MATERIALS AND METHODS

Patients

Paired colorectal mucosal biopsies from 13 patients with IBD (nine with UC, four with CD) were collected at routine colonoscopy. IBD had been confirmed previously by conventional histological, endoscopic, and radiological criteria.

Five patients undergoing colonoscopy, and found to have endoscopically and histologically normal mucosa, served as controls; their diagnoses were haemorrhoids (one), irritable bowel syndrome (two), and previous colorectal carcinoma (two).

For normal controls and patients with IBD and involvement of the sigmoid colon, biopsies were taken from the sigmoid. For patients with CD without disease in the sigmoid, biopsies were taken from the involved area. Table 1 gives details of age, sex, disease type and distribution, and drug treatment.

All patients gave written informed consent and the studies were approved by the East London and City Health Authority research ethics committee.

Assessment of disease activity

Disease activity in UC was assessed endoscopically using a standard score.¹⁷ This score was adapted and used to assess endoscopic disease activity in patients with CD because of the lack of availability of a standard score. Active disease was defined as scores 2 and 3 (contact or spontaneous mucosal bleeding), and inactive disease as 0 and 1 (normal or oedematous mucosa).

Disease activity was also assessed histologically in haematoxylin and eosin (H&E) stained sections of biopsies from patients with both UC and CD using a semiquantitative score, as previously described.¹⁸

Tissue processing

Biopsies collected at colonoscopy were immediately snap frozen in liquid nitrogen and then stored at −70°C until processed for immunostaining. Paired biopsies were placed in formalin for routine Gill's H&E staining.

Immunohistochemistry for TXS

Frozen sections of the biopsies were cut at 6 μm, picked up on silane treated microscope slides, allowed to air dry, and fixed in acetone for two minutes. A monoclonal antibody against human thromboxane synthase (Tü 300),¹⁹ as previously used in studies of placental tissue,²⁰ was applied at a dilution of 1/100 (in antibody diluent) and incubated with the tissue overnight at 4°C. Preliminary optimisation studies using an incubation time of one hour, although showing positive staining in the placenta, were unsuccessful with the colonic tissue. Bound antibody was detected by a standard biotin–streptavidin–peroxidase system using a Vector Elite kit (Vector Labs, Peterborough, UK).

In each immunohistochemistry run, placental bed tissue served as a positive control²⁰ and omission of the primary antibody served as a negative control. Appropriate non-specific staining blocks, including incubation with normal serum (twice for the double staining method), endogenous peroxidase block, intestinal alkaline phosphatase block, and avidin/biotin block were also used as controls. For the double staining method, additional blocks were incorporated in the form of levamisole, an inhibitor of alkaline phosphatase added to the substrate solution to eliminate background.

Test and control slides were stained in one run, both for single and double staining. The amount of TXS signal in the double staining method was comparable to that of TXS alone. Controls were always included. All the staining was performed by one person (CN).

Analysis of staining

All tissue was assessed on each slide. The proportion of lamina propria cells showing positive staining, regardless of the intensity, was determined by an experienced gastointestinal histopathologist (RMF), who was blinded to the diagnosis and disease activity.

Double immunostaining for CD68 and TXS

Double staining using the antibody to TXS as above, together with a monoclonal antibody to the human macrophage antigen CD68, KP-1 clone (Dako Ltd, Ely, Cambridgeshire, UK), was performed. Tissue sections were first incubated overnight with anti-TXS, as described previously, washed thoroughly in distilled water, then incubated for one hour with anti-CD68, diluted 1/1000 (in antibody diluent). CD68 was detected using an alkaline phosphatase detection system, Vector ABC-AP (Vector Labs). Placental bed tissue served as a positive control for the demonstration of TXS and normal appendix served as control for CD68. Omission of the primary stage served as a negative control for each antibody.

Statistical analysis

Statistical comparisons between groups were made using the Mann-Whitney U test (two tailed). Correlations were assessed by Spearman's rank correlation (two tailed).

RESULTS

Immunostaining of TXS in patients with IBD and controls

Immunohistochemical expression of TXS in lamina propria cells was cytoplasmic and often granular. No pattern of distribution within the lamina propria was discernable. The proportion of cells positive for TXS ranged from 1% to 9.5% in controls and 15% to 90% in active IBD.

The proportion of lamina propria cells staining for TXS was higher in patients with active IBD than in those with inactive IBD or controls (p = 0.02 and p = 0.002, respectively; figs 1 and 2).

Correlation of immunostaining and disease activity

There was a direct correlation between the proportion of lamina propria cells staining for TXS and disease activity, as determined both by endoscopic score (p = 0.001) and by histological score on H&E stained biopsies (p = 0.001; fig 3).

Double immunostaining

Double immunostaining for TXS and CD68 showed that a proportion of the cells expressing TXS were macrophages (fig 4).

DISCUSSION

We have demonstrated, for the first time, excess immunostaining for TXS in the cells of the lamina propria, including macrophages, of colorectal mucosal biopsies from patients with IBD. This upregulation of TXS expression, which correlated with disease activity, measured by endoscopic score and histology, may account for the recognised increased production of thromboxanes by inflamed intestinal mucosa^7–11 and in the blood of patients with IBD.²¹

In vitro studies have shown that thromboxanes have proinflammatory actions. Thromboxane induces the activation of neutrophils,²² the production of leukotriene B4,²³ and enhanced endothelial adhesion²⁴ and subsequent diapedesis²⁵ of neutrophils. Thromboxanes also induce apoptosis²⁶ and modulate T cell function,²⁷ and may have a role in ischaemia reperfusion injury.²⁸

“Preliminary studies of thromboxane synthase inhibitors have shown some benefits in patients with ulcerative colitis”

Vasoconstriction²⁹ and platelet aggregation¹ are also induced by thromboxanes. In patients with active IBD, platelets are activated³⁰ and show abnormal aggregation.²¹ Furthermore, platelet aggregates have been identified in the capillaries in inflamed rectal biopsies in IBD,³¹ and microvascular thrombosis has been proposed as an early pathogenic factor in CD.³² It has therefore been proposed that thromboxanes may play a role in the pathogenesis of IBD.²

Preliminary studies of thromboxane synthase inhibitors have shown some benefits in patients with UC^14–16; however, the results of large randomised controlled trials are awaited.

TXS has been purified and is a cytochrome P450 enzyme.³³ It has been identified in kidney,³⁴ spleen,³⁵ brain,³⁶ and placenta,²⁰ in addition to platelets,³⁷ macrophages,³⁸ lung fibroblasts,³⁹ and other cell types. To our knowledge, this is the first study to investigate TXS immunostaining in intestinal mucosa.

The double staining technique confirmed that many cells in the lamina propria staining positively for TXS were also positive for the macrophage marker CD68. This is in accordance with studies in the placenta, where macrophages were identified as a source of TXS,^{20, 40} and indicates that at least some of the excess TXS in inflamed IBD mucosa is located in macrophages. However, it is possible that other types of lamina propria cells, including lymphocytes and endothelial cells, were also expressing TXS in the cases studied here.

Our study has shown that increased TXS expression in lamina propria cells occurs in active IBD. As a result, increased thromboxane synthesis may occur and contribute to mucosal inflammation and intramucosal thrombogenesis.