Original Article
Fibrinogen-like protein 2 prothrombinase may
contribute to the progression of inflammatory
bowel disease by mediating immune coagulation
Xiu-Li Dong1*, Hai-Hua Lin2*, Ren-Pin Chen1, Huan-Dong Zhou3, Wan-Dong Hong1, Xiang-Rong Chen1, Qing-Ke Huang1, Xue-Cheng Sun1, Zhi-Ming Huang1
1Department of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical University, Ouhai District,
Wen-zhou, Zhejiang, China; 2Department of Pediatrics, The Children’s Hospital, Zhejiang University School of Medicine,
Binjiang District, Hangzhou, China; 3Department of Vascular Surgery, Hangzhou Third Hospital, Shangcheng
District, Hangzhou, China. *Equal contributors and co-first authors.
Received November 19, 2017; Accepted December 2, 2017; Epub March 1, 2018; Published March 15, 2018
Abstract: Inflammation and coagulation are interdependent processes that enable each process to activate and propagate the other in inflammatory bowel disease (IBD). Thus, we investigated the role of a novel immune co -agulant, fibrinogen-like protein 2 prothrombinase (FGL2), in patients and mice with IBD. 83 IBD patients and 40 normal controls were enrolled, and trinitro-benzene-sulfonic acid (TNBS)-induced colitis mice were used. Expression of FGL2 in the intestine was detected by immunohistochemistry. Using serial sections, staining was performed to detect tumor necrosis factor α (TNF-α) expression, and to demonstrate co-localization of FGL2 with macrophages and fibrin. Correlations between FGL2 expression with some common laboratory parameters were examined. FGL2 was seen primarily in inflammatory infiltrating cells, mainly macrophages, and microvascular vessels and had a strong co-localization with fibrin deposition. IBD patients and mice had increased expression of FGL2 compared with controls. Furthermore, FGL2 expression was correlated with intestinal and plasmatic TNF-α expression, mean platelet volume (MPV), platelet count (PLT), platelet-crit (PCT), and fibrinogen. Our data indicate that FGL2 may me -diate immune coagulation in IBD patients. It may be considered as a novel molecule that contributes to the onset and development of IBD.
Keywords: Inflammatory bowel disease, fibrinogen-like protein 2 prothrombinase, coagulation, inflammation
Introduction
Inflammatory bowel disease (IBD) comprises a group of chronic and relapsing-remitting im-mune-mediated disorders of the gastrointesti -nal tract, which mainly includes Crohn’s dis -ease (CD) and ulcerative colitis (UC). It has been shown that the coagulation cascade and fibrinolysis are activated in patients with IBD and increased thromboembolic events are extra-intestinal and potentially life-threatening complications [1-3]. The prothrombotic state in IBD may be promoted by the inflammatory response and cytokines [4]. As subsequent anticoagulant mechanisms fail to control the clotting process, the pro-inflammatory state is maintained or even increased [5-7]. There is growing evidence that inflammation and coagu
-lation are two interdependent processes, which is crucial for the progression of IBD [8, 9].
Fibrinogen-likeprotein 2 prothrombinase (FG-L2), also called fibroleukin, has been identified as a new member of fibrinogen-related protein superfamily. FGL2 has been shown previously to have the attributes to a serine protease capable of directly cleaving prothrombin into thrombin and then initiate a cascade coagulat-ing reaction [10]. Several studies have indicat-ed that FGL2 is involvindicat-ed in fulminant hepatitis, spontaneous abortion and xenograft rejection by mediating “immune coagulation”, such as fibrin deposition and microthrombus formation [11-15].
the mediators that link inflammation and coag -ulation remain poorly defined. Therefore, we propose that FGL2 may contribute to the inflam -mation-induced coagulation or thrombosis in IBD through a clotting-dependent pathway.
Materials and methods
Patients and observation
The study was performed at the Department of Gastroenterology and Hepatology of the First Affiliated Hospital of Wenzhou Medical Un-iversity from December 2006 to December 2015. All the patients were followed up for 6-12 months. Informed consent was obtained for each patient included in the study. The research protocol was reviewed and approved by the clin -ical research Ethics Committee of the First Affiliated Hospital of Wenzhou Medical Un-iversity.
