Systemic sclerosis (SSc; scleroderma) is a complex inflammatory, fibrogenic disorder in which vascular dysfunction underlies the major clinical manifestations (1). In common with other connective tissue disorders, the autoantibodies elaborated in this disease are predictive of characteristic disease phenotypes (2). Well-recognized associations include anticentromere antibodies (ACAs) with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias) variant of SSc, and anti-topoisomerase I antibodies with the diffuse subset of SSc and pulmonary fibrosis (for review, see refs.3–5).
Recent studies on the connective tissue disorders have implicated modification of autoantigen structure during cell death in the selection of target molecules of the autoimmune response (6, 7). Relevant pathways include those occurring during both caspase-dependent and caspase-independent modes of cell death. One property increasingly recognized as unifying many autoantigens across the spectrum of autoimmunity (including SSc, systemic lupus erythematosus, Sjögren's syndrome, inflammatory muscle disease, and Rasmussen's encephalitis) is susceptibility to modification by proteases in the cytotoxic lymphocyte granule pathway. In this regard, numerous autoantigens are susceptible to proteolytic cleavage by granzyme B (GB) during cytotoxic lymphocyte granule-mediated cell death, generating unique fragments not observed during other forms of cell death (8–10). A smaller number of autoantigens targeted in systemic autoimmunity are also directly cleaved by granzyme A (11–13).
While several autoantigens are cleaved with similar efficiencies (but at different sites) by both GB and caspases during cell death, other autoantigens are exclusively or preferentially cleaved by GB (9). Prominent among the latter molecules are autoantigens targeted in scleroderma, including CENP-B and fibrillarin (which are cleaved by GB but not by caspases) as well as topoisomerase I (which is cleaved by GB ∼100-fold more efficiently than by caspases). Interestingly, in the one instance in which this has been studied, autoantibodies reactive against uniquely modified forms of autoantigens appear to be predictive of clinical phenotype. Specifically, recognition of the caspase-cleaved form of U1-70 kd by autoantibodies was associated with lupus skin disease (14). Since different modes of apoptotic cell death generate distinct modified forms of autoantigens, the demonstration of an association between reactivity against specifically modified autoantigens and unique clinical phenotypes may implicate a particular apoptotic cell death pathway in generating disease expression.
Of particular interest in this regard is the SSc phenotype of severe digital ischemia. Although vascular abnormalities are prominent and universal features in SSc patients (15, 16), only a distinct subset develops critical tissue ischemia resulting in ischemic digital loss (IDL) (17). There are 3 recognized predictors of this unique phenotype (17–23): limited cutaneous involvement, anti-endothelial cell antibodies (AECAs), and ACAs.
Since CENP-B (a major component of the centromere antigen ) is cleaved by GB but not by caspases, we addressed whether autoantibodies from patients with limited SSc and digital loss preferentially immunoblot GB-cleaved forms of autoantigens. We demonstrated a striking association between recognition of GB-cleaved autoantigens and IDL in this population. Interestingly, the presence of both ACAs and antifragment antibodies was highly specific for IDL. In several cases, autoantibodies preferentially recognized the cleaved fragment over the intact form of the antigen. Moreover, in 1 subject with serum available prior to the onset of IDL, these autoantibodies preceded digital loss, suggesting that antibodies recognizing GB-generated fragments may predict subsequent phenotype. Taken together, these studies strongly implicate the cytotoxic lymphocyte granule pathway in the generation of the immune response associated with this specific clinical phenotype.
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- PATIENTS AND METHODS
Modification of autoantigen structure during different forms of cell death has been implicated in the selection of molecules as targets of the autoimmune response. By demonstrating a striking association between recognition of GB-cleaved autoantigens and a specific clinical phenotype (IDL in limited scleroderma), this study strongly implicates the cytotoxic lymphocyte granule pathway in the generation of this phenotype and its associated autoantibody response. Furthermore, this is the first direct demonstration that a subgroup of phenotype-associated autoantibodies preferentially recognizes the cleaved form of autoantigens, indicating that GB-mediated cleavage plays an important role in unmasking cryptic B cell epitopes. Finally, since there are very few predictors of the development of digital loss in patients with limited scleroderma, these findings have potential clinical relevance for identifying patients who are at particularly high risk for developing IDL.
GB is a serine protease expressed in cytoplasmic granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It induces apoptosis in target cells during granule exocytosis-induced cytotoxicity by catalyzing the cleavage and activation of several caspases, as well as through caspase-independent pathways. Unlike most forms of apoptotic death, which occur in noninflammatory contexts and actively suppress the initiation of primary immune responses (36), the GB pathway has a major function at the proinflammatory host-pathogen interface, where it plays a central role in clearance of intracellular infections (37–39).
