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Effect of ulcerative colitis treatment on transforming growth factor β1 in plasma and rectal mucosa
Regulatory Peptides 113 (2003) 57 – 61 www.elsevier.com/locate/regpep
Effect of ulcerative colitis treatment on transforming growth factor h1 in plasma and rectal mucosa Alicja Wiercin´ska-Drapalo *, Robert Flisiak, Danuta Prokopowicz Department of Infectious Diseases, Intestinal Diseases Unit, Medical Academy of Bialystok, 15-540 Bialystok, Zurawia str., 14 Poland Received 25 June 2002; received in revised form 3 December 2002; accepted 12 December 2002
Abstract The aim of this study was to evaluate the effect of active ulcerative colitis (UC) treatment on transforming growth factor h1 (TGF-h1) concentration in plasma and rectal mucosa measured in 28 patients. The highest plasma values were observed in patients with the severe course of the disease (74.2 F 14.0 ng/ml), and they were significantly higher than in the group with mild one (43.7 F 5.6 ng/ml). Mean TGFh1 measured in mucosal samples from patients with severe UC (563 F 146 pg/mg protein) doubled values from patients with mild UC (286 F 65 pg/mg protein). Plasma and mucosal TGF-h1 correlated significantly with disease activity index (DAI) and clinical activity index (CAI). Plasma TGF-h1 correlated additionally with scored endoscopic degree of mucosal injury. Treatment caused significant decrease of plasma and mucosal TGF-h1 concentrations. Patients who responded completely had higher baseline plasma and mucosal TGF-h1 that decreased significantly after the treatment. These results show that plasma and mucosal concentrations of transforming growth factor h1 are strongly associated with ulcerative colitis activity, and successful treatment of the disease results with decrease of their levels. More effective response to the treatment can be achieved in patients with higher baseline concentrations of TGF-h1. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Cytokines; TGF-h1; Inflammatory bowel diseases; Treatment
1. Introduction Ulcerative colitis (UC) is a chronic bowel inflammation caused by abnormal immune response and seems to be related to diminished ability of mucosal protection and regeneration. These processes are controlled by signalling between epithelial cells involving complex network of cytokines, growth factors and other bioactive substances responsible for cell proliferation and differentiation as well as regulation of immune response [1 –3]. Successful treatment of UC-related mucosal injury stimulates cells proliferation, extracellular matrix (ECM) formation, angiogenesis and tissue restitution. Transforming growth factor-h1 (TGFh1) affects all these mechanisms leading to mucosal healing and is also known as a regulator of immune response playing a principal role in UC pathogenesis [4 – 6]. Enhanced expression of TGF-h1 in inflammatory bowel diseases indicates role of this growth factor in the patho-
* Corresponding author. Tel./fax: +48-85-7416921. E-mail address: [email protected] (A. Wiercin´ska-Drapalo).
genesis of UC [7– 9]. As demonstrated recently, release of TGF-h1 by intestinal myofibroblasts is enhanced in patients with ulcerative colitis [10]. The aim of this study was to evaluate effect of the treatment of patients with active ulcerative colitis on TGFh1 concentration measured in plasma and rectal mucosa.
2. Materials and methods 2.1. Patients TGF-h1 concentration was measured in the plasma and mucosal samples, before and after treatment of 28 patients (11 females and 17 males) with active ulcerative colitis (UC), in age ranging from 22 to 72 (mean: 48.1 F 2.7). All patients had history of diagnosed ulcerative colitis that required typical clinical and endoscopical signs of distal part of bowel involvement. After admission to the hospital, they received treatment with sulphasalazine (3 g/day) or 5aminosalicylic acid (1.5 g/day). Oral prednisone (0.5/day) was administered in patients with severe form of present
0167-0115/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-0115(02)00300-2
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Fig. 1. Mean plasma concentration of TGF-h1 in patients with mild or severe form of ulcerative colitis (UC). Statistical significance between groups or before and after treatment is indicated by arrows connecting bars; statistical significance in comparison with normal values is indicated by asterisks.
