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Transforming growth factor-β1 and its receptors in patients with ulcerative colitis
International Immunopharmacology 9 (2009) 761–766
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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p
Transforming growth factor-β1 and its receptors in patients with ulcerative colitis Antoni Stadnicki a,b,⁎, Grzegorz Machnik c, Ewa Klimacka-Nawrot a, Anna Wolanska-Karut d, Krzysztof Labuzek e a
Department of Basic Biomedical Sciences, Medical University of Silesia, Katowice, Poland Department of Gastroenterology, St Barbara's Hospital, Sosnowiec, Poland Department of Biotechnology and Genetic Engineering, Medical University of Silesia Katowice, Poland d Department of Histopathology, Medical University of Silesia Katowice, Poland e Department of Pharmacology, Medical University of Silesia, Katowice, Poland b c
a r t i c l e
i n f o
Article history: Received 30 April 2008 Received in revised form 24 February 2009 Accepted 27 February 2009 Keywords: Ulcerative colitis TGF-β1 TGFβ receptors Immunohistochemistry
a b s t r a c t Transforming growth factor-β1 (TGF-β1) plays a role in the pathogenesis of ulcerative colitis (UC) by activating its specific receptors (TβRI–TβRIII). We investigated the expression of genes encoding for TGF-β1 and TβRI–III using RT-QPCR in patients with active and inactive UC and non-IBD controls. The localization and level of TGF-β1 protein in intestinal tissue was estimated by immunohistochemistry, and serum TGF-β1 concentrations were determined using ELISA. We found a significant increase in TGF-β1 gene expression and increase in the expression of genes encoding receptor TβRI in patients with active UC when compared with controls. The expression of genes encoding TβRII was found to be higher in patients with both active and inactive UC when compared to controls. Specific staining for TGF-β1 in fibroblasts was significantly greater in both active and inactive UC as compared to controls. The serum concentration of TGF-β1 was significantly higher in patients with active UC when compared with controls as well as in UC patients with left side/total colonic extension when compared with those with disease limited to rectum/rectosigmoid area. However, no correlation between TGF-β1 serum concentrations and UC activity index was found. Increases in TGF-β1 gene expression and its protein level, associated with altered TGF-β1 receptor profile indicate a functional role for TGF-β1 in intestinal inflammatory/repair processes in UC. Increases in TGF-β1 serum concentrations correlate with extension of disease. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The major forms of inflammatory bowel disease (IBD) i.e. Crohn's disease (CD) and ulcerative colitis (UC) result from the interaction of genetic and environmental factors that promote immunopathogenic responses either by abnormality of the effector cells activity or poorly controlled regulatory mechanisms. High levels of TNF-α have been associated with the intestinal inflammation found in IBD. Infliximab, a monoclonal antibody against TNF-α, administered with steroids, has been found to be effective in inducing responses and maintaining remissions in patients with moderate-to-severe stages of IBD [1]. The intestinal epithelium creates a barrier to potentially immunogenic and noxious factors, including microorganisms and dietary components, within the intestinal lumen. Healing of the intestinal surface is regulated by a complex mechanism that involves growth factors, cytokines, as well as intracellular matrix proteins and blood clotting factors to preserve homeostasis and integrity of the intestinal mucosa [2,3]. ⁎ Corresponding author. Department of Basic Biomedical Sciences, Medical University of Silesia, 3, Kasztanowa Street PL-41-205 Sosnowiec, Poland. Tel.: +48 32 3682603; fax: +48 32 2945548. E-mail address: [email protected] (A. Stadnicki). 1567-5769/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2009.02.014
Over the past decade, nitric oxide (NO) and reactive oxygen species have been investigated as possible etiological factors in the initiation and/or propagation of inflammatory processes, because of their detrimental effect on intestinal barrier. Interestingly, free radicals are inducers of TNF-α production [4]. Once the intestinal epithelial barrier is damaged, highly immunogenic bacterial antigens from the intestinal lumen can enter the normally sterile submucosal layers and thus may play a role in the pathogenesis of IBD. This may also contribute to how antibiotics can be effective as adjunctive therapy in IBD [5]. Among the regulatory peptides that are expressed within the intestinal mucosa, transforming growth factor-β (TGF-β) and epidermal growth factor family peptides (EGFs) play especially important roles in the pathogenesis of IBD. EGFs, potent stimulators of intestinal epithelial cell proliferation, are produced mainly in submaxillary salivary glands, Brunner's glands in the duodenal submucosa, and within the exocrine pancreas [4,6]. TGF-β is capable of regulating growth, differentiation, and function of immune cells by the switching of B cells and inhibiting of T helper cell function. Importantly, TGF-β1 counteracts TNF-α and acts as a negative regulator of mucosal inflammation that is essential for wound healing [7]. TGF-β1 directly stimulates angiogenesis in vivo, and this stimulation can be blocked by TGF-β1 antibodies [8]. Intestinal epithelial wound healing and tissue
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repair may also take place through a TGF-β1 independent pathway, involving cell interactions and blood coagulation factors. Previous data suggests that the interaction between Factor XIIIa and its natural plasmatic substrates e.g. fibronectin and α-2 plasmin inhibitor, play a role in the healing of mucosal lesions in UC [9,10]. Consequently, Factor XIIIa infusions have been shown to promote intestinal wound healing in both UC and CD patients [9,11]. Three isoforms of TGF-β, namely: TGF-β1, TGF-β2, and TGF-β3, have been detected in the gut. Functional distinctions between TGF-β isoforms are still unclear. However, TGF-β1 mRNA is mainly expressed in gut tissues [13]. TGF-β1 initiates signaling through a complex transmembrane serine/threonine kinase consisting of TβRI and TβRII receptors, whereas TβRIII receptor participates in storage of the protein, and may present TGF-β1 to TβRII [12]. The ligand TGF-β1 signals from the receptor to the nucleus using a set of proteins termed Smads [14]. Overproduction of TGF-β can result in deposition of scar tissue and fibrosis in part by stimulating the production of collagen and other constituents of the extracellular matrix. Both TGF-β1 gene expression and synthesis of TGF-β1 protein are increased in fibrotic diseases including segmental glomerulonephritis, lupus nephritis, diabetic nephropathy as well as systemic sclerosis, and inhibitors of TβR may reduce or abolish fibrosis [15]. Some recent investigations indicate that activation of TGF-β1 in CD may contribute to intramural fibrosis and intestinal obstruction [16,17]. However, a role for TGF-β1 in UC is not well established. Over the past decade, the functional role of TGF-β1 in the pathogenesis of neoplasm and chronic inflammation has been defined [12]. In the gut, TGF-β1 has been found to inhibit proliferation of intestinal epithelial cells. Paradoxically, TGF-β1 seems to play a vital role in promoting reepithelialization after mucosal wounding [18]. The lack of TGF-β dependent control, because of the TβRII gene mutation, was revealed in the majority of UC patients with associated bowel carcinoma [19,20]. The development of enterocolitis in TGF-β1 deficient mice supports the concept that ongoing inflammation can result from the failure to down-regulate inflammation [21], but the relevance of this observation to human IBD remains unclear. The aim of the present study was to investigate the expression of genes encoding TGF-β1 and its receptor subtypes as well as the distribution and concentration of the TGF-β1 protein in inflamed tissues in patients with active and inactive UC compared with non-IBD controls. Changes in both the mRNA distribution and protein levels of TGF-β1 coexisting with altered TGF-β1 receptor profile in UC patients may indicate a functional role for TGF-β1 in inflammatory processes. The specific reaction for TGF-β1 staining in colonic fibroblasts was significantly higher in active UC when compared with non-IBD controls, indicating a role for fibroblasts as a source of TGF-β1 synthesis in UC, which may contribute to the mucosal repair. Increased serum TGF-β1 concentrations were associated with UC extension. However, we did not find any correlation between UC activity index and serum TGF-β concentrations.
