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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the formation of antinuclear autoantibodies. Increased apoptosis and reduced clearance of apoptotic material have been assigned a role in the pathogenesis of SLE, but the underlying mechanisms remain elusive. During apoptosis apoptotic blebs are formed in which autoantigens are clustered. The cellular remnants after blebbing are referred to as apoptotic cell bodies. We undertook this study to compare the effects of apoptotic blebs and apoptotic cell bodies on maturation of dendritic cells (DCs) and their T cell stimulatory capacity in a murine setting.

Methods

The uptake by DCs of apoptotic blebs and apoptotic cell bodies was analyzed by flow cytometry and confocal microscopy. DC maturation and DC-induced T cell activation were determined by measuring expression of costimulatory molecules using flow cytometry and by measuring production of cytokines using enzyme-linked immunosorbent assay.

Results

DCs internalized apoptotic blebs more efficiently than apoptotic cell bodies. Incubation of DCs with apoptotic blebs resulted in increased CD40 and CD86 expression and increased interleukin-6 (IL-6) and tumor necrosis factor α production, while apoptotic cell bodies had no stimulatory effects. Using chloroquine, apoptotic bleb–induced DC maturation was shown to be independent of Toll-like receptors 3, 7, and 9. Interestingly, in cocultures with allogeneic T cells, bleb-matured DCs induced production of IL-2, interferon-γ, and, in particular, IL-17, suggesting a Th1/Th17 response.

Conclusion

Apoptotic blebs, in contrast to apoptotic cell bodies, induce DC maturation, thereby providing DCs with increased Th17 cell stimulatory capacity. These data imply that apoptotic bleb–induced DC maturation represents an important driving force in the autoimmune response in SLE.

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).

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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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Cell culture.

BALB/c (H-2d) and CBA (H-2k) mice ages 8–10 weeks were purchased from Charles River (Maastricht, The Netherlands) and maintained under specific pathogen–free conditions and handled according to the guidelines of the local ethics committee of the Radboud University Nijmegen. Bone marrow–derived DCs were obtained by culturing bone marrow from BALB/c mice as previously described (30, 31). Briefly, bone marrow was flushed from femur and tibia and cultured for 8 days in 6-well plates (1 × 106 cells/well; Corning Costar, Badhoevedorp, The Netherlands) containing RPMI 1640 DM medium (Invitrogen Life Technologies, Breda, The Netherlands) with 10% fetal calf serum (FCS; Greiner Bio One, Alphen aan den Rijn, The Netherlands), 1% pyruvate, 1% Glutamax, 1% penicillin/streptomycin (all from Invitrogen Life Technologies), and 20 ng/ml granulocyte–macrophage colony-stimulating factor (Pepro-Tech, London, UK). Murine 32D clone 3 (32Dcl3) cells (H-2k) (32, 33) were cultured in complete RPMI 1640 DM medium supplemented with 15% WEHI-3B–conditioned medium as a source of murine IL-3 (DSMZ, Braunschweig, Germany). Cells were routinely tested for mycoplasma (Gen-Probe, San Diego, CA), and the results were consistently negative.

Induction and measurement of apoptosis and isolation of apoptotic blebs and cell bodies.

Apoptosis was induced in 32Dcl3 cells by incubating the cells with 10 μM 4-nitroquinoline 1-oxide (Sigma-Aldrich, Zwijndrecht, The Netherlands) for 24 hours. Apoptosis was routinely determined by staining cells with annexin V–fluorescein isothiocyanate and propidium iodide (PI) (BioVision, Palo Alto, CA) and analyzed by flow cytometry using a FACSCalibur instrument (BD PharMingen, Alphen aan den Rijn, The Netherlands) following the protocol of the manufacturer (11). Apoptotic cell bodies were isolated from apoptotic cell culture by centrifugation for 10 minutes at 1,550g at room temperature. Subsequently, apoptotic blebs were isolated from the resulting supernatant by centrifugation for 50 minutes at 15,700g at room temperature. Pelleted blebs or apoptotic cell bodies were gently resuspended in RPMI 1640 DM medium. The concentrations of blebs and apoptotic cell bodies were determined with the bicinchoninic acid protein determination assay (Sigma-Aldrich) and expressed as protein equivalents. FCS was added to the bleb and apoptotic cell body preparations to achieve a final concentration of 10%. Equal amounts in protein equivalents of blebs and apoptotic cell bodies contained approximately equal amounts of nucleic acids as determined by measurements of absorbance at 260 nm (A260 nm) and A280 nm, respectively.

