Rosetting, phagocytosis and immune red cell damage

Observations on rosette formation of red blood cells sensitized with a known number of IgG1 or IgG3 polyclonal or monoclonal anti-Rh(D) antibodies with monocytes, lymphocytes and granulocytes have provided further evidence for the essential role of monocytes/macrophages in red cell destruction. The rosette test with monocytes and the monocyte-monolayer assay (MMA) have confirmed, on a quantitative basis, the greater binding ability of IgG3 than of IgG1 antibodies and that the rate of interaction with red cells increases in proportion to the level of their sensitization. These observations have suggested that the MMA may reflect haemolysis in vivo. It has appeared that the MMA is suitable for assessing haemolysis in many cases of haemolytic disease of the newborn, in autoimmune haemolytic anaemia and in patients with alloantibodies to be transfused. Since in individual cases a clear correlation between the MMA results and haemolysis in vivo was not observed, the factors which may influence such discrepancies are discussed.     

Mechanisms of Immune Haemolysis

The mechanisms of red blood cell destruction in 2 patients with penicillin-induced immune haemolytic anaemia were investigated. Anti-penicillin antibodies of the IgG subclass were found in the patients' sera and in the eluates of their direct antiglobulin positive red blood cells. Using a rapid ⁵¹Cr in vitro assay it was shown that fresh peripheral blood mononu-clear phagocytes and granulocytes but not lymphocytes from both patients lysed and phagocytosed autologous red blood cells previously treated in vivo or in vitro by penicillin and autologous anti-penicillin antibody. Antibody-dependent cell-mediated cytotoxicity (ADCC) as well as antibody-dependent phagocytosis (ADPh) were proportional to serum concentration and to the number of attacking cells. Anti-penicillin antibody from 1 patient activated the complement system in vitro but failed to induce lysis of penicillin-treated red blood cells in the presence of complement. These results suggest that ADCC as well as ADPh participate in the destruction of red blood cells in penicillin-induced haemolysis in vivo.     

Ex vivo antigen preparation for the serological detection of drug-dependent antibodies in immune haemolytic anaemias

S ummary . TWO patients with severe, intravascular haemolysis due to drug-dependent antibodies are described. The antibodies were directed against presumptive metabolites of buthiazide (International Non-proprietary Name, butizide) and nomifensine. Their detection was possible only in the presence of ex vivo antigens (i.e. fresh serum of volunteers after ingestion of the drugs) while in vitro antigen preparations yielded inconclusive results. Both antibodies lysed normal red cells in the presence of ex vivo antigens and complement. The buthiazide-related antibody was IgG (subclass IgGl), the nomifensine-related antibody was IgM. We conclude that the use of ex vivo antigens is of great importance in the serological evaluation of cases with suspected drug-dependent immune haemolysis.     

Phagocytosis of Erythrocytes Sensitized with Known Amounts of IgGl and IgG3 Anti-Rh Antibodies

Phagocytosis was investigated using human peripheral monocytes and erythrocytes sensitized with known amounts of subclass-specific IgG anti-Rh antibodies. The erythrocyte-bound IgG was quantitated by a radiometric antiglobulin test. This evaluation revealed the following: (1) there is a relationship between phagocytosis and the number of erythrocyte-bound IgG molecules; (2) phagocytosis is IgG subclass-dependent, since a similar degree of phagocytosis is observed with fewer IgG3 than IgG1 molecules and also the minimum number of IgG3 molecules for phagocytosis is 150-640, whilst for IgG1 the minimum is 1,230-4,020; (3) the minimum levels of sensitization for phagocytosis should be detectable by the serological antiglobulin test; (4) the phagocytosis assay is no more sensitive than the monocyte rosette assay for the detection of anti-Rh alloantibodies, and (5) phagocytosis of adherent erythrocytes observed by video-enhanced microscopy indicated that erythrocytes may adhere to monocytes for a considerable time before phagocytosis, but that phagocytosis itself was rapid.     

