During homeostasis, billions of cells die through apoptosis each day, and as a potential source of autoantigens, these cells must be efficiently cleared in an immunologically silent manner to prevent pathologic autoimmune reactions (). Defective clearance of apoptotic cells (ACs) has been demonstrated both in vitro and in vivo in systemic lupus erythematosus (SLE), an autoimmune disease characterized by the generation of antibodies against multiple nuclear antigens ([2-4]). This is demonstrated by the high incidence of SLE in patients with a genetic deficiency of C1q, a complement component involved in the opsonization and clearance of ACs. Like humans with SLE, transgenic mice deficient in complement C1q develop autoantibodies or a lupus-like disease (), and mice deficient in tyrosine receptor kinases necessary for AC phagocytosis also develop severe autoimmunity (). Furthermore, immunization of mice with ACs results in autoantibody production and autoimmune disease ().
Anti-AC antibodies have previously been identified in serum samples from patients with SLE and have been noted to be bound to glomerular apoptotic nucleosomes in kidneys from patients with lupus nephritis (). Anti-AC antibodies from SLE patients can exercise pathogenic functions by promoting phagocytosis of ACs ([9, 10]), resulting in the engagement of intracellular Toll-like receptors, which leads to the release of type I interferon and other proinflammatory cytokines ([11-13]). While studies of IgG anti-AC antibodies in SLE have been focused on their influence on phagocytosis ([3, 10, 14]), systematic studies of their prevalence and significance are lacking. Similarly, the nature of IgG anti-AC antibodies and the processes leading to their generation and selection in SLE remain unclear. Of note, IgM anti-AC antibodies have been associated with protection against renal disease in SLE ().
In this study, we systematically investigated the presence of IgG and IgM antibody binding to ACs in SLE patients by use of a flow cytometry–based assay, and we determined the contribution of antibodies bearing the 9G4 idiotype (9G4+) to this autoreactivity. The study of intrinsically autoreactive 9G4+ antibodies encoded by the VH4–34 gene is informative in SLE, since due to defective germinal center editing, these antibodies represent 10–40% of all serum IgG ([16, 17]). The relevance of understanding the antigenic forces that underpin the expansion of 9G4+ antibodies in SLE is further illustrated by their high degree of specificity for SLE and their correlation with disease activity and specific clinical manifestations, including lupus nephritis ([18-21]). Our results indicated that the presence of 9G4+ anti-AC antibodies is common in SLE and showed that patients with elevated levels of 9G4+ anti-AC antibodies are more likely to have active disease. These findings demonstrate that reactivity to AC antigens contributes significantly to the expansion of a major autoreactive B cell population that is specifically expanded in SLE and provide the experimental basis for a better understanding of the antigenic forces involved in the pathogenesis of this disease.
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Antinuclear antibodies are not uncommon in otherwise healthy individuals (), which demonstrates that a breakdown in tolerance is insufficient for the occurrence of autoimmune disease and that tolerance breakdown alone cannot explain why a person develops a specific autoimmune disease. Instead, selection by disease-specific self antigens is an important factor in determining how autoimmunity is manifested. These considerations are of central significance in SLE, a systemic autoimmune disease characterized by the presence of multiple autoantibodies, some of which (anti-dsDNA, anti-Sm, and anti–ribosomal P antibodies) are highly specific for the disease. Anti-AC antibodies constitute another relevant autoantibody system, as SLE is characterized by defective clearance of ACs, which express a high density of SLE immunogens.
Anti-AC antibodies can mediate multiple pathogenic mechanisms, including tolerance breakdown, epitope spreading, and further induction of autoantibodies capable of both amplifying inflammation and directly inducing tissue damage ([8, 12, 37-39]). Elevated levels of 9G4+ antibodies are also specific for SLE and are prevalent in patients with active SLE (). The canonical intrinsic autoreactivity of 9G4+ antibodies is imparted by a germline-encoded hydrophobic patch that mediates binding to N-acetyl-lactosamine (NAL) sugar and accounts for the striking anti–B cell autoreactivity of 9G4+ antibodies in vivo and in vitro (). NAL sugar chains have been shown to be exposed on the surface of ACs ([40, 41]), and furthermore, our preliminary results ([27, 28]) indicated the potential of 9G4+ antibodies to bind ACs. Additionally, defective censoring leads to the accumulation of 9G4+ B cells in SLE germinal centers (), sites of accumulation of uncleared ACs ().
In this study, we tested the hypothesis that defective tolerance and the localization of 9G4+ B cells in germinal centers would result in selection of 9G4+ antibodies with anti-AC antibody reactivity that would contribute to the global anti-AC antibody response in SLE. The results presented here support this hypothesis. Elevated levels of 9G4+ anti-AC antibodies were present in the majority of our unselected patients with SLE (60%). Moreover, the 9G4+ anti-AC antibody MFI correlated with the degree of disease activity as measured by the SLEDAI, and patients with high levels of 9G4+ anti-AC antibodies were significantly more likely to have active disease and lupus nephritis. While the concentrations of IgG anti-AC antibodies, serum 9G4+ IgG, and anti-dsDNA antibodies also correlated with disease activity, patients with high values for these correlates who also had high levels of 9G4+ anti-AC antibodies were more likely to have active disease than were those with lower levels of 9G4+ anti-AC antibodies. Taken together, our results identify 9G4+ anti-AC antibodies as an important marker of disease activity and possibly of disease severity, an intriguing finding whose clinical implications need to be conclusively validated by ongoing longitudinal studies.
