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

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.     

Antigenicity,storage, and aging: physiologic autoantibodies to cell membrane and serum proteins and the senescent cell antigen

Summary Normal human serum contains autoantibodies to a wide range of cellular and serum proteins. IgG autoantibodies to cell membrane proteins spectrin, syndein (Band 2.1), Band 3 degradation products, and the senescent cell antigen are among them. Physiologic autoantibodies to the senescent cell antigen, a 62 000 dalton glycopeptide derived from Band 3, initiate removal of senescent, damaged, and stored cells in vivo. The senescent cell antigen is one of the two Band 3 degradation products (Mr 66 000 and 62 000) observed in freshly prepared ghosts.Since the senescent cell antigen is observed on red cells aged in situ, data suggest that Band 3 undergoes proteolysis in situ. IgG eluted from blood stored for transfusion binds to the senescent cell antigen. The amount of IgG on red cells increases during storage suggesting accumulation of the senescent cell antigen. Autoantibodies to other cell and serum proteins are discussed as possible regulators of homeostasis. The effect of age on physiologic autoantibodies is reviewed.     

Malaria: a vaccine concept based on sickle haemoglobin's augmentation of an innate autoimmune process to band 3

The protection from malaria afforded by sickle haemoglobin (and certain other haemoglobinopathies) suggests that it may be possible to utilise a common property that their erythrocytes share with both malaria-infected erythrocytes and senescent erythrocytes to develop a vaccine. All three conditions cause clustering of a specific protein molecule, band 3, on their erythrocyte's surface and this protein, when present on senescent erythrocytes at least, results in the immune recognition and removal of these by naturally occurring antibodies. It is hypothesised that if an up-regulated immune response to this protein on sickle cells is responsible for the benefit afforded to malaria patients then a vaccine using antigenic band 3 peptides may provide similar protection.     

Erythrocyte aging in sickle cell disease.

Physiological removal of old erythrocytes from the circulation by macrophages is initiated by binding of autologous IgG to senescent cell antigen (SCA). SCA is generated from the anion exchanger band 3. This process is accompanied by a number of alterations in the function and structure of band 3. We measured these aging-related parameters in erythrocytes from individuals with sickle cell anemia. Most sickle erythrocytes have characteristics that are also found in senescent normal erythrocytes, such as an increased density and considerable concentrations of cell-bound IgG. Together with the concomitant changes in structure and function of band 3, these data suggest that most sickle erythrocytes have undergone a process of accelerated aging. Preliminary results indicate that this process is reversed upon vitamin E supplementation. These data show that the erythrocyte aging paradigm may provide a useful conceptual framework for the study of the pathophysiology and the evalution of therapeutic intervention in sickle cell disease, and support the view that oxidation can generate neoantigens that are recognized by autoantibodies.     

Naturally occurring anti-band 3 antibodies and complement in phagocytosis of oxidatively-stressed and in clearance of senescent red cells

Treatment of human red blood cells with diamide and opsonization with whole serum enhanced their phagocytosis by mononuclear phagocytes. Opsonization of diamide-treated red cells with whole serum containing 20-100 times the physiologic concentration of naturally occurring anti-band 3 antibodies further increased the extent of phagocytosis. Enhanced phagocytosis was due to an anti-band 3 mediated binding of C3b to red cells via the alternative pathway. Red cell-bound anti-band 3 was slightly elevated on diamide-treated cells and elicited a C3 binding that exceeded the amount of bound antibody by two orders of magnitude. Pretreatment of red cells with a monoclonal anti-CR1 did not significantly inhibit opsonization and phagocytosis if cells were opsonized at elevated anti-band 3 concentrations. On the other hand, phagocytosis of mildly oxidized (20 microM diamide) red cells was completely inhibited by blocking CR1 if cells were opsonized with serum containing physiologic concentrations of anti-band 3. The results suggest that two types of opsonization mediate in vitro phagocytosis: one operating at physiologic anti-band 3 concentrations with mildly oxidized red cells (IC-like mechanism) and one that operates with either heavily oxidized (greater than 200 microM diamide) red cells at physiologic anti-band 3 concentrations, or with mildly oxidized cells opsonized at elevated concentration of anti-band 3. The latter mechanism is relevant in vivo. It is most likely that it starts by Fab-dependent binding of anti-band 3 to diamide-induced band 3 protein oligomers. Complement activation may occur by assembly of an alternative convertase on C3b covalently bound to red cell-associated anti-band 3. This mechanism is also likely to mediate clearance of senescent red cells, as it was primarily from senescent red cells that we could isolate complexes containing IgG covalently bound to C3b.     

