activation of complement cascade via the antibody-mediated classical pathway can initiate red blood cell (RBC) destruction, causing transfusion reactions and hemolytic anemia. In the present study, we have assessed the ability of a human recombinant soluble form of complement receptor 1 (sCR1) to inhibit complement-mediated RBC destruction in vitro and in vivo. Using an in vitro alloimmune incompatibility model, sCR1 inhibited complement activation and prevented hemolysis. Following transfusion of human group O RBCs into mice lacking detectable pre-existing antibodies against the transfused RBCs, systemic coadministration of 10 mg/kg sCR1, a dose well tolerated in human subjects for prevention of tissue injury, completely inhibited the in vivo clearance of the transfused RBCs and surface C3 deposition in the first hour after transfusion, correlating with the half-life of sCR1 in the circulation. Treatment with sCR1 increased the survival of transfused human group A RBCs in the circulation of mice with pre-existing anti-A for 2 hours after transfusion by 50%, reduced intravascular hemolysis, and lowered the levels of complement deposition (C3 and C4), but not immunoglobulin G (IgG) or IgM, on the transfused cells by 100-fold. We further identified potential functional domains in CR1 that can act to limit complement-mediated RBC destruction in vitro and in vivo. Collectively, our data highlight a potential use of CR1-based inhibitors for prevention of complement-dependent immune hemolysis.     

Complement inhibitors to treat IgM-mediated autoimmune hemolysis

Complement activation in autoimmune hemolytic anemia may exacerbate extravascular hemolysis and may occasionally result in intravascular hemolysis. IgM autoantibodies as characteristically found in cold autoantibody autoimmune hemolytic anemia, in cold agglutinin disease but also in a considerable percentage of patients with warm autoantibodies are very likely to activate complement in vivo. Therapy of IgM-mediated autoimmune hemolytic anemia mainly aims to decrease autoantibody production. However, most of these treatments require time to become effective and will not stop immediate ongoing complement-mediated hemolysis nor prevent hemolysis of transfused red blood cells. Therefore pharmacological inhibition of the complement system might be a suitable approach to halt or at least attenuate ongoing hemolysis and improve the recovery of red blood cell transfusion in autoimmune hemolytic anemia. In recent years, several complement inhibitors have become available in the clinic, some of them with proven efficacy in autoimmune hemolytic anemia. In the present review, we give a short introduction on the pathogenesis of autoimmune hemolytic anemia, followed by an overview on the complement system with a special focus on its regulation. Finally, we will discuss complement inhibitors with regard to their potential efficacy to halt or attenuate hemolysis in complement-mediated autoimmune hemolytic anemia.     

Autoimmune hemolytic anemia.

Warm-type autoantibodies of autoimmune hemolytic anemia (AIHA) are usually IgG but may be IgM or IgA. They are usual Rh specific. Cold-type antibodies are IgM or IgG (Donath-Landsteiner [DL] antibody). IgM antibodies are usually anit-l (occasionally anti-i) and DL antibodies anti-P. The warm IgG antibodies do not fix complement (C); they cause red blood cell (RBC) destruction predominantly in the spleen as the result of interaction between fixing; they cause RBC destruction either by intravascular lysis (complement sequence completed) or by interaction between C3-coated RBCs and phagocytes in liver and spleen. Gentic factors, immunoglobulin deficiency, somatic mutation, viral infections and drugs, and failure of T-lymphocyte function, all probably play a part in breaking immunological tolerance and the development of AIHA.     

Effect of C3b inactivator on monocyte-bound C3-coated human erythrocytes

As a model of IgM-induced hemolytic anemia in man, human erythrocytes were sensitized with IgM antibody and coated with complement components, including C3 and C4, using human serum as a source of complement. These coated red cells were then interacted with monolayers of human mononuclear phagocytic cells (monocytes). Complement-coated red cells so bound could be displaced from their monocyte attachment site in a dose- and time-dependent manner by serum factors, including C3b inactivator (C3bINA). These factors were more efficient in inactivating red cell-bound complement components prior to interaction of the coated cells with monocytes. With large amounts of complement per erythrocyte, measured as membrane-bound C3, the ability of the serum inactivating factor(s) to remove the complement-coated red cells from the monocyte surface was compromised and persistently bound red cells were progressively phagocytosed. These studies implicate C3bINA in the displacement of complement-coated erythrocytes, formed from the interaction of IgM antibody and serum complement, from the hepatic macrophage in IgM-induced immune hemolysis. They suggest that both the concentration of complement components, especially on the erythrocyte surface, and the level of C3bINA and perhaps other inactivators may be important features regulating hemolysis in this disorder.     

