Behçet's disease (BD) is a chronic, multisystem disorder characterized by a recurrent inflammatory reaction. Serious complications, such as blindness or intestinal perforations, can occur. Because of the lack of definitive diagnostic tests for BD, the diagnosis is based solely on the clinical manifestations. The pathogenesis of BD remains obscure. Clinically, BD patients experience recurrent thrombophlebitis, thrombosis, and cutaneous vasculitis (1). Histopathologic changes consisting of perivascular mononuclear cellular infiltrates, endothelial cell swelling or necrosis, partial obliteration of the vessel lumen, and occasional fibrinoid necrosis of vessels have been observed. In more severe forms, small to medium-sized arteries, veins, and capillaries may be affected by a necrotizing or granulomatous vasculitis (2, 3).
Anti–endothelial cell antibody (AECA) was first detected in various inflammatory diseases by indirect immunofluorescence using a mouse kidney substrate (4). AECA has been demonstrated in the serum of patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), mixed connective tissue disease, scleroderma, and dermatomyositis, as well as patients with Wegener's granulomatosis (WG), primary retinal vasculitis, Kawasaki disease, acute hemolytic uremic syndrome, diabetes mellitus, organ transplants, hypoparathyroidism, multiple sclerosis, thrombotic thrombocytopenic purpura, angioedema, and Buerger's disease (5, 6). AECA has also been demonstrated in the serum of BD patients (7–10) and has been associated with disease activity and vasculitis symptoms (6, 7).
Even though a large number of molecules on the surface of endothelial cells have been characterized, only a few reports have studied the characteristics of the antigen recognized by AECA. In a previous study (9), we used an enzyme-linked immunosorbent assay (ELISA) to detect circulating antibodies against human dermal microvascular endothelial cells (HDMECs) in the serum of patients with BD. Serum IgM antibody against HDMEC was detected in 49 of 131 BD patients. On Western blots, IgM-positive BD serum reacted with the 44–50-kd HDMEC surface antigen, whereas IgM-positive SLE serum reacted with the 81-kd HDMEC surface antigen (9).
Proteomics is a field of science that evaluates a large number of proteins expressed from a given cell line or organism. The technology of proteomics has been used as a method of discovering the target protein specific to a particular disease by searching for the expression or modification of the protein (11, 12). To characterize the AECA-binding HDMEC antigen, which is closely related to the pathogenesis of BD, we first identified a target protein by 2-dimensional (2-D) gel electrophoresis and immunoblotting, and then searched for a similar protein after the amino acids were sequenced by mass spectrometry. We next searched for the DNA sequence of the target protein at the National Center for Biotechnology Information (NCBI) and purified the recombinant target protein by gene cloning. We then investigated the reactivities of the recombinant target protein in BD.
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- PATIENTS AND METHODS
Although AECAs have been detected in a variety of inflammatory diseases, there are a few reports of the association of AECA with the clinical manifestations of vasculitis. It has been shown that when vasculitis symptoms improve in patients with RA, AECA titers tend to decrease (15). Moreover, high titers of AECA in patients with SLE are associated with an active lesion of the kidney and vasculitis, and it has therefore been suggested that the AECA titer could be used as a measure of renal involvement (16).
Microvascular endothelial cells carry a specific antigen profile that is quite different from the profiles of the large vessels (17, 18). In BD, vasculitis mainly involves capillaries and small vessels (2, 3). Direct immunofluorescence of the lesions of BD patients mainly shows deposition of immunoglobulin on the walls of small veins (19). One study reported a higher prevalence of AECA when sera from BD patients were tested against microvascular endothelial cells as compared with human umbilical vein endothelial cells as the substrate (8). This finding provided the rationale for choosing HDMECs for the present experiments.
AECA-positive BD patients have a significantly higher frequency of active ocular lesions and acute thrombosis than do AECA-negative BD patients (7, 8). We have also previously demonstrated that AECA-positive BD patients have a higher frequency of thrombophlebitis than do AECA-negative BD patients (20).
