The chronic inflammatory bowel diseases (IBDs), Crohn's disease (CD) and ulcerative colitis (UC), are typically characterized by episodes of acute flares and remission.1, 2 Depending on disease location and extent, exacerbation leads to diarrhea, abdominal pain, and systemic symptoms such as fatigue and weight loss.3–5 Disease activity indices have been developed as outcome measures in clinical trials.6, 7 They may help to reproducibly and validly assess the patients' status and to support therapeutic decision-making.6 Variables of disease activity indices comprise frequency of bowel movements, severity of abdominal pain, general well-being, occurrence of extraintestinal manifestations, and laboratory parameters.8
One of the most important protein biomarkers increased during the inflammatory state is C-reactive protein (CRP). However, elevation of serum CRP levels is not observed in certain inflammatory diseases. While serum CRP levels are highly increased in CD and rheumatoid arthritis (RA) patients and widely used for monitoring disease activity, only modest to absent CRP responses are observed in systemic lupus erythematosus (SLE), dermatomyositis, Sjogren's syndrome, or UC, although active inflammation is present.9–11 In UC, endoscopic disease activity may predict future clinical symptoms,12 but direct endoscopic or radiologic visualization of the degree of inflammation is rarely performed in outpatients with inactive or mild disease. Therefore, alternative biomarkers, which can conveniently and precisely monitor disease activity during therapy in inflammatory diseases, are required for the determination of adequate treatment.
By using a quantitative proteomic approach, we have previously reported that serum levels of leucine-rich alpha-2 glycoprotein (LRG) were elevated in patients with active RA and serum LRG levels were correlated with disease activity of not only RA but also CD, suggesting that serum LRG is a serological biomarker for monitoring disease activity.13 LRG is an ≈50 kDa glycoprotein and contains repetitive sequences with a leucine-rich motif, first purified from human serum.14, 15 LRG has been reported to be expressed by the liver cells and neutrophils16, 17; however, its function remains unclear. To date, the relationship between serum LRG levels and disease activity in UC has not been assessed. In this study we investigated serum LRG expression levels in UC patients and evaluated their correlation with clinical disease activity. Serum LRG levels were significantly increased in the active UC patients. LRG expression was upregulated in the inflamed colonic mucosa of UC possibly through stimulation by various cytokines including tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-22, the expression of which are increased in active UC. Moreover, we show that serum LRG correlates more strongly than CRP with disease activity in UC. Therefore, serum LRG may be a useful disease activity biomarker for UC.
- Top of page
- MATERIALS AND METHODS
- Supporting Information
In this study we first demonstrated that serum LRG levels were significantly increased in sera of active UC patients compared with patients in remission and HC. Serum LRG is likely elevated in diverse racial groups, because we detected increased serum LRG levels not only in Japanese patients (Fig. 1A)13 but also in Caucasian patients with UC (Fig. 1C,D) and CD (data not shown). In addition, levels of serum LRG were significantly correlated with disease activity in UC and the correlation was stronger than CRP. Moreover, by analyzing ROC curve and AUC, serum LRG levels showed higher AUC than CRP and serum LRG levels represented superior sensitivity and specificity to CRP for remission and active of UC by CAI (Fig. 2D), indicating that LRG is a useful marker to evaluate disease activity in UC. In the normal state, serum LRG is thought to be produced from liver and LRG is abundantly found in the sera of HC. In colonic inflammation, we found that the expression of LRG is increased in the inflamed mucosa of UC patients and mice with DSS colitis, suggesting that inflamed tissues can be a source for production of LRG (Fig. 3). The increased expression of LRG in inflamed tissue has previously been observed in appendix during acute appendicitis.28 Moreover, in acute inflammatory disorders, including appendicitis and diverticulitis, increased expression of serum LRG was observed (Fig. 1A). These results indicate that the elevated expression of LRG at inflamed sites and in sera occurs in various acute and chronic inflammatory disorders. Therefore, increased serum LRG levels are not suitable for use as a specific diagnostic marker of IBD.
CRP is the most common serum marker used to evaluate disease activity in inflammatory diseases. However, serum CRP is primarily dependent on liver production induced by circulating IL-6. Compared with CD and RA, only modest to absent CRP responses are observed in UC, despite active inflammation in colon.9 Indeed, our cohort of 82 UC patients, analyzed in this study, included five patients with normal value of CRP while having active disease (Fig. 2A). However, our study demonstrated that serum LRG levels were significantly increased in active UC patients' sera and correlated better with disease activity of UC than CRP levels (Figs. 1A, 2A). Particularly, in the group of patients with negative CRP (CRP <0.2), significant correlation was observed between serum LRG levels and CAI (Supporting Fig. 2C). Similarly, among CRP-negative patients serum LRG levels were significantly elevated in those with endoscopically active UC, compared with UC in remission (Supporting Fig. 1B). In addition, serum LRG levels were decreased after therapy (Fig. 2C), suggesting that LRG is a useful serological biomarker for evaluating disease activity and therapeutic effect in UC.
Better correlation of serum LRG levels with disease activity of UC than CRP might be explained in part by the differences in induction mechanisms between LRG and CRP. While the expression of CRP is essentially dependent on IL-6, several cytokines may compensate for the absence of elevated IL-6 in induction of LRG expression. Accordingly, expression of LRG in COLO205 cells was induced not only by IL-6 but also by TNF-α and IL-22 (Fig. 4B), all of which were increased in sera of UC patients (Fig. 4A). Expression of LRG was strongly induced by IL-22 in COLO205 cells, correlating with enhanced STAT3 (Tyr705) phosphorylation by IL-22 compared with IL-6 (data not shown). Thus, inflammatory cytokines such as TNF-α and IL-22 may mediate LRG expression in the absence of IL-6. Moreover, using DSS-induced colitis in IL-6-deficient mice we could demonstrate an IL-6-independent pathway for LRG induction (Fig. 5E). Because promoter regions of human and mouse LRG share high sequence homology and contain putative binding sites for transcription factors such as C/EBP, MZF1, and STAT,17 it is conceivable that the similar IL-6-independent mechanisms of LRG induction are also involved in humans. Future studies are required to fully elucidate the induction mechanisms of LRG in both humans and mice.
In the three disease categories of UC based on extent of disease, serum LRG levels tended to be low in proctitis compared with extensive colitis and left-sided colitis (Fig. 1B). In addition, correlation between serum LRG levels and disease activity did not reach significance in proctitis (Fig. 2B). Although the low number of patients with active proctitis may preclude the proper evaluation of LRG levels, limited inflamed area of proctitis may also be a reason for slight increases of serum LRG levels in these patients. Given the increased production of LRG in inflamed colonic mucosa, fecal LRG might be a more sensitive disease biomarker for UC including proctitis. Optimization for the measurement of fecal LRG is currently under way in our laboratory.
This study also highlights the potential usefulness of LRG in evaluating murine colitis. Our results indicate that serum LRG levels increase as the disease progresses in a DSS-induced colitis model (Fig. 5B,C). In addition, the LRG expression is significantly upregulated in the colon with DSS-induced colitis (Fig. 5D). Thus, LRG in mice can be an objective disease activity marker for colitis models and may be useful for preclinical studies of IBD.
In conclusion, serum LRG levels reflect disease activity of UC better than CRP, especially in patients with low CRP. In the inflammatory condition, LRG is expressed in the inflamed tissue and expression of LRG is regulated by mechanisms different from that of CRP. These findings suggest that serum LRG is a novel and potential serologic biomarker for evaluating disease activity of UC.