Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the formation of autoantibodies against nuclear self antigens. The formation of these autoantibodies was shown to be T cell dependent (1). SLE is a prototype of an immune complex–mediated disease, in which the deposition of chromatin–antichromatin immune complexes in the basement membrane of, for example, skin and kidney can elicit a local inflammatory reaction (2). Chromatin, in particular mononucleosomes, can be detected in the circulation of patients with SLE and lupus mice (3, 4). Most likely, this chromatin is released from apoptotic cells through the action of apoptosis-activated endonucleases (5). Both an increased rate of apoptosis and an insufficient clearance of apoptotic cells/material have been implicated as key processes leading to the presence of chromatin in the circulation of SLE patients and to the subsequent development of the antichromatin immune response. Indeed, experimental interference with apoptosis or with the clearance of apoptotic material can lead to the development of antinuclear autoantibodies and lupus disease manifestations like glomerulonephritis in mice (6). Accordingly, the clearance of apoptotic material by phagocytes appears to be impaired both in lupus mice and in SLE patients (7, 8).
Apoptosis leads to dramatic morphologic and biochemical changes of cells. The formation of apoptotic blebs is one of the characteristic cellular features during apoptosis. Autoantigens that are involved in SLE are clustered in the segregating apoptotic blebs (9), and these blebs are specifically recognized by autoantibodies (10, 11). We define the apoptotic cell body as the cellular remnant after the blebbing process has ended (see Figure 1). Apoptotic cell bodies also have been termed late apoptotic or secondary necrotic cells. During apoptosis, various modifications of autoantigens (e.g., chromatin) may take place, including cleavage by proteinases, caspases, and/or endonucleases and specific posttranslational modifications (6, 11–15). These findings suggest that apoptotic blebs are a source of (modified) autoantigens and that they may play a key role in the development of the antichromatin response in SLE. Normally, early apoptotic cells are efficiently removed by macrophages, nonprofessional phagocytes, and dendritic cells (DCs), which prevents the release of blebs containing (modified) autoantigens. We hypothesize that in SLE, modified chromatin derived from apoptotic cells, and in particular from blebs, has escaped a proper clearance. Modified chromatin can then act as a danger signal leading to maturation of DCs. These matured DCs can present the altered self antigens to T cells (6, 16–18).
Figure 1. Scheme defining apoptotic blebs and apoptotic cell bodies. Left, Healthy cell. Middle, Actively blebbing apoptotic cell. Right, Remaining apoptotic cell body (ACB) that has finished blebbing with separated apoptotic blebs.
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Whether the exposure of the immune system to an (auto)antigen will result in tolerance or (auto)immunity depends on the maturation status of the DCs, in particular that of myeloid DCs. In the immature state DCs capture antigens, including self antigens like apoptotic cells. Immature DCs loaded with self antigens can induce tolerance, which is associated with the production of antiinflammatory cytokines like transforming growth factor β (TGFβ) and interleukin-10 (IL-10). In contrast, fully matured and activated DCs are the most efficient antigen-presenting cells that induce antigen-specific immunity, associated with the production of proinflammatory cytokines like tumor necrosis factor α (TNFα) and IL-6 (18). Several reports describe that in particular late apoptotic cells and/or secondary necrotic cells lead to maturation of DCs, while early apoptotic cells do not exert such a maturation effect (19–21).
Recently, we have demonstrated that apoptosis-induced histone acetylation is a trigger for the immune system in SLE. Histone acetylation appeared to be pathogenic in prediseased lupus mice, whereas hyperacetylated nucleosomes led to maturation of myeloid DCs and subsequent syngeneic T cell activation (11). Other studies have shown that administration of apoptotic cells to lupus-prone mice, alone or in combination with DCs, leads to the development of autoimmunity (22–26). Notably, another type of DC, the plasmacytoid DC (PDC), responds specifically to RNA/DNA-containing immune complexes by producing the type I interferon interferon-α (IFNα), which seems crucially involved in the pathogenesis of SLE (27, 28). However, ingestion of apoptotic cells by PDCs is unlikely to occur (29).