IBD patients were diagnosed according to the article written by Lennard-Jones JE [16]. 83 Patients with active disease enrolled in our study had not received any steroid medications for their disease. For patients with CD, severity of disease was classified in accordance with the CDAI (activity indices of CD) score [17]. Active disease was defined as a CDAI score ≥150, in inactive disease <150. Disease activi -ty in UC patients (activi-ty indices of UC, UCAI) was evaluated by using of the Truelove-Witts criteria [18]. For statistical purposes, the clas -sification was quantitatively modified [19]. Active disease was defined as score >3 and inactive disease as a score ≤3. Disease activity was evaluated at the time of sample collection. 40 normal controls from the normal mucosal areas of healthy subjects or the patients with colonic polyp were included.
The characteristics of patients and controls are shown in Table 1.
Laboratory parameters of patients and con-trols
Venous blood samples were collected from all the participants with tubes containing 3.8% tri-sodium, then centrifuged (2000 rpm) for 10 min at 4°C, and finally plasma was separated. Plasma levels of ESR and C-reaction protein (CRP), PLT, MPV, PCT, prothrombin time (PT), activated partial thromboplastin time (APTT), TNF-α, and fibrinogen were routinely me-asured.
TNBS-induced colitis in mice
6- to 8-week-old BALB/c male mice were pur -chased from Shanghai SLAC Laboratory Animal Co., Ltd (License Number: SCXK (Shanghai) 2012-0002). Mice were kept under specific pathogen-free conditions (Laboratory Animal Research Center of Wenzhou Medical Uni-versity), and all procedures were conducted ethically according to the Guide for the Care and Use of the Administration Committee of Experimental Animals of Wenzhou Medical University. After a week of adaptive feeding, mice were weighed and anesthetized by intra -peritoneal injection with 80 ml of ketamine/ xylazine solution per 10 g body weight. TNBS was dissolved in alcohol (50:50 vol/vol). 100 μl of TNBS solution (vol (TNBS)/vol (alcohol) was 50:50) was administered intra-rectally (via 3.5 F-catheter) to mice maintained for 60 s in a ver -tical position. The dose was 100 mg/kg body weight. The insert depth was 4 cm proximal to the anus. Control mice received 100 μl of 0.9% saline intra-rectally [20]. Sample size in each group was 6.
Clinical and macroscopic analysis of colitis in mice
[image:2.612.90.288.96.189.2]The clinical analysis of colitis in mice was moni -tored daily for body weight, diarrhea, and hemafecia [25]. Loss of body weight was calcu -lated as percent difference relative to initial body weight. Diarrhea was scored as follows: 0, normal; 2, loose stools; and 4, diarrhea that remained adhesive to the anus. Bleeding scores were assessed as follows: 0, negative hemoccult; 2, positive hemoccult; and 4, obvi-ous bleeding [23]. Mice were euthanized 3 days after TNBS administration. The colon was removed and opened longitudinally. The mac -roscopic damage was measured by a blinded observer with the following score system [5, Table 1.Characteristics of the patients and
control groups
Characteristics NC IBD
Number 40 83
Male 22 42
Female 18 41
Mean age, years 42 (20-79) 41.7 (15-79)
Smoking 10 29
Oral contraceptive use 4 13
26]: 0, normal; 1, hyperemia, edema, no ulcer; 2, hyperemia, edema, small linear ulcers or petechiae; 3, hyperemia, edema, wide ulcers, necrosis, or adhesions; 4, hyperemia, edema, megacolon, stenosis, or perforation.