relapse. Endoscopic picture scored according to Meyers et al. [11], the disease activity index (DAI) according to Schroeder et al. [12], clinical activity index (CAI) according to Truelove and Witts [13] and basic laboratory indices of UC activity such as C-reactive protein (CRP) and sedimentation rate (SR) were analysed in all patients before and after treatment. They were compared with concentrations of TGF-h1 measured in plasma and rectal mucosa. Mucosal biopsy for histological confirmation of UC was taken before treatment, and for TGF-h1 measurement before and after treatment. Patients were divided into two groups in respect to severity of the disease. They were qualified to the group with severe UC if values of endoscopic score, DAI and CAI, exceeded middle of range of all three scales that was respectively: 8, 6 and 10. Severe form of ulcerative colitis was diagnosed in 12 patients and
mild one in 16. Moreover, patients were evaluated in respect to efficiency of the treatment. Improvement in all three scoring systems was considered as a complete response, whereas lack of improvement in at least one among these classifications was considered as an uncomplete response. Results of plasma TGF-h1 measurement were also compared with 13 healthy controls (mean age: 47.5 F 1.7 years). The study was approved by the Bioethical Committee of the Medical Academy in Bialystok. Informed consent was obtained from each patient. 2.2. TGF-b1 measurement Venous blood was collected on ice using vacutainer tubes with EDTA as an anticoagulant and centrifuged at 1.000 g for 30 min of collection. Obtained plasma was
Fig. 2. Mean mucosal concentration of TGF-h1 in patients with mild or severe form of ulcerative colitis (UC). Statistical significance before and after treatment is indicated by arrows connecting bars.
A. Wiercin´ska-Drapalo et al. / Regulatory Peptides 113 (2003) 57–61 Table 1 Indices of ulcerative colitis activity before (I) and after (II) treatment of patients with mild or severe form of the disease
Endoscopic score DAI CAI CRP SR
59
Table 3 Plasma and mucosal TGF-h1 concentration before (I) and after (II) treatment in respect to its effect
Mild UC n = 16
Severe UC n = 12
Response to the treatment
I
II
I
II
Complete (n = 22)
8.3 F 0.6 5.8 F 0.4 6.8 F 0.5 23.1 F 4.9 21.4 F 4.1
4.8 F 0.6** 3.8 F 0.4** 4.9 F 0.3* 11.9 F 1.8* 23.8 F 4.3
12.0 F 0.6 8.3 F 0.3 12.1 F 0.4 33.5 F 4.5 25.4 F 3.6
7.8 F 0.8** 5.6 F 0.3** 8.5 F 0.5** 19.8 F 4.7* 31.4 F 2.7
Plasma TGF-h1 [ng/ml] Mucosal TGF-h1 [pg/mg protein]
Uncomplete (n = 6)
I
II
I
II
60.8 F 8.5
25.3 F 6.1**
42.0 F 12.4
27.2 F 9.5*
431 F 90
111 F 24*
262 F 71
153 F 48
Statistical significance between both evaluations: *< 0.05, **< 0.001.
Statistical significance between both evaluations: *< 0.05, **< 0.001.
additionally centrifuged at 10.000 g for 10 min at 2 –8 jC for complete removal of platelets, and to avoid release of platelet-derived TGF-h1 . Biopsy specimens were mashed with homogenizer, and the suspension was divided and used to determine TGF-h1 and protein concentration. To activate latent TGF-h1 to immunoreactive TGF-h1, 0.1 ml of a mixture consisting of 2.5 N acetic acid and 10 M urea was added to 0.1 ml of platelet-poor plasma and incubated for 10 min at room temperature. Acidified sample was neutralized by adding 0.1 ml of mixture consisted of 2.7 N NaOH and 1 M HEPES. Activated plasma samples were four-fold diluted and assayed in duplicate with the quantitative sandwich enzyme immunoassay (EIA) technique using recombinant human TGF-h soluble receptor Type II (ThRII) as a solid phase precoated onto a microplate (Quantikine R&D Systems, Minneapolis, USA), as described previously [14]. Optical density was read with a microtitre plate photometer Stat FaxR 2100 (Alab, Poland) at 450 nm and corrected by subtraction of readings at 540 nm. The concentration of TGF-h1 in a sample was determined by interpolation from a standard curve, prepared with standard samples supplied by the manufacturer and expressed as ng/ml. Mucosal TGF-h1 concentration was expressed as pg/ml of protein that was measured by the method of Lowry et al. [15].
and linear regression was performed. Values of p < 0.05 were considered to be significant.