Antibody-dependent cell-mediated cytotoxicity and phagocytosis of autologous red blood cells in alphamethyldopa-induced haemolysis

7 alphamethyldopa (AMD)-treated patients with positive direct antiglobulin test (DAT) were investigated. Peripheral blood mononuclear phagocytes of 2 patients suffering from haemolysis caused lysis and phagocytosis of autologous DAT-positive red blood cells (RBC). Eluates from RBC of both patients contained antibodies of IgG1 subclass and supported antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADPh) of the patients' RBC after remission. Both cytotoxic activities were proportional to the serum concentration and to the number of attacking cells. The 5 patients without overt haemolysis did not show in vitro lysis or phagocytosis of autologous RBC. These results suggest that ADCC as well as ADPh participate in the destruction of RBC in AMD-induced haemolysis in vivo.     

Erythrocyte autoantibodies, the monocyte monolayer assay and in vivo haemolysis

Summary. Monocyte monolayer assays (MMA) were performed using red cells (RBCs) from 159 patients with a positive direct antiglobulin test and monocytes from healthy individuals. The results of the MMA reflected, to a great extent, in vivo haemolysis; 56/63 patients with a positive result, but only 10/96 with a negative one, had evidence of in vivo RBC destruction. Positive MMAs were observed with autoantibodies of all types: warm (42 cases), cold (14 cases), mixed (seven cases), but never in patients with only C3d on their RBCs and no detectable autoantibodies. With warm autoantibodies, the MMA results depended, in part, on the level of IgG sensitization; additional C3d coating acted synergistically to lower the amount of IgG necessary for monocyte interaction to occur. The presence of IgG3 as well as IgG 1 on the RBCs was usually associated with a high MMA result; most of these cases, however, also exhibited larger amounts of cell-bound IgG. When the MMA results were analysed in respect of the various types of autoantibodies, differences in the phagocytosis: adherence ratio were observed; in patients with warm autoantibodies, there was a preponderance of phagocytosis, whereas in those with cold and mixed types, adherent RBCs predominated or were found alone.     

Quantitative Aspects of the Destruction of Red Cells Sensitized with IgG1 Autoantibodies: An Application of Flow Cytofluorometry

S ummary . The purpose of this study was to determine whether quantitative or qualitative factors are of major importance in the destruction of red cells sensitized with incomplete warm autoantibodies of subclass IgG1.
To that end, the relative amount of IgG1 antibody present on the red cells of patients with autoantibodies of this subclass only, was measured by means of continuous flow cytofluorometry. This method appeared to give an idea of the amount of antibody on red cells and was reproducible. The intensity of the fluorescence of patient's red cells, measured after incubation with a FITC-labelled anti-IgG1, was compared with the presence or absence of signs of increased haemolysis in vivo and the cytotoxic activity of normal monocytes towards these red cells in vitro .
It appeared that it was predominantly the amount of IgGl autoantibody that determined whether or not these antibodies induced haemolysis in vivo or cytotoxicity of monocytec in vitro . This was also true with methyldopa-induced IgG1 autoantibodies.     

Enhanced phagocytosis of ring-parasitized mutant erythrocytes: a common mechanism that may explain protection against falciparum malaria in sickle trait and beta-thalassemia trait

High frequency of erythrocyte (red blood cell [RBC]) genetic disorders such as sickle cell trait, thalassemia trait, homozygous hemoglobin C (Hb-C), and glucose-6-phosphate dehydrogenase (G6PD) deficiency in regions with high incidence of Plasmodium falciparum malaria and case-control studies support the protective role of those conditions. Protection has been attributed to defective parasite growth or to enhanced removal of the parasitized RBCs. We suggested enhanced phagocytosis of rings, the early intraerythrocytic form of the parasite, as an alternative explanation for protection in G6PD deficiency. We show here that P falciparum developed similarly in normal RBCs and in sickle trait, beta- and alpha-thalassemia trait, and HbH RBCs. We also show that membrane-bound hemichromes, autologous immunoglobulin G (IgG) and complement C3c fragments, aggregated band 3, and phagocytosis by human monocytes were remarkably higher in rings developing in all mutant RBCs considered except alpha-thalassemia trait. Phagocytosis of ring-parasitized mutant RBCs was predominantly complement mediated and very similar to phagocytosis of senescent or damaged normal RBCs. Trophozoite-parasitized normal and mutant RBCs were phagocytosed similarly in all conditions examined. Enhanced phagocytosis of ring-parasitized mutant RBCs may represent the common mechanism for malaria protection in nonimmune individuals affected by widespread RBC mutations, while individuals with alpha-thalassemia trait are likely protected by a different mechanism.     