The following findings of several recent studies have strongly implicated the GB pathway in the pathogenesis of systemic autoimmune diseases. First, CTLs are present and express an activated phenotype with markedly up-regulated granzyme expression in several diseases, including scleroderma (40), Sjögren's syndrome (41, 42), and autoimmune myositis (43). Second, GB is expressed at high levels by several cell types (including T cells and NK cells) within the synovium in rheumatoid arthritis (RA) (44, 45). Third, levels of GB in joint fluid and serum appear to be predictive of the subsequent development of erosive joint disease in RA (46, 47). Fourth, GB specifically cleaves most autoantigens at sites not recognized by caspases (9), potentially revealing cryptic T cell epitopes. While this last property is strongly associated with a molecule's autoantigen status, previous studies have not demonstrated that recognition of GB-cleaved autoantigens associates with particular phenotypes, nor has there been any demonstration of preferential recognition by autoantibodies of GB-cleaved forms of antigens over the intact molecules (9).
This study demonstrates that autoantibody recognition of GB-cleaved antigens is strongly associated with IDL in limited SSc. The mechanism underlying this association is not yet known, but may involve a direct role for GB in altering the immunogenicity of these molecules (see below) or may identify another structural feature that influences immunogenicity.
Although they frequently coexist with ACAs, the antibodies that recognize cleaved antigens contribute independently to the prediction of digital loss. Approximately one-third of sera from patients with IDL recognized a 60-kd fragment arising from GB-mediated cleavage of CENP-C. Of note, antibodies to CENP-C preferentially recognized the fragment rather than the intact form of the antigen. Although both CENP-B and CENP-C are susceptible to cleavage by GB, proteolysis of these two proteins differed in 3 distinct ways. First, CENP-C was susceptible to efficient cleavage by several caspases as well as by GB, with generation of distinct fragments, while CENP-B was exclusively cleavable by GB. Second, GB cleaved CENP-C significantly more efficiently than CENP-B. Third, GB-generated fragments of CENP-C were preferentially recognized over the intact form by sera from patients with IDL, while fragments of CENP-B were not preferentially bound. Thus, among the numerous autoantigens that are cleaved by GB, CENP-C represents the first autoantigen in which fragment-preferential autoantibodies have been demonstrated (9).
Fragment-specific autoantibodies may arise when cleavage generates or reveals novel structure that is cryptic in the native molecule. This might include exposure of the new termini generated by cleavage, or other conformational modifications distant from the cleavage site. The presence of such fragment-specific antibodies argues strongly that the cleaved form of the antigen is responsible for driving the immune response in patients with this phenotype. The fact that the majority of autoantibodies targeted across the spectrum of autoimmune diseases do not preferentially recognize cleaved forms of their target molecules strongly suggests that the B cell epitopes in these molecules are not hidden in the intact molecule, while the relevant T cell epitopes are (and hence might require cleavage in order to be revealed). By demonstrating preferential recognition of GB-induced fragments in some individuals manifesting a unique clinical phenotype, this study shows the first in vivo evidence that the cytotoxic lymphocyte granule pathway plays a role in the selection of targets for an autoantibody response. Demonstration that a similar principle applies to the generation of T cell epitopes for many other autoantigens is a high priority.
The fact that most patients with limited scleroderma and without digital loss did not recognize GB-cleaved antigens, but did manifest other clinical features of limited SSc, suggests that the GB pathway plays a role in generating some aspects of this phenotype, but that it is not a universal component of pathogenesis in this disease. Previous studies have demonstrated that vascular cell apoptosis may be an early event in SSc (21), but the inducers of this cell death in vivo are not yet known. Potential candidates include cytotoxic lymphocytes (including NK cells and CTLs), antibody-mediated cytotoxicity (e.g., AECAs), and recurrent ischemia-reperfusion. The potential for additional autoantigen modifications (e.g., metal-catalyzed oxidation, glutathiolation) during the last two of these processes has been emphasized (48, 49). It will be important to determine whether the autoantigens that are shared between patients with and without IDL and that also are not cleaved by GB are otherwise modified during apoptotic cell death. Defining such a unifying process will focus attention on additional pathogenic pathways of universal importance in this disease spectrum. In this regard, it is noteworthy that pyruvate dehydrogenase E2, the major autoantigen in primary biliary cirrhosis, which is sometimes associated with limited SSc, is oxidatively modified during apoptotic death in a cell type-specific manner (49).
In summary, we have demonstrated a striking and (in some cases) preferential recognition of GB-generated autoantigen fragments in sera from patients with limited SSc and the unique phenotype of IDL. These findings constitute the first in vivo evidence that antibodies against GB-generated centromeric peptide fragments identify a distinct clinical subset. Furthermore, the identification of serologic risk factors for IDL in limited scleroderma may allow the opportunity for early targeted intervention in at-risk patients.