Phagocytosis of apoptotic blebs or apoptotic cell bodies.

First, 32Dcl3 cells were labeled with 10 μM PKH26 (Sigma-Aldrich) before the induction of apoptosis. Next, PKH26-labeled apoptotic cell bodies or blebs (10 or 100 μg/ml) were added to 0.5 × 106/ml DCs labeled with 1 μM 5,6-carboxyfluorescein succinimidyl ester (CFSE; Invitrogen Life Technologies) with a final volume of 200 μl, and subsequently incubated at 0°C or 37°C for 2 hours. Internalization of blebs or apoptotic cell bodies by DCs was visualized with confocal laser scanning microscopy using a Leica TCS NT system (Leica Lasertechnik, Heidelberg, Germany). In addition, the percentage of CFSE/PKH26 double-positive cells was analyzed by flow cytometry.

Determination of DC phenotype by flow cytometry and cytokine enzyme-linked immunosorbent assay (ELISA).

For analysis of DC phenotype, 0.5 × 106 immature DCs per ml were incubated in medium alone or medium supplemented with blebs or apoptotic cell bodies. Addition of 1 μg/ml lipopolysaccharide (LPS) (L4391; Sigma-Aldrich) was used as a positive control for maturation of DCs. After 14 hours of incubation, cells were harvested and subjected to direct or indirect fluorescence staining of cell surface markers, essentially as described previously (31). Briefly, cells were stained with anti-CD40 (clone FGK45.5; Miltenyi Biotec, Utrecht, The Netherlands) or isotype-matched control antibodies (R35-95; BD PharMingen), followed by phycoerythrin (PE)–conjugated goat anti-F(ab′)2 anti-rat (Beckman Coulter, Bedfordshire, UK) and Alexa 647–conjugated anti-CD11c (N418; Serotec, Oxford, UK), or with PE–conjugated anti-CD86 (PO3.1; eBioscience, Malden, The Netherlands) and Alexa 647–conjugated anti-CD11c. Samples were analyzed using a FACSCalibur instrument, and data were processed using CellQuest software (BD PharMingen). Supernatant was collected for determination of levels of TNFα, IL-1β, IL-6, IL-23 (all from eBioscience), and IFNα (R&D Systems, Abingdon, UK) in sandwich ELISA according to the protocols provided by the manufacturers.

Bleb-induced DC maturation was investigated for its dependency on Toll-like receptors (TLRs) 3, 7, and 9 by incubating DCs with chloroquine (10 μM; Invivogen, San Diego, CA) alone or in combination with apoptotic blebs (100 μg/ml), LPS (1 μg/ml), or CpG-containing oligonucleotide 1826 (1 μg/ml; Invivogen) as positive control. Maturation was examined by measuring levels of IL-6 in supernatants by ELISA.

Mixed leukocyte reaction (MLR).