The Role of Adherence to Human Mononuclear Phagocytes in the Destruction of Red Cells Sensitized with Non-Complement Binding IgG Antibodies

In patients with IgG incomplete non-complement binding warm autoantibodies, the subclass composition of the antibodies was studied in relation to the occurrence of increased haemolysis in vivo and the adherence of the patients red cells to peripheral blood monocytes (PBM) in vitro. The presence of IgG3 autoantibodies was almost always accompanied by haemolytic anaemia, but the presence of IgG1 autoantibodies only in some patients but not in others. IgG2 and IgG4 autoantibodies were not associated with increased red cell destruction. A relation identical to that between subclass composition and increased haemolysis was found between subclass composition and adherence of the patients erythrocytes to PBM and thus a strong correlation between positive adherence in vitro and increased red cell destruction in vivo. These results support an important role of adherence to mononuclear phagocytic cells in the destruction of red cells sensitized with non-complement binding IgG antibodies. Strong indications were found that IgG1 autoantibodies are of two kinds, only one of which causes adherence to phagocytes and thus increased red cell destruction.     

In vitro Functional Activity of IgG1 and IgG3 Polyclonal and Monoclonal anti-D

Background and objectives: IgG anti-D is generally restricted to IgG1 and IgG3; it mediates red cell destruction through interactions with IgG Fc receptors (FcγR) on effector cells. The relative ability of these two IgG subclasses of anti-D to mediate haemolysis in vitro by monocytes and K cells was investigated. Materials and methods: Anti-D was affinity purified from 5 preparations of prophylactic anti-D immunoglobulin, and IgG subclasses quantified by ELISA; mean levels were 86.5% IgG1, 1.4% IgG2, 11.6% IgG3 and 0.4% IgG4. IgG1 and IgG3 polyclonal anti-D were further purified separately from some of the anti-D by removal of either IgG3 using magnetic beads coated with anti-IgG3, or of IgG1 using protein A. These preparations were compared with monoclonal anti-D (BRAD-3 and BRAD-5) for their ability to lyse red cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Results: Monocyte-mediated lysis of red cells coated with IgG3 anti-D was approximately twice that of cells coated with IgG1 anti-D at similar sensitization levels, and anti-D preparations containing 10% or more IgG3 gave similar lysis. By contrast, in the K cell ADCC, IgG1 anti-D was 2–4 times more haemolytic than IgG3 anti-D. Polyclonal IgG1 and IgG3 anti-D promoted about 20% more lysis than BRAD-5 (IgG1) and BRAD-3 (IgG3), respectively, in the K cell ADCC, although no difference was observed between polyclonal and monoclonal anti-D in the monocyte ADCC. Conclusions: These experiments demonstrated a functional dichotomy between these two subclasses of anti-D; IgG3-coated red cells were lysed preferentially by monocytes mediated predominantly through FcγRI interactions, whereas haemolysis of IgG1-sensitized cells was mediated mainly by FcγRIII on K cells.     

