Scleroderma (systemic sclerosis [SSc]) is characterized by circulating autoantibodies to nuclear and nucleolar antigens, an obliterative vasculopathy, and widespread cutaneous and visceral fibrosis. The vascular complications include Raynaud's phenomenon, telangiectasia formation, digital ulceration, pulmonary hypertension, and renal crisis and are characterized by proliferative intimal lesions of the small arteries and widespread capillary dropout (1). SSc is a complex autoimmune disorder in which a variety of genes interact with as yet unknown environmental factors to produce the disease phenotype (2). There is increasing evidence that endothelial cell (EC) apoptosis, which is initiated by immunoglobulin binding, is an early feature of the disease (3, 4), but it is still unclear whether immune alterations precede or follow endothelial changes (5, 6).
In SSc, antinuclear antibodies (ANAs) are present in more than 95% of patients with anticentromere antibodies (ACAs), and anti–topoisomerase I (or, anti–Scl-70) antibodies being those most commonly observed (7). ACAs are typically found in patients with limited skin and internal organ involvement who are more likely to develop severe Raynaud's phenomenon, digital ulcers, and pulmonary hypertension (8). Anti–Scl-70 antibodies occur in SSc patients who are more likely to develop diffuse cutaneous disease and pulmonary fibrosis (9). Recent reports suggest a role of these autoantibodies in vascular disease associated with SSc. ACAs derived from the sera of SSc patients with ischemic loss of digits have been shown to recognize fragments of centromeric proteins (CENP-C), thus, potentially implicating anti–CENP-C antibodies in the pathogenesis of ischemic loss of digits (10). Another study demonstrated reactivity of ACAs with an 18–19-kd epitope from membrane extracts of human umbilical vein endothelial cells (HUVECs) (11) suggesting that ACAs may be among the heterogeneous group of anti–endothelial cell antibodies (AECAs) detected in SSc sera that are capable of binding to and inducing EC pathology.
While anti–Scl-70 antibodies have not been directly implicated in SSc vascular dysfunction, several studies suggest a role of these antibodies in disease development. Studies of twins have shown anti–Scl-70 antibody production in SSc-affected twins but not in unaffected twins, suggesting that anti–Scl-70 may be linked to the development of SSc (12, 13). A recent study demonstrated the direct binding of purified human anti–Scl-70 to the cell surface of fibroblasts, suggesting a role for anti–Scl-70 antibodies in fibroblast dysfunction (14). Finally, IgG titers of anti–Scl-70 antibodies have been shown to fluctuate with disease activity and skin fibrosis, implying a pathogenic role of these autoantibodies in SSc (7).
While the etiology of the generation of SSc-specific autoantibodies is not known, data suggest that autoimmunity may result from posttranslational modifications of autoantigens during apoptosis, which exposes cryptic epitopes as targets for autoantibody generation (15). Apoptosis-specific posttranslational modifications of a subgroup of autoantigens have been described, including proteolysis by caspases and granzyme B (16–18). The presence of autoantibodies to fibrillin 1 (a major component of microfibrils in the extracellular matrix) has also been reported in the sera of most SSc patients (19), and these antibodies appear to be specific for SSc (20). Whether fibrillin 1 antibodies are pathogenic has yet to be proven, but one could hypothesize that distinct AECAs are present in the sera of patients with limited and diffuse SSc that are capable of inducing EC apoptosis (4, 21) and contributing to the respective clinical phenotype.
In this study, we hypothesized that SSc sera containing autoantibodies to centromere or topoisomerase I contain subsets of AECAs that trigger distinct pathways of apoptosis and gene expression in normal adult human dermal endothelial cells (HDECs). Vascular cell apoptosis could, in turn, expose autoantigens to immune surveillance, evoking an autoimmune response and perpetuating autoimmunity to blood vessels in SSc. The data from this study provide evidence that distinct patterns of HDEC gene expression are induced by AECAs from patients with limited and diffuse SSc, respectively, that culminate in apoptosis associated with increased caspase 3 protease activity and aberrant expression of the microfibrillar protein fibrillin 1.