2.3. Statistical methods Values were expressed by their mean and standard error of the mean ( F S.E.M.). The significance of the difference was calculated by two-tailed Student’s t-test. For correlation analysis, the Pearson product moment correlation was used
Table 2 Correlation expressed through r-value and its significance (*p < 0.05) between analysed indices of the disease activity and plasma or mucosal TGF-h1 concentration in all patients with ulcerative colitis Endoscopic score DAI CAI CRP SR
Plasma TGF-h1
Mucosal TGF-h1
0.47* 0.52* 0.46* 0.26 0.01
0.33 0.48* 0.49* 0.18 0.14
3. Results Plasma mean concentration of TGF-h1, measured before the treatment in all patients with active UC, was significantly elevated (56.8 F 7.2 ng/ml) in comparison to healthy controls (18.3 F 1.6 ng/ml). The highest values were observed in patients with the severe course of the disease (74.2 F 14.0 ng/ml), and they were significantly higher ( p = 0.03) than in group with mild one (43.7 F 5.6 ng/ml), (Fig. 1). Mean levels of TGF-h1 measured in mucosal samples from patients with severe UC (563 F 146 pg/mg protein) doubled values from patients with mild UC (286 F 65 pg/mg protein), but the difference was not significant (Fig. 2). As demonstrated in Table 1, treatment resulted with significant improvement of analysed indices of the disease activity except sedimentation rate in both groups. Treatment of patients with both mild and severe UC resulted with significant decrease of plasma TGF-h1 concentration to values (19.9 F 3.6 and 33.5 F 10.9 ng/ml, respectively) below the half of initial level (Fig. 1). This trend was even more intensive in respect to TGF-h1 concentration in rectal mucosa that was three times lower after treatment in both groups (94 F 23 and 168 F 37 pg/mg protein, respectively), (Fig. 2). As shown in Table 2, plasma TGF-h1 levels analysed in all UC patients revealed significant positive correlations with scored endoscopic degree of mucosal injury, DAI and CAI. Mucosal TGF-h1 correlated significantly only with values of DAI and CAI (Table 2). There was no correlation with sedimentation rate in respect to both plasma and mucosal TGF-h1 concentration. As demonstrated in Table 3, patients who responded completely had higher plasma and mucosal TGF-h1 that decreased significantly after treatment. In patients who did not respond completely, significant decrease after treatment was related only to plasma TGF-h1 (Table 3).
