Departments of 1Internal Medicine, 2Histology and Embryology, and 3Molecular Biology, Biochemistry, and Biopharmacy, Medical University of Silesia, Katowice, Poland; and 4Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania
Submitted 13 August 2004 ; accepted in final form 22 March 2005
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ABSTRACT |
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ulcerative colitis; Crohn's disease; bradykinin
In the past two decades, the role of plasma kallikrein in chronic inflammation has been well delineated (4). In our models of inflammatory arthritis and enterocolitis induced by proteoglycan-polysaccharide (PG-APS), activation of plasma kallikrein was documented in the genetically susceptible Lewis rat but not in the resistant Buffalo rat (12). This activation is not specific for PG-APS since it can be induced by indomethacin in Lewis rats (23). A selective plasma kallikrein inhibitor has been shown to attenuate acute (19) and chronic (22) enterocolitis in the Lewis rat at low concentrations, which selectively inhibits plasma kallikrein. Activation of plasma kallikrein has been documented in patients with the active phase of ulcerative colitis (UC; see Ref. 20). Agents that block BK receptors also modulate experimental inflammatory arthritis induced by PG-APS (23, 24). Recently, we have shown that experimental enterocolitis is 60% inhibited in rats deficient in HK and LK in a Lewis genetic background (8a).
Less attention has been paid to the role of tissue kallikrein (TK; see Ref. 6) in the pathogenesis of inflammatory bowel disease (IBD). We have identified TK in goblet cells of the normal and inflamed rat colon (18), and we postulated that intestinal tissue kallikrein (ITK) was secreted at higher levels in inflammation. Recently we localized human TK and the TK inhibitor kallistatin (5) immunochemically in the intestinal mucosa of patients with IBD and normal subjects (21). The amount of immunoreactive TK and kallistatin was measured, and mRNA was assessed for TK and kallistatin. Because the tissue level of TK and its inhibitor (kallistatin) was decreased, we concluded that ITK was secreted during human IBD inflammation.
The aim of this study was to ascertain the concentrations of the B1R and bradykinin receptor 2 (B2R) proteins and the expression of genes encoding of the B1R and B2R in inflamed tissues compared with controls. The alterations in both the distribution and levels of B1R and B2R proteins and mRNAs in patients with IBD compared with controls suggest a possible role of the kinins in the inflammatory process. Increased expression of B1R in active IBD provides a structural basis for kinin function.
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MATERIALS AND METHODS |
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Blood was obtained from all patients and controls by venipuncture from the antecubital vein and anticoagulated in 3.8% sodium citrate [9:1 (vol/vol)]. All samples were centrifuged at 3,000 g for 15 min. Plasma 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 Hospitals. The patients gave written, informed consent.
Immunolocalization of kinin receptors in human colonic tissue. The specificity of rabbit anti-human polyclonal antibody reactivity to B2 (mix AS28783) and B1 (mix AS434) used by us in all experiments has been reported. Antibodies were kindly donated by Dr. Werner Muller-Esterl, Institute of Biochemistry II, The University of Mainz (Mainz, Germany). Eight samples of normal colonic tissue were obtained from patients undergoing partial colectomy for colon cancer, and inflammatory intestinal tissue samples were obtained after surgery from UC or CD patients. Tissue samples were fixed overnight in 10% neutral-buffered formalin (phosphate buffer). The samples were subsequently passed through graded alcohol solutions, processed three times in xylene, and finally embedded in paraffin blocks. Slices of 5 µm thickness were placed on Apes-coated slides, deparaffinized, and rehydrated. To unmask the antigen, sections were boiled in 0.01 M citrate buffer (pH 6.0) in a microwave oven for 10 min at 800 watts. Thereafter, sections were allowed to stand undisturbed for 20 min and washed two times (5 min each) in Tris-buffered saline (TBS, 0.05 M, pH 7.4, and 0.85% NaCl). For quenching of endogenous peroxidase activity, tissue sections were blocked with 3% (vol/vol) H2O2 for 10 min. Before incubation with the primary antibody, the sections were washed two times (5 min each) in TBS and were pretreated with normal goat serum for 30 min. The primary rabbit polyclonal antihuman antibodies to B2R and B1R were added, and the sections were incubated overnight (4°C). This step was followed by incubation with biotinylated secondary antibody, and final visualization was achieved by using the avidin-biotin complex peroxidase kit (Vector Laboratories, Burlingame, CA), freshly prepared 3,3'-diaminobenzidine tetrahydrochloride, and hydrogen peroxide according to the protocol provided by the manufacturer. Normal rabbit serum instead of anti-B2R or anti-B2R antibodies was used as a control (9, 21).