Histology in patients and mice
For histological analysis, mucosal biopsy speci -mens, from macroscopically inflamed areas of patients, and normal controls from the normal areas of healthy subjects were obtained. Patients with a colonic polyp were seen at the Department of Gastroenterology and Hepa-tology of First Affiliated Hospital of Wenzhou Medical University, and samples were collected in this study from December 2006 to December 2015. Furthermore, the colons of mice were removed and divided into fragments after sacri -fice. The colonic fragments (0.5 cm) of patients,
4°C for 16 h. After washing with PBS, sections were incubated with immunoperoxidase-conju-gated rabbit IgG fraction to mouse IgG Fc (Zhongshan Company, Beijing, China) at 37°C for 30 min, followed by three washes in PBS. Finally, the sections were incubated with 3,3’-diaminobenzidine chromagen and counter -stained with hematoxylin. Negative control was set in the experiment. For the evaluation of FGL2 expression, a total of 10 random fields across each section were selected to conduct the semi-quantitative analysis of the mean absorbance at a magnification of ×200.
Fibrin deposition in local intestine
[image:3.612.91.372.73.376.2]For fibrin detection, serial sections were stained with a rabbit-anti-fibrinogen Ab (Abcam, Ca-mbridge, USA, at a dilution of 1:100). This reagent is known to react with fibrinogen and
Figure 1.Mice were treated with TNBS (100 mg/kg), then observed every day until euthanasia. (A) Body weight changes (percentage of original body weight); (B) Bleeding score; (C) Diarrhea score; (D) Macroscopic score. Rep
-resentative macroscopic images of normal control mice (I) and IBD mice
(J). **P<0.001, n=6 per each group. Representative H&E-stained colon sections were shown: (E) Colon sections from the control patients (original magnification, ×200). (F) Colon sections from IBD patients (original magnifi -cation, ×200). (G) Colon sections from the control mice (original magnifica -tion, ×100). (H) Colon sections from IBD mice (original magnification, ×100).
controls and mice were fixed in 4% paraform, dehydrated, embedded in paraffin, sec -tioned (4 μm thickness) and stained with hematoxylin and eosin. The pathological slices were observed under a light microscope by two blinded pathologists.
Immunohistochemistry stain-ing FGL2 expression in local intestine
fibrin in mouse and human tissues. The tech -nique used for detection of fibrin was the meth -od as previously described.
TNF-α expression in local intestine
Serial sections were stained with a rabbit-anti-TNF-α Ab (Abcam, Cambridge, USA, at a dilution of 1:150). The technique used for detection of TNF-α was the method as previously described. Immunoperoxidase staining of macrophages
A CD68 Ab (ProteinTech, at a dilution of 1:100) was used to detect macrophages using the similar methodology described above.
Statistical analysis
Statistical Analysis was carried out by SPSS16.0 software. All results were presented as mean ±
SEM and P<0.05 were considered significant. The associations between FGL2 expression and disease activity indices or other Laboratory parameters, including CRP, ESR, PLT, MPV, PCT, fibrinogen, D-dimer, PT, APTT, TNF-α were exam -ined by non-parametric correlation (Spearman r).
Results
TNBS-induced mice develop serious clinical manifestation with general and histological changes similar to IBD patients
[image:4.612.91.522.74.429.2]The TNBS-induced colitis mice acquired a severe sickness characterized by body weight loss, diarrhea, defecate occult blood (OB) or hemafecia after TNBS administration (Figure 1A-C). Subsequently, the colonic samples were generally observed and scored by a blinded Figure 2.Immunohistochemical staining of various indicators. TNF-α in IBD mice and patients (B and F, ×400), and normal controls (A and E ×400). FGL2 in IBD mice and patients (C and J, ×400), and normal controls (D ×200 and
observer (Figure 1D, 1I, 1J). We observed that the colon of IBD model was accompanied by hyperemia, edema, ulcers, necrosis, and adhe -sion. The clinical manifestation and general changes were similar to patients with IBD. Simultaneously, histological examination sh-owed that the changes of TNBS-induced mice covered decrease of goblet cells, loss of crypts, damage of crypts, infiltration by inflammatory cells, and extensive destruction of the mucosal layer (Figure 1H) compared with normal con-trols (Figure 1G). Similarly, IBD patients had the same histopathology (Figure 1E, 1F).