4. Discussion Transforming growth factor h1 has several important functions in the pathogenesis of inflammatory bowel dis-
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eases that include control of epithelial cells proliferation and differentiation, immunosuppresion and finally, regulation of extracellular matrix (ECM) formation. In the initial phase of bowel inflammation, TGF-h1 inhibits proliferation and stimulates differentiation of epithelial cells that aids in the repair process of the mucosa following the damage [1]. Immunosuppressive effect is related to inhibition of both B and T cells activity that was strongly supported by the study on TGF-h null mice, which died because of multifocal inflammation [16]. Inhibitory effect of TGF-h1 on Th1-mediated experimental colitis through suppression of interleukin-12 signalling and interferon-gamma transcription suggests its possible immunosuppressive activity in Crohn disease considered as Th1-mediated disorder [5]. Unfortunately, we were not able to carry out the study in patients with Crohn disease because of extremely low incidence of the disease in this part of Europe. Since it has been demonstrated by Seder et al. [17] that IL-4 and IL-10 stimulate TGF-h1 production by CD4 + cells, its increased concentration may reflect Th2mediated T cell response in UC patients. Additionally, TGFh1 affects inflammation in bowel through induction of cyclooxygenase-2 and subsequent increased prostaglandin production [18]. As we demonstrated previously, plasma and mucosal concentrations of prostaglandin E2 correlate significantly with the degree of mucosal injury [19]. However, the most important role of TGF-h1 is related to fibrosis stimulation and wound healing. Role of transforming growth factor h1 in the pathogenesis of diseases associated with enhanced ECM formation has been established recently [14,20 –22]. Fibrosis is a major complication of inflammatory bowel diseases and according to Lawrance et al. [23] collagen secretion from mucosal fibroblasts is stimulated by growth factors, including TGF-h1. Because of this effect, TGF-h1 seems to be implicated in stricture formation and muscle hypertrophy occurring sometimes as a complication in inflammatory bowel diseases [8,18]. Lack of TGF-h dependent control, because of the receptor ThRII gene mutation, was demonstrated in patients with UC-associated bowel neoplasma and therefore, TGF-h should be considered as the major suppressor of bowel carcinogenesis [24]. According to Grainger et al. [25], the most important factors affecting measurement of TGF-h1 in human is preparation of samples with minimal contamination from platelets as a powerful source of this cytokine and methodology that enables to use one particular assay for detection of multiple latent TGF-h1 complexes. The Quantikine ELISA System is recommended mostly because of relatively quick and simple activation with acid and urea that disrupt majority of TGF-h1 complexes [26]. Mean plasma concentration of TGF-h1 measured in our healthy controls with this method was consistent with range from more than 20 studies reviewed by Grainger et al. [25]. In this study, we demonstrated a significant increase of TGF-h1 concentration in plasma of patients with ulcerative colitis that rised depending on the degree of mucosal injury. Additionally, this association was confirmed with other
classifications (DAI and CAI) that are frequently used for evaluation of UC activity. We also demonstrated significant correlation between these indices and TGF-h1 concentration in rectal mucosa. The possible source of this growth factor can be cells located closely to the bowel lamina propria of affected mucosa that have been demonstrated as a place of enhanced expression of TGF-h1 [7]. McKaig et al. [10] found cultured intestinal myofibroblasts as a possible source of this cytokine in humans with UC. According to Monteleone et al. [6], blockade of TGF-h1 caused by Smad7 activation helps to maintain the chronic production of proinflammatory cytokines responsible for the inflammation in UC. As demonstrated recently by Hahm et al. [27], deficiency of TGF-h1 signalling contributes to the development of inflammatory bowel disease. Therefore, insufficient response to TGF-h1 due to Smad7 effect can be a reason of enhanced TGF-h1 production. However, up to now there was almost no information about a possible association between plasma TGF-h1 and the disease activity. Sturm et al. [9] demonstrated unsignificant tendency towards increased TGF-h1 values in ulcerative colitis. In the study of Sambuelli et al. [28], circulating TGF-h1 was significantly higher than in controls but results are not reliable because the measurement was performed in serum, so unpredictable part of TGFh1 could be released from platelets. However, even in this study, elevated values at the beginning of the treatment were observed only in patients who responded and they significantly decreased after the treatment. In our study, patients with uncomplete response to the treatment had also lower baseline levels of TGF-h1 both in plasma and mucosa than complete responders. It is consistent with mentioned research on the role of insufficient TGF-h1 signalling in the pathogenesis of UC [6,27] and recent observations of Whiting et al. [29] who proved low levels of active TGF-h as a possible factor contributing to unresolved experimental colitis. Taken together, plasma and mucosal concentrations of transforming growth factor h1 are strongly associated with ulcerative colitis activity, and successful treatment of the disease results with decrease of their levels. More effective response to the treatment can be achieved in patients with higher baseline concentrations of TGF-h1.