2. Materials and methods UC was diagnosed according to commonly accepted criteria i.e. past and present clinical symptoms, sigmoidoscopy with biopsy, colonoscopy or barium enema. Clinical UC activity was assessed using a modified Truelove and Witts UC activity index [22]. Patients with a score of less than or equal to 2 were classified as inactive UC, those with a score from 3 to 9 were regarded as active UC. Exclusion criteria included systemic infections and other serious cardiopulmonary, liver, or renal diseases as well as patients with fulminant colitis. Blood was obtained from all patients and non-IBD controls by venipuncture from the antecubital vein. All samples were centrifuged at 3000 ×g for 15 min. Serum samples were stored at −70 °C until tested. Patients were studied in accordance with the protocol approved by the Institutional Committee on Human Subjects of the
Medical University of Silesia, Katowice, Poland. All patients gave written, informed consent. 2.1. Expression of TGF-β1 mRNA and TβRI, TβRII, and TβRIII mRNA Tissue specimens were obtained during colonoscopy from 36 patients with UC and 14 non-IBD patients who served as a control group. There were 22 patients with active UC (10 women, 12 men with median age of 44 years) as well as 14 patients with inactive disease (6 women, 8 men with median age of 39 years). The control subjects (with no macroscopic and histological changes in the large bowel during colonoscopy) were compatible with both subgroups (active and inactive UC patients) and matched for age and sex. Mucosal disease activity of UC patients was assessed using the modification of endoscopic index according to Baron et al. [23], taking into consideration the mucosal vascular pattern, erythema, friability, erosions, and ulcerations. Patients with UC were given conventional medical treatment. All UC patients received sulfasalazine or its derivatives. Seven patients with active UC were given moderate doses of corticosteroids and four patients (two in the active stage and two in the inactive stage) were on azathioprine. The extent of colonic disease was variable. Colonic biopsies were taken from all subjects. The diagnosis of UC was confirmed histologically; colonic biopsy specimens were stained with hematoxylin and eosin to determine the histological grade of inflammation. For molecular evaluation, colonic biopsy specimens of approximately 4 × 4 mm were removed by endoscopic forceps and immediately frozen at − 70 °C until use. Expression of TGF-β1 gene and its receptor (TβRI, TβRII and TβRIII) genes in biopsy specimens were quantitatively evaluated as a number of mRNA copies per microgram of total RNA. Constitutively expressed beta-actin gene was used as a control of the reaction. For RNA extraction, a single step method by modified guanidinum thiocyanate–phenol–chloroform extraction was employed [24]. After extraction, RNA samples were resuspended in RNase free water and were treated with RNase free DNase I (Fermentas, Lithuania) in order to remove any residual DNA. RNA extracts were size-fractionated by electrophoresis in 1% agarose gel. RNA concentrations in each sample were measured spectrophotometrically. The numbers of mRNA molecules for TGF-β1 and for its receptors TβRI-III in 1 µg of total RNA were detected with ABI PRISM 7700 Sequence Detector (Applera INC) as two-step RT-QPCR. For quantification of mRNA molecules we utilized SYBR-Green as described by Ponchel et al. [25]. Appropriate primers for reverse transcription (RT) and, subsequently, for quantitative polymerase chain reactions (QPCR) were used. We utilized complementary oligonucleotides as follows: For TGFβ: TGFβ1f: 5′-TGA ACC GGC CTT TCC TGC TTC TCA TG-3′ TGFβr 5′-GCG GAA GTC AAT GTA CAG CTG CCG C-3′; for TGFβ Receptor I: TGFR1f: 5′-ACT GGC AGC TGT CAT TGC TGG ACC AG-3′; TGFR1r: 5′-CCT GAG CCA GAA CCT GAC GTT GTC ATA TCA3′; for TGFβ Receptor II: TGFR2f 5′-GGC TCA ACC ACC AGG GCA TCC AGA T-3′, TGFR2r: 5′-CTC CCC GAG AGC CTG TCC AGA TGC T-3′; for TGFβ Receptor III: TGFR3f: 5′-ACC GTG ATG GGC ATT GCG TTT GCA-3′, TGFR3r: 5′-GTG CTC TGC GTG CTG CCG ATG CTG T-3′; and for betaactin gene: B_for: 5′-TCA CCC ACA CTG TGC CCA TCT ACG A-3′, B_rev: 5′-CAG CGG AAC CGC TCA TTG CCA ATG G-3′. For reverse transcription reaction (RT) we used MasterAMP Tth DNA/RNA polymerase (Epicentre Technology INC.). Reaction parameters (in 10 μl of reaction mixture) were performed according to manufacturer's instructions. Thereafter, 1/5 volume of RT mixture (i.e. 2 μl) served as template for QPCR. Quantitative PCR was performed in duplicates for each sample using fluorescent dye SYBR-Green (2× QuantiTect SYBR-Green PCR Kit, Qiagen GmbH, Germany) [25]. To confirm the specificity of QPCR, each sample was examined by calculation of melting temperature (Tm). All samples gave uniform melting temperature which was specific for its designed PCR product (data not shown). Additionally, randomly selected reaction samples were separated on a 6%
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polyacrylamide gel and the specific products were visualized by silver staining (the product length for TGFβ1, TβRI, TβRII, TβRIII and β-actin were as follows: 151, 200, 138, 172 and 295 bp). Single bands were visible on the gels and confirmed the specificity of the reactions (data not shown). 2.2. Immunolocalization of TGF-β1 protein in human colonic tissue Patients and control subjects: The immunolocalization of TGF-β1 protein was assessed in 10 patients with active UC (4 women, 6 men with mean age of 52 years), 10 patients with inactive UC (4 women, 6 men with mean age of 45 years) and 10 normal controls (5 women, 5 men with mean age of 45 years). The immunolocalization of TGF-β1 was performed in inflamed colonic tissue samples obtained after surgery from UC patients and tissue samples obtained from patients undergoing partial colectomy for colon cancer (control group). Tissue samples were prepared and stained for TGF-β1 using the procedure described previously [26]. In the present study, we used the mouse anti-human monoclonal antibodies (NCL-TGFB) to TGF-β1. Briefly, after paraffin embedding, de-waxing and rehydration, the primary mouse anti-human antibodies to TGF-β1 were detected using a biotinylated second antibody, and then avidin–peroxidase complex (Novostain Super ABC Kit, Novocastra). Finally, substrate 3, 3′-diaminobenzidine peroxidase (Substrate Kit; DAB) was used, and the sections were independently analyzed by a masked observer. Normal mouse serum instead of antiTGF-β1 antibody was used as a control. The slides were prepared for transmitted light microscope examination (Olympus with camera Pronis DP 10). The number of cells positively stained for TGF-β1, separately colonocytes or fibroblasts (in the lamina propria), were counted using methods published by Devani et al. [27]. TGF-β1 staining in colonocytes and fibroblasts was scored and subsequently calculated in 50 representative high power fields of each tissue sample. Immunoreactivity in the interstitial compartment was scored as 0 = no immunostaining, + = weak, ++ = moderate, +++ = strong. The evaluation of immunohistochemical pattern was performed in blinded fashion by a pathologist experienced in IBD. 2.3. Measurements of TGF-β1 in serum 2.3.1. Patients and control subjects We measured serum concentrations of TGF-β1 in 55 UC patients (21 women, 34 men with mean age of 47 years) and 20 controls (9 women, 11 men with mean age of 47 years). Patients with UC were divided, according to a Truelove and Witts UC activity index, as 25 UC inactive patients (a score of less than or equal to 2; 11 women and 14 men with mean age of 46 years) and 30 UC active patients (a score from 3 to 9; 10 women and 20 men with mean age of 44 years). In addition, UC patients were divided according to colonic extension as extended to left side/total colitis (19 patients: 10 women and 9 men with mean age of 43 years) or rectum/rectosigmoid colitis (36 patients: 11 women and 25 men with mean age of 47 years). The antigenic levels of TGF-β1 in serum were measured by the enzyme-liked immunoabsorbent assay (ELISA). Monoclonal mouse anti-human TGF-β1 antibody was coupled with biotin, then avidin– peroxidase was added to detect TGF-β1. 2.4. Statistical analysis The significance of differences between the means of each group was determined by the Student's t test. For immunohistochemical data, the means were compared using the Kolmogorov–Smirnov test and the Mann–Whitney U test. Since the mRNA concentrations were distributed logarithmically, we evaluated the significance of the differences between the controls, active UC patients, and inactive UC patients for the measurement of TβRII mRNA and TβRI mRNA
Fig. 1. Gene expression of TGF-β1, TβRI, and TβRII in the colonic tissue. Bars indicate mean ± SEM. A: Gene expression of TGF-β1 in the colonic tissue. B: Gene expression of TβRI in the colonic tissue. C: Gene expression of TβRII in the colonic tissue.
