Dendritic cells (DCs) are potent antigen-presenting cells, which are present in low numbers in many body tissues (1). Immature DCs can ingest antigen, but express low levels of molecules required for antigen presentation and T cell stimulation, e.g., class I and II major histocompatibility complex (MHC) molecules, and costimulatory molecules (2). Following antigen uptake, DCs migrate to secondary lymphoid organs, where they receive maturation signals, which include components of bacteria and viruses, e.g., lipopolysaccharide (LPS), tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and interferon-α (3–5). Mature DCs are less able to ingest antigen but have increased expression of MHC and costimulatory molecules, enabling them to activate antigen-specific T cells and induce primary immune responses (2).
In the immune system, cells usually die by apoptosis, but necrosis may occur in cases of severe injury. Apoptotic and necrotic cells can both be engulfed and degraded by immature DCs, but the outcome with regard to the immune response may vary depending on the type of cell death and whether maturation signals are received (6–12). If apoptotic cells are engulfed by DCs in the absence of a subsequent maturation signal, T cells may become tolerant to the peptides displayed on the surface of the DCs (13). During a viral infection, cells die by apoptosis and necrosis. Both infected apoptotic and necrotic cells are ingested by DCs, and maturation signals received from the infected necrotic cells induce expression of DC-costimulatory molecules, leading to the presentation of viral antigenic peptides from apoptotic cells, T cell activation, and immunity (7, 9).
Gallucci et al have demonstrated in a murine model that, in the absence of foreign substances, signals from necrotic fibroblasts, but not apoptotic or healthy fibroblasts, lead to DC maturation (7). In contrast, Salio et al found that neither necrotic nor apoptotic melanoma cell lines induce human DC maturation (14). A further level of complexity has been added by the suggestion that even without additional maturation signals, high numbers of apoptotic cells (at a ratio of 5 apoptotic cells to 1 DC) are capable of inducing DC maturation and the presentation of intracellular antigens from these apoptotic cells, whereas low numbers of apoptotic cells are disposed of in a noninflammatory manner (15). Furthermore, DCs challenged with low numbers of anti–β2-glycoprotein I–opsonized apoptotic cells present antigen with high efficiency and secrete proinflammatory and maturation factors, e.g., IL-1β and TNFα (16). Opsonization also substantially increases the percentage of DCs engaged in phagocytosis (17). Thus, there is no clearly predictable effect of apoptotic or necrotic cells on DC maturation that can be defined from the current literature, despite the importance such responses may have for development (or lack of development) of tolerance or autoimmunity.
Microscopic polyangiitis (MPA) and Wegener's granulomatosis (WG) are two forms of small-vessel vasculitis in which autoimmune responses are directed toward neutrophil enzymes (18–20). Antineutrophil cytoplasmic antibodies (ANCAs) develop and may bind to proteinase 3 (PR3) (particularly in patients with WG) or to myeloperoxidase (MPO) (particularly in patients with MPA) (21–23). These antibodies, PR3 ANCA and MPO ANCA, are believed to be involved in the initial development of neutrophil-mediated endothelial injury since they can bind to their target antigens when these are expressed on the surface of TNFα-primed neutrophils (24, 25) and also on the surface of apoptotic neutrophils (26). Ligation of PR3 and MPO on primed neutrophils induces neutrophil activation, with a respiratory burst and release of granule contents (27), factors that promote endothelial damage (28, 29). ANCAs also induce accelerated and dysregulated apoptosis of TNFα-primed neutrophils, resulting in a “reduced window of opportunity” for recognition and phagocytosis by macrophages before disintegration (30).
We have been interested in the initial breakdown of tolerance to neutrophil components during vasculitis. We hypothesized that in the event apoptotic or necrotic neutrophils are not cleared by macrophages, DCs may be recruited. In WG, granulomas containing large numbers of freshly activated, apoptotic, and necrotic neutrophils are present in upper airways early in the development of vasculitis (31–33). As neutrophils undergo apoptosis, they express PR3 and MPO on their surface (26, 34). Apoptotic neutrophils injected into rats induce production of ANCA (35). The aim of the present study was to establish how DCs might handle apoptotic or necrotic neutrophils in terms of uptake and effects on DC maturation and T cell–stimulatory ability. Specifically, we investigated whether DC uptake of high numbers of apoptotic neutrophils (15), of antibody–opsonized apoptotic cells (16), or of necrotic cells (7) would increase DC maturation and ability to stimulate T cell proliferation.