References [1] Beck PL, Podolsky DK. Growth factors in inflammatory bowel disease. Inflamm Bowel Dis 1999;5:44 – 60. [2] Fiocchi C. IBD: etiology and pathogenesis. Gastroenterology 1998; 115:182 – 205. [3] Zimmerman CM, Padgett RW. Transforming growth factor h signaling mediators and modulators. Gene 2000;249:17 – 30. [4] Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor h in human disease. N Engl J Med 2000;342:1350 – 8. [5] Kitani A, Fuss IJ, Nakamura K, Schwartz OM, Usui T, Strober W. Treatment of experimental (trinitrobenzene sulfonic acid) colitis by intranasal administration of transforming growth factor (TGF)-beta 1 plasmid: TGF-beta1-mediated suppression of T helper cell type 1
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Effect of ulcerative colitis treatment on transforming growth factor h1 in plasma and rectal mucosa Alicja Wiercin´ska-Drapalo *, Robert Flisiak, Danuta Prokopowicz Department of Infectious Diseases, Intestinal Diseases Unit, Medical Academy of Bialystok, 15-540 Bialystok, Zurawia str., 14 Poland Received 25 June 2002; received in revised form 3 December 2002; accepted 12 December 2002
Abstract The aim of this study was to evaluate the effect of active ulcerative colitis (UC) treatment on transforming growth factor h1 (TGF-h1) concentration in plasma and rectal mucosa measured in 28 patients. The highest plasma values were observed in patients with the severe course of the disease (74.2 F 14.0 ng/ml), and they were significantly higher than in the group with mild one (43.7 F 5.6 ng/ml). Mean TGFh1 measured in mucosal samples from patients with severe UC (563 F 146 pg/mg protein) doubled values from patients with mild UC (286 F 65 pg/mg protein). Plasma and mucosal TGF-h1 correlated significantly with disease activity index (DAI) and clinical activity index (CAI). Plasma TGF-h1 correlated additionally with scored endoscopic degree of mucosal injury. Treatment caused significant decrease of plasma and mucosal TGF-h1 concentrations. Patients who responded completely had higher baseline plasma and mucosal TGF-h1 that decreased significantly after the treatment. These results show that plasma and mucosal concentrations of transforming growth factor h1 are strongly associated with ulcerative colitis activity, and successful treatment of the disease results with decrease of their levels. More effective response to the treatment can be achieved in patients with higher baseline concentrations of TGF-h1. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Cytokines; TGF-h1; Inflammatory bowel diseases; Treatment
1. Introduction Ulcerative colitis (UC) is a chronic bowel inflammation caused by abnormal immune response and seems to be related to diminished ability of mucosal protection and regeneration. These processes are controlled by signalling between epithelial cells involving complex network of cytokines, growth factors and other bioactive substances responsible for cell proliferation and differentiation as well as regulation of immune response [1 –3]. Successful treatment of UC-related mucosal injury stimulates cells proliferation, extracellular matrix (ECM) formation, angiogenesis and tissue restitution. Transforming growth factor-h1 (TGFh1) affects all these mechanisms leading to mucosal healing and is also known as a regulator of immune response playing a principal role in UC pathogenesis [4 – 6]. Enhanced expression of TGF-h1 in inflammatory bowel diseases indicates role of this growth factor in the patho-
* Corresponding author. Tel./fax: +48-85-7416921. E-mail address: [email protected] (A. Wiercin´ska-Drapalo).
genesis of UC [7– 9]. As demonstrated recently, release of TGF-h1 by intestinal myofibroblasts is enhanced in patients with ulcerative colitis [10]. The aim of this study was to evaluate effect of the treatment of patients with active ulcerative colitis on TGFh1 concentration measured in plasma and rectal mucosa.
2. Materials and methods 2.1. Patients TGF-h1 concentration was measured in the plasma and mucosal samples, before and after treatment of 28 patients (11 females and 17 males) with active ulcerative colitis (UC), in age ranging from 22 to 72 (mean: 48.1 F 2.7). All patients had history of diagnosed ulcerative colitis that required typical clinical and endoscopical signs of distal part of bowel involvement. After admission to the hospital, they received treatment with sulphasalazine (3 g/day) or 5aminosalicylic acid (1.5 g/day). Oral prednisone (0.5/day) was administered in patients with severe form of present
0167-0115/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-0115(02)00300-2
58
A. Wiercin´ska-Drapalo et al. / Regulatory Peptides 113 (2003) 57–61
Fig. 1. Mean plasma concentration of TGF-h1 in patients with mild or severe form of ulcerative colitis (UC). Statistical significance between groups or before and after treatment is indicated by arrows connecting bars; statistical significance in comparison with normal values is indicated by asterisks.
