Our results demonstrate that apoptotic cells and necrotic cells trigger divergent signaling responses in BM m
. These results extend previous findings indicating that recognition of apoptotic cells and necrotic cells by m
occurs via distinct mechanisms (5
) and reveal that separate recognition processes are linked to contrasting signaling outcomes. Necrotic cells activate ERK1/2, whereas apoptotic cells inhibit activated ERK1/2, regardless of the activating stimulus. We presented data demonstrating that prior interaction with apoptotic cells precludes subsequent ERK1/2 activation; apoptotic cells also can inhibit previously activated ERK1/2 (data not shown) (17
). Apoptotic cells also activate JNK1/2 and p38, whereas necrotic cells have no detectable effect on these two MAPK modules. We find that the effects of apoptotic cells on signaling responses, like their effects on cytokine secretion (5
), are dominant over those of necrotic cells and can be exerted even when the apoptotic cells are present in reduced numbers. It is important to note that both soluble and membrane-associated material derived from necrotic cells can mimic the effects exerted by whole necrotic cells. Strikingly, our data document that apoptotic cells retain the ability to elicit their characteristic signaling responses throughout the death process, irrespective of membrane integrity. Indeed, the effects of late apoptotic cells are identical in all respects to those of early apoptotic cells. Any intracellular contents released from late apoptotic cells are neutral and not proinflammatory.
Our results may have important implications for our understanding of autoimmunity and tolerance (). Recent studies suggest that necrotic and apoptotic cells have opposing effects on immune homeostasis (24
). Necrotic cells, by virtue of leaking their inflammatory intracellular contents, may provide the costimulation needed for activation of naive T cells and therefore may promote immunity (25
). Apoptotic cells, on the other hand, being actively anti-inflammatory, do not activate T cells, and may even be tolerogenic (26
). According to current models, late apoptotic cells, which have lost membrane integrity and leak their intracellular contents, should behave like necrotic cells and induce an immune response (27
). Thus, delayed or impaired clearance of apoptotic cells, as observed with targeted deletion of the complement protein C1q (14
) or the MER receptor tyrosine kinase (15
), is thought to lead to presentation of apoptotic antigen in the context of inflammatory signals, with resultant autoimmunity ().
Exposure of macrophages to apoptotic cells induces multiple signaling events that contribute to immune homeostasis
The delayed clearance model makes several predictions that we tested directly. First, for necrotic and apoptotic cells to have opposing effects on immune function, the signaling events induced by exposure to these two types of cells must be distinct. This prediction is fulfilled, at least in terms of signaling events involving the ERK, JNK, and p38 MAPK modules. Second, the model predicts that late apoptotic cells, which have lost membrane integrity, should behave like necrotic cells. However, in sharp contrast to this prediction, late apoptotic cells were indistinguishable from early apoptotic cells in all of their effects on m
. Late apoptotic cells inhibited basal and M-CSF-induced ERK1/2 activity, late apoptotic cells activated JNK1/2 and p38, and, as elaborated below in the third prediction of the delayed clearance model, late apoptotic cells were fully dominant over necrotic cells. Third, the model predicts that the signaling events induced by necrotic cells must be dominant over those induced by apoptotic cells. This prediction arises from the following considerations. Because apoptotic cells are continuously generated in vivo
, delayed clearance of apoptotic cells will lead to the coexistence of both early and late apoptotic cells. Immunity will result only if the inflammatory signals in response to the presumed inflammatory intracellular contents of late apoptotic cells can override the anti-inflammatory effects of early apoptotic cells. Because the intracellular contents of necrotic cells should be at least as inflammatory as those of late apoptotic cells, we pitted apoptotic cells against necrotic cells. Again, in sharp contrast to the prediction of the delayed clearance model, the signaling events induced by apoptotic cells were dominant over those induced by necrotic cells. Remarkably, late apoptotic cells were just as dominant as early apoptotic cells in terms of inhibiting ERK1/2 activity and stimulating JNK1/2 and p38. Thus, in striking contrast to the predictions of the delayed clearance model, apoptotic cells appear to be functionally equivalent throughout their existence, irrespective of membrane integrity. Finally, in direct opposition to the delayed clearance model, neither cell-associated nor soluble material recovered from late apoptotic cells was proinflammatory.
Although our results argue against the delayed clearance model of autoimmunity, there is an important caveat. Our studies focused on three specific MAPK signaling cascades and may not generalize to other signal transduction pathways. It is possible that there exist other signaling pathways and/or intermediates for which the predictions of the delayed clearance model hold true and that these other pathways play a more determining role in the balance between tolerance and immunity. This seems unlikely, however, because we have also shown that late apoptotic cells mimic early apoptotic cells with respect to proinflammatory transcriptional activity (10
) and inflammatory cytokine secretion (5
). Moreover, even if delayed clearance contributes to the development of autoimmunity, it is not in and of itself sufficient to produce autoimmunity, because targeted deletion of CD14 or the mannose-binding lectin leads to the in vivo
accumulation of apoptotic cells in the absence of autoimmunity (28
In light of these considerations, we propose that the absence of apoptotic cell-induced signaling events, rather than delayed apoptotic cell clearance per se
, is the critical event leading to the loss of tolerance and the development of autoimmunity. We therefore hypothesize that the signaling pathways induced by the binding and/or uptake of apoptotic cells are critical to the maintenance of tolerance and that deficient or abnormal signaling events within these same pathways can predispose to autoimmunity. Consistent with this hypothesis, we have previously shown that m
from prediseased mice of all the major inbred murine models of spontaneous autoimmunity, including multiple strains that develop systemic lupus erythematosus, as well as the autoimmune diabetes-prone NOD strain, have an identical apoptotic cell-dependent abnormality in the expression of multiple cytokines (22
). Affected systemic lupus erythematosus-prone strains include MRL/+, MRL/lpr, NZB, NZW, NZB/W F1, BXSB, and LG/J. No similar defect in cytokine expression can be found in 16 nonautoimmune strains, including three strains that develop type II (nonautoimmune) diabetes mellitus. Importantly, in the absence of apoptotic cells, cytokine expression by these autoimmune-prone strains is completely normal. Furthermore, cytokine expression is not the sole apoptotic cell-dependent abnormality observed in m
from prediseased autoimmune-prone mice. m
from NOD and the same systemic lupus erythematosus-prone strains also have a reversible defect in the activity of the cytoplasmic G protein Rho, a key regulator of the cytoskeleton, resulting in abnormalities of adhesion and cytoskeletal organization (31
). Again, no similar abnormalities were observed in m
from multiple nonauto-immune control strains. These results provide strong support for our hypothesis that abnormalities in the signaling events induced by apoptotic cells may be causally related to the development of autoimmunity. The downstream consequences of these signaling events, in terms of gene transcription and phentotypic function, are the subject of ongoing investigation.