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. 1998 Sep;43(3):365–374. doi: 10.1136/gut.43.3.365

Kallikrein-kininogen system activation and bradykinin (B2) receptors in indomethacin induced enterocolitis in genetically susceptible Lewis rats

A Stadnicki 1, R Sartor 1, R Janardham 1, I Stadnicka 1, A Adam 1, C Blais 1, R Colman 1
PMCID: PMC1727239  PMID: 9863482

Abstract

Background—The plasma kallikrein-kinin (K-K) system is activated in acute and chronic relapsing intestinal inflammation induced in Lewis rats by intramural injection of exogenous bacterial components. 
Aims—To determine whether this effect is model specific, K-K system activation was investigated in a modified indomethacin induced enterocolitis model, as well as bradykinin 2 (B2) receptor distribution in the normal and acutely inflamed intestine. 
Methods—Lewis rats injected with daily sublethal doses of indomethacin for two days developed acute (two days) and chronic (14 days) intestinal inflammation. Plasma prekallikrein (amidolytic), high molecular weight kininogen (HK, coagulant) and cleavage of HK (western blot) were assayed to detect K-K activation. 
Results—Liver and spleen weights were significantly higher, and body weights and haematocrit values were significantly lower in the indomethacin group than in the control group. During both acute and chronic phases, rats displayed K-K system activation manifested by a significant decrease in plasma prekallikrein and HK functional levels, and by HK cleavage. Plasma T kininogen (a major acute phase protein) was significantly elevated. B2 receptors were identified in both normal and inflammatory intestine with more prominent specific immunohistochemical staining in the acutely inflamed tissue. 
Conclusions—K-K system activation occurs in association with both acute and chronic phases of intestinal injury, regardless of the triggering agent, suggesting that activation of this system is integrally involved in intestinal inflammation in genetically susceptible hosts. Localisation of B2 receptors across intestinal layers provides a structural basis for the kinin function in the intestine. 



Keywords: plasma kallikrein; kininogen; bradykinin 2 receptor; enterocolitis; indomethacin; proteoglycan-polysaccharide

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Figure 1 .

Figure 1

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Histological evidence of enteritis induced by indomethacin in female Lewis rats. (A) At day 2, after subcutaneous injection of 7.5 mg/kg indomethacin for two days, a large ulcer with relatively bland necrosis of the entire mucosa is seen with severe serosal and mesenteric inflammation with neutrophils and macrophages (original magnification ×100). (B) At day 14, there is an extensive ulcer with an active mucosal exudate. A crypt abscess is visible (arrow) and glandular dysplasia with depletion of goblet cells. Transmural inflammation with thickening of the submucosa is evident. The infiltrate consists of both mononuclear cells and neutrophils with fibroblast proliferation and smooth muscle hypertrophy. Collagen indicative of early fibrosis is present (original magnification ×100). 


Figure 2 .

Figure 2

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Gross appearance of intestinal inflammation in indomethacin treated Lewis rats. (A) Thickening of the bowel wall with creeping mesenteric fat (closed arrow) and adhesions (open arrow) is present at day 14 after injection of indomethacin on days 0 and 1. (B) Longitudinal ulcers (arrow) on the mesenteric border are seen in the mid small bowel at day 14. 


Figure 3 .

Figure 3

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Functional assays of contact system proteins. (A) Prekallikrein; (B) factor XI; (C) high molecular weight kininogen. *p<0.05, **p<0.01, ***p<0.001. Solid bars indicate control groups; shaded bars indicate indomethacin treated groups. 


Figure 4 .

Figure 4

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High molecular weight kininogen cleavage in Lewis rats treated with indomethacin shown by western blotting using anti-high molecular weight kininogen antibodies. Lane 1 shows pooled non-activated normal rat plasma (N), and lane 2 pooled rat plasma following exposure to kaolin (K). Lanes 3-5 represent the plasma samples of three control rats, while lanes 6-8 are plasma samples of rats 14 days after treatment with indomethacin. The 120 kDa band represents HK and is visualised across lanes 1 and 3-8, but is absent in lane 2. The broad band localised at about 68 kDa across all lanes indicates cross reacting T kininogen (TK) and albumin. The band situated between albumin and the 43 kDa standard, visualised on lane 2 and lanes 6-8, but not on lane 1 and lanes 3-5, represents the heavy chain of high molecular weight kininogen (HC-HK, 62 kDa). The delicate band situated just above 32 kDa, standard across all lanes, represents a non-specific reaction with alkaline phosphatase that hydrolyses the BCIP/NBT substrate used to develop the colour. 


Figure 5 .

Figure 5

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Plasma T kininogen levels during acute phase (two days) and chronic phase (14 days) of inflammation after indomethacin injection (mean (SEM)). T kininogen levels are expressed as µg/mg plasma protein. ***p<0.001. Solid bars, buffer control rats; shaded bars, rats treated with indomethacin. 


Figure 6 .

Figure 6

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Immunolocalisation of the kinin B2 receptor in rat intestine on day 2. A-C: Rat normal control jejunum (buffer treated). (A) Some focal staining positive for B2 receptor in the villi—V, occasionally in the muscularis mucosa below the crypts—C, in the muscular layer—M, and in the serosa. Original magnification ×100. (B) Intestinal villi. Focal weak immunostaining in the surface of occasional epithelial cells and in cytoplasm (arrow). Original magnification ×400. (C) Some focal immunostaining, cluster of epithelial cells in crypts (large arrow), in the muscular layers (small solid arrows), and in serosal surface (white arrows). Original magnification ×400. D-F: Rat inflamed intestine (day 2 after injection of indomethacin). (D) Focal immunostaining in the villi—V, especially near the area of ulceration - U, in the internal and external muscular layer—M, and in the serosa. Original magnification ×100. (E) Villus near area of ulceration showing strong staining in the epithelial, probably absorptive cells (arrow). Original magnification ×400. (F) Prominent immunostaining in the smooth muscle cells of the muscularis mucosa (solid arrows) and in the external muscular layer (solid arrows). On the serosal surface, staining is also present (white arrows). Original magnification ×400.


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