Senescent cells and macrophages: key players for regeneration?

Over the last decade, our understanding of the physiological role of senescent cells has drastically evolved, from merely indicators of cellular stress and ageing to having a central role in regeneration and repair. Increasingly, studies have identified senescent cells and the senescence-associated secretory phenotype (SASP) as being critical in the regenerative process following injury; however, the timing and context at which the senescence programme is activated can lead to distinct outcomes. For example, a transient induction of senescent cells followed by rapid clearance at the early stages following injury promotes repair, while the long-term accumulation of senescent cells impairs tissue function and can lead to organ failure. A key role of the SASP is the recruitment of immune cells to the site of injury and the subsequent elimination of senescent cells. Among these cell types are macrophages, which have well-documented regulatory roles in all stages of regeneration and repair. However, while the role of senescent cells and macrophages in this process is starting to be explored, the specific interactions between these cell types and how these are important in the different stages of injury/reparative response still require further investigation. In this review, we consider the current literature regarding the interaction of these cell types, how their cooperation is important for regeneration and repair, and what questions remain to be answered to advance the field.     

Characterization of immunoglobulin binding to isolated human erythrocyte membranes: evidence for selective, temperature-induced binding of naturally occurring autoantibodies to the cytoskeleton

Human plasma contains naturally occurring autoantibodies to the predominant components of the erythrocyte membrane: band 3 and spectrin bands 1 and 2 of the cytoskeleton. The titer of cytoskeletal plasma autoantibodies increases in various hemolytic conditions, suggesting that opsonization of the cytoskeleton may play an important role in the clearance of hemolyzed (not senescent) erythrocytes from the circulation. In this study, we use Alexa Fluor 488 goat anti-human IgG conjugate (Molecular Probes, Eugene, OR, USA), to characterize plasma immunoglobulin binding to erythrocyte membranes from osmotically hemolyzed cells ('ghosts'). The results show that exposure of ghosts to plasma results in 4-fold more immunoglobulin binding to the cytoskeleton than is bound to the proteins contained within the lipid bilayer. Preincubation of the ghosts at 37 degrees C causes 8-fold more immunoglobulin binding to the cytoskeleton compared to bilayer proteins. This temperature-induced change resulted from selective immunoglobulin binding to the cytoskeleton, with no change in immunoglobulin binding to bilayer proteins. However, the rate of increase in cytoskeletal antigenicity at 37 degrees C did correlate with the rate of a conformational change in band 3, a transmembrane protein which serves as a major membrane attachment site for the cytoskeleton. The results of this study suggest that the cytoskeleton is the primary target in the opsonization of hemolyzed erythrocyte membranes by naturally occurring plasma autoantibodies. The conformational changes which occur in ghosts at 37 degrees C are associated with selective exposure of new immunoglobulin binding sites on the cytoskeleton, and with a change in the structure of band 3. We propose a model suggesting that opsonization of the cytoskeleton occurs prior to the decomposition of hemolyzed erythrocytes at 37 degrees C.     

Molecular mapping of human band 3 aging antigenic sites and active amino acids using synthetic peptides

An aging antigen, senescent cell antigen appears on old cells and marks them for death by initiating the binding of IgG autoantibody and subsequent removal by phagocytes. This antigen is derived from the major anion transport protein, protein band 3, that is involved in respiration and acid base balance. We use synthetic peptides from the transmembrane, anion transport segment of band 3 to walk band 3 to identify potential aging antigenic sites. A competitive inhibition assay with affinity purified IgG autoantibody from senescent red cells was used. Results indicate that: aging antigenic sites reside on human band 3 residues 538–554, 593–601, and 812–830; and that the smallest residues which act as aging antigenic sites are 593–601 and 813–818. The contribution of lysine and/or arginine to antigenicity is examined by synthesizing peptide analogs in which glycines or arginines are substituted for lysines or arginines. Substitution of neutral glycine for the positively charged amino acids arginine or lysine or both arginine and lysine did not result in a significant difference in antigenicity between the analog and the native band 3 peptide. Substitution of the positively charged arginine for the positively charged lysine resulted in a significant reduction in antigenicity. The chicken sequence of band 3 peptides 538–554 and 812–827 differs from that of the human peptides at several sites. Antigenicity of these chicken analogs were tested and compared to the human peptides. The data suggest that the three-dimensional configuration of band 3 segments plays a dominant role in defining the antigenic determinants reactive with senescent cell IgG autoantibodies.     