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expression using the logarithmic values of each parameter. The means were compared using the Mann–Whitney U test. All values were expressed as mean ± SEM. The correlation between parameters was calculated using the Spearman's rank correlation coefficient.
Table 1 Serum TGF-β1 concentrations.
3. Results
Clinical activity
3.1. TGF-β1, and Tβ-RI, RII, and RIII gene expression
Endoscopic extension
In patients with the active phase of UC, mRNA expression of TGFβ1 gene (81,065 ± 17,357 SE, the number of mRNA copies/μg total RNA) was significantly higher (P b 0.001) when compared with nonIBD controls (17,657 ± 4679). However, TGF-β1 mRNA levels in patients with inactive UC (45,408 ± 14177) did not differ significantly from the controls (Fig. 1A). The expression of genes encoding receptor TβRI was significantly higher (P b 0.005) in patients with active UC
Controls
Group of patients
a b
Serum TGF-β1 concentrations [ng/ml] Active UC Inactive UC Left side/total Rectum/ rectosigmoid
Mean ± SEM
Statistical analysis (P-values)
29.2 ± 5.5 25.4 ± 1.7 30.5 ± 2.0 23.9 ± 2,0
P b 0.01a P b 0.009a P b 0.0001a P b 0.03b
18.4 ± 1.6
vs. controls. Left side/total vs. rectum/rectosigmoid.
(22,776 ± 5665) as compared with non-IBD controls (6873 ± 857) (Fig. 1B). The expression of genes encoding TβRII was found to be higher in patients with active UC (88,032 ± 17,453 SE, P b 0.05) as well as with inactive UC (116,588 ± 16,530 SE, P b 0.002) than in control subjects (27,616 ± 8409) (Fig. 1C). In contrast no significant differences in TβRIII gene expression were observed in all examined groups (data not shown). 3.2. Immunohistochemical localization of TGF-β1 protein We visualized TGF-β1 in normal as well as in inflamed human colon. We demonstrated weak specific reactions for TGF-β1 in colonocytes from both UC patients and controls. Additionally, there was a specific staining reaction for TGF-β in macrophages, granulocytes, and lymphocytes in the colon lamina propria of UC patients. However, strong specific reaction for TGF-β1 was shown in fibroblasts in patients with both active and inactive phases of UC. Thus, the semiquantitative estimation of the specific reaction for TGF-β1 was performed separately in colonocytes and in fibroblasts representing lamina propria cells. The specific immunoreactivity for TGF-β1 was nearly equal in colonocytes in all examined groups. However, the specific reaction for TGF-β1 staining in fibroblasts was significantly higher in active UC (26 points, P b 0.005) as well as in inactive UC (15 points, P b 0.017) when compared with non-IBD controls. Fig. 2A illustrates localization of immunoreactive TGF-β1 in the epithelial cells of the normal human colon, but no positive specific reactions in fibroblasts. Fig. 2B demonstrates TGF-β1 in the inflammatory tissue of the colon lamina propria of inactive UC, mainly in fibroblasts. Fig. 2C shows strong positive specific reactions for TGF-β1 in fibroblasts localized in the colon lamina propria of active UC. 3.3. Serum TGF-β1 concentrations Serum TGF-β1 concentrations were significantly higher (P b 0.01) in active UC patients (29.2 ± 5.5 ng/ml) when compared with nonIBD controls (18.4 ± 1.6 ng/ml). However, TGF-β1 antigenic concentrations in active UC patients did not differ from those in inactive UC patients (25.4 ± 1.7 ng/ml). We were not able to show significant correlation between UC disease activity index and serum TGF-β1 concentrations (r = 0.17, NS). Serum concentrations of TGF-β1 were significantly higher (P b 0.03) in patients with left side/total colonic extension (30.5 ± 2.0) when compared with those limited to rectum/ rectosigmoid (23.9 ± 2.0) (Table 1). 4. Discussion
Fig. 2. Tissue localization of TGF-β1 protein in the colonic mucosa. Immunohistochemical staining for TGF-β1 using the NCL-TGFB antibody (×400 magnification). A: Normal colonic mucosa. Weak staining reaction for TGF-β1 protein in colonocytes (arrow). Please note that there is no staining in the fibroblasts. B: Inactive UC colonic tissue. The lamina propria of the mucosa. Positive staining for TGF-β1 in fibroblasts (arrow). C: Active UC colonic tissue. Please note a strong positive staining for TGF-β1 in fibroblasts (arrow).