During normal homeostasis, the phagocytosis of ACs is generally antiinflammatory (). This tolerogenic antiinflammatory response is altered in SLE patients because of both changes in SLE monocytes () and a shift to Fc receptor internalization through anti-AC antibodies (). SLE anti-AC antibodies alone are sufficient to promote inflammation, as healthy donor monocytes incubated with necrotic cell material and phagocytosis-promoting SLE antibodies produce large amounts of inflammatory cytokines (). The abundance of 9G4+ antibodies in SLE means 9G4+ anti-AC antibodies are likely an important part of this process. Additionally, because 9G4+ antibodies can recognize a diverse array of self antigens ([26, 45]), 9G4+ B cells presenting AC-derived antigens may result in epitope spreading that expands and perpetuates anti-AC responses and enhance antigen presentation of AC-derived T cell epitopes, such as histone peptides from nucleosomes ().
Levels of 9G4+ anti-AC antibodies and anti-dsDNA antibodies correlated with each other and with disease activity but were nonetheless dissociated in a significant proportion of patients. This indicates that these two autoantibody species, while often produced concurrently by patients with active disease, recognize separate antigens. This conclusion is further supported by the colocalization results of the ImageStream and confocal microscopy studies (Figures 1 and 5, respectively), in which 9G4+ antibody binding to ACs did not merge with nucleic acid staining, and many DRAQ5-staining nuclear bodies had no associated 9G4 staining. This suggests that 9G4+ antibody binding to ACs is not mediated by the ability of a fraction of these serum antibodies to recognize DNA. Although it is only correlative, the higher incidence of anti-Ro antibodies in patients with 9G4+ anti-AC antibodies is consistent with our microscopy findings, as Ro localizes to apoptotic blebs () and to the perinuclear space during apoptosis ().
While the precise antigenic targets recognized by 9G4+ antibodies in ACs remains to be determined, their reactivity with other relevant antigens and established structure–function correlations provide important clues. Since 9G4+ idiotype expression, NAL binding, and B cell binding activity are dependent on conservation of the VH4–34 germline–encoded hydrophobic patch in framework region 1 ([26, 49]), the anti-AC antibody reactivity of 9G4+ antibodies could potentially also have similar structural requirements and would therefore segregate with B cell binding activity. This model would point to recognition of antigens shared between the two cellular targets, NAL in particular. Several lines of evidence, however, do not support this possibility. We observed 9G4+ anti-AC antibodies in the absence of B cell binding activity in 23% of patients, and a smaller percentage of patients displayed only 9G4+ B cell binding. Moreover, separate studies of monoclonal antibodies have indicated that the apoptotic and B cell reactivities of 9G4+ antibodies are dependent on different regions of the VH4–34 heavy chain and are likely determined by distinct antigens on the corresponding cellular targets (). That 9G4+ anti-AC antibody reactivity is not largely due to the canonical anti-NAL binding is also supported by the observation that 9G4+ antibodies in the sera of patients infected with the human immunodeficiency virus bind to B cells () but not to ACs (Jenks SA, et al: unpublished observations).
It is possible that 9G4+ anti-AC antibodies could originate from 9G4+ B cells that initially recognize canonical NAL antigens on B cells, red blood cells, or other tissues that are subsequently diversified by somatic hypermutation and selection by ACs. Yet, given the continuous presence of B cells that could exert selective pressure in the germinal centers, this model would also predict the persistence of 9G4+ B cell binding autoreactivity concurrently with the development of anti-AC antibody reactivity. Our results are more consistent with independent triggering and selection of different 9G4+ B cell clones by either B cells or ACs, with the provision that retention of the VH4–34 germline–encoded hydrophobic patch in framework region 1 would endow most AC-reactive 9G4+ antibodies with B cell binding activity as well.
Irrespective of the molecular underpinnings and temporal events of selection, these results demonstrate that like other lupus autoantibodies, while tolerance against AC antigens in a significant proportion of lupus patients is broken, this tolerance breakdown is not a universal phenomenon in SLE. Approximately 20% of patients with elevated 9G4+ IgG levels did not demonstrate anti-AC antibody reactivity, indicating that binding to ACs is not an intrinsic property of antibodies expressing the 9G4+ idiotype. Rather, this property is present in only some 9G4+ antibodies that appear to be selected in a segment of the SLE population.
Taken together, the data presented here strongly suggest that measurement of anti-AC antibodies in general and 9G4+ anti-AC antibodies in particular provide a useful tool with which to assess disease activity in SLE. Moreover, the presence of these antibodies may provide a way to segment lupus patients with different genetic and immunologic defects. Thus, a better understanding of the origin and consequences 9G4+ antibodies against ACs has the potential to increase our understanding of the pathogenesis of SLE and provide new approaches to treatment.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Sanz had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Jenks, Palmer, Sanz.
Acquisition of data. Jenks, Palmer, Marin, Hartson, Chida, Richardson.
Analysis and interpretation of data. Jenks, Palmer, Marin, Sanz.