Spleen cells from CBA mice were isolated by passing spleen tissue through a 70-μm Cell Strainer (BD PharMingen) followed by treatment with erythrocyte lysis buffer (0.15M NH4Cl, 10 mM KHCO3, 0.1 mM Na2-EDTA [pH 7.4]) for 1 minute, and cells were finally washed 3 times in medium (31). In MLRs, 2 × 104 BALB/c DCs were incubated with 1 × 105 CBA splenocytes, as a source for T cells, in 200 μl in 96-well plates at 37°C and 5% CO2. The effects of the addition of blebs or apoptotic cell bodies were analyzed. As controls, splenocytes with or without apoptotic blebs or cell bodies, DCs with or without apoptotic blebs or cell bodies, and LPS-matured DCs were included. Apoptotic cell bodies and blebs were matched for the splenocytes by major histocompatibility complex, which excluded allogeneic T cell stimulation by blebs or apoptotic cell bodies. On day 6 of the MLR, supernatant was collected for determination of IL-2, IL-4, IL-5, IL-10, IL-17, and IFNγ (eBioscience) production in sandwich ELISA according to the corresponding protocols.

Statistical analysis.

All data presented are obtained from at least 3 different experiments using at least 3 different mice. Values are expressed as the mean ± SEM, and significance was determined by Student's t-test using GraphPad Prism version 4 software (GraphPad Software, San Diego, CA). P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Mouse DCs ingest apoptotic blebs more efficiently than apoptotic cell bodies.

Previously, it has been demonstrated that in particular late apoptotic cells and necrotic cells, in contrast to early apoptotic cells, induce maturation of DCs, which increases the T cell stimulatory capacity of these DCs (19, 34). However, in all studies so far, apoptotic cell bodies or a mixture of apoptotic blebs and cell bodies have been used, and consequently the separate effects of blebs and bodies on DC phenotype and function remain elusive. Therefore, we first compared the uptake of purified blebs and apoptotic cell bodies by DCs.

Blebs and apoptotic cell bodies were isolated from cultures of late apoptotic 32Dcl3 cells, as measured by annexin V and PI staining (Figure 2). Apoptotic blebs and apoptotic cell bodies were separated by serial centrifugation. Using confocal laser scanning microscopy we could visualize the uptake of PKH26-labeled apoptotic blebs (Figure 3A) and apoptotic cell bodies (Figure 3B) by CFSE-labeled DCs. The uptake and binding of apoptotic blebs and apoptotic cell bodies was quantified by flow cytometry. Figure 3C depicts a representative example of a flow cytometric analysis of CFSE-labeled DCs that have ingested PKH26-labeled blebs or bodies. To distinguish uptake from binding, experiments were performed at 37°C and at 0°C, respectively, and the uptake was defined as the difference in percentages of double-positive (both PKH26- and CFSE-labeled) cells observed at 37°C and at 0°C. Interestingly, DCs ingested apoptotic blebs more efficiently than apoptotic cell bodies (Figure 3D). In particular, at the highest concentration tested (100 μg/ml), the percentage of DCs that had ingested apoptotic blebs (46%; 54% uptake and binding at 37°C minus 8% binding alone at 0°C) was significantly higher than the percentage that had ingested apoptotic cell bodies (18%; 29% uptake and binding at 37°C minus 11% binding alone at 0°C).

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Figure 2. Induction of apoptosis in culture of 32D clone 3 (32Dcl3) cells. Cells were analyzed for annexin V binding and propidium iodide (PI) staining by flow cytometry. Cells double-positive for annexin V and PI can be considered late apoptotic. A, Analysis of 32Dcl3 cells from a normal culture reveals hardly any binding of annexin V or PI staining. B, Typical analysis of 32Dcl3 cells that were incubated with 10 μM 4-nitroquinoline 1-oxide for 24 hours to induce apoptosis shows that nearly 100% of the cells stain positively for annexin V and PI. C and D, PKH26-labeled apoptotic blebs (C) and apoptotic cell bodies (D) were isolated from late apoptotic 32Dcl3 cell cultures (as presented in B) by serial centrifugation (original magnification × 400).