An Unusual Sialoglycoprotein Associated with the Webb-Positive Phenotype

A 65-year-old white man had severe hemolytic anemia due to a mixture of low-titer IgG lambda and IgM lambda agglutinins showing optimum reactivity at 22 degrees C. The IgG agglutinins were detected by manual indirect antiglobulin test (IAT) using anti-IgG, and had a titer of 1 at 37 degrees C, 128 at 22 degrees C and 16 at 4 degrees C against adult type O red blood cells (RBC). The corresponding titers with cord RBC were 1 (37 degrees C), 64 (22 degrees C) and 8 (4 degrees C). Proteolytic enzyme and neuraminidase treatment of RBC did not decrease these titers. No known specificity could be assigned to these agglutinins. The isolated agglutinins (recovered by cold adsorption, warm elution) were shown by immunoelectrophoresis to be IgG lambda antibodies. They did not bind complement in vitro, consistent with the finding that the patient had negative manual direct antiglobulin test (DAT) by anti-C3d. It could be shown only by automated IAT that patient's serum also contained IgM cold agglutinins which also reacted best at 22 degrees C and appeared to be of lambda light-chain type. The patient responded to corticosteroid therapy and remains well without treatment 14 months after the hemolytic episode. The presence of IgG cold agglutinins may be predictive of a favorable response to corticosteroid therapy.     

Restriction of cell lysis by homologous complement: I. An analysis of membrane attack complex formation on target membranes

The hemolytic efficiency and binding of C9 to homologous and heterologous erythrocytes was evaluated by using a standardized passive sensitization procedure to prepare antigen- and antibody-coated erythrocytes (EA) and human serum for lysis. Heterologous bovine EA were readily lysed by human serum, whereas human EA were quite resistant to lysis. Human EA bound as many C8 and C9 molecules per cell as bovine EA when incubated under identical conditions, but four times as much bound C9 was required to lyse an equal number of human EA compared with bovine EA. The susceptibility of human erythrocytes did not increase when increased volumes of undiluted human serum were used although C9 binding increased to as much as 100,000 molecules per cell. Sodium dodecyl sulfate-resistant polymerized C9 (poly(C9)) was detected on both lysed ghosts and unlysed EA bearing complement proteins C1 through C9 (EAC1-9) after incubation with undiluted human serum; however, the ratio of poly(C9) to monomeric C9 was higher on unlysed cells than on ghosts. Although bovine and human EA bound equal amounts of human C9 at the end point, the rate of lysis and C9 uptake was slower on homologous cells. The rate-limiting step occurred before C9 binding and lysis because the rates of lysis and C9 binding were equal on homologous and heterologous EAC1-8 targets, but the extent of lysis of homologous cells was still lower than lysis of heterologous cells. Human erythrocytes lose restriction against homologous hemolysis during storage in autologous plasma or in isotonic buffers.     

Controlling the complement system for prevention of red cell destruction

PURPOSE OF REVIEW: Complement sensitization of red blood cells (RBCs) can lead to both intravascular and extravascular red cell destruction. Altered levels of naturally occurring complement regulatory proteins on red cells can result in hemolysis, while defective expression of these proteins on immune cells can cause breakdown of tolerance to self antigens and is associated with autoimmune disease. RECENT FINDINGS: To date several complement inhibitors, including recombinant forms of complement regulatory proteins, humanized antibodies, and synthetic molecules have been described that limit complement activation by interfering with different steps in the complement cascade. However, few have been evaluated for prevention of complement-mediated RBC destruction. In this review, possible applications of these complement inhibitors for treatment of complement-mediated hemolysis in specific disease states are described. Furthermore, the implication of the regulatory role of complement in the development of autoimmune hemolytic anemia is discussed. SUMMARY: Complement therapeutics has potential for effective and safe prophylactic use and treatment of hemolytic transfusion reactions and complement-mediated hemolytic diseases. Furthermore, the regulatory function of complement may be exploited to prevent and treat autoimmune hemolytic anemia.     