Growth factors are potential targets for intestinal homeostasis. TGF-β and EGFs are especially relevant for modulation of intestinal inflammation and have a number of important properties in IBD. The role of TGF-β1 in the pathogenesis of IBD was investigated mainly in CD patients. Di Mola et al. [17] reported an overexpression of TGF-β1 and its signaling receptors in epithelium and inflammatory cells
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invading whole intestinal wall in active CD patients. These findings indicated that TGF-β1 might contribute to intestinal fibrosis and subsequent intestinal obstruction. Much less attention was paid to the role of TGF-β1 in the pathogenesis of UC. Chowdhury et al. [28] showed low expression of TGF-β1 mRNA in UC inflammatory intestine suggesting that it may be, at least in part, responsible for UC intractability. In a strict contrast, Babyatsky et al. [29], using semiquantitative Northern blot analysis, reported an increased expression of TGF-β1 mRNA in the affected mucosa of patients with both active UC and CD. In the present study, we have focused our attention on the TGF-β1 system in UC. We demonstrated an increased expression of TGF-β1 mRNA in the affected mucosa in active UC patients which corresponds to findings of Babyatsky et al. [29]. We extended Babyatsky's study showing the expression of genes encoding TGF-β1 and simultaneously its receptors in inflamed UC tissue using quantitative RT-QPCR method. It is known that TGF-β1 initiates activation through ligand-dependent complex consisting of the type I receptor which, in turn, facilitates activation of the associate type II receptor. Interestingly, it has been shown that decreased expression of TβRII gene renders to the cell resistance to the antiproliferative effect of TGF-β1 [30]. Thus, our present observations, showing an overexpression of TβRI and TβRII genes in active UC inflamed intestine, and, interestingly, an increase of TβRII gene expression in inactive colonic UC tissue, indicate a functional role for TGF-β1 in the pathogenesis of UC. A question arises what is a mechanism that leads to TGF-β1 and its receptors overexpression in active UC. In fact, TGF-β1 evokes increases in NO levels, which increased synthesis is present in colonic biopsies and plasma of IBD patients [13]. Nitric oxide may produce feedback inhibition of TGF-β1 [31]. The regulation of TGF-β1 gene expression and the increase in its protein synthesis may also be related to TGF-β1 polymorphism [32]. In addition, intestinal EGF, a promigratory peptide which is present in the gastrointestinal tract, may increase the concentration of bioactive TGF-β [13]. Interestingly, EGF enemas have been proved to be beneficial in UC patients [6]. The current working hypothesis is that UC may be a Th-2 like related disorder with local over-production of IL-13 by natural killer T cells [3]. Importantly, the data from experimental models of colitis indicate that the IL-13 related pathway (by IL α2 receptor) results in the induction of TGF-β1 production. Thus this mechanism may be involved in the pathogenesis of human UC [33]. TGF-β1, a potent immunosuppressive cytokine, is paradoxically induced in the intestine during active inflammation. However, recent data also indicate that defective signaling of TGF-β1 in chronic IBD is related to increase of Smad7, a main inhibitor of TGF-β1 signaling in IBD mucosa [14]. We speculate that TGF-β1 overexpression does not prevent intestinal injury. However, its increased synthesis, combined with upregulation of its receptors, can enhance intestinal repair. In addition to increased TGF-β1 mRNA expression we characterized the cellular localization and concentration of TGF-β1 protein in UC intestine. We demonstrated that TGF-β1 protein is present in the epithelial cells of inflamed human colon and its levels are increased in inflammatory cells within the colonic lamina propria which is in agreement with previous findings [16]. A new observation is that fibroblasts (among other inflammatory cells of the lamina propria of UC intestine) show the most impressive specific reaction for TGF-β1 protein. This observation emphasizes a significance of fibroblasts in the immune responses mediated by TGF-β1 in UC. Previously, Ohtani et al. [34], using immunohistochemical techniques, showed the presence of TβRI and TβRII in fibroblasts in UC lamina propria, but not in organized fibrotic area in CD. More recently, Stallmach et al. [35] demonstrated that fibroblasts isolated from strictures in patients with CD produce significantly more type III collagen than fibroblasts from normal or nonstrictured but inflamed lamina propria. Our data tend to corroborate with the results of Stallmach et al. [35] results and indicate a role for intestinal fibroblasts as a source of TGF-β1 in restoration of the surface continuity in UC.
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However, transmural increase of TGF-β1 and its receptors may enhance collagen III production, and, in turn, implicates stricture formation in CD. In fact, Di Mola et al. [17] showed overexpression of three TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3) transmurally in the active inflammatory CD intestines. Taken together, CD and UC patients may manifest different intestinal TGF-β behavior patterns. Finally, we measured serum TGF-β1 concentrations. Previously, Wiercinska-Drapalo et al. [36] observed a significant positive correlation between increased plasma TGF-β1 concentrations and scored degree of mucosal injury in UC patients. However, Sambuelli et al. [37] demonstrated only a tendency towards increasing serum TGF-β1 concentrations in relation to UC activity. In normal colon, TGF-β1 is present only in epithelial cells, but in UC, it can be found in the inflammatory cells of the lamina propria. Changes in serum TGF-β1 concentrations might have reflected, to some degree, events taking place in inflamed intestine. In the present study, we extended previous findings by showing significantly higher serum TGF-β1 concentrations in UC, related to the degree of colonic extension. However, we did not observe any significant differences in serum TGFβ1 concentrations between active and inactive UC patients, and, consequently, we did not find any correlation between UC activity and serum TGF-β1 concentrations. Thus, our data seems to support previous findings of Sambuelli et al. [37]. In fact, TGF-β1 is synthesized in the intestinal mucosa and appears to act not only locally, but also may be involved in systemic response. Recently, Rezaile et al. [38] have demonstrated significantly higher concentrations of TGF-β1 and NO in saliva of IBD patients, which may indicate an underlying pathology in saliva itself. In addition, elevated concentrations of TGF-β1 and NO in saliva did not significantly differ between UC and CD patients, indicating a pathophysiological role for both TGF-β1 and NO in these diseases. What is the role of TGF-β1 in UC? TGF-β1 enhances the production of the extracellular matrix by fibroblasts and other intestinal cells. Thus it seems to be important for the recovery of tissue integrity. Receptors for these reactions are overexpressed in the human intestinal epithelium during active UC. Thus TGF-β1 can mediate healing processes in the inflamed intestine. However, TGF-β1 may also act systematically as it expresses many proinflammatory actions to stimulate the synthesis of COX-2, the release of eicosanoids and the production of cytokines, mainly IL-8 [39,40], which is known to be important in IBD [3]. In humans, the success of azathioprine and corticosteroids (commonly used as immunosuppressive drugs in IBD) in treating autoimmune hepatitis is, in part, due to the ability of these drugs to reduce serum TGF-β1 concentrations [41]. Thus, the possible role of TGF-β in the treatment of UC requires additional investigation. Acknowledgments This work was supported in part by a grant from the Polish Ministry of Sciences (2PO5B14026; A. Stadnicki) and, in part, by a grant from the Medical University of Silesia (NN-2-047/07; A. Stadnicki). We thank Drs. Z. Krowicki (MediProfile, Inc.) and U. Pisharody (LSUHSC School of Medicine) for helpful advices and manuscript corrections. References [1] Rahimi R, Nikfar S, Abdollahi M. Meta-analysis technique confirms the effectiveness of anti-TNF-alpha in the management of active ulcerative colitis when administered in combination with corticosteroids. Med Sci Monit 2007;13:PI13–8. [2] Podolsky DK. Inflammatory bowel disease. N Engl J Med 2002;347:417–29. [3] Baumgart DC, Carding SR. Inflammatory bowel disease: cause and immunobiology. Lancet 2007;369:1627–40. [4] Jahanshahi G, Motavasel V, Rezaie A, Hashtroudi AA, Daryani NE, Abdollahi M. Alterations in antioxidant power and levels of epidermal growth factor and nitric oxide in saliva of patients with inflammatory bowel diseases. Dig Dis Sci 2004;49: 1752–7. [5] Rahimi R, Nikfar S, Rezaie A, Abdollahi M. A meta-analysis of antibiotic therapy for active ulcerative colitis. Dig Dis Sci 2007;52:2920–5.