FGL2 expression was increased in patients and mice with IBD
Compared with normal controls, there was sig-nificantly up-regulation of FGL2 expression in inflamed mucosal specimens from patients and mice with IBD (Figure 2C, 2D, 2J, 2K). The expression of FGL2 was not only in the areas of inflammatory infiltration and the vessels, but also in the mucosal lamina propria. There was no significant difference between the mean ages of patients and controls. Gender distribu -tion was similar in patients and controls. Cellular source of FGL2 and fibrin deposition in IBD patients
By serial section staining, the majority of CD68+ cells showed high expression of FGL2 protein in patients with IBD (Figure 2K, 2L).
Fibrin deposition was detected in the matrix and around the FGL2-positive infiltrating cells and microvascular vessels (Figure 2G, 2H) and was strongly co-localized with FGL2 expre-ssion.
Correlations between FGL2 expression and inflammation
There was association between FGL2 expres -sion and inflammatory parameters. There was a strong correlation between FGL2 expression and TNF-α expression in the intestine and plas -ma of IBD patients (r=0.628, P<0.001; r=0.425, P<0.001). But the enhanced FGL2 expression was not correlated with ESR levels or CRP lev-els. Moreover, an amount of TNF-α expression was observed in areas of inflammatory infiltra -tion and in the vessels region of IBD patients (Figure 2E, 2F) and colitis mice (Figure 2A, 2B). Spearman-rank correlation coefficient values and the two-tailed significance of the correla -tion between FGL2 expression and inflamma -tion parameters in IBD patients are shown in Table 2.
Correlations between FGL2 expression and coagulation parameters
Intestinal FGL2 expression in patients with IBD negatively correlated with platelet count (r=-0.330, P<0.01), MPV (r=-0.304, P<0.01) and PCT (r=-0.404, P<0.001), but was not correlat -ed with the levels of PT or aPTT. There was cor
-Table 2. Correlation of FGL2 expression with inflammation parameters
Pearson Correlation TNF-α (intestine) TNF-α (plasma) ESR (mm/h) CRP (mg/l)
0.00199 ± 0.00146 84.8 ± 92.2 pg/ml 46.4 ± 34.3 23.8 ± 32.0 FGL2
0.00146 ± 0.00115 0.628*** 0.425*** 0.161 n.s. -0.175 n.s. IBD: Inflammatory bowel disease; ESR: sedimentation rate; CRP: C-reaction protein; TNF-α: tumor necrosis factor α. All results were expressed as mean ± SD. A level of P<0.050 was considered to be statistically significant. ***P<0.001; n.s. = not signifi -cant.
Table 3.Correlation of FGL2 expression with coagulation parameters
Pearson Correlation Plt (10^9/l) MPV (fl) PCT (µg/l) PT (s) aPTT (s) Fibrinogen (g/l) 301.8 ± 119.6 7.77 ± 1.25 0.233 ± 0.097 14.2 ± 1.93 39.2 ± 4.69 4.23 ± 1.22 FGL2
0.00146 ± 0.00115 -0.330** -0.304** -0.404*** 0.005 n.s 0.002 n.s. 0.224*
relation between FGL2 expression and fibrino -gen level (r=0.224, P<0.05). Spearman-rank correlation coefficient values and the two-tailed significance of the correlation between FGL2 expression and coagulation parameters in IBD are shown in Table 3.