Complement-Independent Lysis of Human Red Blood Cells by Cold Hemagglutinins

Hemolysis mediated by human antibodies is generally ascribed to the attack of red blood cells (RBC) by complement. We here extend earlier in vitro observations which indicate that potent cold agglutinins can directly cause lysis of RBC without the participation of complement. We have noted that EDTA plasma taken from patients with cold agglutinin disease is frequently reddish if the plasma is not immediately separated from the cells at 37 degrees C. Moreover, eluates prepared in such cases from plasma or heat-inactivated serum (30 min at 56 degrees C) by absorption (at 4-20 degrees C) and elution (at 37 degrees C) are usually contaminated with hemoglobin, and a large number of RBC used for absorption is lost during the procedure. To characterize this phenomenon further, we examined the effect of different hemagglutinating antibodies in vitro on normal RBC in the absence of complement. Hemolysis (5-17%) of RBC only occurred after treating the cells with potent antibodies at low temperatures (0-20 degrees C). This hemolysis increased 2- to 3-fold when the RBC were treated with an enzyme and decreased with rising temperature. Unlike cells hemolyzed by complement activation, no C5b-9 complexes could be detected on RBC damaged by this mechanism.     

Regulation of complement functional efficiency by histidine-rich glycoprotein.

The modulation of complement functional efficiency by serum histidine-rich glycoprotein (HRG) was investigated. Addition of exogenous HRG to prewarmed diluted serum, followed immediately by sensitized sheep erythrocytes (EA), resulted in enhanced hemolysis. However, when HRG was incubated with diluted serum for 10 minutes at 37 degrees C, inhibition of hemolysis occurred. The biphasic modulation of complement function was also obtained with the complement alternative pathway when HRG was added to diluted serum for hemolysis of rabbit erythrocytes. Partial reduction of complement functional activity was shown when serum was absorbed by an HRG-Sepharose 6MB column. Western blot analysis showed that complement C8, C9, factor D, and S-protein in diluted serum were bound by nylon membrane-immobilized HRG. However, by immunoprecipitation of relatively undiluted serum with anti-HRG IgG beads, HRG was found to coprecipitate with S-protein and plasminogen, which suggested that HRG may complex with these proteins in serum. In functional tests, HRG inhibited C8 hemolytic activity, probably by preventing C8 binding to EAC1-7 cells. HRG also enhanced polymerization of purified C9 as well as the generation of a 45-Kd C9 fragment. Such an effect was even more pronounced in the presence of divalent cations with the reaction mixtures of C9 and HRG. Partial dimerization of C9 was shown when exogenous HRG was added to normal serum. In contrast, polymerization of serum C9 was inhibited by exogenous HRG during poly I:C activation of serum or incubation under low ionic strength conditions. HRG was further shown to inhibit factor D-mediated cleavage of factor B when bound by cobra venom factor. The molecular basis by which HRG regulates serum complement function is not clear. Hypothetically, the tandem repetitions of a consensus histidine-rich penta-peptide sequence in HRG may provide a highly charged area that interacts with complement components.     

Two Examples of Low Ionic Strength-Dependent Autoagglutinins with Anti-Pr1 Specificity

Two low ionic strength-dependent autoagglutinins were studied and found to have anti-Pr1 specificity. This specificity was determined by studies with enzyme-treated and neuraminidase-treated human red blood cells (RBCs), animal RBCs and chemically-modified sialoglycoproteins, all suspended in a low ionic strength solution (LISS). Both IgM complement-binding cold agglutinins had a wide thermal range and caused in vitro hemolysis of some LISS-suspended RBCs at 37 degrees C. Compatible blood was found for these patients by using techniques that did not employ LISS.     