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Figure 3. Dendritic cells (DCs) ingest apoptotic blebs more efficiently than apoptotic cell bodies (ACB). DCs were incubated for 2 hours at 0°C or 37°C with 10 or 100 μg/ml blebs or apoptotic cell bodies. A and B, Ingestion of PKH26-labeled blebs (A) and apoptotic cell bodies (B) by 5,6-carboxyfluorescein succinimidyl ester (CFSE)–labeled DCs was visualized using confocal microscopy (original magnification × 630). C, CFSE-labeled DCs that had ingested/bound PKH26-labeled blebs or apoptotic cell bodies were analyzed by flow cytometry. A representative example is shown. D, Binding (0°C) and uptake and binding (37°C) were quantified by determination of the percentage of CFSE/PKH26 double-positive cells by flow cytometry. Percentage uptake is the difference between uptake and binding (37°C) and binding alone (0°C). Values are the mean and SEM. ∗ = P < 0.05.

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Apoptotic blebs enhance the expression of costimulatory molecules on DCs.

Next, we analyzed the effect of isolated apoptotic blebs and cell bodies on maturation of mouse myeloid DCs by determining the expression of the costimulatory molecules CD40 and CD86. Incubation of DCs with blebs led to an increased expression of CD40 (Figure 4A) and CD86 (Figure 4B) on CD11c-positive cells. In contrast, incubation of DCs with apoptotic cell bodies (10 or 100 μg/ml) did not lead to an increased expression of the costimulatory molecules CD40 and CD86 (Figures 4A and B). Interestingly, the expression of CD40 was even decreased after incubation of DCs with 100 μg/ml apoptotic cell bodies (Figure 4A). In summary, apoptotic blebs induce an increased expression of costimulatory molecules on DCs, whereas apoptotic cell bodies do not result in maturation of DCs.

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Figure 4. Apoptotic blebs induce enhanced expression of the costimulatory molecules CD40 and CD86 by dendritic cells (DCs). DCs were incubated with 10 or 100 μg/ml blebs or apoptotic cell bodies (ACB), or with 1 μg/ml lipopolysaccharide (LPS) or with medium alone. Expression of CD40 (A) and CD86 (B) on CD11c-positive cells was analyzed by flow cytometry. Data are expressed as the mean and SEM mean fluorescence intensity (MFI). ∗ = P < 0.05.

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Apoptotic blebs induce IL-6 and TNFα production by DCs, which is not mediated by TLRs 3, 7, and 9.

Additionally, we examined the response of DCs to apoptotic blebs or apoptotic cell bodies at the level of cytokine production. Incubation of DCs with 10 μg/ml and 100 μg/ml apoptotic blebs resulted in a dose-dependent increase in secretion of the proinflammatory cytokines IL-6 and TNFα compared with control DCs (Figures 5A and B). In contrast, apoptotic cell bodies were not able to induce IL-6 or TNFα secretion by DCs (Figures 5A and B). Even incubation with a 100-fold excess (1 mg/ml) of apoptotic cell bodies over blebs did not lead to IL-6 or TNFα production (not shown). The proinflammatory cytokines IL-1β, IL-23 and IFNα were not detectable in supernatants of DCs incubated with either apoptotic blebs or apoptotic cell bodies (data not shown).

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Figure 5. Apoptotic blebs induce enhanced production of interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) by dendritic cells (DCs) without mediation by Toll-like receptors (TLRs) 3, 7, and 9. DCs were incubated with 10 or 100 μg/ml blebs or apoptotic cell bodies (ACB), or with 1 μg/ml lipopolysaccharide (LPS) or with medium alone. A and B, The production of the proinflammatory cytokines IL-6 (A) and TNFα (B) was determined by enzyme-linked immunosorbent assay. C, The possible involvement of TLRs 3, 7, and 9 in apoptotic bleb–induced DC maturation was examined using the inhibitor chloroquine, with CpG-containing oligonucleotide 1826 as a positive control. Values are the mean and SEM. ∗ = P < 0.05.