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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p
Transforming growth factor-β1 and its receptors in patients with ulcerative colitis Antoni Stadnicki a,b,⁎, Grzegorz Machnik c, Ewa Klimacka-Nawrot a, Anna Wolanska-Karut d, Krzysztof Labuzek e a
Department of Basic Biomedical Sciences, Medical University of Silesia, Katowice, Poland Department of Gastroenterology, St Barbara's Hospital, Sosnowiec, Poland Department of Biotechnology and Genetic Engineering, Medical University of Silesia Katowice, Poland d Department of Histopathology, Medical University of Silesia Katowice, Poland e Department of Pharmacology, Medical University of Silesia, Katowice, Poland b c
a r t i c l e
i n f o
Article history: Received 30 April 2008 Received in revised form 24 February 2009 Accepted 27 February 2009 Keywords: Ulcerative colitis TGF-β1 TGFβ receptors Immunohistochemistry
a b s t r a c t Transforming growth factor-β1 (TGF-β1) plays a role in the pathogenesis of ulcerative colitis (UC) by activating its specific receptors (TβRI–TβRIII). We investigated the expression of genes encoding for TGF-β1 and TβRI–III using RT-QPCR in patients with active and inactive UC and non-IBD controls. The localization and level of TGF-β1 protein in intestinal tissue was estimated by immunohistochemistry, and serum TGF-β1 concentrations were determined using ELISA. We found a significant increase in TGF-β1 gene expression and increase in the expression of genes encoding receptor TβRI in patients with active UC when compared with controls. The expression of genes encoding TβRII was found to be higher in patients with both active and inactive UC when compared to controls. Specific staining for TGF-β1 in fibroblasts was significantly greater in both active and inactive UC as compared to controls. The serum concentration of TGF-β1 was significantly higher in patients with active UC when compared with controls as well as in UC patients with left side/total colonic extension when compared with those with disease limited to rectum/rectosigmoid area. However, no correlation between TGF-β1 serum concentrations and UC activity index was found. Increases in TGF-β1 gene expression and its protein level, associated with altered TGF-β1 receptor profile indicate a functional role for TGF-β1 in intestinal inflammatory/repair processes in UC. Increases in TGF-β1 serum concentrations correlate with extension of disease. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The major forms of inflammatory bowel disease (IBD) i.e. Crohn's disease (CD) and ulcerative colitis (UC) result from the interaction of genetic and environmental factors that promote immunopathogenic responses either by abnormality of the effector cells activity or poorly controlled regulatory mechanisms. High levels of TNF-α have been associated with the intestinal inflammation found in IBD. Infliximab, a monoclonal antibody against TNF-α, administered with steroids, has been found to be effective in inducing responses and maintaining remissions in patients with moderate-to-severe stages of IBD [1]. The intestinal epithelium creates a barrier to potentially immunogenic and noxious factors, including microorganisms and dietary components, within the intestinal lumen. Healing of the intestinal surface is regulated by a complex mechanism that involves growth factors, cytokines, as well as intracellular matrix proteins and blood clotting factors to preserve homeostasis and integrity of the intestinal mucosa [2,3]. ⁎ Corresponding author. Department of Basic Biomedical Sciences, Medical University of Silesia, 3, Kasztanowa Street PL-41-205 Sosnowiec, Poland. Tel.: +48 32 3682603; fax: +48 32 2945548. E-mail address: [email protected] (A. Stadnicki). 1567-5769/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2009.02.014
Over the past decade, nitric oxide (NO) and reactive oxygen species have been investigated as possible etiological factors in the initiation and/or propagation of inflammatory processes, because of their detrimental effect on intestinal barrier. Interestingly, free radicals are inducers of TNF-α production [4]. Once the intestinal epithelial barrier is damaged, highly immunogenic bacterial antigens from the intestinal lumen can enter the normally sterile submucosal layers and thus may play a role in the pathogenesis of IBD. This may also contribute to how antibiotics can be effective as adjunctive therapy in IBD [5]. Among the regulatory peptides that are expressed within the intestinal mucosa, transforming growth factor-β (TGF-β) and epidermal growth factor family peptides (EGFs) play especially important roles in the pathogenesis of IBD. EGFs, potent stimulators of intestinal epithelial cell proliferation, are produced mainly in submaxillary salivary glands, Brunner's glands in the duodenal submucosa, and within the exocrine pancreas [4,6]. TGF-β is capable of regulating growth, differentiation, and function of immune cells by the switching of B cells and inhibiting of T helper cell function. Importantly, TGF-β1 counteracts TNF-α and acts as a negative regulator of mucosal inflammation that is essential for wound healing [7]. TGF-β1 directly stimulates angiogenesis in vivo, and this stimulation can be blocked by TGF-β1 antibodies [8]. Intestinal epithelial wound healing and tissue
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repair may also take place through a TGF-β1 independent pathway, involving cell interactions and blood coagulation factors. Previous data suggests that the interaction between Factor XIIIa and its natural plasmatic substrates e.g. fibronectin and α-2 plasmin inhibitor, play a role in the healing of mucosal lesions in UC [9,10]. Consequently, Factor XIIIa infusions have been shown to promote intestinal wound healing in both UC and CD patients [9,11]. Three isoforms of TGF-β, namely: TGF-β1, TGF-β2, and TGF-β3, have been detected in the gut. Functional distinctions between TGF-β isoforms are still unclear. However, TGF-β1 mRNA is mainly expressed in gut tissues [13]. TGF-β1 initiates signaling through a complex transmembrane serine/threonine kinase consisting of TβRI and TβRII receptors, whereas TβRIII receptor participates in storage of the protein, and may present TGF-β1 to TβRII [12]. The ligand TGF-β1 signals from the receptor to the nucleus using a set of proteins termed Smads [14]. Overproduction of TGF-β can result in deposition of scar tissue and fibrosis in part by stimulating the production of collagen and other constituents of the extracellular matrix. Both TGF-β1 gene expression and synthesis of TGF-β1 protein are increased in fibrotic diseases including segmental glomerulonephritis, lupus nephritis, diabetic nephropathy as well as systemic sclerosis, and inhibitors of TβR may reduce or abolish fibrosis [15]. Some recent investigations indicate that activation of TGF-β1 in CD may contribute to intramural fibrosis and intestinal obstruction [16,17]. However, a role for TGF-β1 in UC is not well established. Over the past decade, the functional role of TGF-β1 in the pathogenesis of neoplasm and chronic inflammation has been defined [12]. In the gut, TGF-β1 has been found to inhibit proliferation of intestinal epithelial cells. Paradoxically, TGF-β1 seems to play a vital role in promoting reepithelialization after mucosal wounding [18]. The lack of TGF-β dependent control, because of the TβRII gene mutation, was revealed in the majority of UC patients with associated bowel carcinoma [19,20]. The development of enterocolitis in TGF-β1 deficient mice supports the concept that ongoing inflammation can result from the failure to down-regulate inflammation [21], but the relevance of this observation to human IBD remains unclear. The aim of the present study was to investigate the expression of genes encoding TGF-β1 and its receptor subtypes as well as the distribution and concentration of the TGF-β1 protein in inflamed tissues in patients with active and inactive UC compared with non-IBD controls. Changes in both the mRNA distribution and protein levels of TGF-β1 coexisting with altered TGF-β1 receptor profile in UC patients may indicate a functional role for TGF-β1 in inflammatory processes. The specific reaction for TGF-β1 staining in colonic fibroblasts was significantly higher in active UC when compared with non-IBD controls, indicating a role for fibroblasts as a source of TGF-β1 synthesis in UC, which may contribute to the mucosal repair. Increased serum TGF-β1 concentrations were associated with UC extension. However, we did not find any correlation between UC activity index and serum TGF-β concentrations.