Cold agglutinin syndrome: nursing management

Although cold agglutinin syndrome rarely results in an acute hemolytic episode, consideration should be given to the potential problems that exist when cold agglutinins are present. This autoimmune response may be present in an acute or chronic form. Viral and bacterial infections, especially mycoplasma pneumonia, may precipitate acute CAS, whereas, chronic CAS may be idiopathic or associated with malignant lymphoma. Both acute and chronic CAS may result in RBC hemolysis as the IgM antibody reacts with the I-antigen on the RBC, setting the complement cascade into action. Therefore, screening for cold agglutinins should be done before any procedure that would introduce cold fluids into the body, e.g., blood transfusions and cold cardioplegia during coronary artery bypass surgery.     

Suppression of chemotactic activity of human polymorphonuclears by monoclonal IgMs with and without biological activity

The influence of monoclonal IgMs on migration of human polymorphonuclears was studied at various temperatures by the use of 19 sera with monoclonal IgMs from patients with macroglobulinemia of Waldenstrom (MW) without obvious biological activity, 29 sera with monoclonal IgM cold agglutinins (18 with anti-I and 11 with anti-i IgMs) and 3 sera with monoclonal IgM rheumatoid factor (RF). Under-agarose migration method and modified Boyden chamber method with double filters and 51Cr-PMNs were used. In under-agarose method, chemotactic differentials for controls, MW, anti-I, and anti-i groups were, respectively, 57 +/- 8 mm, 39 +/- 9 mm, 44 +/- 14 mm, and 32 +/- 16 mm at 37 degrees C and 47 +/- 18 mm, 22 +/- 11 mm, 17 +/- 9 mm, and 15 +/- 12 mm at 24 degrees C. All three sera with IgM RF inhibited chemotaxis. The differences between all groups and controls were significant at p less than 0.01. Random migration was inhibited at 24 degrees C (p less than 0.01) but not at 37 degrees C. Inhibitory concentrations of IgM in the sera tested were equal or less than 0.5 mg/ml. Thirteen sera were tested by the modified Boyden chamber method. At 37 degrees C 8 of 13 sera and at 24 degrees C 11 of 13 sera inhibited significantly chemotaxis at a concentration of IgM of 1 mg/ml. The lowest inhibitory concentration of IgM was 25 micrograms/ml. Eleven chromatographically pure IgMs were tested in the under-agarose assay. At concentrations of 0.4-3.7 mg/ml, eight IgMs inhibited chemotactic differential at 37 degrees C and nine inhibited it at 24 degrees C. At concentrations of 0.6-2.0 mg/ml, all seven pure IgMs tested by the Boyden chamber method significantly inhibited chemotaxis at 24 degrees C and 37 degrees C. Some IgMs inhibited chemotaxis at concentrations as low as 25 micrograms/ml. Ten IgM CA were eluted from the red blood cells. Eluates inhibited strongly chemotaxis at 24 degrees C and 37 degrees C. Heat inactivation did not alter inhibitory activity of IgM, however pepsin digestion or reduction and alkylation of purified IgMs did abolish their inhibitory activity. Inhibition of chemotaxis was not related to the light chain type, the titre, or the thermoamplitude of cold agglutination. However, monoclonal IgMs with anti-i cold agglutinin activity were stronger inhibitors than anti-I. Since 75% of IgMs tested inhibited chemotaxis at 37 degrees C, it is possible that monoclonal IgMs, especially those with anti-i cold agglutinin activity, inhibit PMN migration in vivo.     