- Top of page
- MATERIALS AND METHODS
The association of serum ACAs and anti–Scl-70 antibodies with distinct clinical phenotypes and outcomes in SSc patients led us to hypothesize that SSc sera containing autoantibodies to centromere or topoisomerase I contain subsets of AECAs that trigger distinct pathways of apoptosis and gene expression in normal adult HDECs. Indeed, in this study using adult HDECs, the overall gene expression pattern observed in response to stimulation with SSc sera containing ACAs and anti–Scl-70 antibodies was a down-regulation of numerous genes, including those responsible for angiogenesis and a proadhesive EC phenotype (VCAM-1, ICAM-1, ICAM-2) and an up-regulation of genes for apoptosis (e.g., caspase 3) and the SSc autoantigen fibrillin 1. Furthermore, studies with fractionated SSc sera provide additional support for the role of AECAs in SSc serum–mediated HDEC pathology and demonstrated distinct gene expression profiles for serum from patients with limited SSc (ACA positive) and diffuse SSc (anti–Scl-70 positive).
The dysregulation of EC genes related to angiogenesis or involved in pulmonary vessel development, for example, by serum from patients with limited SSc (and ACAs), bring to mind the telangiectasia/digital ulcer formation and pulmonary hypertension associated with limited SSc. Similarly, one could argue that there are distinct AECAs in patients with diffuse SSc (and anti–Scl-70) that dysregulate EC genes, for example, that signal cytotoxic T lymphocytes to dermal antigens or that inhibit angiogenesis leading to the diffuse skin and lung fibrosis seen in patients with diffuse SSc. Investigations using sera from larger cohorts of SSc patients are currently under way to confirm the association of these gene expression profiles with clinical phenotype in SSc.
While AECAs from SSc (24) and SLE (26, 27) patients have been reported to induce EC activation, other studies have demonstrated EC activation in the absence of AECAs (28) or, instead, have demonstrated the induction of apoptosis by AECAs (29). The down-regulation of genes favoring a proadhesive EC phenotype are inconsistent with the findings of Carvalho et al (24) and may reflect differences in the source of ECs used (umbilical cord versus adult skin) Interestingly, IL-6 (interferon-β2) and IL-18 (interferon-α–inducing factor) were up-regulated independently of cellular adhesion molecules, reminiscent of the proinflammatory phenotype triggered in HUVECs exposed to AECAs from vasculitis sera (30). One possible explanation for these findings would be that there are different subsets of AECAs in autoimmune diseases that have a proclivity for the vasculature (SSc, SLE, vasculitis) that can induce a proinflammatory state and/or proadhesive state.
While distinct differences in gene expression could be demonstrated in HDECs stimulated with sera from patients with limited or diffuse SSc, exposure to either serum resulted in apoptosis. Caspase 3 is a terminal executor of programmed cell death, and significant protease activity was demonstrated in HDECs exposed to SSc sera independently of clinical disease or SSc autoantibody status. Caspase 3 inhibition led to partial blockade of the apoptosis-inducing activity of either SSc or SLE sera, suggesting the additional involvement of other caspase or noncaspase pathways in autoimmune sera–mediated programmed cell death. Alternatively, the increased activity of caspase 3 may be a reflection of its role in the cleavage of class 4 substrates, rather than autoimmune serum–mediated EC apoptosis (31). Interestingly, class 4 substrates include topoisomerase I and the 70-kd protein in U1 small nuclear RNP, both of which are autoantigens to which autoantibodies are known to develop in SSc and SLE, respectively.
Our investigation into EC autoantigen expression yielded similar results when HDECs were stimulated with sera from patients with limited or diffuse SSc, namely, the overexpression of fibrillin 1. Since adult HDECs do not express fibrillin 1, this investigation is, to our knowledge, the first to show the induction of fibrillin 1 in adult HDECs stimulated with SSc sera. The gene encoding fibrillin 1 has been implicated in the pathogenesis of human SSc (32, 33), and disease-specific anti–fibrillin 1 antibodies have been reported in the sera of most SSc patients independently of SSc autoantibody status (19, 20). Fibrillin 1 is a 350-kd glycoprotein that is a major constituent of microfibrils in the extracellular matrix (34) and is believed to play a critical role in fibrosis through its regulation of transforming growth factor β (TGFβ) signaling in the extracellular matrix through TGFβ sequestration and activation (35). From a vascular perspective, fibrillins have been demonstrated in anchoring filaments from human lymphatic vessel ECs and transmit chemical/mechanical stimuli from the EC cytoskeleton to the extracellular matrix (36). They are also found in association with type VI collagen in human vascular subendothelium (37) and in association with fibronectin in the filaments extending from ECs in the developing mouse aorta (38).
