Previous studies demonstrated that 20% of haemoglobin is lost from circulating erythrocytes during their total lifespan by vesiculation. To study whether removal molecules other than membrane-bound haemoglobin were present in erythrocyte-derived vesicles, flow cytometry and immunoblot analysis were employed to examine the presence of phosphatidylserine (PS) and IgG, and senescent cell antigens respectively. It was demonstrated that 67% of glycophorin A-positive vesicles exposed PS, and that half of these vesicles also contained IgG. Immunoblot analysis revealed the presence of a breakdown product of band 3 that reacted with antibodies directed against senescent erythrocyte antigen-associated band 3 sequences. In contrast, only the oldest erythrocytes contained senescent cell antigens and IgG, and only 0.1% of erythrocytes, of all ages, exposed PS. It was concluded that vesiculation constitutes a mechanism for the removal of erythrocyte membrane patches containing removal molecules, thereby postponing the untimely elimination of otherwise healthy erythrocytes. Consequently, these same removal molecules mediate the rapid removal of erythrocyte-derived vesicles from the circulation.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2141.2008.07055.x

Erythrocyte vesiculation: a self-protective mechanism?

Macrophages tightly control the production and clearance of red blood cells (RBC). During steady state hematopoiesis, approximately 10(10) RBC are produced per hour within erythroblastic islands in humans. In these erythroblastic islands, resident bone marrow macrophages provide erythroblasts with interactions that are essential for erythroid development. New evidence suggests that not only under homeostasis but also under stress conditions, macrophages play an important role in promoting erythropoiesis. Once RBC have matured, these cells remain in circulation for about 120 days. At the end of their life span, RBC are cleared by macrophages residing in the spleen and the liver. Current theories about the removal of senescent RBC and the essential role of macrophages will be discussed as well as the role of macrophages in facilitating the removal of damaged cellular content from the RBC. In this review we will provide an overview on the role of macrophages in the regulation of RBC production, maintenance and clearance. In addition, we will discuss the interactions between these two cell types during transfer of immune complexes and pathogens from RBC to macrophages.

/pdf/of-macrophages-and-red-blood-cells-a-complex-love-story-322fzhxm4r.pdf

Of macrophages and red blood cells; a complex love story

All cells require inorganic sulfate for normal function. Sulfate is among the most important macronutrients in cells and is the fourth most abundant anion in human plasma (300 μM). Sulfate is the m...

https://www.physiology.org/doi/pdf/10.1152/physrev.2001.81.4.1499

Physiological roles and regulation of mammalian sulfate transporters.

Erythrocyte transfusion is essential in conditions of large blood loss, of inadequate bone marrow production and of increased erythrocyte breakdown. The structural and biochemical changes that erythrocytes go through during storage, probably associated with the disappearance of up to 30% of the erythrocytes within 24 h after transfusion, are likely to contribute to the transfusion side effects: iron overload, erythrocyte adhesion to the endothelial surface with proinflammatory consequences, autoantibody formation, endothelial damage by released erythrocyte constituents, a hampered microcirculation and oxygen delivery. In vivo, senescent erythrocytes are marked for removal by binding of autologous immunoglobulin G to ageing antigens, which arise by changes in the conformation of the membrane domain of band 3. Also, vesicle formation has been described as an integral part of the erythrocyte ageing process. Comparable changes occur during erythrocyte storage. This review describes the current state of knowledge of the mechanism of erythrocyte ageing in vivo, ageing-related changes occurring during erythrocyte storage in blood bank conditions and their possible relation with the transfusion side effects. In view of the key position of band 3 in the maintenance of erythrocyte structure and function, elucidation of the pathways that control posttranslational modification of band 3 during storage may lead to new approaches towards maintaining ATP concentration and cellular integrity. This review concludes with the challenge to further explore the underlying processes of erythrocyte ageing in order to provide physiologically relevant tools for assessing and predicting erythrocyte homeostasis in vitro and in vivo and thereby to contribute to the development of rational transfusion protocols for various patient categories.

Erythrocyte ageing in vivo and in vitro: structural aspects and implications for transfusion.

