Mechanisms by which Incompatible Red Cells may be Cleared from Circulation
Introduction
One of the leading causes of transfusion-associated mortality is related to haemolytic transfusion reactions.1 Many factors that account for haemolytic transfusion, and complement activation is the most widely studied and documented feature linked with such reactions and fatalities, and many researchers have focused on the role of complement initiation, activation and regulation with respect to these reactions. Others have focused on the underlying strategies involved in mitigating the complement modulation, in the event of an incompatible red blood cell transfusion.1 Many reports have stated that incompatible red blood cell (RBC) transfusions are the leading cause of death in haemolytic transfusion reactions (HTRs). In most cases, incompatible RBCs are cleared from the system; however, in some instances, a few incompatible RBCs remain the circulatory system with the presence of RBC-specific antibodies. This manly occurs due a condition in the system known as antigen modulation.1
Mechanisms by which incompatible red cells may be cleared from circulation
Complement activation is one of the most widely studied mechanisms and comprises three events: activation, classic and alternate events.1,3 The alternative and lectin pathways of the complement activations play an important role in the immune system. The initiation of the complement activity is presented by the antibodies at the stage of haemolytic transfusion. Each pathway works independently, irrespective of the initiating stimulus to converge and form the enzyme complex that converts the complement component C3 into active products, such as C3a and C3b. These active components, especially C3b, initiate a cascade reaction to form a membrane attack complex initiating lysis.1,3 The initial transfusion leads to antigen-antibody interactions that initiate the first component of the classical pathway, known as C1q. A cascade of conformational changes occurs in the serine protease C1r, which eventually cleaves C1s to release the active C1s protease. The release of C1s is bound to cleave C2 and C4 to target C2b and C4b, in order to release the soluble products C2a and C4a. The C3 convertase is formed by the binding of the C2b and C4b, which form the complex.1,3 The C3 convertase complex cleaves C3, in order to form C3a and C3b. The C3a possesses antimicrobial activity and provides a complement regulator for a range of immunologic pathways, such as the activation of phagocytes and mast and endothelial cells. The complement cascade continues with the help of C3b, which is bound to the target membrane with the help of a reactive thioester. The conversion of C5 to C5a and C5b is initiated by the bound C3b, which is attached with an additional C3b and C2b. C5a plays a role similar to C3a and regulates a range of factors in the immune system. The C5b is associated with the regulation of the C6, C7, and C8 components that result in the formation of C9. The C9-pore leads to the osmotic lysis of the target.1,3
Fcg Receptor Ligation: Antigen Modulation and Protection of RBCs from Antibodies
Based on current evidence, ligation of the FcgRs or fixation of the complement are observed in activities of alloantibodies and autoantibodies that damage self-tissue and transplanted tissue. Many researchers have reported that many pathways may constitute immune self-tissues that are resistant to the complement activation, in order to protect against such antibodies.4 It is not well understood if parallel pathways co-exist to provide protection from antibodies in the case of FcgR ligation. It is essential to understand different and novel pathways through which cell surface antigens are decreased to the presence of Ag modulation. In addition, Ag modulation is associated with the recognition of cells for Fcg-dependent clearance.4 FcgR ligation is required for Ag modulation. These pathways, in conjugation with the presence of Ag modulation, provide a scientific and molecular basis for self-protective cascades for FcgR-mediated antibody damage.4,5 One recent study used the retransfer procedure wherein the presence of an FcgR-dependent process resulted in RBCs resistant to clearance. The experiment confirmed the presence of Ag modulation and its relationship to the clearance of incompatible RBCs.5 However, there were some technical limitations; researchers could not conclude whether the Ag modulation was completely responsible for the resistance.5
Novel Mechanism: Human blood group glycophorin A (hGPA)
Compatibility testing is recommended during transfusion therapy, to avoid HRTs and other fatal conditions. Researchers in a murine-model study analysed RBC survival with respect to incompatible transfusions. Monoclonal antibodies were utilized for Duffy (also known as fusion proteins or HOD antigens) and human blood group glycophorin A (hGPA). RBCs that expressed HOD were cleared from the system with the help of anti-Duffy antibodies and Fc-receptors.5 Researchers also observed that these RBCs were cleared from the system with help of anti-hGPA antibodies through a unique biphasic mechanism.5 The first stage of the bi-phase mechanism is the agglutination of these RBCs via anti-hGPA antibodies. In the second phase, incompatible RBCs are removed using phagocytic cells irrespective of the presence of complement activation and Fc-receptors. Based on evaluations of the release or clearance of RBCs, a cytokine burst was observed with the presence of Fc-receptors. This mechanism suggests that there are two factors involved in the clearance of RBCs: cytokine secretion and phagocytosis.