relapse. Endoscopic picture scored according to Meyers et al. [11], the disease activity index (DAI) according to Schroeder et al. [12], clinical activity index (CAI) according to Truelove and Witts [13] and basic laboratory indices of UC activity such as C-reactive protein (CRP) and sedimentation rate (SR) were analysed in all patients before and after treatment. They were compared with concentrations of TGF-h1 measured in plasma and rectal mucosa. Mucosal biopsy for histological confirmation of UC was taken before treatment, and for TGF-h1 measurement before and after treatment. Patients were divided into two groups in respect to severity of the disease. They were qualified to the group with severe UC if values of endoscopic score, DAI and CAI, exceeded middle of range of all three scales that was respectively: 8, 6 and 10. Severe form of ulcerative colitis was diagnosed in 12 patients and
mild one in 16. Moreover, patients were evaluated in respect to efficiency of the treatment. Improvement in all three scoring systems was considered as a complete response, whereas lack of improvement in at least one among these classifications was considered as an uncomplete response. Results of plasma TGF-h1 measurement were also compared with 13 healthy controls (mean age: 47.5 F 1.7 years). The study was approved by the Bioethical Committee of the Medical Academy in Bialystok. Informed consent was obtained from each patient. 2.2. TGF-b1 measurement Venous blood was collected on ice using vacutainer tubes with EDTA as an anticoagulant and centrifuged at 1.000 g for 30 min of collection. Obtained plasma was
Fig. 2. Mean mucosal concentration of TGF-h1 in patients with mild or severe form of ulcerative colitis (UC). Statistical significance before and after treatment is indicated by arrows connecting bars.
A. Wiercin´ska-Drapalo et al. / Regulatory Peptides 113 (2003) 57–61 Table 1 Indices of ulcerative colitis activity before (I) and after (II) treatment of patients with mild or severe form of the disease
Endoscopic score DAI CAI CRP SR
59
Table 3 Plasma and mucosal TGF-h1 concentration before (I) and after (II) treatment in respect to its effect
Mild UC n = 16
Severe UC n = 12
Response to the treatment
I
II
I
II
Complete (n = 22)
8.3 F 0.6 5.8 F 0.4 6.8 F 0.5 23.1 F 4.9 21.4 F 4.1
4.8 F 0.6** 3.8 F 0.4** 4.9 F 0.3* 11.9 F 1.8* 23.8 F 4.3
12.0 F 0.6 8.3 F 0.3 12.1 F 0.4 33.5 F 4.5 25.4 F 3.6
7.8 F 0.8** 5.6 F 0.3** 8.5 F 0.5** 19.8 F 4.7* 31.4 F 2.7
Plasma TGF-h1 [ng/ml] Mucosal TGF-h1 [pg/mg protein]
Uncomplete (n = 6)
I
II
I
II
60.8 F 8.5
25.3 F 6.1**
42.0 F 12.4
27.2 F 9.5*
431 F 90
111 F 24*
262 F 71
153 F 48
Statistical significance between both evaluations: *< 0.05, **< 0.001.
Statistical significance between both evaluations: *< 0.05, **< 0.001.
