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- MATERIALS AND METHODS
Background: Identification of a disease- and organ-specific autoantigen can potentially contribute to understanding molecular mechanisms involved in Crohn's disease (CD) and lead to development of clinically useful markers. The aim was to identify potential intestinal autoantigens specific to patients with CD and evaluate their diagnostic value.
Methods: A cDNA expression library from normal terminal ileum was created and screened with pooled sera from 3 randomly selected patients with CD. For evaluation of the diagnostic value of antibody screening, serum samples obtained from 39 patients with CD, 28 patients with ulcerative colitis (UC), and 60 healthy controls were examined for IgG against the strongest clone.
Results: We identified an intestinal cDNA clone encoding ubiquitination factor E4A (UBE4A), a U-box-type ubiquitin-protein ligase. The prevalence of anti-UBE4A IgG in patients with CD was significantly higher than that in patients with UC or healthy controls (46.2% versus respectively 7.1%, P = 0.0006; 3.3%, P < 0.0001). Anti-UBE4A-positive patients with CD were more likely to require surgery (P = 0.0013). The level of anti-UBE4A IgG was correlated with disease activity (r = 0.777, P < 0.0001) and associated with stricturing or penetrating disease (P = 0.0028). Immunohistochemical studies showed upregulation of UBE4A in enteroendocrine cells of inflamed ileal mucosa with CD.
Conclusions: Anti-UBE4A antibodies are potentially useful markers for detection and prediction of clinical activity and outcome in patients with CD.
Crohn's disease (CD) and ulcerative colitis (UC) are the 2 major forms of inflammatory bowel disease (IBD). CD is an inflammatory disease of the gut with a chronic relapsing and remitting course. CD is characterized by transmural inflammation of the gastrointestinal tract, most often affecting the ileocecal region, but which may affect any part of the digestive tract from the mouth to the anus.1 The etiology of CD is unknown, but current hypotheses suggest that an inappropriate mucosal immune response to the gut lumen microflora leading to an excessive Th1-mediated chronic inflammation may be the critical component of CD.2 Genetic factors such as NOD2/CARD15 mutations may have an important role in determining susceptibility to CD.3, 4 A large number of genetically modified mice develop spontaneous intestinal inflammation.5 Many of these animal models develop colitis under conventional conditions but not under germ-free conditions, emphasizing a role for microbial flora in the pathogenesis of chronic mucosal inflammation.6
The hypothesis that CD might be an autoimmune reaction has been considered for several decades, but it seems unlikely that CD is a simple autoimmune disease directly caused by autoantibodies or self-reactive lymphocytes.7 However, involvement of the autoimmune process in the pathogenesis of CD cannot be excluded.8 It has been suggested that a number of autoimmune diseases are caused, or triggered, by microbes.9, 10 Extraintestinal manifestations in CD such as inflammation of skin, eye, and joint support the hypothesis that CD is a systemic disease.11 Several autoantibodies have been described for patients with CD such as anti-lymphocytes,12 anti-cytoskeletal proteins,13 and pancreatic autoantibodies.14
Although there is no conclusive evidence for a direct pathogenic role for any autoantibody in CD, detection of autoantibodies may have clinical and research potential. Identification of CD-related autoantigens may contribute to understanding the underlying dysregulation of the intestinal immune system, crossreactivity with enteric microbial antigens, and immunological alterations related to genetic susceptibility.15 Organ-specific autoantigens may shed light on the novel mechanisms involved in the sustained intestinal inflammation. Only 2 organ-specific autoantigens have been reported in IBD: epithelial cell-associated components (ECAC)16 and human tropomyosin isoform 5 (hTM5).17 ECAC-specific reactivity was present in both CD and UC, whereas autoantibodies against hTM5 were found in UC, but not CD patients.
The aim of the present study was to identify specific autoantigens potentially contributing to the pathogenesis in CD and which may serve as useful serological markers for the clinical diagnosis of CD. By screening a cDNA library from normal terminal ileum with sera from patients with CD, we identified 1 strongly immunoreactive cDNA clone that encodes the C-terminal subunit of ubiquitination factor E4A (UBE4A), a U-box-type ubiquitin-protein ligase. To evaluate anti-UBE4A serum immunoreactivity, we measured specific IgG levels in patients with CD and compared them with those in patients with UC and in healthy controls. We then evaluated the predictive value of anti-UBE4A IgG level for clinical and phenotypic characteristics of CD. Finally, we investigated the expression of UBE4A protein in normal and inflamed ileal mucosa by immunohistochemistry.