Discussion
Despite numerous studies, to date, the exact pathogenesis of IBD has not been clearly eluci -dated. The most prevailing factors include host genetic susceptibility and immune abnormali -ties [21-23]. However, recently, a large number of studies that focus on the role of endothelial damage and coagulation disturbances as IBD-triggering factors [24-27]. Inflammation and coagulation are interdependent processes that enable each process to activate and propagate the other [6-8]. The persistent pro-coagulant state likely involves endothelial cells, leuko -cytes, and platelets, which are activated in response to the inflammatory stimulus. The activated endothelial cells, leukocytes, and platelets during inflammation phase exhibit an increased tissue factor expression, downregu -lation of the anticoagulant protein C pathway, and generation/activation of coagulation fac -tors (e.g., factor Xa), as well as enhanced throm -bin production [28-30]. In addition, several find -ings have demonstrated a role for cytokines in mediating inflammation-induced coagulation or thrombosis, such as interleukin (IL)-1β, TNF-α and IL-6. Blocking antibodies of IL-1β or TNF-α, or lacking the gene for the TNF-α receptor (TNF r-/-) largely abolish the extra-intestinal thrombo -sis associated with experimental IBD [31, 32]. While all these studies have clearly shown that distinct pro-inflammatory stimuli activates the clotting cascade, parallels have also indicated that coagulation in turn propagates the inflam -matory state, by activating of signaling path -ways, or recruiting more inflammatory cells to the inflamed tissue [7-9].
FGL2 prothrombinase (FGL2) is a 64-70kDa, type 2 transmembrane protein containing a C-terminal FRED (fibrinogen related extracellu -lar domain). It was classified as a member of fibrinogen superfamily due to its homology (36%) with fibrinogen β and γ chains. It has been demonstrated that FGL2 is significantly up-regulated in activated macrophages, T cells, and endothelial cells under the stimuli of lipo
-polysaccharide or cytokines like TNF-α, IL-2. The direct prothrombinase activity of FGL2 has implicated in the pathogenesis of several inflammatory disorders including fulminant hepatitis and severe hepatitis, allo- and xeno-graft rejection, as well as murine and human cytokine induced fetal loss [10-15]. FGL2 con -tributes to these diseases by mediating “immune coagulation” such as fibrin deposition and microthrombus formation. Furthermore, neutralizing antibodies to FGL2 prevent both fibrin deposition and death from MHV-3 infec -tion. Gene-targeted FGL2-deficient (FGL2-/-) mice or targeted FGL2 gene with antisense FGL2 result in the prevention of MHV-3-induced fibrin deposition, liver injury, and death [11]. The observation that xenografts from FGL2-/-mice transplanted into rats are devoid of throm -bosis also supports FGL2 as a coagulant [13].
Our study first demonstrates the role of FGL2 in IBD patients. We found that there was signifi -cantly higher FGL2 expression in patients and mice with IBD than normal controls. The up-regulated expression of FGL2 in IBD patients may be promoted by a inflammatory response or pro-inflammatory cytokines like TNF-α [32]. This is consistent with our result that there was parallel high expression of intestinal TNF-α. FGL2 was primarily seen in macrophages and endothelial cells in IBD patients and was co-localization with fibrin deposition. This con -forms to the cellular source and coagulable characteristic of FGL2 as previous studies [11-15]. Disease activity indices, ESR, and CRP are important for disease assessment in IBD. Platelet count, MPV, PCT, PT, and APTT are also biomarkers of coagulation and fibrinolysis as measures of disease activity in active IBD [9]. In our results, correlation of FGL2 expression with CDAI or UCAI, ESR, and MPV, PCT, as well as fibrinogen was found. All these findings sup -port our assumption that FGL2 may particip-ate in inflammation-induced coagulation and thrombosis in IBD through a clotting-dependent pathway.
helpful biomarker in the pathogenesis and dis -ease assessment of IBD. To confirm this, we have now initiated further investigation of the role of FGL2 in patients with IBD and in experi -mental IBD models.
Acknowledgements
The authors are grateful to the Department of Surgical Laboratory, the First Affiliated Hospital of Wenzhou Medical University, for assistance with technical support and cons- ulting. This work was supported by the Scien-ce and Technology project of Wenzhou, China (Y20150163) and the Natural Science Foun-dation of China (81570495).
Disclosure of conflict of interest
None.
Address correspondence to: Zhi-Ming Huang, De- partment of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical University, South Bai-xiang Street, Shangcai Village, Ouhai District, Wen-zhou, Zhejiang, China. Tel: +86-13706658620; Fax: +86-577-55578033; E-mail: wzhospitalhzm@126. com
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