Another study analysed the two antigens involved in the clearance pathways of RBCs with respect to the survival of RBCs in vivo.6 Researchers determined that not all HOD RBCs and hGPA were cleared from the system with the help of the two antigens (anti-hGPA and anti-Duffy). In some cases, a multitude of RBCs were resistant to the hGPA and HOD antigens.6 Furthermore, C3 or the competent pathway was not required for the clearance of RBCs, suggesting resistance to hGPA or HOD RBCs. These findings contradict the initial ones that suggest the biphasic mechanism of hGPA and HOD antigens.6 Research on the different mechanisms also suggest that there could be a spectrum of RBCs that are not susceptible to the antigens, while a majority of them state that the bi-phasic mechanism is a novel and unique factor in the clearance of incompatible RBCs.5,6
Anti-Band 3 Antibodies
The anion transport protein (known as Band 3) on the RBCs is altered in the 55-kDa chymotryptic fragment by naturally found anti-Band 3 antibodies. The selective clearance of incompatible, oxidative and senescent RBCs takes place through the binding of these anti-Band 3 antibodies.8 However, such NAbs are known to be present in lower concentrations and to have a low affinity that prevents them from binding on second sites. The two binding proteins, such as Syk kinase and hemichromes on the Band 3 protein, are known to result in the detachment of the Band 3 proteins from the cytoskeleton.8 This occurs after the cascade of biochemical events following oxidative damage or cellular senescence. Bivalent binding of the anti-Band 3 antibodies occurs due to the clustering of the Band 3 proteins. Stimulating the C3b deposition occurs with the bivalent binding of anti-Band 3 antibodies through the generation of C3b2-IgG complexes, which are known to act as C3 convertase precursors similar to the complement pathway.8 Induced anti-lactoferrin antibodies increase the number of antibodies binding and the oligomerized Band 3 proteins. The 38-kDa fragment of Band 3 proteins and the lactoferrin carbohydrates are bound by polylactosaminyl oligosaccharide. The anti-lactoferrin antibodies a have anti-neutrophil cytoplasmic antibodies in patients with HRT and autoimmune disease.8
PS Exposure
Immunoglobulin G (IgG)-mediated haemolysis occurs when there is a mismatch of transfused RBCs and recipient RBC antibodies. This phenomenon may occur in a reaction known as a delayed haemolytic transfusion reaction (DHTR). In this case, sensitized RBCs are cleared from the system with the help of the complement activation or the release of macrophages. Researchers suggest that many of DHTR aetiologies remain unclear, since they occur in the absence of the alloantibody or the RNC autoantibody.9 There are three types of DHTR, and that many mechanisms have been proposed to examine these types, which may include reactive haemolysis by hyperactive macrophages or the bystander. In the case of patients with sickle cell disease, the clearance of sickled erythrocytes occurs via macrophages, due to the exposure of phosphatidylserine (PS). This is a novel phenomenon and mechanism in the case of abnormal RBC structures and their clearance during transfusion.9
Researchers in a murine-modelled clinical study confirmed that PS exposure is closely associated with DHTR and the clearance of RBCs in the event of antigen-matched RBC with the help of macrophages and haemolysis. The clearance takes place irrespective of the presence of alloantibodies and autoantibodies. The study also concluded that an increased exposure of PS results in increased haemolysis in the case of incompatible RBCs during DHTR.9
CD-47 in RBC Clearance
RBC clearance is induced by the interplay of CD47 on RBC, which is closely associated with the signal regulatory protein alpha (SIRPalpha protein) and the receptor macrophages. Increased exposure of PS has also been linked with RBC clearance. CD47 (integrin-associated protein) protects RBCs from phagocytosis.10 The binding of he SIRPα on macrophages in the process of engulfment is prevented by the CD47 protein, which is also reflected as the ‘Do not engulf/destroy me’ signal. However, recent clinical studies have identified CD-47 as a promoter for phagocytosis of incompatible RBCs in the systems. These RBCs have a conformational difference because the CD-47 induces phagocytosis with the help of thrombospondin-1 and SIRPα. These mechanisms of the CD-47 are known to induce phagocytosis, and the clearance of RBCs is of prime importance to researchers.10
SC Receptors and Incompatible RBC Clearance
SC receptors are widely known as scavenger receptors and aim to recognise modified low-density lipoprotein (LDL), either by acetylation or oxidation. SC receptors clean or remove foreign objects like incompatible RBCs from the body.11 The removal mechanism is based on the ligand specificity. A modified ligand on the incompatible receptor induces a cascade of reactions, eventually leading to their destruction and clearance via SC receptors.11
Based on current scientific evidence, there are three types of SC receptors, A, B, and C, differentiated by their structures.11 Class A receptors comprise six main domains: collage, cysteine, space, alpha-helical, transmembrane, and cytosol.11 These receptors comprise nearly 70% of incompatible RBC removals, due to their high binding affinity towards the modified ligands on such RBCs. Class B receptors (SCARB) have two transmembrane regions that clear RBCs by macrophage activity. The third class, SCARC receptors, has a transmembrane protein that plays a vital role in RBC clearance. In short, RC receptors play a vital role in the clearance of incompatible RBCs.11
Conclusion
Based on current evidence on the clearance of incompatible RBCs during therapeutic transfusions, three main mechanisms were identified: complement activation, Fc-receptor-linked mechanisms and bi-phasic mechanisms (anti-hGPA and anti-Duffy). This paper provided an overview of the key mechanisms involved in the clearance of incompatible RBCs, including those resistant to other mechanisms. Further studies are needed to confirm the use of these mechanisms or the probability of combining them for the clearance of incompatible RBCs during transfusion therapy.5,6,7
References
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