2. Materials and methods UC was diagnosed according to commonly accepted criteria i.e. past and present clinical symptoms, sigmoidoscopy with biopsy, colonoscopy or barium enema. Clinical UC activity was assessed using a modified Truelove and Witts UC activity index [22]. Patients with a score of less than or equal to 2 were classified as inactive UC, those with a score from 3 to 9 were regarded as active UC. Exclusion criteria included systemic infections and other serious cardiopulmonary, liver, or renal diseases as well as patients with fulminant colitis. Blood was obtained from all patients and non-IBD controls by venipuncture from the antecubital vein. All samples were centrifuged at 3000 ×g for 15 min. Serum samples were stored at −70 °C until tested. Patients were studied in accordance with the protocol approved by the Institutional Committee on Human Subjects of the
Medical University of Silesia, Katowice, Poland. All patients gave written, informed consent. 2.1. Expression of TGF-β1 mRNA and TβRI, TβRII, and TβRIII mRNA Tissue specimens were obtained during colonoscopy from 36 patients with UC and 14 non-IBD patients who served as a control group. There were 22 patients with active UC (10 women, 12 men with median age of 44 years) as well as 14 patients with inactive disease (6 women, 8 men with median age of 39 years). The control subjects (with no macroscopic and histological changes in the large bowel during colonoscopy) were compatible with both subgroups (active and inactive UC patients) and matched for age and sex. Mucosal disease activity of UC patients was assessed using the modification of endoscopic index according to Baron et al. [23], taking into consideration the mucosal vascular pattern, erythema, friability, erosions, and ulcerations. Patients with UC were given conventional medical treatment. All UC patients received sulfasalazine or its derivatives. Seven patients with active UC were given moderate doses of corticosteroids and four patients (two in the active stage and two in the inactive stage) were on azathioprine. The extent of colonic disease was variable. Colonic biopsies were taken from all subjects. The diagnosis of UC was confirmed histologically; colonic biopsy specimens were stained with hematoxylin and eosin to determine the histological grade of inflammation. For molecular evaluation, colonic biopsy specimens of approximately 4 × 4 mm were removed by endoscopic forceps and immediately frozen at − 70 °C until use. Expression of TGF-β1 gene and its receptor (TβRI, TβRII and TβRIII) genes in biopsy specimens were quantitatively evaluated as a number of mRNA copies per microgram of total RNA. Constitutively expressed beta-actin gene was used as a control of the reaction. For RNA extraction, a single step method by modified guanidinum thiocyanate–phenol–chloroform extraction was employed [24]. After extraction, RNA samples were resuspended in RNase free water and were treated with RNase free DNase I (Fermentas, Lithuania) in order to remove any residual DNA. RNA extracts were size-fractionated by electrophoresis in 1% agarose gel. RNA concentrations in each sample were measured spectrophotometrically. The numbers of mRNA molecules for TGF-β1 and for its receptors TβRI-III in 1 µg of total RNA were detected with ABI PRISM 7700 Sequence Detector (Applera INC) as two-step RT-QPCR. For quantification of mRNA molecules we utilized SYBR-Green as described by Ponchel et al. [25]. Appropriate primers for reverse transcription (RT) and, subsequently, for quantitative polymerase chain reactions (QPCR) were used. We utilized complementary oligonucleotides as follows: For TGFβ: TGFβ1f: 5′-TGA ACC GGC CTT TCC TGC TTC TCA TG-3′ TGFβr 5′-GCG GAA GTC AAT GTA CAG CTG CCG C-3′; for TGFβ Receptor I: TGFR1f: 5′-ACT GGC AGC TGT CAT TGC TGG ACC AG-3′; TGFR1r: 5′-CCT GAG CCA GAA CCT GAC GTT GTC ATA TCA3′; for TGFβ Receptor II: TGFR2f 5′-GGC TCA ACC ACC AGG GCA TCC AGA T-3′, TGFR2r: 5′-CTC CCC GAG AGC CTG TCC AGA TGC T-3′; for TGFβ Receptor III: TGFR3f: 5′-ACC GTG ATG GGC ATT GCG TTT GCA-3′, TGFR3r: 5′-GTG CTC TGC GTG CTG CCG ATG CTG T-3′; and for betaactin gene: B_for: 5′-TCA CCC ACA CTG TGC CCA TCT ACG A-3′, B_rev: 5′-CAG CGG AAC CGC TCA TTG CCA ATG G-3′. For reverse transcription reaction (RT) we used MasterAMP Tth DNA/RNA polymerase (Epicentre Technology INC.). Reaction parameters (in 10 μl of reaction mixture) were performed according to manufacturer's instructions. Thereafter, 1/5 volume of RT mixture (i.e. 2 μl) served as template for QPCR. Quantitative PCR was performed in duplicates for each sample using fluorescent dye SYBR-Green (2× QuantiTect SYBR-Green PCR Kit, Qiagen GmbH, Germany) [25]. To confirm the specificity of QPCR, each sample was examined by calculation of melting temperature (Tm). All samples gave uniform melting temperature which was specific for its designed PCR product (data not shown). Additionally, randomly selected reaction samples were separated on a 6%
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polyacrylamide gel and the specific products were visualized by silver staining (the product length for TGFβ1, TβRI, TβRII, TβRIII and β-actin were as follows: 151, 200, 138, 172 and 295 bp). Single bands were visible on the gels and confirmed the specificity of the reactions (data not shown). 2.2. Immunolocalization of TGF-β1 protein in human colonic tissue Patients and control subjects: The immunolocalization of TGF-β1 protein was assessed in 10 patients with active UC (4 women, 6 men with mean age of 52 years), 10 patients with inactive UC (4 women, 6 men with mean age of 45 years) and 10 normal controls (5 women, 5 men with mean age of 45 years). The immunolocalization of TGF-β1 was performed in inflamed colonic tissue samples obtained after surgery from UC patients and tissue samples obtained from patients undergoing partial colectomy for colon cancer (control group). Tissue samples were prepared and stained for TGF-β1 using the procedure described previously [26]. In the present study, we used the mouse anti-human monoclonal antibodies (NCL-TGFB) to TGF-β1. Briefly, after paraffin embedding, de-waxing and rehydration, the primary mouse anti-human antibodies to TGF-β1 were detected using a biotinylated second antibody, and then avidin–peroxidase complex (Novostain Super ABC Kit, Novocastra). Finally, substrate 3, 3′-diaminobenzidine peroxidase (Substrate Kit; DAB) was used, and the sections were independently analyzed by a masked observer. Normal mouse serum instead of antiTGF-β1 antibody was used as a control. The slides were prepared for transmitted light microscope examination (Olympus with camera Pronis DP 10). The number of cells positively stained for TGF-β1, separately colonocytes or fibroblasts (in the lamina propria), were counted using methods published by Devani et al. [27]. TGF-β1 staining in colonocytes and fibroblasts was scored and subsequently calculated in 50 representative high power fields of each tissue sample. Immunoreactivity in the interstitial compartment was scored as 0 = no immunostaining, + = weak, ++ = moderate, +++ = strong. The evaluation of immunohistochemical pattern was performed in blinded fashion by a pathologist experienced in IBD. 2.3. Measurements of TGF-β1 in serum 2.3.1. Patients and control subjects We measured serum concentrations of TGF-β1 in 55 UC patients (21 women, 34 men with mean age of 47 years) and 20 controls (9 women, 11 men with mean age of 47 years). Patients with UC were divided, according to a Truelove and Witts UC activity index, as 25 UC inactive patients (a score of less than or equal to 2; 11 women and 14 men with mean age of 46 years) and 30 UC active patients (a score from 3 to 9; 10 women and 20 men with mean age of 44 years). In addition, UC patients were divided according to colonic extension as extended to left side/total colitis (19 patients: 10 women and 9 men with mean age of 43 years) or rectum/rectosigmoid colitis (36 patients: 11 women and 25 men with mean age of 47 years). The antigenic levels of TGF-β1 in serum were measured by the enzyme-liked immunoabsorbent assay (ELISA). Monoclonal mouse anti-human TGF-β1 antibody was coupled with biotin, then avidin– peroxidase was added to detect TGF-β1. 2.4. Statistical analysis The significance of differences between the means of each group was determined by the Student's t test. For immunohistochemical data, the means were compared using the Kolmogorov–Smirnov test and the Mann–Whitney U test. Since the mRNA concentrations were distributed logarithmically, we evaluated the significance of the differences between the controls, active UC patients, and inactive UC patients for the measurement of TβRII mRNA and TβRI mRNA
Fig. 1. Gene expression of TGF-β1, TβRI, and TβRII in the colonic tissue. Bars indicate mean ± SEM. A: Gene expression of TGF-β1 in the colonic tissue. B: Gene expression of TβRI in the colonic tissue. C: Gene expression of TβRII in the colonic tissue.