additionally centrifuged at 10.000 g for 10 min at 2 –8 jC for complete removal of platelets, and to avoid release of platelet-derived TGF-h1 . Biopsy specimens were mashed with homogenizer, and the suspension was divided and used to determine TGF-h1 and protein concentration. To activate latent TGF-h1 to immunoreactive TGF-h1, 0.1 ml of a mixture consisting of 2.5 N acetic acid and 10 M urea was added to 0.1 ml of platelet-poor plasma and incubated for 10 min at room temperature. Acidified sample was neutralized by adding 0.1 ml of mixture consisted of 2.7 N NaOH and 1 M HEPES. Activated plasma samples were four-fold diluted and assayed in duplicate with the quantitative sandwich enzyme immunoassay (EIA) technique using recombinant human TGF-h soluble receptor Type II (ThRII) as a solid phase precoated onto a microplate (Quantikine R&D Systems, Minneapolis, USA), as described previously [14]. Optical density was read with a microtitre plate photometer Stat FaxR 2100 (Alab, Poland) at 450 nm and corrected by subtraction of readings at 540 nm. The concentration of TGF-h1 in a sample was determined by interpolation from a standard curve, prepared with standard samples supplied by the manufacturer and expressed as ng/ml. Mucosal TGF-h1 concentration was expressed as pg/ml of protein that was measured by the method of Lowry et al. [15].
and linear regression was performed. Values of p < 0.05 were considered to be significant.
2.3. Statistical methods Values were expressed by their mean and standard error of the mean ( F S.E.M.). The significance of the difference was calculated by two-tailed Student’s t-test. For correlation analysis, the Pearson product moment correlation was used
Table 2 Correlation expressed through r-value and its significance (*p < 0.05) between analysed indices of the disease activity and plasma or mucosal TGF-h1 concentration in all patients with ulcerative colitis Endoscopic score DAI CAI CRP SR
Plasma TGF-h1
Mucosal TGF-h1
0.47* 0.52* 0.46* 0.26 0.01
0.33 0.48* 0.49* 0.18 0.14
3. Results Plasma mean concentration of TGF-h1, measured before the treatment in all patients with active UC, was significantly elevated (56.8 F 7.2 ng/ml) in comparison to healthy controls (18.3 F 1.6 ng/ml). The highest values were observed in patients with the severe course of the disease (74.2 F 14.0 ng/ml), and they were significantly higher ( p = 0.03) than in group with mild one (43.7 F 5.6 ng/ml), (Fig. 1). Mean levels of TGF-h1 measured in mucosal samples from patients with severe UC (563 F 146 pg/mg protein) doubled values from patients with mild UC (286 F 65 pg/mg protein), but the difference was not significant (Fig. 2). As demonstrated in Table 1, treatment resulted with significant improvement of analysed indices of the disease activity except sedimentation rate in both groups. Treatment of patients with both mild and severe UC resulted with significant decrease of plasma TGF-h1 concentration to values (19.9 F 3.6 and 33.5 F 10.9 ng/ml, respectively) below the half of initial level (Fig. 1). This trend was even more intensive in respect to TGF-h1 concentration in rectal mucosa that was three times lower after treatment in both groups (94 F 23 and 168 F 37 pg/mg protein, respectively), (Fig. 2). As shown in Table 2, plasma TGF-h1 levels analysed in all UC patients revealed significant positive correlations with scored endoscopic degree of mucosal injury, DAI and CAI. Mucosal TGF-h1 correlated significantly only with values of DAI and CAI (Table 2). There was no correlation with sedimentation rate in respect to both plasma and mucosal TGF-h1 concentration. As demonstrated in Table 3, patients who responded completely had higher plasma and mucosal TGF-h1 that decreased significantly after treatment. In patients who did not respond completely, significant decrease after treatment was related only to plasma TGF-h1 (Table 3).
