Systemic sclerosis (SSc) is a complex autoimmune disease characterized by inflammation, immune activation, excessive extracellular matrix deposition, fibrosis, and vascular obliteration in the skin and internal organs. SSc can be divided into 2 major subsets, limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc), according to the extent to which the skin is affected. The lcSSc form is defined by the presence of skin thickening in areas solely distal to the elbows and knees, with or without involvement of the face. Patients with lcSSc present with various clinical features of CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasias), the name by which lcSSc was previously known (1, 2). Patients with dcSSc have skin involvement proximal to the elbows and knees, tend to have earlier and more rapid involvement of the skin, and are at increased risk of cardiac disease, interstitial lung disease, renal crisis, and early death.
While the initial event leading to the development of SSc is still poorly understood, one characteristic feature of the disease is the production of a variety of autoantibodies to nuclear antigens. Specific autoantibodies associated with SSc are anticentromere antibodies (ACAs), anti–topoisomerase I antibodies (anti–topo I; also known as anti–Scl-70), and antinucleolar antibodies (ANoA) (3–5). ACAs are typically associated with lcSSc, whereas anti–topo I and ANoA are mostly observed in dcSSc, although some specificities, such as anti-Th/To, may be observed in lcSSc (3–5). The presence of these specific autoantibodies is further correlated with disease severity: ACAs have been associated with a reduced risk of pulmonary fibrosis, whereas anti–topo I have been associated with interstitial lung disease, more severe skin progression, and early death (3, 6, 7).
Although autoantibodies to (ribo)nucleoprotein particles have frequently been considered to be epiphenomena, it has recently been shown in systemic lupus erythematosus (SLE) that following binding to their cognate antigens and incubation with peripheral blood mononuclear cells (PBMCs) in vitro, these autoantibodies induce high concentrations of interferon-α (IFNα), a cytokine that is strongly implicated in disease pathogenesis (for review, see ref.8). Furthermore, evidence of type I IFN stimulation of PBMCs has recently been demonstrated by expression arrays in several other systemic autoimmune disorders (Sjögren's syndrome, polymyositis, and SSc) (9). In SSc, increased expression of type I IFN genes was found to be increased in PBMCs (10), and we recently reported similar findings in monocytes from SSc patients, as well as direct evidence of IFNα production in the skin (11). IFNα is a potent immune adjuvant that functions through activation of antigen-presenting cells as well as B and T lymphocytes (for review, see ref.12).
Since autoantibodies in SSc target a diverse population of nucleoprotein antigens that are associated with different clinical subsets of disease, we sought to determine whether differences in IFNα-inducing capacity were associated with different autoantibody populations and clinical manifestations in SSc. We observed that anti–topo I antibodies were more potent at inducing IFNα and that ACA-containing sera rarely did so.
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- PATIENTS AND METHODS
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The main findings of this study are that SSc sera with autoantibody specificities associated with diffuse or systemic, but not limited scleroderma, had IFNα-inducing properties. As observed for autoantibodies in SLE (9), IFNα induction required ingestion through FcγR, endosomal transport, and stimulation of plasmacytoid DCs. These findings could, in part, explain the IFNα signature found in many patients with SSc (10), the increase in SIGLEC1, an IFN-induced gene on monocytes from SSc patients (30), as well as the IFNα mRNA detected in the skin of some SSc patients (31). Since evidence of immunoglobulin secretion by B cells has also been found in skin samples obtained from patients with SSc (31), local production of autoantibodies may contribute to immune complex–mediated cytokine production.
The highest IFNα-inducing activity was observed in anti–topo I–containing sera. Anti–topo I antibodies are found in ∼20% of patients with SSc and are much more frequent in those with dcSSc. These antibodies bind to topoisomerase I, an abundant nuclear protein that catalyzes DNA relaxation and SR protein phosphorylation (32). In addition to its well-known role in preventing supercoiling during DNA synthesis, topo I is also physically associated with 36 nuclear proteins, several of which are involved in either RNA splicing, processing, or metabolism (33). This observation likely explains the greater inhibitory effect of RNase compared with DNase on IFNα induction, although the combination of both enzymes reduced IFNα to baseline levels. We confirmed that IFNα induction was produced by the IgG fraction, rather than by other serum components, and there was a strong correlation between anti–topo I levels and IFNα production. However, we cannot formally exclude the possibility that other autoantibodies in the anti–topo I–containing sera contributed to IFNα stimulation.
ACAs are observed in 20–30% of SSc patients and bind 1 or more of the 6 centromeric proteins, CENP-A through CENP-F (3). The 80-kd CENP-B is the most common autoantigen of the 3 centromere proteins, and it binds to an α-satellite DNA that comprises tandem repeat units of 171 bp and functions as a cis element for centromere-specific nucleosome assembly (34, 35). Intriguingly, CENP-B binding within the α-satellite DNA is relatively specific for a 17-bp sequence called the CENP-B box, which contains 2 CpG dinucleotides (34). CENP-C also binds to α-satellite DNA (36) and has recently been shown to be a dual DNA/RNA binding protein that is implicated in assembly of the nucleolus during mitosis (37). In striking contrast to the anti–topo I–containing sera, sera containing ACAs rarely induced IFNα (or tumor necrosis factor α [results not shown]). Immunoblotting with ACAs confirmed the presence of CENP antigens in the soluble HeLa nuclear extracts; this was likely due to inclusion of a micrococcal nuclease-like enzyme during preparation of the extract. To further mimic situations in vivo, such as apoptosis or necrosis, that might allow the release of nucleosomes or random DNA fragments, respectively, cells were induced to undergo cell death by a variety of manipulations, and their extracts were tested for stimulatory activity with ACAs. No increased IFNα-inducing activity was observed.