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Immature DCs are capable of efficiently phagocytosing both apoptotic and necrotic cells (7, 9, 14). This study is the first to provide direct evidence that immature human monocyte-derived DCs can ingest human apoptotic and necrotic neutrophils. We also hypothesized that uptake of either high numbers of apoptotic neutrophils, antibody-opsonized apoptotic neutrophils, or necrotic neutrophils might sensitize DCs for T cell stimulation. These hypotheses were of interest with regard to the development of antineutrophil-directed autoimmune responses in systemic vasculitis, a group of disorders in which aberrant interactions between neutrophils and ANCAs lead to endothelial cell and vascular injury (28, 29). Our results did not confirm these hypotheses since they did not provide evidence that aberrant uptake of neutrophils by DCs triggers an autoimmune response leading to generation of ANCAs. The data also did not support the contention that ANCAs may help perpetuate an immune response through their previously demonstrated effects on accelerating neutrophil apoptosis. ANCAs induce accelerated and dysregulated apoptosis of TNFα-primed neutrophils, resulting in a reduced likelihood of recognition and phagocytosis by macrophages before disintegration through secondary necrosis (30). However, uptake of secondarily necrotic neutrophils by DCs led to a reduced, not enhanced, capacity of DCs to stimulate an MLR.
As has previously been described (15), if DCs are fed with increasing numbers of apoptotic cells they take up increasing numbers of cells, and this is also true for apoptotic neutrophils. We tested whether high numbers of apoptotic or necrotic neutrophils could induce DC maturation. High numbers of apoptotic T cells from a murine T cell line have been shown to induce DC maturation as assessed by secretion of IL-1β and TNFα and up-regulation of class II MHC, CD86, and CD40 (15). In the present study, the addition of apoptotic neutrophils to DCs did cause an increase in CD83 (a DC maturation-specific marker) and class II MHC expression, which would suggest maturation. However, high numbers of apoptotic neutrophils actually caused a down-regulation of the costimulatory molecules CD80 and CD86, and of CD40, which is required for T cell activation. This down-regulation corresponds to a decreased ability of DCs treated with high numbers of apoptotic neutrophils to stimulate T cell proliferation in an MLR. These results are consistent with the findings of previous studies showing that when DCs are cocultured with apoptotic cells prior to the addition of a maturation stimulus, they fail to mature phenotypically, and in fact their surface expression of CD83 and CD86 was lower than that in control DCs, as was the ability to induce allogeneic T cell responses (43, 44).
Sauter et al have found that certain tumor cell lines that have undergone 4 cycles of freeze–thawing are able to stimulate DC maturation as assessed by induction of CD83 and lysosome-associated membrane glycoprotein expression, up-regulation of CD86, HLA, and CD40 expression, and ability to stimulate allogeneic and superantigen-stimulated syngeneic T cell proliferation, but with no release of TNFα or IL-1β (9). Subjecting neutrophils to 4 cycles of freeze–thawing resulted in complete cellular fragmentation. Addition of necrotic neutrophils did cause DC maturation as assessed by CD83 and class II MHC expression; however, there was down-regulation of CD80 and CD86 as seen with apoptotic neutrophils, and an even greater down-regulation of CD40. Sauter and colleagues demonstrated DC maturation only when necrotic cell lines were used, and not with necrotic primary cells. Furthermore, they did not examine whether primary necrotic cells can cause a down-regulation of costimulatory molecules and CD40 (9). Corresponding to the very low level of CD40 expression on DCs following uptake of necrotic neutrophils, DCs were less able to stimulate T cells in an MLR when treated with necrotic cells compared with apoptotic neutrophils.
These results would suggest that neutrophils, whether apoptotic, secondarily necrotic, or 4 freeze–thawed necrotic, do not per se shift the outcome from tolerance to autoimmunity in ANCA-associated vasculitis. In accordance with the findings of Sauter et al (9), we found that necrotic B-LCL did induce DC maturation as assessed by an increase in expression of CD40, CD80, and CD86 on the DC surface, and also had an increased ability to stimulate allogeneic T cell proliferation. Urban et al showed that necrotic cells derived from primary cell isolates did not lead to DC maturation, but unlike apoptotic cells, they also did not prevent LPS-induced maturation (43).
