The principal findings of our study were that merkd mice developed autoimmunity to nuclear antigens, accompanied later on by rheumatoid factor. A defect in the rate of apoptotic cell clearance was apparent in vivo, yet only anti-phospholipid autoantibody production was accelerated upon administration of apoptotic cells in vivo, indicating that endogenous sources of apoptotic cells were sufficient to provide the stimulus for most autoantibodies. It was noteworthy that polyclonal B cell activation was not a feature of the autoimmune syndrome of the c-mer–deficient mice. This may reflect the specific stimulus of autoantigen in provoking autoantibodies, without concurrent stimulation of nonautoreactive B cells.
Vast numbers of apoptotic cells are generated through cell senescence, maturation, and turnover. Multiple macrophage scavenger receptors serve the important function of recognizing and promoting engulfment and removal of apoptotic cells (2
). Several recent reports have emphasized that defective macrophage clearance of apoptotic debris may lead to autoimmunity. For instance, absence of C1q or of the CR2 receptor lead to autoimmunity, as does knocking out the gene encoding serum amyloid protein and deletion of DNase I (16
). It has been proposed that these genetic manipulations, by altering the efficiency of clearance of apoptotic cells, promote self-immunization with nuclear antigens exposed in blebs on the surface of dying cells (17
). While direct proof of such self-immunization is not available, the hypothesis is appealing and has been widely disseminated.
In support of the notion that apoptotic cells represent a vehicle for potential autoimmunization, investigators have documented modest transient antibody production in animals immunized with apoptotic cells (13
). In other systems, T cell reactivity to transfected antigens contained within apoptotic cells in vivo and in vitro has been observed, and is enhanced when appropriate costimulatory molecules and cytokines are present (18
). The present findings are consistent with the hypothesis that apoptotic cells represent a potential source of autoantigens.
It was of interest that the effect of immunization with apoptotic cells was evident only in increased anti-phospholipid antibody titers, with little effect on anti-DNA or anti-chromatin. This finding suggests that the continuous burden of endogenous apoptotic cells is sufficient to drive the immunization process in merkd
mice; additional apoptotic cells may add little to the immunization process. The increase in anti-phospholipid antibodies may indicate a special role for mer in the clearance of debris containing phospholipids, perhaps via binding by mer (via GAS6) of phospholipid-rich apoptotic bodies as has been suggested (20
While absence of certain scavenger receptors results in autoimmunity, mice without other receptors (SRS A, CD36) do not develop an autoimmune phenotype despite in vitro defects. What accounts for the autoimmunity in merkd
mice? One possibility is that the c-mer receptor is more relevant for certain autoantigens than some of the others. This hypothesis is supported by the observation of accumulated apoptotic cells in merkd
mice but not in SRS-A–deficient animals in vivo. Another possibility is that c-mer binds to apoptotic cells at a stage at which they are more immunogenic. The expression of phosphatidylserine on apoptotic cells is an early event, and is believed to mediate binding via growth arrest specific protein 6 (GAS 6; reference 21
) to c-mer. A failure to eliminate apoptotic cells at this very early stage may lead to accumulation of late apoptotic cells and of cells undergoing secondary necrosis. These cells may present a more immunogenic challenge and may thus provoke autoantibody formation.
Another consideration for understanding autoimmunity in merkd mice concerns the nature of our knockout mouse. Because only the cytoplasmic domain of c-mer has been removed, it is possible that merkd macrophages retain the ability to bind apoptotic cells but not to internalize them. Macrophage-bound apoptotic cells adherent to c-mer may lead to cross priming and self-immunization. We think this possibility unlikely because we have seen little if any c-mer surface expression in merkd mice (unpublished data).
A final explanation for the autoimmune phenotype of merkd
mice deserves serious consideration. As initially reported, c-mer serves an important role in regulating the cytokine profile of macrophages. Macrophages from merkd
mice secrete larger amounts of TNFα than do control macrophages, leading to increased LPS sensitivity (5
). Others have emphasized the key role of cytokine expression in macrophages ingesting apoptotic cells. In contrast to their secretion of inflammatory cytokines when confronted with necrotic cells, macrophages ingesting apoptotic cells produce a panel of noninflammatory cytokines such as TGFβ and IL-10, thereby reducing the immunogenicity of apoptotic cells (22
macrophages, in addition to their impaired clearance of apoptotic cells, may generate an inflammatory pattern of macrophage cytokine production. The mice may therefore have both an increased burden of apoptotic cells and a macrophage environment conducive to the induction of T cell immunity to apoptotic cell antigens. The combination of macrophage activation in an inflammatory mode, together with persistence of late apoptotic cells and possibly cells undergoing secondary necrosis, may present a sufficient autoimmunogenic stimulus so that tolerance to nuclear antigens is broken and autoimmunity ensues.
In support of the idea that macrophage activation is crucial in promoting autoimmunity to apoptotic debris is a recent report emphasizing the lymphoproliferative and autoimmune phenotype of mice lacking c-mer, axl, and tyro-3. These animals develop apparently higher levels of autoantibodies to DNA than c-mer single knockouts, together with other autoantibody specificities (23
). The more severe autoimmunity in these animals may reflect their intense macrophage activation, or may be due to a more severe apoptotic clearance defect in the absence of all three members of this receptor tyrosine kinase family.
The finding of autoimmunity in merkd
mice opens the way to new avenues to study the role of apoptotic debris as an immunogenic stimulus in autoimmune disease. Macrophages from these mice, because of their delayed phagocytosis of apoptotic cells, may make available nuclear antigens to dendritic cells, as has been described in the MHC class I pathway (24
), and thereby generate immunogenic self-peptide complexes in the context of MHC class II molecules. We are currently devising model systems to assess disposition of apoptotic cell derived antigens in merkd
mice. These experiments should allow deduction of the role of apoptotic cell uptake and macrophage cytokine production on the presentation of nuclear autoantigens relevant to SLE.