Our data demonstrating the reexpression of this glycoprotein in adult HDECs exposed to SSc sera are reminiscent of tenascin expression in skin fibroblasts (39). Tenascin is transiently expressed during embryonic development, but is absent from most normal adult tissues. However, in healing wounds and in fibrotic diseases such as SSc, tenascin is reexpressed and is up-regulated in SSc fibroblasts by high levels of IL-4. Similarly, the development of AECA-mediated vasculopathy in SSc may trigger the reexpression of fibrillin 1 in HDECs. The absence of fibrillin 1 expression in HDECs undergoing apoptosis in response to SLE sera or to treatment with camptothecin suggests that fibrillin 1 induction is specific to SSc and is less likely a secondary response of HDECs undergoing programmed cell death. The induction of this protein in apoptotic ECs could unmask cryptic epitopes and perpetuate an immune response (e.g., anti–fibrillin 1 antibody). One could speculate about a role of these anti–fibrillin 1 antibodies (generated during vascular apoptosis in SSc) in binding to the microfibrils of the extracellular matrix and triggering fibrosis through the activation of sequestered TGFβ, as was described recently (40). Such speculation is intriguing, since it might explain why fibrosis in patients with SSc is preceded by vasculopathy (e.g., Raynaud's phenomenon, nailfold capillary dropout, and telangiectasia formation).
Our demonstration of SSc AECA–dependent HDEC apoptosis is consistent with previous investigations demonstrating apoptosis of HUVECs upon exposure to SSc AECAs (4, 21) and the in vivo apoptosis-inducing effects of AECAs in the microvasculature of normal chicken embryos following intravenous injections of AECA-positive sera obtained from UCD-200 chickens (an avian model of SSc) (41). However, a recent study demonstrated that SSc serum alone is incapable of inducing either HDEC or HUVEC apoptosis and that activated natural killer cells are required, in addition to SSc sera, to trigger apoptosis in HDECs via CD95 (42). Possible factors that may explain these differences include differences in EC stimulation media (<5% serum versus 15% serum) or the source of microvascular ECs (foreskin versus abdominal skin).
Because an investigation evaluating the activity of SSc AECAs against ECs from the micro- and macrovasculature demonstrated significantly higher binding to the microvasculature (thus emphasizing the distinct differences in ECs obtained from different tissues ), we used ECs obtained from the abdominal skin of a normal adult female as a substrate for AECA detection. With this method, we have shown, for the first time, the colocalization of AECAs with blebs resulting from autoimmune sera–mediated HDEC apoptosis which, in the case of SSc sera, is additionally associated with fibrillin 1 expression. The inability of control sera and AECA-negative sera (from SSc patients with anti–Scl-70 and minimal vascular disease) to induce apoptosis suggests that pathogenic AECAs are absent in control sera and are distinct from SSc autoantibodies in their ability to induce HDEC apoptosis. This is consistent with a recent study that demonstrated the binding of purified human SSc anti–Scl-70 antibodies to fibroblasts but not to HUVECs or dermal microvascular ECs (14).
In conclusion, the investigations described herein demonstrate that distinct AECA subsets exist in the sera of patients with limited SSc (with ACAs) and patients with diffuse SSc (with anti–Scl-70 antibodies) and can differentially modulate EC gene expression. Despite these serum-specific differences in HDEC gene expression, both types of SSc sera trigger apoptosis and, as identified herein for the first time, are also associated with increased caspase 3 activity and the reexpression of EC fibrillin 1. This pathologic recapitulation of fibrillin 1 expression in the setting of apoptosis may provide autoantigenic substrates that lead to autoimmunity in SSc.