Histopathologically, BD has a characteristic vasculitis, presenting as an infiltration of CD4+ T lymphocytes around the vessels (21). Pretreatment of human endothelial cells with biologic response modifiers, such as interleukin-1α (IL-1α) and tumor necrosis factor α (TNFα), leads to an increase in the expression of intercellular cell adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1, and E-selectin molecules and, consequently, to an increase in the adhesion of T lymphocytes (14, 22). In a previous study, we found that AECA-positive sera from BD patients led to changes in the expression of adhesion molecules on the cell surface of HDMECs and promoted the adherence of T lymphocytes to HDMECs and, thus, initiated or amplified inflammatory vascular injury (10). Our findings also suggested that IgM AECAs could play a pathogenetic role in BD by activating EC directly, and not via the production of TNFα or IL-1α by HDMECs. In addition, as a signal transduction pathway, extracellular signal–regulated kinases 1 and 2 were involved in the expression of ICAM-1 on HDMECs stimulated by IgM AECA (23).
AECA has been detected at various frequencies in several diseases, but few studies have characterized the surface antigens against AECA. Moreover, there have been no other reports elucidating the antigens against AECA in BD. The human genome was completely sequenced recently, and as a result, many studies analyzing the function of various human genes are now in progress. Proteomics is the most suitable method for these studies, because proteomics can analyze proteins by electrophoresis, and these results can be analyzed by several methods that identify the protein (24–26).
In this study, we produced an HDMEC protein map with ∼2,000 protein spots on 2-D electrophoresis and an 18-cm IPG strip. We observed that many protein spots produced by BD patient sera, which on the 2-D immunoblot were between pH 5.0 and pH 8.0 (isoelectric point) and 44–50 kd, reacted with AECA-positive serum. Using the peptide fingerprint of the protein spot obtained by mass spectrometry, we were able to compare the protein with human α-enolase and found that 7 of 17 peptides coincided within 30 ppm. Due to the high coincidence of 41% of the amino acid sequences, we were able to conclude that the protein reacting with AECA was a human α-enolase of molecular weight 47 kd and of isoelectric point 7.0. Initially, we expected that the protein coinciding with AECA would be a previously unidentified protein. However, the protein was identified as α-enolase, which has a known DNA sequence. Consequently, by making use of its known DNA sequence, it was not difficult to separate and refine the human α-enolase using gene engineering.
The glycolytic enzyme α-enolase is a homodimer of 47–48 kd. There is >90% homology among the α-enolases of mammals (27). The α-enolase enzyme catalyzes the dehydration of 2-phosphoglycerate to phosphoenolpyruvate and is usually found in the cytoplasm. It exists as a multienzyme complex with other glycolytic enzymes, such as pyruvate kinase, phosphoglycerate mutase, muscle creatine kinase, and aldolase, in the cytoplasm (28, 29). Although the mechanisms of α-enolase expression in the cell membrane are not clear, enolase is expressed on the cell surface after exposure to an inflammatory stimulus. Enolase functions as the plasminogen receptor on the surface of various cells, such as epithelial cells, endothelial cells, and hematopoietic cells. The consequent binding of plasminogen to α-enolase plays a crucial role in fibrinolysis. The role of this enzyme in systemic or invasive autoimmune disease was recently reported (30). Certain properties of α-enolase, especially those related to surface expression and plasminogen binding, indicate that enolase may play an important role in the initiation of the disease process by modulating the pericellular and intravascular fibrinolytic system.
Antibodies against α-enolase have been found in various inflammatory and immune disorders, such as cancer-associated retinopathy, ANCA-positive vasculitis, inflammatory bowel disease, discoid lupus erythematosus, SLE, systemic sclerosis, endometriosis, primary membranous nephropathy, mixed connective tissue disease, and in autoimmune liver diseases (27, 30, 31). In SLE with nephritis, mixed cryoglobulinemia, and diffuse systemic sclerosis, antienolase antibodies react readily with renal and endothelial cell antigens, which express abundant α-enolase and, thus, induce injury to those cells (27). The anti–α-enolase antibodies in the sera of patients with ANCA-positive vasculitis and inflammatory bowel disease react with antigens in the cytoplasm of neutrophils. In the present study, we found that antibodies against HDMEC α-enolase were present in 37.5% of sera from 40 patients with BD. We also found that antibodies against HDMEC α-enolase are present in patients with other systemic rheumatic diseases. Thus, the antibody response to HDMEC α-enolase does not seem to be restricted to BD.