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- MATERIALS AND METHODS
In recent years, several antibody markers such as antibodies to microbial antigens (ASCA, antibodies against E. coli outer membrane porin C [anti-OmpC], against a CD-related protein from Pseudomonas fluorescens [anti-I2], and against bacterial flagellin [anti-CBir1]) and pancreatic autoantibodies have been found to be useful for the diagnosis and differentiation of CD. ASCA has been shown to be found in about two-thirds of patients with CD.20, 21 Anti-OmpC, anti-I2, and anti-CBir1 were detected in approximately half of CD patients.22–24 Pancreatic autoantibodies which are present in about one-third of patients with CD have a high degree of specificity for CD,25 but most of the consistently detectable antibodies associated with CD do not correlate with disease activity.15, 23, 24 Anti-UBE4A levels are significantly correlated with CDAI, and also associated with requirement of surgery and stricturing or penetrating behavior; therefore, anti-UBE4A may be helpful in predicting disease severity. It should be noted that anti-UBE4A level was determined on a single sample for each patient in this study. A longitudinal study of anti-UBE4A levels over time in relationship to clinical activity or behavior changes needs to confirm the relationship. In addition, although the prevalence is relatively low, anti-UBE4A is highly disease-specific. The combined search for anti-UBE4A and ASCA may help increase the diagnostic precision and detection percentage for CD, because anti-UBE4A was not associated with ASCA status.
UBE4A is a mammalian homolog of yeast Ufd2, a U-box-type ubiquitin-protein ligase (E3). The ubiquitination-mediated protein degradation has been shown to play a significant role in multiple cellular processes such as cell cycle regulation, cellular signaling in response to stress and to extracellular signals, morphogenesis, secretory pathway, DNA repair, and organelle biogenesis.26, 27 When a cellular protein degrades, a ubiquitin-activating enzyme (E1) forms a thiolester bond with ubiquitin and then transfers the activated ubiquitin to the target protein via a ubiquitin-conjugating enzyme (E2) and an E3. The polyubiquitylated target proteins are recognized by the 26S proteasome and degraded. Ubiquitin on the target are removed by deubiquitylating enzymes and recycled. The U-box proteins were originally described as an E4 ubiquitination factor,28 but mammalian U-box proteins are thought to constitute a third family of E3 enzymes, in addition to the HECT and RING-finger families.29 UBE4A also might play diverse roles in collaboration with molecular chaperone systems. Mouse UFD2b, a homolog of human UBE4A, binds to DnaJc7,30 which appears to interact with Hsp90.31 DnaJc7 possesses 2 functional domains related to chaperone function: a TPR domain, which interacts with the COOH-terminal region of Hsp90 or Hsp70 (EEVD motif),32 and a J domain, which mediates the interaction with Hsc70.33
UBE4A is reported to be expressed in the skeletal muscle, kidney, and liver at high levels, but the expression levels of UBE4A in small and large intestine are low.34 Immunohistochemical analysis in the present study revealed that upregulation of UBE4A in enteroendocrine cells was seen in the inflamed ileum of CD but not in the normal ileum. Generation of antibodies to UBE4A in patients with CD might be the result of upregulation of UBE4A in inflamed intestinal mucosa.
The various peptide hormones produced by enteroendocrine cells control important physiological functions including growth and repair of the gut epithelium and motility of the gut wall.35 An increased number of enteroendocrine cells has been demonstrated in CD36 and TNBS-induced ileitis.37 Since secretory products from enteroendocrine cells could contribute to mucosal repair and protection from further epithelial injury in inflamed mucosa of CD, upregulation of UBE4A in these cells may be associated with altered secretion of gut hormones. However, the physiological role of UBE4A in production of gut hormones requires further study.
Another possibility of the generation of anti-UBE4A antibodies is the result of crossreactivity with enteric microbial antigens. Mounting evidence exists that serum antibody responses to self-antigens in IBD are likely to be triggered by crossreactivity with luminal antigens.38, 39 Epitope-specific crossreactivity between microbial antigens and self-antigens has been shown in some animal models to initiate autoimmunity.40 Identification of an immunodominant epitope of C-terminal UBE4A might provide information on the mimicking microbial antigen.
In conclusion, UBE4A was identified as a novel autoantigen in CD. The presence of autoantibodies against UBE4A in sera was significantly specific to CD and had a significant association with disease activity, behavior, and requirement of surgery. Our results suggest that anti-UBE4A antibodies are potentially useful markers to diagnose and predict disease severity in patients with CD. Whether UBE4A plays a role in the etiopathogenesis of CD remains to be determined.