HUMAN COMPLEMENT ACTIVATION BY SELF-ASSOCIATED IgG RHEUMATOID FACTORS

IgG rheumatoid factors (IgG–RFs) undergo concentration-dependent self-association into dimers and higher polymers, as previously reported. The interactions of purified IgG–RF from the plasma of 3 patients with rheumatoid arthritis, with guinea pig and human complement were studied. Self-associating IgG–RFs were isolated by affinity columns and gel filtration. These preparations contained no detectable IgM and were composed only of IgG subclasses known to fix complement. Complement utilization of IgG–RF was compared with that of monomeric IgG, heat-aggregated IgG, and soluble rabbit IgG immune complexes. Although incubation of IgG–RF or monomeric IgG with 3 units of guinea pig or human complement resulted in decreased hemolysis of sheep erythrocytes sensitized with IgM hemolysin, these substances were less than 100 times as effective as heat-aggregated IgG or soluble immune complexes. The ability of human or guinea pig complement that had been incubated with IgG–RF to restore hemolytic activity to C4-deficient guinea pig serum served to distinguish Clq binding from complement cascade activation. IgG–RFs and monomeric IgG did not activate guinea pig complement cascade in contrast to aggregated IgG. IgG–RFs, however, activated human complement cascade; monomeric IgG only bound human Clq. These results indicate that self-associated IgG–RFs can activate human complement in fluid phase, but less effectively than aggregated IgG or large-latticed immune complexes.     

A new human monoclonal cold agglutinin Sa recognizing terminal N- acetylneuraminyl groups on the cell surface

A human homogeneous IgM/K cold agglutinin (CA) Sa is described, whose corresponding antigen on erythrocytes (RBC) was abolished by neuraminidase. This indicated that the antigen was related to N- acetylneuraminic acid, similar to Pr and Gd antigens. In contrast, this antigen was only partially destroyed by proteases, whereas Pr antigens are completely destroyed and Gd antigens are not influenced by proteases. Sa antibody activity was inhibited by sialyllactose NeuAc (alpha 2 leads to 3) (alpha 2 leads to 6) Gal (beta, 1 leads to 4) Glc like anti-Gd but in contrast to anti-Pr. The corresponding antigen was associated with an RBC membrane glycoprotein fraction like Pr, Sa is one of a spectrum of human monoclonal CA against cell surface neuraminyl groups.     

Autoimmune Hemolytic Anemia Associated with an IgG Cold Incomplete Antibody1

Abstract. Serologic studies are reported on a patient with severe autoimmune hemolytic anemia, whose red cells were strongly coated with IgG and with α2D component of C3. Direct and indirect Donath-Landsteiner reactions were negative. In addition to a typical IgM anti-I cold agglutinin of modest titer, the serum contained a lambda IgG incomplete antibody which bound more strongly to normal red cells at 4°C (1/64) than at 37°C (1/4). The IgG antibody did not require complement for binding but could bind complement and cause hemolysis with papainized red cells. Once bound, the IgG antibody did not dissociate appreciably at room temperature. The antibody eluted poorly from the patient's cells at 56°C but was readily dissociable from cell stroma at pH 3.5. The eluate exhibited no blood group specificity with a diagnostic red cell panel. In particular, equally strong reactions were obtained with O, A₂, B, A₁B, P + P₁-negative, Rh-null, adult and cord red cells. Reactivity was not impaired by prior papainization of the cells. Although the patient improved following splenectomy, neither IgG nor IgM antibodies were detectable in a concentrated splenic extract. The properties of the present cold IgG incomplete antibody are compared with those previously reported; possible clinical implications of these antibodies are briefly discussed.     

Rheumatoid factor inhibition of in vitro binding of IgG complexes in the human glomerulus

We studied the effects of rheumatoid factor (RF) on binding of immune complexes to activated C3 (C3b) receptors in vitro. IgM fraction of serum containing RF activity (IgM-RF), IgM isolated from pooled normal human serum and having no RF activity (IgM-control), bovine serum albumin, and Veronal buffered saline solutions were used in a C3b assay system consisting of aggregated human IgG (AggHuIgG) coupled to sheep erythrocytes (SRBC) with guinea pig and normal human serum complement. The number of glomerular bound AggHuIgG-SRBC with IgM-control and bovine serum albumin or Veronal buffered saline was similar, while the number of bound cells with IgM-RF was reduced significantly. This effect was seen with both guinea pig and normal human serum complements. Supernatant hemolytic complement activity was maintained with IgM-RF, but reduced with control solutions. The blocking factor was shown to be RF by serial dilutions of IgM-RF resulting in inverse correlations with latex flocculation and inhibition of SRBC binding, absorption of blocking from IgM-RF with insolubilized AggHuIgG, and failure of IgM-control to block binding. IgM-RF did not directly interfere with activation of complement, but blocked attachment of C3 to AggHuIgG and formation of C3b capable of reacting with glomerular receptors. These results showed that IgM-RF can inhibit binding of AggHuIgG complexes to human glomeruli. This in vitro phenomenon may represent a possible protective mechanism of RF in vivo in diseases with immune complexes.     