In the presence of apoptotic peripheral blood lymphocytes, PBMCs and monocytes have been found to produce increased amounts of antiinflammatory cytokines and decreased amounts of proinflammatory cytokines (45). Macrophage responses have been shown to differ after uptake of apoptotic or necrotic neutrophils. Phagocytosis of apoptotic neutrophils induced macrophages to secrete antiinflammatory cytokines and actively inhibited the production of proinflammatory cytokines (46). Similarly, late apoptotic neutrophils ingested by macrophages did not trigger the release of proinflammatory cytokines (47), whereas necrotic neutrophils induced macrophages to secrete inflammation mediators (48) and were able to efficiently costimulate T cells due to rapid up-regulation of CD40 (49). Clearly, these results and the results of the current study emphasize the differential effects of apoptotic and necrotic cells on macrophages and DCs and highlight the need for study of other types of primary cells.
Opsonization of low numbers of apoptotic cells has been shown to substantially increase the percentage of DCs engaged in phagocytosis (17) and to induce DC maturation as assessed by secretion of IL-1β and TNFα (16). Opsonization of apoptotic neutrophils with IgG from ANCA-positive vasculitis patients increased the numbers of neutrophils phagocytosed by the DCs but did not induce DC maturation. In fact, the results obtained with opsonized apoptotic neutrophils were equivalent to those obtained with nonopsonized apoptotic neutrophils.
This study shows that the negative effects of apoptotic or necrotic neutrophils on DC phenotype and function can be partially counteracted by TNFα, a proinflammatory cytokine present at sites of inflammation. The down-regulation of CD80, CD86, and CD40 seen when necrotic neutrophils were added to DCs was overcome very slightly by concurrent addition of TNFα, but the levels of expression were still below those seen when DCs were untreated (i.e., immature DCs). This was mirrored in the ability of DCs to stimulate T cell proliferation, which was still reduced. It is conceivable that future studies that could be undertaken with varying dose ratios of necrotic neutrophils to TNFα might yield results that would highlight more subtle influences here. The down-regulation of CD80, CD86, and CD40 expression on DCs following addition of apoptotic neutrophils was overcome following addition of TNFα and apoptotic neutrophils, and addition of TNFα and apoptotic neutrophils caused a slight enhancement in the expression of CD40. Once again the level of CD40 expression correlated with T cell proliferation since the proliferation seen was greater than that observed with DCs only.
Thus, TNFα was able to partially overcome the effects of apoptotic cells on DCs, but was unable to rescue the necrotic cell effect, suggesting that apoptotic and necrotic cells may have subtle differences in recognition mechanisms or in strength of suppressive signal. In addition, TNFα may act on the small population of living cells in the apoptotic fraction, leading to the release of proinflammatory agents, which activate DCs.
There is evidence for enhanced transcription of the TNFα gene in PBMCs from patients with systemic vasculitis compared with controls (50). Furthermore, TNFα and IL-1β have been detected, by immunocytochemistry, polymerase chain reaction, and in situ hybridization, in the kidneys of patients with ANCA-positive systemic vasculitis, demonstrating that cytokines are produced in these patients (51). Circulating T cells from patients with active WG secrete increased amounts of TNFα compared with T cells from healthy controls (52). The production of TNFα, IL-1, and IL-8 by macrophages is significantly up-regulated when they are incubated with ANCA-opsonized apoptotic neutrophils compared with normal IgG–opsonized or nonopsonized apoptotic neutrophils (34, 53). TNFα levels prior to development of vasculitis have not been studied, but, since many patients have a history of chronic infection before the onset of vasculitis (54) and intercurrent infection often precedes relapse (55), it is likely that synthesis of TNFα is increased. If this is the case, it is conceivable that uptake of apoptotic neutrophils by DCs within an environment rich in TNFα might encourage development of an autoimmune response toward neutrophil PR3 or MPO.
Contrary to our hypotheses prior to these studies, uptake of apoptotic and especially necrotic neutrophils appears to have a tolerogenic effect on DCs. However, cytokines found in vivo at sites of inflammation, such as airway granulomata prior to the development of overt autoimmune systemic vasculitis, may act as maturation factors for DCs, allowing the uptake of apoptotic neutrophils to push the outcome toward autoimmunity instead of tolerance. Clearly, future studies need to address the ability of healthy or patient-derived DCs to take up apoptotic or necrotic neutrophils in the presence or absence of TNFα and to present PR3 or MPO antigens to antigen-specific T cells.