We hypothesize that the ingestion of apoptotic blebs and/or apoptotic cell bodies by myeloid DCs leads to maturation of DCs and subsequent stimulation of T cells. In this study in mice, we have separated apoptotic blebs from apoptotic cell bodies, and we have evaluated their ingestion by myeloid DCs and their capacity to induce maturation of myeloid DCs. Furthermore, we have determined the T cell stimulatory capacity of these DCs after exposure to apoptotic blebs or apoptotic cell bodies.
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We observed in a murine system that the percentage of DCs that ingested apoptotic blebs was about 2–3-fold higher than the percentage of DCs that ingested apoptotic cell bodies. Our data extend some of the observations made by Frisoni et al (35), who described an increased uptake by DCs of a preparation enriched in rather large apoptotic blebs compared with a nonfractionated apoptotic cell preparation, especially in the presence of opsonizing antibodies. Seemingly in contrast with Frisoni et al's data and ours, Ip and Lau showed that late apoptotic cells induced maturation of DCs (19). However, in their study apoptotic cell bodies and apoptotic blebs were not separated or defined, suggesting that the observed effects were mediated by apoptotic blebs and not by apoptotic cell bodies.
We found that the addition of apoptotic blebs, but not of apoptotic cell bodies, to mouse DC cultures resulted in maturation of DCs, as measured by an up-regulated expression of the costimulatory molecules CD40 and CD86 and an increased production of the proinflammatory cytokines IL-6 and TNFα. Interestingly, elevated levels of endogenous IL-6 have been described in SLE (36). There are several candidate molecules residing in the blebs that may specifically trigger the maturation of DCs. It has been shown that besides nucleosomes, which are comprised of histones and DNA, many other SLE-associated autoantigens are clustered in blebs, such as Sm, RNP, and Ro 60, containing U1 and Y1–Y5 RNAs, respectively (9, 37). Indeed, maturation of DCs is a known effect of nucleosomes (38), mammalian DNA (39), and RNA (37).
The failure of chloroquine to inhibit bleb-induced maturation of DCs, as shown here, indicates that TLRs 3, 7, and 9 are not involved, suggesting that DNA and RNA do not mediate the bleb-induced maturation of DCs. However, nucleosomes could very well be responsible for the observed DC maturation, since normal nucleosomes can lead to maturation of DCs in a myeloid differentiation factor 88/TLR–independent way (38). It has also been suggested by others that the initiation phase of SLE, mediated by uptake of apoptotic material by DCs, is TLR independent, while the amplification phase, with uptake of TLR ligands derived from self antigens (principally nucleic acids) complexed with autoantibodies, seems to be TLR dependent (40). Once maturation of DCs has been induced, it can amplify further uptake of apoptotic blebs, since an increased antigen uptake has been described in the early phase of DC maturation (41).
An additional factor triggering the maturation of DCs may be the presence of apoptosis-induced modifications on the nucleosomes residing in apoptotic blebs. We have recently identified apoptosis-induced acetylation of nucleosomes as a pathogenic factor in SLE and found that hyperacetylated nucleosomes were superior in maturation of DCs when compared with normal nucleosomes (11). These apoptosis-induced chromatin modifications were predominantly located in the apoptotic blebs and to a lesser extent in apoptotic cell bodies, which could explain the higher maturation potential of the blebs compared with the apoptotic cell bodies. Even at extremely high concentrations of apoptotic cell bodies, we did not observe any maturation of DCs.
There are several ways to explain how DC maturation by blebs can be a pathogenic factor in the development of SLE. As shown here, uptake of apoptotic blebs will lead to an immunogenic presentation of native and modified autoantigens by DCs to T cells, possibly resulting in a Th17 response (see below). Two routes may then lead to the development of full-blown autoimmunity: 1) direct activation of autoreactive T cells that recognize the autoantigens presented by bleb-matured DCs, and 2) activation of T cells that recognize cryptic epitopes, or modified self antigens, with subsequent activation of autoreactive B and T cells via epitope spreading.