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expression using the logarithmic values of each parameter. The means were compared using the Mann–Whitney U test. All values were expressed as mean ± SEM. The correlation between parameters was calculated using the Spearman's rank correlation coefficient.
Table 1 Serum TGF-β1 concentrations.
3. Results
Clinical activity
3.1. TGF-β1, and Tβ-RI, RII, and RIII gene expression
Endoscopic extension
In patients with the active phase of UC, mRNA expression of TGFβ1 gene (81,065 ± 17,357 SE, the number of mRNA copies/μg total RNA) was significantly higher (P b 0.001) when compared with nonIBD controls (17,657 ± 4679). However, TGF-β1 mRNA levels in patients with inactive UC (45,408 ± 14177) did not differ significantly from the controls (Fig. 1A). The expression of genes encoding receptor TβRI was significantly higher (P b 0.005) in patients with active UC
Controls
Group of patients
a b
Serum TGF-β1 concentrations [ng/ml] Active UC Inactive UC Left side/total Rectum/ rectosigmoid
Mean ± SEM
Statistical analysis (P-values)
29.2 ± 5.5 25.4 ± 1.7 30.5 ± 2.0 23.9 ± 2,0
P b 0.01a P b 0.009a P b 0.0001a P b 0.03b
18.4 ± 1.6
vs. controls. Left side/total vs. rectum/rectosigmoid.
(22,776 ± 5665) as compared with non-IBD controls (6873 ± 857) (Fig. 1B). The expression of genes encoding TβRII was found to be higher in patients with active UC (88,032 ± 17,453 SE, P b 0.05) as well as with inactive UC (116,588 ± 16,530 SE, P b 0.002) than in control subjects (27,616 ± 8409) (Fig. 1C). In contrast no significant differences in TβRIII gene expression were observed in all examined groups (data not shown). 3.2. Immunohistochemical localization of TGF-β1 protein We visualized TGF-β1 in normal as well as in inflamed human colon. We demonstrated weak specific reactions for TGF-β1 in colonocytes from both UC patients and controls. Additionally, there was a specific staining reaction for TGF-β in macrophages, granulocytes, and lymphocytes in the colon lamina propria of UC patients. However, strong specific reaction for TGF-β1 was shown in fibroblasts in patients with both active and inactive phases of UC. Thus, the semiquantitative estimation of the specific reaction for TGF-β1 was performed separately in colonocytes and in fibroblasts representing lamina propria cells. The specific immunoreactivity for TGF-β1 was nearly equal in colonocytes in all examined groups. However, the specific reaction for TGF-β1 staining in fibroblasts was significantly higher in active UC (26 points, P b 0.005) as well as in inactive UC (15 points, P b 0.017) when compared with non-IBD controls. Fig. 2A illustrates localization of immunoreactive TGF-β1 in the epithelial cells of the normal human colon, but no positive specific reactions in fibroblasts. Fig. 2B demonstrates TGF-β1 in the inflammatory tissue of the colon lamina propria of inactive UC, mainly in fibroblasts. Fig. 2C shows strong positive specific reactions for TGF-β1 in fibroblasts localized in the colon lamina propria of active UC. 3.3. Serum TGF-β1 concentrations Serum TGF-β1 concentrations were significantly higher (P b 0.01) in active UC patients (29.2 ± 5.5 ng/ml) when compared with nonIBD controls (18.4 ± 1.6 ng/ml). However, TGF-β1 antigenic concentrations in active UC patients did not differ from those in inactive UC patients (25.4 ± 1.7 ng/ml). We were not able to show significant correlation between UC disease activity index and serum TGF-β1 concentrations (r = 0.17, NS). Serum concentrations of TGF-β1 were significantly higher (P b 0.03) in patients with left side/total colonic extension (30.5 ± 2.0) when compared with those limited to rectum/ rectosigmoid (23.9 ± 2.0) (Table 1). 4. Discussion
Fig. 2. Tissue localization of TGF-β1 protein in the colonic mucosa. Immunohistochemical staining for TGF-β1 using the NCL-TGFB antibody (×400 magnification). A: Normal colonic mucosa. Weak staining reaction for TGF-β1 protein in colonocytes (arrow). Please note that there is no staining in the fibroblasts. B: Inactive UC colonic tissue. The lamina propria of the mucosa. Positive staining for TGF-β1 in fibroblasts (arrow). C: Active UC colonic tissue. Please note a strong positive staining for TGF-β1 in fibroblasts (arrow).
