Protective effects of neurokinin-1 receptor during colitis in mice: role of the epidermal growth factor receptor

Abstract

1. The role of substance P and its high affinity neurokinin-1 receptor in colitis has not been fully elucidated. We assessed the participation of neurokinin-1 receptor in colitis using the 2,4,6,-trinitrobenzensulphonic acid and dextran sulphate-induced animal models of colitis and genetically-engineered, neurokinin-1 receptor-deficient mice. 2. Clinical signs, macroscopic and histologic damage associated with 2,4,6,-trinitrobenzensulphonic acid (12 days) and dextran sulphate (5 days) colitis were more severe in neurokinin-1 deficient than in wild-type mice, while immunoreactivities for epidermal growth factor and its receptor were similar in the colon of both mice strains before and after colitis. 3. Substance P, dose-dependently induced intestinal fibroblast proliferation and enhanced epidermal growth factor-induced proliferation in intestinal fibroblasts isolated from wild-type, but not from neurokinin-1 receptor deficient mice. 4. Substance P-induced intestinal fibroblast proliferation required the presence of epidermal growth factor receptor with kinase activity. Furthermore, substance P induced epidermal growth factor tyrosine phosphorylation and activation in normal intestinal fibroblasts. 5. Our results indicate that in mice lacking the neurokinin - 1 receptor, substance P plays a protective role in prolonged experimental colitis.

Figure 1

Increased severity of colitis in NK-1R^−/− mice. TNBS or DSS colitis was induced in wild-type (±/±) (B and C) and NK-1R^−/− mice (−/−) (panels E & F) as described in Methods. Animals were sacrificed after 5 days (DSS) or 12 days (TNBS) and the colon was removed, fixed in 4% PFA, and full thickness sections stained with H&E. Colon from control animals exhibited normal architecture in both wild-type (A) and NK-1R^−/− mice (D). In contrast, animals treated with TNBS (B and E) showed damage characterized by necrosis, disruption of the mucosal architecture, granulocytic infiltrate, and mucosal ulcers. However ulcers and inflammatory damage associated with TNBS colitis were more severe in NK-1R^−/− mice than in wild-type (compare B and E). During DDS colitis (C and F) inflammatory infiltration and superficial ulcerations were evident. However, ulcerative lesions were more pronounced in NK-1R^−/− mice. Original magnification×160.

Figure 2

Expression of EGF during colitis in NK-1R^−/− and wild-type mice. TNBS (12 days) or DSS (5 days) colitis was induced in wild-type (±/±) (B and C, respectively) and NK-1R^−/− (−/−) (E and F, respectively) mice and colonic sections were processed for detection of EGF-like immunoreactivity as described in Methods. Confocal microscopy showed little EGF-like immunoreactivity in the normal colon of wild-type (A) and NK-1R^−/− mice (D). EGF-like immunoreactivity increased in cells localized in the colonic lamina propria during TNBS (B and E) and DSS (C and F) colitis in wild-type and in NK-1R^−/− mice. Results are representative of three experiments for each experimental condition. Original magnification×300.

Figure 3

Expression of EGF-R during colitis in NK-1R^−/− and wild-type mice. TNBS (12 days) or DSS (5 days) colitis was induced in wild-type (±/±) (B and C, respectively) and NK-1R^−/− (−/−) (E and F, respectively) mice and colonic sections were processed for immunohistochemical detection of EGF-R as described in Methods. Sections were examined by confocal microscopy. Note the presence of modest signal for EGFR in the normal colon of wild-type (A) and NK-1R^−/− mice (D). EGFR-immunoreactivity increases in the colonic mucosa both in wild-type and NK-1R^−/− mice during TNBS (B and E, respectively) and DSS colitis (C and F, respectively). Results are representative of three experiments for each experimental condition. Original magnification×300.

Figure 4

SP enhances EGF-R-mediated DNA synthesis in intestinal fibroblasts. Intestinal fibroblasts from wild-type (A) and NK-1R^−/− (B) mice were plated in 12-well plates (2.5×10⁴ cells cm⁻²) pre-coated with fibronectin as described in Methods. After 48 h cells were serum-starved for 24 h and then incubated with SP (100–0.1 nM), EGF (100–1 ng ml⁻¹) or SP and EGF (1 nM and 1 ng ml⁻¹, respectively) in the presence of [³H]-thymidine (0.5 μCi well⁻¹). After 12 h, cells were collected and [³H]-thymidine incorporation was determined. (A) In fibroblasts isolated from NK-1R^±/± mice, SP and EGF dose dependently increased DNA synthesis. Co-incubation of intestinal fibroblasts with SP and EGF, at doses ineffective to induce DNA synthesis, significantly increased DNA synthesis. (B) In fibroblasts obtained from NK-1R^−/− mice EGF caused a proliferative response, which was similar to that obtained from NK-1R^±/± mice. SP, however, failed to stimulate a significant [³H]-thymidine uptake and had no synergistic effect on EGF (1 ng ml⁻¹) - induced [³H]-thymidine uptake. Results are expressed as % change of DNA synthesis over control and are representative of three different experiments each with triplicate determinations. *, and ** denote P<0.05 and P<0.01 vs SP-treated cells, respectively;±±, denotes P<0.01 vs EGF alone (1 ng ml⁻¹)-treated cells.

Figure 5

SP-induced DNA synthesis in intestinal fibroblasts requires transactivation of the EGF-R. Mock or pCHER transfected fibroblasts were serum-starved and incubated with SP (10 nM) or EGF (10 ng ml⁻¹) in the presence of [³H]-thymidine (0.5 μCi well⁻¹) for 6 h. Cells were collected and placed in scintillation vials to determine [³H]-thymidine incorporation. Results were expressed as per cent change of DNA synthesis over control. SP and EGF increased DNA synthesis in mock-transfected cells while pCHER-transfected cells, SP- and EGF-induced DNA synthesis was significantly inhibited. Data are expressed as mean±s.e.m. and are representative of three different experiments each with triplicate determinations. ** indicates P<0.01 vs SP-treated mock transfected cells and ++ indicates P<0.01 vs EGF-treated mock transfected cells.

Figure 6

SP induces tyrosine phosphorylation of EGF-R and the formation of an activated EGF-R complex. Intestinal fibroblasts incubated with SP (10 nM) or EGF (20 ng ml⁻¹) for the indicated time periods were lysed in RIPA buffer. Soluble proteins were immunoprecipitated with an anti EGF-R antibody and then fractionated on a 7.5% SDS–PAGE gel. Proteins were detected using an anti-phosphotyrosine antibody. SP, time dependently, induces increased tyrosine phosphorylation of EGF-R and multiple co-precipitating proteins. Data are representative of four different experiments that gave similar results.