However, a remaining key question is whether apoptotic blebs can also be found in vivo. Indeed, particles representing apoptotic blebs, sometimes referred to as microparticles, can be found in the circulation of SLE patients and also in healthy controls (42, 43). Whether apoptotic blebs found in SLE patients are able to induce maturation of DCs is currently unknown. Nevertheless, the concentrations of nucleosomes, also residing in the apoptotic blebs, that are found in the circulation of patients with SLE correspond to the bleb concentrations that induce maturation of DCs in our study (38). This suggests that the in vivo concentrations of apoptotic blebs/nucleosomes are sufficient for induction of maturation of DCs.
We also observed that DCs matured by apoptotic blebs, in contrast to DCs exposed to apoptotic cell bodies, obtained an increased ability to stimulate allogeneic T cells. Upon stimulation with bleb-matured DCs, T cells produced IFNγ and especially high levels of IL-17, which suggests a mixed Th1/Th17 response. Previously, most autoimmune diseases were thought to be Th1 mediated. However, the recently discovered Th17 cell population, characterized by the production of IL-17, has been shown to be involved in several autoimmune diseases.
Th17 cells are present in both mice and humans; however, along with many similarities of these species with regard to induction and characteristics, there are some differences. The development of Th17 cells in mice depends on the presence of IL-6 and TGFβ, after which IL-21 produced by the Th17 cells acts as an autocrine factor inducing expansion. However, in humans, IL-1β seems to be an important factor in the development of Th17 cells from naive T cells. IL-23 is suggested to be important in sustaining the Th17 response (44). A recent report also indicates a mixed Th1/Th17 response in patients with SLE, as measured by the concentrations of the Th1-promoting cytokine IL-12, the Th17-maintaining cytokine IL-23, and the Th1 chemokine CXCL10 (45). Th17 cells have been linked to the development of autoimmunity (44, 46), and there is also evidence for a role of these cells in patients with SLE (45). The tolerance that can be induced in lupus-prone mice with low-dose peptide is associated with a reduction of Th17 cells (47). Furthermore, a genetic association has been found between SLE and polymorphisms of IL-21, an important cytokine for the development of Th17 cells (48). Blocking IL-21 in a lupus-prone mouse model reduces disease progression (49).
Altogether, these data indicate that Th17 cells may have a central role in the development of SLE, and possibly they are activated by bleb-matured DCs. It is tempting to speculate which constituent of the blebs induces DCs to produce high amounts of IL-6 and to activate and differentiate T cells into IL-17–producing cells in this system. Recently, it was shown that DCs matured by peptidoglycans are potent inducers of Th17 differentiation, compared with DCs matured by LPS or CpG (44). It is a challenging task to determine whether modified chromatin is part of this causative constituent. Irrespective of the causative constituent, extrapolation of our in vitro data toward an in vivo setting suggests that a defective clearance of apoptotic cells by phagocytes results in the release of apoptotic blebs that can be taken up by DCs. This can lead to a Th1/Th17-driven immune response against (modified) self antigens including chromatin, finally resulting in SLE. However, the physiologic relevance of bleb-induced DC maturation in the development of SLE should be examined in vivo and in a human setting and is the subject of our ongoing research. In addition to the activation of Th17 cells and the inhibition of Treg cells, IL-6 produced by bleb-matured DCs may facilitate autoantibody production by autoreactive B cells.
It was recently described that apoptotic blebs, as defined by us (Figure 1), stimulate human PDCs to produce IFNα (50). This may indicate a maturing effect of blebs on PDCs in addition to that on myeloid DCs. Nevertheless, uptake of apoptotic bodies/blebs by PDCs has never been demonstrated (29). Notably, we did not observe any IFNα production (data not shown), excluding the involvement of PDCs in our experiments.
In conclusion, our in vitro data show that apoptotic blebs generated by apoptosis induce the maturation of DCs that subsequently acquire a Th1 and strong Th17 stimulatory capacity. This bleb-induced DC maturation, and the following Th1/Th17 response, may represent an important driving force in the autoimmune response in SLE and a novel target for developing therapeutic strategies.