The role of senescent T cells in immunopathology

The development of senescence in tissues of different organs and in the immune system are usually investigated independently of each other although during ageing, senescence in both cellular systems develop concurrently. Senescent T cells are highly inflammatory and secrete cytotoxic mediators and express natural killer cells receptors (NKR) that bypass their antigen specificity. Instead they recognize stress ligands that are induced by inflammation or infection of different cell types in tissues. In this article we discuss data on T cell senescence, how it is regulated and evidence for novel functional attributes of senescent T cells. We discuss an interactive loop between senescent T cells and senescent non‐lymphoid cells and conclude that in situations of intense inflammation, senescent cells may damage healthy tissue. While the example for immunopathology induced by senescent cells that we highlight is cutaneous leishmaniasis, this situation of organ damage may apply to other infections, including COVID‐19 and also rheumatoid arthritis, where ageing, inflammation and senescent cells are all part of the same equation.     

Inflammatory signaling and cellular senescence

Inflammation acts as a double-edged sword in the pathogenesis of cancer. Inflammatory responses play a key role in eliminating potentially cancerous cells; however, an inflammatory microenvironment also promotes the development of cancer. Proinflammatory cytokines, the key mediators of inflammation, also play a dual role in oncogenesis. While they can promote neoplastic progression, recent studies have revealed an unexpected function of the inflammatory pathways in inhibiting cancer development. These studies demonstrate that cells undergoing senescence, a cellular program serving as a barrier to cancer development, produce increased amount of inflammatory cytokines. These inflammatory cytokines play an essential role in the initiation and maintenance of cellular senescence, and are responsible for triggering an innate immune response that clears the senescent tumor cells in vivo. The purpose of the present review is to discuss the dual roles of the inflammatory cytokines produced by senescent cells in the pathogenesis of cancer, and the signaling pathway mediating their role in cellular senescence.     

Identification of pancreatic glycoprotein 2 as an endogenous immunomodulator of innate and adaptive immune responses

Pancreatic autoantibodies are Crohn disease-specific serologic markers. The function and immunological role of their recently identified autoantigen, glycoprotein 2 (GP2), are unknown. We therefore investigated the impact of GP2 on modulation of innate and adaptive immune responses to evaluate its potential therapeutic use in mucosal inflammation. Our data indicate a previously unknown function for GP2 as an immunomodulator. GP2 was ubiquitously expressed on cells vital to mucosal immune responses. The expression of GP2 was upregulated on activated human T cells, and it was further influenced by pharmaceutical TNF-α inhibitors. Recombinant GP2 significantly decreased human intestinal epithelial cells, mucosal and peripheral T cell proliferation, apoptosis, and activation, and it distinctly modulated cytokine secretion. Furthermore, intestinal epithelial cells stimulated with GP2 potently attracted T cells. In conclusion, we demonstrate a novel role for GP2 in immune regulation that could provide a platform for new therapeutic interventions in the treatment of Crohn disease.     

Erythrocyte aging: a more than superficial resemblance to apoptosis?

In physiological circumstances, erythrocyte aging leads to binding of autologous IgG followed by recognition and removal through phagocytosis, mainly by Kupffer cells in the liver. This process is triggered by the appearance of a senescent erythrocyte-specific antigen. The functional and structural characteristics of senescent erythrocytes strongly suggest that this antigen originates on band 3, probably by calcium-induced proteolysis. Generation of vesicles enriched in denatured hemoglobin is an integral part of the erythrocyte aging process. These vesicles are also removed by Kupffer cells, with a major role for exposure of phosphatidylserine. Moreover, senescent erythrocyte-specific antigens are present on vesicles. Thus, vesicles and senescent erythrocytes may be recognized and removed through the same signals. These and other, recent data support the theory that erythrocyte aging is a form of apoptosis that is concentrated in the cell membrane, and provide the context for future studies on initiation and regulation of the erythrocyte aging process. Insight into the normal aging mechanism is essential for understanding the fate of erythrocytes in pathological circumstances and the survival of donor erythrocytes after transfusion.     