Growth factors are potential targets for intestinal homeostasis. TGF-β and EGFs are especially relevant for modulation of intestinal inflammation and have a number of important properties in IBD. The role of TGF-β1 in the pathogenesis of IBD was investigated mainly in CD patients. Di Mola et al. [17] reported an overexpression of TGF-β1 and its signaling receptors in epithelium and inflammatory cells
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invading whole intestinal wall in active CD patients. These findings indicated that TGF-β1 might contribute to intestinal fibrosis and subsequent intestinal obstruction. Much less attention was paid to the role of TGF-β1 in the pathogenesis of UC. Chowdhury et al. [28] showed low expression of TGF-β1 mRNA in UC inflammatory intestine suggesting that it may be, at least in part, responsible for UC intractability. In a strict contrast, Babyatsky et al. [29], using semiquantitative Northern blot analysis, reported an increased expression of TGF-β1 mRNA in the affected mucosa of patients with both active UC and CD. In the present study, we have focused our attention on the TGF-β1 system in UC. We demonstrated an increased expression of TGF-β1 mRNA in the affected mucosa in active UC patients which corresponds to findings of Babyatsky et al. [29]. We extended Babyatsky's study showing the expression of genes encoding TGF-β1 and simultaneously its receptors in inflamed UC tissue using quantitative RT-QPCR method. It is known that TGF-β1 initiates activation through ligand-dependent complex consisting of the type I receptor which, in turn, facilitates activation of the associate type II receptor. Interestingly, it has been shown that decreased expression of TβRII gene renders to the cell resistance to the antiproliferative effect of TGF-β1 [30]. Thus, our present observations, showing an overexpression of TβRI and TβRII genes in active UC inflamed intestine, and, interestingly, an increase of TβRII gene expression in inactive colonic UC tissue, indicate a functional role for TGF-β1 in the pathogenesis of UC. A question arises what is a mechanism that leads to TGF-β1 and its receptors overexpression in active UC. In fact, TGF-β1 evokes increases in NO levels, which increased synthesis is present in colonic biopsies and plasma of IBD patients [13]. Nitric oxide may produce feedback inhibition of TGF-β1 [31]. The regulation of TGF-β1 gene expression and the increase in its protein synthesis may also be related to TGF-β1 polymorphism [32]. In addition, intestinal EGF, a promigratory peptide which is present in the gastrointestinal tract, may increase the concentration of bioactive TGF-β [13]. Interestingly, EGF enemas have been proved to be beneficial in UC patients [6]. The current working hypothesis is that UC may be a Th-2 like related disorder with local over-production of IL-13 by natural killer T cells [3]. Importantly, the data from experimental models of colitis indicate that the IL-13 related pathway (by IL α2 receptor) results in the induction of TGF-β1 production. Thus this mechanism may be involved in the pathogenesis of human UC [33]. TGF-β1, a potent immunosuppressive cytokine, is paradoxically induced in the intestine during active inflammation. However, recent data also indicate that defective signaling of TGF-β1 in chronic IBD is related to increase of Smad7, a main inhibitor of TGF-β1 signaling in IBD mucosa [14]. We speculate that TGF-β1 overexpression does not prevent intestinal injury. However, its increased synthesis, combined with upregulation of its receptors, can enhance intestinal repair. In addition to increased TGF-β1 mRNA expression we characterized the cellular localization and concentration of TGF-β1 protein in UC intestine. We demonstrated that TGF-β1 protein is present in the epithelial cells of inflamed human colon and its levels are increased in inflammatory cells within the colonic lamina propria which is in agreement with previous findings [16]. A new observation is that fibroblasts (among other inflammatory cells of the lamina propria of UC intestine) show the most impressive specific reaction for TGF-β1 protein. This observation emphasizes a significance of fibroblasts in the immune responses mediated by TGF-β1 in UC. Previously, Ohtani et al. [34], using immunohistochemical techniques, showed the presence of TβRI and TβRII in fibroblasts in UC lamina propria, but not in organized fibrotic area in CD. More recently, Stallmach et al. [35] demonstrated that fibroblasts isolated from strictures in patients with CD produce significantly more type III collagen than fibroblasts from normal or nonstrictured but inflamed lamina propria. Our data tend to corroborate with the results of Stallmach et al. [35] results and indicate a role for intestinal fibroblasts as a source of TGF-β1 in restoration of the surface continuity in UC.
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However, transmural increase of TGF-β1 and its receptors may enhance collagen III production, and, in turn, implicates stricture formation in CD. In fact, Di Mola et al. [17] showed overexpression of three TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3) transmurally in the active inflammatory CD intestines. Taken together, CD and UC patients may manifest different intestinal TGF-β behavior patterns. Finally, we measured serum TGF-β1 concentrations. Previously, Wiercinska-Drapalo et al. [36] observed a significant positive correlation between increased plasma TGF-β1 concentrations and scored degree of mucosal injury in UC patients. However, Sambuelli et al. [37] demonstrated only a tendency towards increasing serum TGF-β1 concentrations in relation to UC activity. In normal colon, TGF-β1 is present only in epithelial cells, but in UC, it can be found in the inflammatory cells of the lamina propria. Changes in serum TGF-β1 concentrations might have reflected, to some degree, events taking place in inflamed intestine. In the present study, we extended previous findings by showing significantly higher serum TGF-β1 concentrations in UC, related to the degree of colonic extension. However, we did not observe any significant differences in serum TGFβ1 concentrations between active and inactive UC patients, and, consequently, we did not find any correlation between UC activity and serum TGF-β1 concentrations. Thus, our data seems to support previous findings of Sambuelli et al. [37]. In fact, TGF-β1 is synthesized in the intestinal mucosa and appears to act not only locally, but also may be involved in systemic response. Recently, Rezaile et al. [38] have demonstrated significantly higher concentrations of TGF-β1 and NO in saliva of IBD patients, which may indicate an underlying pathology in saliva itself. In addition, elevated concentrations of TGF-β1 and NO in saliva did not significantly differ between UC and CD patients, indicating a pathophysiological role for both TGF-β1 and NO in these diseases. What is the role of TGF-β1 in UC? TGF-β1 enhances the production of the extracellular matrix by fibroblasts and other intestinal cells. Thus it seems to be important for the recovery of tissue integrity. Receptors for these reactions are overexpressed in the human intestinal epithelium during active UC. Thus TGF-β1 can mediate healing processes in the inflamed intestine. However, TGF-β1 may also act systematically as it expresses many proinflammatory actions to stimulate the synthesis of COX-2, the release of eicosanoids and the production of cytokines, mainly IL-8 [39,40], which is known to be important in IBD [3]. In humans, the success of azathioprine and corticosteroids (commonly used as immunosuppressive drugs in IBD) in treating autoimmune hepatitis is, in part, due to the ability of these drugs to reduce serum TGF-β1 concentrations [41]. Thus, the possible role of TGF-β in the treatment of UC requires additional investigation. Acknowledgments This work was supported in part by a grant from the Polish Ministry of Sciences (2PO5B14026; A. Stadnicki) and, in part, by a grant from the Medical University of Silesia (NN-2-047/07; A. Stadnicki). We thank Drs. Z. Krowicki (MediProfile, Inc.) and U. Pisharody (LSUHSC School of Medicine) for helpful advices and manuscript corrections. References [1] Rahimi R, Nikfar S, Abdollahi M. Meta-analysis technique confirms the effectiveness of anti-TNF-alpha in the management of active ulcerative colitis when administered in combination with corticosteroids. Med Sci Monit 2007;13:PI13–8. [2] Podolsky DK. Inflammatory bowel disease. N Engl J Med 2002;347:417–29. [3] Baumgart DC, Carding SR. Inflammatory bowel disease: cause and immunobiology. Lancet 2007;369:1627–40. [4] Jahanshahi G, Motavasel V, Rezaie A, Hashtroudi AA, Daryani NE, Abdollahi M. Alterations in antioxidant power and levels of epidermal growth factor and nitric oxide in saliva of patients with inflammatory bowel diseases. Dig Dis Sci 2004;49: 1752–7. [5] Rahimi R, Nikfar S, Rezaie A, Abdollahi M. A meta-analysis of antibiotic therapy for active ulcerative colitis. Dig Dis Sci 2007;52:2920–5.
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