The proteome of red cell membranes and vesicles during storage in blood bank conditions

Our current studies focus on the molecular changes induced by aging. During aging, changes in proteins occur that alter their function and render them immunogenic. These "neoantigens" are recognized by physiologic autoantibodies. Physiologic autoantibodies and their corresponding antigens offer therapeutic strategies for disease intervention through the innate immune response. Early studies done in the 1970s showed humans and animals to have physiologic antibodies that bind to a neoantigen called senescent cell antigen (SCA), which appears on senescent and damaged cells and initiates their removal by macrophages. These studies led to the discovery that oxidation can generate a new antigen in situ. Oxidation accelerated aging of red cells, generated SCA and IgG binding, and triggered removal of red cells by macrophages. Since then, a number of laboratories have found that oxidation can generate other neoantigens. For example, oxidized LDL (OxLDL) induces antibodies that can modify the natural progression of atherosclerosis. Apoptotic cells express oxidatively modified moieties on their surfaces that are involved in macrophage recognition and phagocytosis. Physiologic autoantibodies were used to isolate SCA from brain tissue. HPLC and fast atom bombardment ionization mass spectrometry (FAB-MS) of the isolated antigen suggested that the aging antigen is a subset of band 3, a family of proteins also called anion exchange proteins (AE1-3). FAB-MS results indicate that residues matching all three band 3 isoforms (AE1, AE2, and AE3) are detected in aging antigen fractions. Among the fragments identified with FAB-MS was a sequence corresponding to an aging epitope, human band 3 sequence LFKPPKYHPDVPYVKR, residue 812-830 in AE1; HHPDVTYVK, residue 1144-1152 in AE2; or HHPEQPYVTK, residue 1135-1144 in AE3. A residue that is close to that region was identified in mouse AE1 ASGPGAAAQIQEVK, residue 762-775. The potential for altering the natural progression of diseases using select peptide-defined epitopes within or overlapping the aging antigenic site (547-553 and 824-829) is discussed using the innate immune response to band 3 in malaria as an example.

Immunoregulation of cellular life span.

Physiologic autoantibodies, that is, those with an active physiologic role, are an important part of the normal human immune system and are essential in maintaining homeostasis. Evidence suggests that the body uses autoantibodies to prevent disease and to self-treat diseases once started. This suggests a potential therapeutic role for autoantibodies, or, even better, a way to use them to prevent disease. Their capacity to remove aged, damaged cells is well established. Immunoglobulin (Ig) G autoantibodies bind to senescent cell antigen (SCA), which is an altered band 3 anion exchanger protein found mainly on aged cells. Once bound, IgG triggers the removal of the senescent cells by macrophages. Band 3 is altered primarily by oxidation, which in turn generates SCA. These studies demonstrated that oxidation can generate neoantigens that the immune system will recognize. Band 3 isoforms are ubiquitous: they have been found in all mammalian cells and species so far examined. The innate immune response to band 3 membrane proteins, and their regulation of cellular lifespan and therapeutic potential will be presented. Examples of other potential innate and physiologic autoantibodies include neuroprotective antibodies to amyloidgenic toxic peptides and antibodies to oxidized LDL (OxLDL), which modify the natural progression of atherosclerosis.

Physiologic autoantibody and immunoglobulin interventions during aging.

Posttranslational changes in band 3 in adult and aging brain following treatment with ergoloid mesylates, comparison to changes observed in alzheimer' s disease

Molecular recognition of senescent cells involves oxidation of a crucial membrane protein leading to generation of a neoantigen, called ‘senescent cell antigen’ (SCA), and binding of physiologic autoantibodies. These IgG autoantibodies trigger macrophage removal of the cell prior to its lysis at a time when anion transport has decreased but the membrane is still grossly intact. The neoantigen SCA is generated by oxidation of a major anion transport protein called band 3 or anion exchange protein. In this study, we use IgG physiologic autoantibodies from senescent red cells to isolate SCA from brain, and HPLC and fast atom bombardment ionization mass spectrometry (FAB-MS) to compare brain SCA to band 3. HPLC peptide maps of band 3 and SCA showed substantial homology, suggesting that SCA is a subset of band 3, and includes an estimated ≥45% of the band 3 molecule. FAB-MS results indicate that residues matching all three band 3 isoforms (AE1, AE2 and AE3) are detected in SCA fractions. These findings suggest that other isoforms of band 3 may undergo the same aging changes that AE1 on red blood cells undergoes to generate SCA. This provides confirmation that SCA is on non-erythroid cell types. Implications of these studies to the generation of neoantigens by oxidation and their recognition by autoantibodies to them are discussed. Copyright © 2003 John Wiley & Sons, Ltd.

Mapping of senescent cell antigen on brain anion exchanger protein (AE) isoforms using HPLC and fast atom bombardment ionization mass spectrometry (FAB-MS).

Marguerite Kay

Papers

Immunoregulation of cellular life span.

Physiologic autoantibody and immunoglobulin interventions during aging.

Posttranslational changes in band 3 in adult and aging brain following treatment with ergoloid mesylates, comparison to changes observed in alzheimer' s disease

Mapping of senescent cell antigen on brain anion exchanger protein (AE) isoforms using HPLC and fast atom bombardment ionization mass spectrometry (FAB-MS).