Correlation of mononuclear phagocyte assay results and in vivo haemolytic rate in subjects with a positive direct antiglobulin test

A mononuclear phagocyte assay has been used to measure the in vitro interaction of normal donor monocytes with the red cells from subjects with a positive direct antiglobulin test (DAT). The relationship between the in vivo haemolytic rate in these subjects and the results obtained in the assay, expressed as the number of red cells associated with 100 monocytes (ARC value), has been examined. From the results of assays in which normal donor monocytes were incubated with normal (DAT negative) donor red cells it was calculated that ARC values of greater than 2 could be considered significantly elevated. Assays performed on 24 patients with a positive DAT and warm autoimmune haemolytic anaemia resulted in a mean ARC value of 42 (range 1-212). Assay results for two of the patients in this group, however, were not significantly elevated from normal. The red cells from three patients with a negative DAT who were suspected of having autoimmune haemolytic anaemia showed no association with normal donor monocytes in the assay. The mean ARC value obtained for 14 non-haemolysing subjects with a positive DAT was 7 (range 1-47). Assay results for only 10 of the 14 subjects in this non-haemolysing group fell within the normal range. The correlation of assay results and in vivo haemolysis was strengthened when the assays were performed with autologous monocytes. A possible explanation for the unexpectedly low ARC values obtained in some cases of autoimmune haemolytic anaemia is discussed.     

Receptor Sites on Human Monocytes for Complement: BINDING OF RED CELLS SENSITIZED BY COLD AUTOANTIBODIES

S ummary Receptor sites on isolated human monocytes were assessed by the demonstration of rosette formation by sensitized red cells around monocytes. This type of binding to monocytes occurred using red cells sensitized with a typical monoclonal IgM cold agglutinin but (in contrast to IgG antibodies) only if the red cells were also coated with complement (C) components. Prior treatment of monocytes with trypsin (1 mg/ml) inhibited the detection of the C receptor but not the detection of the IgG receptor. Although this reaction resembled the phenomenon of immune adherence to some extent, it required the presence of Mg²⁺ ions and was inhibited by EDTA. The effects of temperature variation on C-dependent rosette formation and on haemolysis were comparable. The binding of sensitized erythrocytes to monocytes, however, required significantly less antibody than for haemolysis. C-coated red cells remained reactive with mononuclear phagocytes after elution of the cold antibody as well as under conditions when C was bound to red cells without antibody.     

Mild Course of Fetal Rh D Haemolytic Disease due to Maternal Alloimmunisation to Paternal HLA Class I and II Antigens

The patient is a pregnant women of African origin with a prior history of spontaneous abortion and newborn dystrophy. The investigation showed an anti-Rh D antibody (IgG isotypes 1 and 3) with an indirect antiglobulin tube test (IAT) titre of 1:512. The monocyte monolayer assay (MMA) proved clearly the interaction of Fc receptors with the maternal anti-D, and so a clinical significance was expected. In spite of this, no signs of severe haemolysis in the Rh-D-positive and direct antiglobulin test-positive fetus could be observed. Furthermore, two HLA class I and II alloantibodies (anti-A10, anti-DR13) directed against paternal and fetal antigens were detected in the serum of the gravida. Both antibodies showed an inhibitory effect on the in vitro phagocytosis capacity of mononuclear cells expressing at least one of the corresponding HLA antigens (immunophagocytosis inhibition (IPI) test). Thus, the mild course of haemolytic disease may be explained by an effective inhibition of the fetal mononuclear phagocyte system by maternal HLA class I and/or class II antibodies resulting in a diminished destruction of anti-D-coated fetal red blood cells.     

Antibodies against human neutrophil antigens in non-transfused women with red blood cell alloimmunisation induced by pregnancy