A case of hemoglobin SC disease with cold agglutinin-induced hemolysis

Children with sickle cell disease commonly require red blood cell (RBC) transfusion. We report the first case of hemoglobin (Hb) SC disease with development of severe anemia induced by cold agglutinin hemolysis after Mycoplasma infection. Complete blood count (CBC) showed falsely decreased RBC count and hematocrit and falsely elevated MCV and MCHC. Peripheral blood smear showed RBC clumping at room temperature; this disappeared after warming to 37 degrees C. Anti C3b-C3d was present on red cells, and indirect antiglobulin test revealed a circulating cold agglutinin. Furthermore, anti-Mycoplasma pneumoniae IgM antibody was detected in serum. Careful evaluation of CBCs and peripheral blood smears is required in cases of worsening anemia among sickle cell patients and consideration should be given to cold hemagglutinin disease as an etiology.     

A Human Monoclonal IgM k Cold Agglutinin Recognizing Oligosaccharides with Immunodominant Sialyl Groups Preferentially at the Blood Group M-Specific Peptide Backbone of Glycophorins: Anti-PrM

A kappa-monotypic IgM high titer cold agglutinin reacting like anti-Pr at low, like anti-M at higher temperatures, is described. It recognizes tetra- and/or trisaccharides with immunodominant sialyl groups on glycophorins A, B, C like anti-Pr. Its affinity to the oligosaccharides is, however, approximately 10-fold increased when they are attached to the M-specific peptide backbone of glycophorin A. The antibody, termed anti-PrM, occurred in a blood group MN patient with chronic cold agglutinin disease and caused autoimmune hemolytic anemia.     

Auto-immune haemolytic anemia complicating infectious mononucleosis in a patient with hereditary elliptocytosis.

Auto-immune haemolytic anemia complicating infectious mononucleosis occurred in a patient with hereditary elliptocytosis. A cold antibody of IgM anti-i specificity with narrow thermal amplitude was identified in the serum and the erythrocytes were found to be coated with complement. Significantly excessive erythrophagocytosis was demonstrated in samples of the patient's blood which had been chilled and then incubated at 37 degrees C. The patient recovered spontaneously. The elliptocytosis does not appear to have contributed to the episode of haemolytic anaemia; the other elliptocytic member of the family (her father) had no history and no present evidence of haemolysis.     

A Combination of IgG and IgM Autoantibodies in Chronic Cold Agglutinin Disease: Immunologic Studies and Response to Splenectomy

A patient with chronic cold agglutinin disease and anti-I antibody was studied. The titer of the patient's serum at 4 degrees C was 700 with adult I RBC, 256 with cord RBC, 256 with adult i RBC. At room temperature the titers were decreased and the serum also reacted at a titer of 4,000 with enzyme treated adult I RBC. Dithiothreitol treatment of the serum and purified antibody decreased reactivity only slightly, indicating that the antibody was IgG. The heat eluate contained 1.2 mg/ml IgG kappa, but only 80 micrograms/ml IgM kappa. A hybridoma made by fusing the patient's peripheral blood lymphocytes with a human myeloma cell line contained only IgM kappa (30 micrograms/ml) anti-I activity. The IgM fraction of the heat eluate reacted similarly to the hybridoma supernatant. The IgG fraction resembled the serum and heat eluate. In this case study, IgG kappa and IgM kappa immunoglobulins, both possessing cold agglutinin activity, were present in the patient's serum. This finding is unusual in idiopathic cold agglutinin disease and in view of the predominance of an IgG cold agglutinin, splenectomy was recommended for treatment.