The CENP-B box contains CpG motifs that may stimulate the production of IFNα by plasmacytoid DCs (for review, see ref.38). We synthesized a 21-mer ODN to which CENP-B is known to bind and tested both the naturally occurring PDE-linked ODN as well as the relatively DNase-resistant ODN with a PT backbone for their IFNα-inducing capacity. The sense PT–containing dsDNA induced IFNα without the requirement for a transfection reagent. Intriguingly, the PT dsDNA was much more stimulatory than the PDE dsDNA following transfection, suggesting that intracellular DNase is crucial for protection from IFNα stimulation. Since demethylation promotes the binding of CENP-B to the CENP-B box (19) and would also be expected to enhance the stimulatory activity of CpG-containing DNA, we treated cultures with a demethylating agent, but we were unable to show induction of IFNα with ACAs. Thus, although the CpG motifs within CENP-B–binding DNA can stimulate IFNα, especially when protected from DNase activity, we have been unable to reproduce in vitro a condition that stimulates IFNα production.
The potent stimulatory effects of IFNα on immune function are now well described and include enhanced antigen presentation, homing to lymphoid organs, activation of immune cells, and stimulation of a Th1 response (for review, see ref.12). Type I IFNs also stimulate the development of CD8+ T cells, and these cells have been implicated in pulmonary injury in patients with SSc (39). In addition, type I IFNs decrease the threshold for activation of B cells via the B cell receptor and enhance differentiation, antibody production, and immunoglobulin isotype class switching. This positive feedback loop may contribute to the relentless course of disease seen in many patients with dcSSc. It should also be noted that IFNα induction by SSc sera was not as high as that observed in SLE sera and that variations in the levels of IFNα induced by different PBMC donors was also observed. The source of variation is currently under study.
A second mechanism by which immune complex–mediated IFNα induction may cause pathologic changes is through vascular injury, a key component of SSc (40, 41). It has been suggested that vasospasm-induced ischemia (42) or antiendothelial autoantibodies (43) lead to apoptosis, thereby providing a source of nucleoprotein-containing antigens. Administration of IFNα has been associated with the temporal development of Raynaud's phenomenon (44) as well as SSc (45). IFNα is known to have an antiangiogenic effect, and it retards wound healing (46, 47). The antiangiogenic effects have previously been explained by IFNα-mediated antiproliferative activity or induction of apoptosis of endothelial cells, whereas more recent studies have shown that after prolonged exposure, IFNα induces replicative senescence of endothelial cells (48). Many other factors have been implicated in vascular injury in SSc, but our results should encourage further studies to determine whether prolonged local exposure of vessels to IFNα may be a contributory factor to the loss of small vessels that is characteristic of this disease.
We also observed higher IFNα-inducing activity in patients with lung fibrosis as compared with those without lung fibrosis. This observation is consistent with the higher frequency of anti–topo I–containing sera in the group with lung fibrosis (55%) as compared with the group without lung fibrosis (23%) (3). The association between IFNα-inducing activity and lung fibrosis is similar to the recently reported association between Jo-1 and lung fibrosis observed in polymyositis patients (49) and further supports a role of IFNα in the promotion of fibrosis. It is of considerable interest that a trend toward a decline in forced vital capacity and a reduction in diffusing capacity for carbon monoxide was observed in SSc patients receiving treatment with IFNα (50). Whether fibrosis is the end result of a vasculopathic effect, as discussed above, an exuberant immune response, or has some as-yet-unknown effect on profibrotic factors remains to be determined. Despite the fact that IFNα-inducing activity was higher in patients with lung fibrosis, sera from some patients with lung fibrosis did not induce IFNα in the bioassay, which suggests that other factors also contribute to lung fibrosis. Immune complexes may also engage FcγR on other cell types, releasing inflammatory cytokines.
We conclude that SSc sera containing anti–topo I induce IFNα by plasmacytoid DCs in vitro and likely contribute to IFNα production, as recently observed in ex vivo blood and tissue samples obtained from patients with SSc. The ability to induce IFNα is positively correlated with clinical disease, especially lung fibrosis. These results shed light on the immunologic differences between dcSSc and lcSSc and help to elucidate the molecular mechanisms responsible for the pathogenesis of SSc and other autoimmune diseases characterized by antibodies directed against autoantigens associated with nucleic acids. The nature and availability of antigen and the relationship between IFNα and fibrogenic stimuli, particularly in the lung, are key questions to address in future studies.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Elkon 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 design. Kim, Peck, Santer, Patole, Schwartz, Elkon.
Acquisition of data. Kim, Peck, Patole, Molitor, Arnett.
Analysis and interpretation of data. Kim, Peck, Santer, Patole, Schwartz, Molitor, Arnett, Elkon.
Manuscript preparation. Kim, Peck, Santer, Schwartz, Molitor, Arnett, Elkon.
Statistical analysis. Kim, Peck, Elkon.