Expression of B1R mRNA and B2R mRNA kinin receptors. In the first series of the experiment, specimens of rectal biopsies were obtained from patients with UC (n = 28) and noninflamed controls (n = 27) during colonoscopy. There were 12 UC men and 16 women, with a median age of 44 yr. The control subjects (healthy subjects or individuals with gastrointestinal disturbances) were compatible with both subgroups (active and inactive UC patients) and matched for age and sex. Mucosal disease activity of UC patients was assessed as active (n = 17 UC patients) or inactive (n = 11 UC patients) using the modification of endoscopic index of Baron et al. (2), taking into consideration the mucosal vascular pattern, erythrema, friability, granulation, erosions, and ulcerations. The patients were given conventional medical treatment. All UC patients received sulfasalazine or its derivatives. Five UC active patients and two UC inactive patients were taking moderate doses of corticosteroids, and three of the UC patients (two in the active stage and one in the inactive stage) were on azathioprine. The UC colonic extent of disease was variable; however, the specimens were taken from the rectum in all cases. The diagnosis of UC was confirmed histologically; rectal biopsy specimens were stained with hematoxylin and eosin to determine the grade of histological inflammation.
For molecular evaluation, rectal biopsy specimens of 4 x 4 mm were removed by endoscopic forceps. The tissue samples were immediately frozen at 70°C until use. The expression of B1 and B2 kinin receptor genes in biopsy specimens was evaluated as a number of mRNA copies by QRT-PCR using Sequence Detector ABI PRISM 7700 by Perkin-Elmer. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction was employed.
The numbers of mRNA molecules for B1R and B2R in 1 µg total RNA were detected with an ABI PRISM 7700 Sequence Detector and with the primers and probe designed with the use of Primer Express 10 software. This was done on the basis of mRNA sequences obtained from the internet databases of http://www.ebi.activation.uk and http:www.ncbi.nlm.nih.gov/PubMed in relation to the RT-PCR for -actin, a marker of the number of copies for all genes studied in each reaction. We used the following complementary oligonucleotides: B2R, FBR2: 5'-CAC GGT GCT AGT CCT GGT TGT GCT-3', RBR2: 5'-AGG TCC GCA GTG TGC CCA TG-3', and SBR2: 5'-TCC AGC TGC CAG GAC GAG CGC ATC A-3'; B1R, FBR1: 5'-CTG CAC AGA GTG CTG CCG ACA TT-3', RBR1: 5'-ACA CCA GAT CAG AGG CTG CCA GG-3', and SBR1: 5'-CCG GCG GCA ACT GAC GTG GCA GAA-3'. Probes used for the quantitative RT-PCR analysis were coupled with the following fluorescent adducts: 5'-end with 6-carboxyfluorescein, 3'-end with 6-carboxy tetramethylrodamine. The number of mRNA molecules for B1R and B2R in 1 µg total RNA were quantified using real-time PCR (TaqMan). First, a standard curve was constructed after amplification of the five concentrations of
-actin standards (26, 260, 2,600, 5,200, and 26,000 copies) using a
-actin Control Reagent Kit (Applied Biosystems). A standard curve was created for each separate experiment. As constructed, a 10-fold increase in the number of copies for
-actin mRNA contributes to the increase of twofold of the threshold cycle (Ct). The Ct is the number of cycles before the fluorescence emitted reaches a fixed limit. The log10 of the number of targets initially present in the sample is proportional to the Ct value and can be measured using the standard curve. In addition, for each sample, a number of mRNA
-actin (constitutive gene) copies was evaluated as an endogenous control of real-time PCR reaction. Finally, the number of B1 and B2 mRNA molecules present in each examined sample was calculated for 1 µg total RNA. Total RNA level was measured spectrophotometrically using the program GeneQuant DNA/RNA Calculator (Pharmacia).
Kinin receptor levels in intestinal tissue samples. In the second series of the investigation, colonic tissue specimens were taken from 15 UC active patients (10 men and 5 women, 50 yr average age), 15 UC inactive patients (8 men and 7 women, 44 yr age average), and 12 normal control subjects (6 men and 6 women, 49 yr age average). These biopsy samples for immunohistochemical analysis were transferred directly to tubes containing 10% buffered formalin, fixed for 4 h, and prepared as described above.