BackgroundAlloantibodies against human neutrophil antigens (HNA) resulting from allogeneic exposure may be associated with transfusion-related acute lung injury and immune neutropenia. Understanding the risk factors for the formation of such antibodies could have a great impact on the adoption of measures to prevent potentially fatal transfusion reactions. The aim of the study was to determine the prevalence of anti-HNA alloantibodies in non-transfused pregnant women with and without red blood cell (RBC) alloantibodies.Materials and methodsHNA alloantibodies were investigated in blood samples from 147 pregnant women with RBC alloimmunisation induced by pregnancy as the only allogeneic stimulus (group 1). The control group (group 2) consisted of 563 women with at least one pregnancy without RBC alloimmunisation. Both groups were investigated for the presence and identity of HNA alloantibodies using granulocyte agglutination tests, white blood cell immunofluorescence testing, and the bead-based LABScreen Multi Kit. Genotyping was performed to confirm the specificity of the HNA alloantibodies.ResultsGroup 1 women had a statistically higher number of HNA alloantibodies compared to group 2 women (9/147 [6.1%] vs 9/563 [1.6%]; p=0.005, OR=4.01; 95% CI 1.5–10.3). Considering only multiparous women, there was a higher statistical significance for the difference in the presence of HNA alloantibodies between the two groups (7/82 [8.5%] vs 9/493 [1.8%]; p=0.002, OR=5.02; 95% CI 1.8–13.9).DiscussionOur data show that RBC alloimmunisation is significantly associated with the development of anti-HNA alloantibodies, corroborating the hypothesis that some individuals are better immune responders and react strongly to allogeneic exposure. The presence of RBC alloantibodies can, therefore, facilitate the identification of individuals with a higher risk of alloimmunisation to antigens from other cells, also acting as a tool to avoid potentially fatal transfusion reactions.     

Erythrocyte autoantibodies in paediatric patients with sickle cell disease receiving transfusion therapy: frequency, characteristics and significance

The formation of erythrocyte autoantibodies following transfusion therapy has been described in case reports and small series. To determine the frequency, serological characteristics, and clinical significance of this phenomenon in paediatric patients with sickle cell disease, we analysed the patient database at the Duke University Pediatric Hematology Clinic. We identified children who received multiple erythrocyte transfusions, then reviewed clinical records to identify children who developed erythrocyte autoantibodies in association with transfusions. Among 184 paediatric patients who received multiple erythrocyte transfusions, 14 children (7.6%) developed warm (IgG) erythrocyte autoantibodies. Median transfusion exposure at the time of autoantibody formation was 24 erythrocyte units, range 3-341 units. The autoantibody reacted as a panagglutinin in 11 cases but had anti-e specificity in three patients. Surface complement also was detected in five patients. Clinically significant haemolysis was documented in four patients, each of whom had both surface IgG and C3 detected. The development of erythrocyte autoantibodies was associated with the presence of erythrocyte alloantibodies. Formation of warm erythrocyte autoantibodies in association with transfusions is not rare in paediatric patients with sickle cell disease. Clinicians should be aware of this complication and recognize that the presence of surface C3 is often associated with significant haemolysis.     

Human phagocyte respiratory burst by Mycobacterium bovis BCG and M. leprae: functional activation by BCG is mediated by complement and its receptors on monocytes.

We have measured the role of serum components on two parameters of the phagocytosis reaction: a) the chemiluminescence (CL) response associated with the oxidative respiratory burst in response to Mycobacterium bovis BCG and M. leprae, and b) the uptake of these two mycobacteria by healthy human monocytes. Pre-incubations of fresh or heat-inactivated serum or serum containing EGTA or EDTA indicate that these two mycobacteria activate the alternative complement pathway. Monoclonal antibodies against CR1 and CR3 inhibit the responses of M. bovis BCG and M. leprae, demonstrating that complement receptors mediate the phagocytosis of these two mycobacteria. Thus, complement and its receptors on the surface of the monocytes (CR1 and CR3) are important in the functional activation of phagocytosis of M. bovis BCG and M. leprae.     

Triamterene-induced immune haemolytic anaemia with acute intravascular haemolysis and acute renal failure.

Acute intravascular haemolysis and renal failure developed while a patient was taking triamterene. A direct antiglobulin test with a polyvalent reagent was positive. Serum caused agglutination of normal red cells in the presence of triamterene and caused an increase of partial haemolysis of both trypsin-treated erythrocytes and red cells from a patient with paroxysmal nocturnal haemoglobinuria (PNH) in the presence of complement. From the results of antibody-neutralization test and treatment with 2-mercaptoethanol, the presence of IgM antibody with lambda light chain could be demonstrated. The triamterene seemed to bind strongly to the red cells in vitro but in vivo there was no detectable adsorption to red cells. Haptenic inhibition was not demonstrated. From these results, it was assumed that this antibody was found to cross-react with methotrexate which has a structure similar to that of triamterene.     