Functional Characterization of Autoantibodies against Complement Component C3 in Patients with Lupus Nephritis

Lupus nephritis (LN) is a complication of the autoimmune disease systemic lupus erythematosus. Because the complement system plays a critical role in orchestrating inflammatory and immune responses as well as in the clearance of immune complexes, autoreactivity to complement components may have considerable pathological consequences. Autoantibodies against the central complement component C3 have been reported in systemic lupus erythematosus, but their molecular mechanism and functional relevance are not well understood. The objective of this study was to evaluate the frequency and the functional properties of the anti-C3 autoantibodies. Anti-C3 autoantibodies were measured in plasma of 39 LN patients, and identification of their epitopes on the C3 molecule was performed. By using surface plasmon resonance, we analyzed the influence of patient-derived IgG antibodies on the interaction of C3b with Factor B, Factor H, and complement receptor 1. The capacity of these antibodies to dysregulate the C3 convertase on the surface of endothelial cell was measured by flow cytometry. Here we report that the frequency of anti-C3 autoantibodies in LN is ∼30%. They inhibited interactions of the negative complement regulators Factor H and complement receptor 1 with C3b. An enhanced C3 deposition was also observed on human endothelial cells in the presence of C3 autoantibodies. In addition, anti-C3 autoantibody levels correlated with disease activity. In conclusion, the anti-C3 autoantibodies in LN may contribute to the autoimmune pathology by their capacity to overactivate the complement system.     

Band-3 polymers and aggregates, and hemoglobin precipitates in red cell aging

As part of our systematic ongoing studies of mechanisms of cellular and molecular aging, we developed a "biochemical profile" of senescent human red cells. This "red cell aging" panel allows us to assess functional red cell age independent of chronologic age. The panel used to obtain this profile includes IgG binding, phagocytosis, enzyme activity, anion transport, ankyrin binding, and immunoblotting with antibodies to band 3. We used this panel to compare the biochemical profile of glucose 6-phosphate dehydrogenase-deficient and hemoglobin K?ln cells containing high molecular weight protein polymers or hemoglobin precipitates with that of normal senescent cells. We found no evidence in support of the concept that aggregation of band 3 plays a role in the mechanism for generating senescent cell antigen. Observations such as these support the hypothesis that degradation of band 3, rather than aggregation is a critical event in IgG binding and normal erythrocyte aging.     

Potential involvement of the immune system in the development of endometriosis

This article presents an overview of immunological factors and their role in the development of endometriosis, with emphasis on inflammatory cytokines, growth and adhesion factors. Although retrograde menstruation is a common phenomenon among women of reproductive age, not all women who have retrograde menstruation develop endometriosis. The development of endometriosis is hypothesised to be a complex process, which may be facilitated by several factors, including the quantity and quality of endometrial cells in peritoneal fluid (PF), increased inflammatory activity in PF, increased endometrial-peritoneal adhesion and angiogenesis, reduced immune surveillance and clearance of endometrial cells, and increased production of autoantibodies against endometrial cells. Potential biomarkers like cytokines and autoantibodies upregulated during development of endometriosis may be useful in the development of a non-surgical diagnostic tool. Although endometriosis can be treated using hormonal suppression, there is need for non-hormonal drugs, which can inhibit the development of endometriosis and alleviate pain or infertility without inhibition of ovulation. New molecules that modulate immune function in endometriosis should be the targets for future research.     

Modulation of dendritic cell maturation and function by B lymphocytes

Investigating the signals that regulate the function of dendritic cells (DC), the sentinels of the immune system, is critical to understanding the role of DC in the regulation of immune responses. Accumulating lines of evidence indicate that in addition to innate stimuli and T cell-derived signals, B lymphocytes exert a profound regulatory effect in vitro and in vivo on the Ag-presenting function of DC. The identification of B cells as a cellular source of cytokines, chemokines, and autoantibodies that are critically involved in the process of maturation, migration, and function of DC provides a rationale for immunotherapeutic intervention of autoimmune and inflammatory conditions by targeting B cells. Conversely, efficient cross-presentation of Ags by DC pulsed with immune complexes provides an alternative approach in the immunotherapy of cancer and infectious diseases.     