4. Discussion Transforming growth factor h1 has several important functions in the pathogenesis of inflammatory bowel dis-
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eases that include control of epithelial cells proliferation and differentiation, immunosuppresion and finally, regulation of extracellular matrix (ECM) formation. In the initial phase of bowel inflammation, TGF-h1 inhibits proliferation and stimulates differentiation of epithelial cells that aids in the repair process of the mucosa following the damage [1]. Immunosuppressive effect is related to inhibition of both B and T cells activity that was strongly supported by the study on TGF-h null mice, which died because of multifocal inflammation [16]. Inhibitory effect of TGF-h1 on Th1-mediated experimental colitis through suppression of interleukin-12 signalling and interferon-gamma transcription suggests its possible immunosuppressive activity in Crohn disease considered as Th1-mediated disorder [5]. Unfortunately, we were not able to carry out the study in patients with Crohn disease because of extremely low incidence of the disease in this part of Europe. Since it has been demonstrated by Seder et al. [17] that IL-4 and IL-10 stimulate TGF-h1 production by CD4 + cells, its increased concentration may reflect Th2mediated T cell response in UC patients. Additionally, TGFh1 affects inflammation in bowel through induction of cyclooxygenase-2 and subsequent increased prostaglandin production [18]. As we demonstrated previously, plasma and mucosal concentrations of prostaglandin E2 correlate significantly with the degree of mucosal injury [19]. However, the most important role of TGF-h1 is related to fibrosis stimulation and wound healing. Role of transforming growth factor h1 in the pathogenesis of diseases associated with enhanced ECM formation has been established recently [14,20 –22]. Fibrosis is a major complication of inflammatory bowel diseases and according to Lawrance et al. [23] collagen secretion from mucosal fibroblasts is stimulated by growth factors, including TGF-h1. Because of this effect, TGF-h1 seems to be implicated in stricture formation and muscle hypertrophy occurring sometimes as a complication in inflammatory bowel diseases [8,18]. Lack of TGF-h dependent control, because of the receptor ThRII gene mutation, was demonstrated in patients with UC-associated bowel neoplasma and therefore, TGF-h should be considered as the major suppressor of bowel carcinogenesis [24]. According to Grainger et al. [25], the most important factors affecting measurement of TGF-h1 in human is preparation of samples with minimal contamination from platelets as a powerful source of this cytokine and methodology that enables to use one particular assay for detection of multiple latent TGF-h1 complexes. The Quantikine ELISA System is recommended mostly because of relatively quick and simple activation with acid and urea that disrupt majority of TGF-h1 complexes [26]. Mean plasma concentration of TGF-h1 measured in our healthy controls with this method was consistent with range from more than 20 studies reviewed by Grainger et al. [25]. In this study, we demonstrated a significant increase of TGF-h1 concentration in plasma of patients with ulcerative colitis that rised depending on the degree of mucosal injury. Additionally, this association was confirmed with other
classifications (DAI and CAI) that are frequently used for evaluation of UC activity. We also demonstrated significant correlation between these indices and TGF-h1 concentration in rectal mucosa. The possible source of this growth factor can be cells located closely to the bowel lamina propria of affected mucosa that have been demonstrated as a place of enhanced expression of TGF-h1 [7]. McKaig et al. [10] found cultured intestinal myofibroblasts as a possible source of this cytokine in humans with UC. According to Monteleone et al. [6], blockade of TGF-h1 caused by Smad7 activation helps to maintain the chronic production of proinflammatory cytokines responsible for the inflammation in UC. As demonstrated recently by Hahm et al. [27], deficiency of TGF-h1 signalling contributes to the development of inflammatory bowel disease. Therefore, insufficient response to TGF-h1 due to Smad7 effect can be a reason of enhanced TGF-h1 production. However, up to now there was almost no information about a possible association between plasma TGF-h1 and the disease activity. Sturm et al. [9] demonstrated unsignificant tendency towards increased TGF-h1 values in ulcerative colitis. In the study of Sambuelli et al. [28], circulating TGF-h1 was significantly higher than in controls but results are not reliable because the measurement was performed in serum, so unpredictable part of TGFh1 could be released from platelets. However, even in this study, elevated values at the beginning of the treatment were observed only in patients who responded and they significantly decreased after the treatment. In our study, patients with uncomplete response to the treatment had also lower baseline levels of TGF-h1 both in plasma and mucosa than complete responders. It is consistent with mentioned research on the role of insufficient TGF-h1 signalling in the pathogenesis of UC [6,27] and recent observations of Whiting et al. [29] who proved low levels of active TGF-h as a possible factor contributing to unresolved experimental colitis. Taken together, plasma and mucosal concentrations of transforming growth factor h1 are strongly associated with ulcerative colitis activity, and successful treatment of the disease results with decrease of their levels. More effective response to the treatment can be achieved in patients with higher baseline concentrations of TGF-h1.
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