In the third series of the study, the colonic tissue samples were obtained from 10 active CD patients undergoing partial intestinal resection (4 men and 6 women, 39 yr average age) and 10 controls (normal colonic samples) matched according to sex and age. All UC patients were treated with sulfasalazine, five active and two inactive patients with corticosteroids, and four active and four inactive patients with azathioprine. Six CD patients had corticosteroids, with three of these additionally having azathioprine and two azathioprine alone.
Tissue samples were prepared and stained with rabbit anti-human polyclonal antibodies for B2R and B1R, as described above. The number of positively stained epithelial cells for B2R and B1R in eight different areas (173 x 130 µm) of each tissue sample was determined using computer-assisted image analysis. In each enterocyte, the intensity of staining was measured as the optical density of the reaction product, as described previously (13), with the program KS 100 VIDAS video image analyzer served by IBAS 2.5 system and a Panasonic WV-CL700 digital camera (Kontron Elektronic, Warsaw, Poland) at a magnification of 25-fold (10, 11, 21). For each analyzed area, 173 x 130 µm average optical density per unit area was calculated. Finally, the arithmetic mean was calculated from 160 microscopic images for controls (20 tissue samples x 8 areas), 120 for UC, and 120 for CD.
Statistical analysis. The significance of differences between the means of each group was determined by the Student's t-test. In the immunochemical data, the means were compared using the Kolmogorov-Smirnov test and the Mann-Whitney U-test. Because the mRNA concentration was distributed logarithmically, we evaluated the significance of the differences between the controls, active UC patients, and inactive UC patients for the measurement for B2R mRNA and B1R mRNA expression using the logarithmic values of each parameter. The means were compared using the Mann-Whitney U-test. All values were expressed as means ± SE.
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RESULTS |
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Quantitation of kinin receptors in intestinal samples. Positive immunohistochemical reaction for both B1R and B2R was observed in a vast majority of enterocytes in both normal and inflamed colon. To compare the level of both B1R and B2R in normal and inflamed intestinal tissue, we quantified the intensity of staining in mucosal tissue samples using image analysis. The units are optical density per unit area (see Kinin receptor levels in intestinal tissue samples). The mean level of B1R in the apical part of enterocytes in active UC [124.5 ± (SE) 11 SE] and inactive UC (107.9 ± 7.7) was significantly (P < 0.0001) higher compared with noninflamed control (50.5 ± 1.9; Table 1). However, there was no statistical difference in the mean level of B1R in the apexes of enterocytes between the UC active and UC inactive patients. In contrast, image analysis revealed the significant decrease (P < 0.0001) of B1R in the basal part of enterocytes in UC active inflammatory specimens (69.8.6 ± 3.2) and UC inactive specimens (62.4 ± 3.2) compared with normal controls (106.9 ± 9). Finally, the total level of B1R was significantly higher (P < 0.003) in enterocytes of patients with the active phase of UC (110.8 ± 2.5) compared with controls (87.9 ± 2.7; Table 2).
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Similarly, the B1R and B2R levels were calculated in tissue samples of active CD patients (Table 3). The mean level of B1R was found to be significantly higher (P < 0.001) in the apical part (130.6) and basal part (101.3 ± 13.8 of enterocytes in CD patients compared with controls; 107.4 and 101.3, respectively). In contrast, the mean level of B2R significantly decreased (P < 0.001) in the apical part of CD enterocytes (143.9 ± 25.0) compared with controls (165.4 ± 22), whereas in the basal part of CD enterocytes, the B2 level was higher (128.2 ± 20, P < 0.001) than in controls (109.2 ± 18.6).
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DISCUSSION |
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We postulated that, if human IBD resembled the rat, many of the same findings would occur in human IBD as in experimental rat enterocolitis. We extended the findings and enhanced them by characterizing the cellular localization, concentration, and mRNA of kallistatin in addition to ITK. The demonstration that ITK is in the goblet cells of normal and inflamed human colon and macrophages of intestine for IBD (21) is in agreement with our previous findings in rats (18).
