Systemic sclerosis (SSc), or scleroderma, is an autoimmune disease characterized by excessive extracellular matrix (ECM) deposition, fibrosis, and vascular obliteration in connective tissues of the skin, lungs, gastrointestinal tract, heart, and kidneys (1). Following a still-undefined initial event, mononuclear cells such as lymphocytes and monocytes are recruited into connective tissues, where the subsequent release of inflammatory cytokines leads to fibroblast activation, collagen accumulation, and tissue hypoxia (2).
One of the hallmarks of SSc is the presence, in >90% of patient sera, of highly specific autoantibodies against a variety of nuclear proteins (i.e., antinuclear autoantibodies [ANAs]) (3). The most common ANAs associated with SSc include anti–DNA topoisomerase I (anti–topo I), anti–centromeric protein B (anti–CENP-B), anti–RNA polymerase III, and anti-Th/To autoantibodies. ANAs have been associated with different clinical manifestations and various degrees of SSc severity. For example, anti–topo I are usually associated with more diffuse cutaneous SSc (4, 5), whereas anti–CENP-B are detected predominantly in patients with limited cutaneous SSc (4, 6, 7). Also, anti–topo I are strongly associated with pulmonary fibrosis in SSc (6, 8), while pulmonary arterial hypertension is more common in patients with anti–CENP-B (9). In spite of the prevalence of ANAs and their association with different SSc subsets and selective visceral involvement, their direct or indirect role in the pathophysiology of SSc is still unclear.
Recent in vitro studies have demonstrated that some SSc autoantibodies directed against cell surfaces could display direct pathogenic effects. For example, anti–endothelial cell antibodies (AECAs), which are present in 25–85% of SSc patients (10), have been shown to trigger antibody-dependent cellular cytotoxicity in the presence of leukocytes (11, 12), while antifibroblast antibodies (AFAs), which are present in 26–58% of SSc patients (13, 14), have been shown to induce a proadhesive and proinflammatory phenotype in fibroblasts upon binding (13, 15). Endothelial cell apoptosis and dysregulated fibroblast activation are cornerstones of SSc pathophysiology, the former being one of the earliest events observed during the disease (16) and the latter being directly responsible for the increased and continuous deposition of ECM, notably type I collagen, in tissues (17–19). Although there is now evidence that AECAs and AFAs are directly implicated in the pathophysiology of SSc, no such evidence yet exists for ANAs.
In a previous study, we demonstrated that anti–topo I from SSc patients could bind specifically to the cell surface of fibroblasts via an unknown surface target (14). The aims of the present study were therefore 1) to identify the fibroblast surface target of anti–topo I and 2) to characterize the consequences of anti–topo I binding to fibroblasts on the mobilization and activation of monocytes.
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- MATERIALS AND METHODS
ANAs are well-established markers for SSc. They are present in up to 90% of SSc patient sera and are generally associated with specific SSc subtypes (35–37). However, their role in the pathogenesis of SSc is still unclear, and few studies have directly addressed this subject (for review, see ref. 37). One of the reasons for this situation is that ANAs in SSc are traditionally considered bystander products of an immune system that has gone awry. Sequestered in cell nuclei, ANA targets are presumed to be inaccessible to circulating SSc autoantibodies, therefore leading to the notion that ANAs cannot play an active part in the disease etiology. Our recent findings shed new light on the possible role of some ANAs in SSc pathogenesis. Indeed, we demonstrate for the first time that topo I binds to fibroblast surfaces, recruits SSc anti–topo I, and subsequently induces monocyte adhesion and activation.
Topo I is a 100-kd nuclear enzyme responsible for DNA relaxation during transcription and replication (38). During apoptosis, topo I is cleaved into major fragments of 70 and 80 kd (30, 39) and is redistributed into blebs and apoptotic bodies (30). These apoptotic cell remnants should normally be rapidly cleared by phagocytes. However, in the event of a sudden increase in apoptotic cell numbers, the capacity of normal clearance mechanisms may be exceeded, resulting in the accumulation and progress of apoptotic cells toward a late apoptotic state (i.e., secondary necrosis), thereby allowing the release of apoptotic body contents to the medium. This may be the case in SSc, in which endothelial cell apoptosis is detected simultaneously in several tissues, notably, around small blood vessels (16, 40).
As we have shown above, topo I can be released from apoptotic endothelial cells, probably due to the increased permeability of secondary necrotic bodies. Hence, this released topo I could bind to nearby cells in SSc tissues. Since topo I has a specific affinity for fibroblast surfaces, it could bind to bystander fibroblasts and recruit circulating anti–topo I, present only in SSc patients, which would then induce adhesion and activation of circulating monocytes. Ultimately, this chain of events could lead 1) to amplification of the immune responses due to proinflammatory cytokines released by activated monocytes and 2) to fibrosis due to local secretion of profibrotic cytokines by activated fibroblasts. Thus, the presence of anti–topo I in SSc patients would be directly associated with increased immune responses and fibrosis.
The hypothesis that anti–topo I could directly contribute to the pathogenesis of fibrosis in SSc is supported by several indirect findings. First, anti–topo I autoantibodies are associated with a more severe SSc phenotype, notably, pulmonary fibrosis (8, 41–44). Second, in patients with anti–topo I, these antibodies are almost invariably present very early in the disease course (43). Third, anti–topo I titers correlate with disease severity (42). Moreover, anti–topo I titers are higher in patients with active disease than in those with inactive disease (42). Some SSc patients even display anti–topo I titers that fluctuate in parallel with the total skin score (42). Strikingly, SSc patients who have lost anti–topo I experience significant improvements in pulmonary function and survival compared with those in whom anti–topo I persist (44). Although convincing, all of these findings remain indirect, and an in situ localization of anti–topo I in SSc tissues will be important to further establish their pathogenic role.
Anti–topo I are not the only autoantibodies that bind to the cell surface via a nuclear autoantigen. For example, nucleosomes, or DNA–histone complexes, have been shown to recruit anti–double-stranded DNA autoantibodies to the surface of various cell types (45). In that particular case, an unidentified 94-kd protein has been pinpointed as the target of nucleosomes on cell surfaces. It is therefore likely that topo I binds to cells via a surface protein or protein complex that remains to be identified. Interestingly, this ligand seems to be more abundant on fibroblasts, independently of their origin, than on other cell types. Experiments to identify the cell surface ligand of topo I on fibroblasts are currently in progress in our laboratory.
The pathogenesis of SSc, like that of all systemic autoimmune diseases, is extremely complex (2). The presence of specific autoantibodies is only one immune abnormality among several others in this disease. However, the association of specific ANAs with different SSc subsets and with different patterns of organ involvement suggests that these autoantibodies are directly implicated in the pathogenesis of the disease. Anti–topo I could therefore drive the disease toward a more severe phenotype. A better understanding of the pathogenic role of ANAs in SSc could lead to new therapeutic strategies to improve the survival of patients.