Human monoclonal anti-D antibodies: II. THE RELATIONSHIP BETWEEN IgG SUBCLASS, Gm ALLOTYPE AND Fc MEDIATED FUNCTION

Eight monoclonal antibodies (mabs) to the Rh antigen D produced by Epstein-Barr virus transformed B-lymphoblastoid cell lines from two individuals have been compared for their behaviour in in vitro cell-mediated assays. Three IgG1 Glm(1,17) and two IgG3 G3m(21) mabs from one donor and three IgG1 Glm(3) mabs from another were used. IgG3 anti-D mabs induced greater adherence and phagocytosis of sensitized red cells by U937 monocytes than IgG1 anti-D mabs or the polyclonal anti-D. Minimum sensitization levels for rosetting and phagocytosis by U937 monocytes were 2,000 molecules IgG/cell for IgG3 and 5,000 molecules/cell for IgG1 mabs; maximum rosetting mediated by both IgG1 and IgG3 mabs was obtained at 15,000-20,000 molecules/cell. The IgG3 anti-D mabs were comparable to polyclonal anti-D in mediating binding of sensitized red cells to gamma-interferon stimulated monocyte-derived cultured macrophages and were markedly more effective than the IgG1 anti-D mabs. However, in lymphocyte ADCC assays, only anti-D mabs which were IgG1 Glm(3) were effective in mediating high levels of lysis of sensitized red cells, unlike the IgG1 Glm (1,17) or IgG3 G3m(21) mabs. Minimum sensitization levels required for this lymphocyte-mediated red cell lysis were found to be approximately 5,000 molecules/cell with one IgG1 Glm(3) mab; maximum lysis with this mab was obtained at 10,000 molecules/cell. Polyclonal anti-D containing both IgG1 and IgG3 was effective in all three assays. These observations suggest that different isotypes and allotypes of anti-D antibodies mediate red cell removed or destruction by monocyte or lymphocyte effector cell through functionally dissimilar Fc receptor interactions.     

Monocyte-Erythrocyte Interaction in Autoimmune Haemolytic Anaemia in Relation to the Number of Erythrocyte-Bound IgG Molecules and Subclass Specificity of Autoantibodies

Monocyte-erythrocyte interaction in patients with autoimmune haemolytic anaemia (AIHA) was assessed by phagocytosis and rosette assays. In most patients, a relationship was observed between haemolysis and the phagocytosis of their own erythrocytes by allogenic peripheral monocytes. An evaluation of the number of immunoglobulins on patient erythrocytes and IgG subclasses of autoantibodies shows that in patients with only IgG1 antibody or with additional IgG2 or/and IgG4, phagocytosis was always observed when the number of erythrocyte-bound IgG molecules was above 2,000. On the other hand, in all patients where IgG3 was detectable, phagocytosis was observed even if the amount of IgG was as low as 230 molecules per erythrocyte. Similar observations were made in the rosette assay. Generally, the number of erythrocyte-bound IgG and the presence of phagocytosis were correlated with the degree of haemolysis, but there were exceptions, i.e. the amount of IgG and phagocytosis were high but there was no evidence of haemolysis, or where there was little IgG, no phagocytosis but haemolysis was present. Our data do not indicate that erythrocytes from AIHA are preferentially bound to autologous monocytes.     

Increased phagocytosis of Plasmodium falciparum-infected erythrocytes with haemoglobin E by peripheral blood monocytes

Erythrocytes from subjects with homozygous and heterozygous haemoglobin E (HbE) infected with Plasmodium falciparum in vitro were phagocytosed to a greater extent by normal human monocytes than infected erythrocytes from normal subjects. Susceptibility to phagocytosis was maximal when the parasites developed to trophozoite and schizont stages in both normal and patients' erythrocytes. The increased susceptibility of P. falciparum-infected HbE erythrocytes to phagocytosis by monocytes may play a role in protection against malaria.