A new observation was that plasma cells on the border of the granuloma contain large amounts of TK. This observation emphasizes the close relationship between the immune responses important in IBD and inflammatory mediators, including the kallikrein-kinin system. We again find that TK levels were significantly decreased in inflamed intestinal tissue, and we observed the decrease of intestinal kallistatin concentration in IBD using two different methods. In normal colon, kallistatin is physically separate from TK, but, during inflammation, the inhibitor is available to neutralize TK. This process is reflected in the plasma where kallistatin is decreased in both UC and CD relative to normal subjects, similar to the findings in the rat model. Kininogen has been demonstrated in both normal and inflamed human colon; thus, TK can generate BK.
In the present study, we investigated for the first time the expression of genes encoding kinin receptors and the precise localization of these two proteins in normal and human inflamed intestinal tissue. Using specific immunostaining and image analysis for quantification, we observed in the normal intestine not only constitutive B2R but also inducible B1R, which suggests that both receptors may play a role in intestinal physiology. In general, in the normal condition, B1R is present in most tissue only in minimal amounts but is induced during the inflammatory process upon exposure to proinflammatory cytokines, especially IL-1 and endotoxins. Recently, Sawant et al. (16) assessed immunoreactive kinin receptors in normal and inflamed gastric mucosa. In contrast to our data, they observed only B2R, but not B1, in unchanged tissue, whereas B1R protein was upregulated during gastritis. However, in normal rectum (in contrast to healthy gastric mucosa), a moderately increased leukocyte infiltration may reflect the physiological response to normal microbial stimuli. Thus normal gut is not inflamed; but, in the presence of inflammatory cells such as leukocytes, this also can be considered normal in rectal tissue.
In inflamed active IBD tissue, we documented a significantly higher B1R protein level compared with the controls, in all probability stimulated by endotoxin, which is detectable in almost all active IBD patients and, in turn, may mediate endothelial perturbation that results in leukocyte stimulation with cytokine release. We also observed a colocalization of TK and kallistatin as well as kinin receptor proteins in the endothelium of intestinal vessels. In macrophages forming granulomas, we found positive staining again for TK kallistatin and the B1R, but not the B2R, indicating the significance of the B1R-related pathway in Crohn's inflammation.
Despite a moderate increase of B1R protein level in active inflammatory tissue, the mRNA level for both B1R and B2R was found to be reduced in inflamed colon compared with the normal control. The discrepancy between the receptor protein concentration and its mRNA level is difficult to explain. In UC, local homeostasis is disrupted, leading to active inflammation to epithelial cell loss and ulcer formation. However, in the present study, we calculated gene expression for kinin receptors in relation to -actin gene expression. The
-actin gene is unchanged in inflammation and thus serves as an endogenous denominator of gene expression from the same cell. Thus the decrease of both B1 and B2 kinin receptor encoding genes is the result of an alteration of transcription rather than an effect of cell loss. It is well recognized that exposure to endotoxins may cause, in vitro and in vivo, a refractory state to the synthesis of cytokines and nitric oxide. Alternatively, an abundance of protein for both kinin receptors in the vast majority of enterocytes may feed back to inhibit new kinin receptor synthesis and lower mRNA level in inflamed tissue. In addition, the B2R may be recycled several times in the same enterocytes after internalization (1a). This process is supported by the appearance of B2R intracellularly in some enterocytes in UC intestine (17). In contrast, the B1R is thought to not be internalized, but rather redistributed, to caveolae-related lipid rafts in the cell membrane (14). Regardless of transcriptional and/or translational modification, the most striking of our results is the increase in the ratio of B1R to B2R gene expression in relation to the degree of intestinal inflammation. Thus the increase of B1R gene expression (relative to B2R gene expression) is concomitant with an increase of B1R protein concentration in UC active intestine, indicating that B1R is a major structural background for kinin function in IBD. These observations provide insight in the pathogenesis of IBD and suggest that selective B1R inhibitors may have potential in a therapeutic trial. However, we have found (24) that B1R antagonists in rat enterocolitis appear to aggravate the inflammation, suggesting that further studies are needed.
What is the role of kinins in the inflamed colon? BK not only has many proinflammatory actions, as indicated earlier, but also can stimulate release of mediators for endothelial, epithelial, and white blood cells, such as thromboxanes (12), nitric oxide (8), and cytokines, known to be important in IBD. Receptors for these reactions are present in the human intestinal epithelium; thus, BK can initiate these inflammatory reactions in the inflamed ileum and colon.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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