Natural IgM anti-leukocyte autoantibodies attenuate excess inflammation mediated by innate and adaptive immune mechanisms involving Th-17

Little is known about the function of natural IgM autoantibodies, especially that of IgM anti-leukocyte autoantibodies (IgM-ALA). Natural IgM-ALA are present at birth and characteristically increase during inflammatory and infective conditions. Our prior clinical observations and those of other investigators showing fewer rejections in renal and cardiac allografts transplanted into recipients with high levels of IgM-ALA led us to investigate whether IgM-ALA regulate the inflammatory response. In this article, we show that IgM, in physiologic doses, inhibit proinflammatory cells from proliferating and producing IFN-γ and IL-17 in response to alloantigens (MLR), anti-CD3, and the glycolipid α-galactosyl ceramide. We showed in an IgM knockout murine model, with intact B cells and regulatory T cells, that there was more severe inflammation and loss of function in the absence of IgM after renal ischemia reperfusion injury and cardiac allograft rejection. Replenishing IgM in IgM knockout mice or increasing the levels of IgM-ALA in wild-type B6 mice significantly attenuated the inflammation in both of these inflammatory models that involve IFN-γ and IL-17. The protective effect on renal ischemia reperfusion injury was not observed using IgM preadsorbed with leukocytes to remove IgM-ALA. We provide data to show that the anti-inflammatory effect of IgM is mediated, in part, by inhibiting TLR-4-induced NF-κB translocation into the nucleus and inhibiting differentiation of activated T cells into Th-1 and Th-17 cells. These observations highlight the importance of IgM-ALA in regulating excess inflammation mediated by both innate and adaptive immune mechanisms and where the inflammatory response involves Th-17 cells that are not effectively regulated by regulatory T cells.     

Comparative proteomics of erythrocyte aging in vivo and in vitro

During aging in vivo and in vitro, erythrocytes display removal signals. Phagocytosis is triggered by binding of autologous IgG to a senescent cell antigen originating on band 3. Erythrocytes generate vesicles as an integral part of the aging process in vivo and in vitro, i.e. during storage. These vesicles display senescent cell antigens as well as phosphatidylserine, that is recognized by scavenger receptors. Recent comparative proteomic analyses of erythrocytes and their vesicles support the hypothesis that aging is accompanied by increased binding of modified hemoglobins to band 3, disruption of the band 3-mediated anchorage of the cytoskeleton to the lipid bilayer, vesicle formation, and antigenic changes in band 3 conformation. Proteomic data also suggest an, until then unknown, involvement of chaperones, stress proteins, and proteasomes. Thus, the presently available comparative proteomic analyses not only confirm previous immunochemical and functional data, but also (1) provide new clues to the mechanisms that maintain erythrocyte homeostasis; (2) open new roads to elucidate the processes that regulate physiological erythrocyte aging and removal, and thereby; (3) provide the foundation for rational interventions to prevent untimely erythrocyte removal, and unwanted interactions between the erythrocyte and the immune system, especially after transfusion.     

Exosomal vesicles enhance immunosuppression in chronic inflammation: Impact in cellular senescence and the aging process

Exosomes represent an evolutionarily conserved signaling pathway which can act as an alarming mechanism in responses to diverse stresses, e.g. chronic inflammation activates the budding of exosomal vesicles in both immune and non-immune cells. Exosomes can contain both pro- and anti-inflammatory cargos but in chronic inflammation, exosomes mostly carry immunosuppressive cargos, e.g. enzymes and miRNAs. The aging process is associated with chronic low-grade inflammation and the accumulation of pro-inflammatory senescent cells into tissues. There is clear evidence that aging increases the number of exosomes in both the circulation and tissues. Especially, the secretion of immunosuppressive exosomes robustly increases from senescent cells. There are observations that the exosomes from senescent cells are involved in the expansion of senescence into neighbouring cells. Interestingly, the age-related exosomes contain immune suppressive cargos which enhance the immunosuppression within recipient immune cells, i.e. tissue-resident and recruited immune cells including M2 macrophages, myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg). It seems that increased immunosuppression with aging impairs the clearance of senescent cells and their accumulation within tissues augments the aging process.