However, ELISA showed that the IgM antibody response to HDMEC α-enolase was present only in the sera of BD and WG patients, and not in the sera of RA and SLE patients. Furthermore, all BD patients with anti–α-enolase antibodies were negative for RF, ANA, and ANCA, a finding that is inconsistent with the common antibody profiles observed in other rheumatic diseases. We might well conclude that this antibody response found in the sera of BD patients might be useful for the diagnosis of BD, since at present, there is no definitive diagnostic test for BD.
Three isoforms of enolase that display tissue-specific distributions have been described. Beta-enolase is found in skeletal and cardiac muscles, γ-enolase is expressed in neural tissue, and α-enolase, the embryonic form, is ubiquitous, although it is most highly expressed in the kidney and thymus (32). Beta-enolase and γ-enolase share ∼83% homology with α-enolase. Despite the high degree of homology among the 3 enolase isoforms, the antibodies react only with the α isomer, which suggests that these antibodies recognize a unique epitope on α-enolase (33, 34). In the present study, BD patient sera that were positive for anti–α-enolase antibody did not react with human γ-enolase. These results suggest that antibodies from BD patients react with a specific epitope structure of human α-enolase.
Several previous reports have suggested that in autoimmune diseases, AECA is directed against class I and class II major histocompatibility complex (MHC) or ABO blood group antigens (35, 36). However, antibodies against the MHC antigen in normal persons and in some autoimmune disease patients are often observed only during pregnancy or following transfusion. Yet, in the present study, many male patients were positive for this antibody, and there was no history of transfusion or pregnancy among any of the antibody-positive BD patients. Previous studies have shown higher IgM positivity compared with IgG positivity for AECA in BD (20), and direct immunofluorescence has shown a predominance of IgM class among the immunoglobulins deposited in the venular walls of BD lesions (19, 37). The present study also showed that the reactivity to HDMEC α-enolase was detected only in IgM-positive BD sera. These results suggest an important role for IgM class AECAs in the pathogenesis of vasculitis in BD.
The presence of α-enolase on the surface of streptococci may play a crucial role in the induction of autoimmune disease caused by streptococci (38). Serum samples from patients with acute rheumatic fever contain elevated levels of antibodies that react with both streptococcal and human α-enolase. Also, Hsp48, one of the heat-shock proteins (HSPs), was identified as α-enolase (39). Because HSPs are immunogenic molecules and can be expressed on cell membranes, their role in autoimmune and inflammatory diseases has been examined. A cellular immune response of T cells and a humoral response with the production of antibodies against HSP have been observed to occur in the course of those diseases (40). Autoantibodies may bind to the enolase on the surface of neutrophils and interfere with phagocytosis. Human enolase is expressed on the immune cell surface after an inflammatory stimulus, and it may react with antienolase antibodies. Generally, self-tolerance prevents an autoimmune response, but if this fails, the reaction of the enolase on the surface of immune cells with antibody may lead to opsonization or cell destruction, an increased inflammatory reaction, and tissue damage. Moreover, the high homology between streptococcal enolase and human enolase may facilitate the initiation and development of autoimmune reactions.
We cannot describe the precise role of α-enolase in the pathogenesis of BD, nor can we explain the significance of the existence of antibody to α-enolase in the sera of BD patients. However, we believe that this antigen can be used as a marker of BD. This is the first study to show that autoantibodies to the α-enolase of HDMEC are present in the sera of BD patients. Possible explanations for the role of α-enolase in BD include the histopathologic features of vasculitis, frequent retinal involvement, frequent reports of preceding bacterial infections (e.g., Streptococcus), the role of HSPs, and positive reactivity to ANCA. Thus, further studies on the relationship between α-enolase and the pathogenesis of BD may yield important information about its pathogenesis. Moreover, it is hoped that our findings might aid in the development of new treatments for BD and be of value in studies of other autoimmune diseases in which vasculitis is a component. Further studies are necessary to elucidate the roles of this protein and circulating autoantibodies in BD, to determine the differences between BD and other autoimmune diseases in which vasculitis is a component, and to establish the role of Streptococcus in the pathogenesis of BD.