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Subsequently, we evaluated the possible involvement of TLRs in apoptotic bleb–induced maturation of DCs. We focused on TLRs 3, 7, and 9, since normally these TLRs can be triggered by various (endogenous) nucleic acid–containing compounds, which may be present in these apoptotic blebs. Therefore, we applied the endosome acidification inhibitor chloroquine to block signal transduction through TLRs 3, 7, and 9. However, addition of chloroquine in combination with 100 μg/ml apoptotic blebs did not inhibit IL-6 production by DCs, while the IL-6 production induced by CpG-containing oligonucleotide 1826 (a TLR-9 ligand) was completely inhibited (Figure 5C).

In summary, apoptotic blebs, but not apoptotic cell bodies, induce the production of the proinflammatory cytokines IL-6 and TNFα by DCs. Bleb-induced cytokine production by DCs appears to be independent of TLRs 3, 7, and 9.

Apoptotic bleb–matured DCs stimulate the production of IL-2, IFNγ, and IL-17 by activated T cells.

As a functional test, we compared T cell stimulatory capacities between DCs incubated with apoptotic blebs, DCs incubated with apoptotic cell bodies, or DCs alone in an allogeneic MLR with splenocytes as responders. We used IL-2 production as a measure of T cell proliferation, and we measured the production of cytokines specific for Th1 (IFNγ), Th2 (IL-4, IL-5, IL-10), or Th17 (IL-17) responses. IL-10 production may also indicate the involvement of Treg cells.

Bleb-matured DCs showed an increased ability to activate T cells as measured by IL-2 production (Figure 6A). In contrast to bleb-matured DCs, DCs exposed to apoptotic cell bodies induced IL-2 production in MLR similar to that induced by control DCs. Moreover, IFNγ and IL-17 production were significantly increased in MLR with bleb-matured DCs compared with their production in MLR either with DCs exposed to apoptotic cell bodies or with control DCs (Figures 6B and C). Notably, IL-17 production in MLR with bleb-matured DCs was several times higher than that in MLR with LPS-matured DCs, while IFNγ production was about the same in both conditions. As a result, the IL-17:IFNγ ratio was ∼5 after stimulation with bleb-matured DCs compared with a ratio of only ∼1.5 after stimulation with LPS-matured DCs. These data suggest a mixed Th1/Th17 response of responder cells toward bleb-matured DCs. No secretion could be detected of the Th2 cytokines IL-4, IL-5, and IL-10 in any of the conditions tested in MLR (data not shown), which suggests the absence of a Th2 response. The absence of IL-10 also suggests that Treg cells are not involved. In summary, bleb-matured DCs induce in vitro a mixed Th1/Th17 response, whereas DCs exposed to apoptotic cell bodies have no effect on cytokine production in MLR.

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Figure 6. Apoptotic bleb-matured dendritic cells (DCs) are able to induce Th1/Th17 T cell cytokine production in a mixed leukocyte reaction (MLR). BALB/c (H-2d) DCs (20,000) were incubated with 10 or 100 μg/ml blebs or apoptotic cell bodies (ACB), or with 1 μg/ml lipopolysaccharide (LPS) or with medium alone, and were used in an MLR with CBA (H-2k) splenocytes (100,000) as a source for T cells. After 6 days, supernatants were isolated and examined by enzyme-linked immunosorbent assay for production of interleukin-2 (IL-2) (A), interferon-γ (IFNγ) (B), and IL-17 (C). No IL-2, IFNγ, or IL-17 production was found when splenocytes or DCs alone were incubated with apoptotic blebs (data not shown). Values are the mean and SEM. ∗ = P < 0.05.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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. Berden 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. Fransen, Hilbrands, van der Vlag, Berden.

Acquisition of data. Fransen, Ruben, Stoffels.

Analysis and interpretation of data. Fransen, Hilbrands, Adema, van der Vlag, Berden.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We thank Dr. J. Greenberger (University of Pittsburgh Cancer Institute, Pittsburgh, PA) and Dr. S. Baker (Temple University, Philadelphia, PA) for providing the 32D clone 3 cell line. Mrs. Claudia Koëter (Department of Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands) is acknowledged for her technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES