Reduced susceptibility to dextran sulphate sodium-induced colitis in the interleukin-2 heterozygous (IL-2) mouse

Abstract

Summary Mice homozygous for an inactivation of the interleukin-2 (IL-2) gene develop a T-cell dependent colitis. Heterozygous (IL-2+/-) mice are clinically healthy but have been shown to express reduced levels of IL-2 in the colon. Splenocytes from the IL-2+/- mice had a poorer proliferative response to polyclonal T-cell activation and these mice have reduced numbers of intestinal regulatory T cells (CD4+ CD25+ cells) when compared to wild type mice. When exposed to dextran sulphate sodium (DSS) IL-2+/- mice showed a markedly reduced susceptibility to DSS-induced colitis. While DSS treatment caused a marked increase in both CD4+ and CD8+ colonic T cells expressing increased levels of IL-2, IL-4, and IL-10 in wild type mice none of these changes were seen in IL-2+/- mice. On the contrary, cytokine expression in intestinal T cells of IL-2+/- mice was actually reduced after DSS treatment. These results suggest that reduced levels of IL-2 leads to attenuated activation and function of intestinal T cells in IL-2+/- mice and a failure to react adequately to DSS exposure.

Figure 1

T cells of IL-2^+/− mice exhibit reduced proliferative response to stimulation. Splenocytes of IL-2^+/+ (WT) and IL-2^+/− (HZ) mice were subjected to polyclonal T cell stimulation by anti-CD3 plus anti-CD28 mAbs or left untreated for 24, 48, and 72 hr. Proliferation was estimated as incorporation of ³H-labelled thymidine. Bars indicate mean + 1SD of triplicate cultures. Splenocytes isolated from IL-2^+/− mice had a significantly reduced proliferative response when compared to the IL-2^+/+ mice at all time points. **P < 0·01 and *P < 0·05.

Figure 2

Clinical presentation of DSS induced colitis in IL-2^+/+ and IL-2^+/− mice. (a) Twelve IL-2^+/+ (WT) and 12 IL-2^+/− (HZ) mice were subjected to DSS exposure in the drinking water. The mice were weighed daily and their weight expressed as a percentage (%) of their weight before DSS treatment. Six of the mice in both groups were sacrificed at day 3. Initially no weight reduction could be observed in the two groups of mice. From day 3 and onwards mice from both groups display a rapid loss of weight. (b) Frequency of mice with rectal bleeding upon exposure to DSS. Bleeding was observed from day two and onwards and by day four all of the IL-2^+/+ mice had significant rectal bleeding, whereas the IL-2^+/− mice developed rectal bleeding at a slower rate. (c) Frequency of mice with loose stools upon exposure to DSS. Both groups of mice displayed symptoms of looser stool consistency and initially no difference between the groups could be observed. However, from day 3 and onwards the IL-2^+/− mice clearly displayed less loose stools than the IL-2^+/+ mice. (d) Frequency of mice with diarrhoea upon exposure to DSS. Diarrhoea was defined as uniform watery forms of feces. The IL-2^+/+ also developed diarrhoea at a faster rate than the IL-2^+/− mice. P-values in (b) and (c) were obtained by Fisher's exact test comparing IL-2^+/− and IL-2^+/+ mice.

Figure 3

Histology of colonic mucosa in IL-2^+/+ and IL-2^+/− mice after DSS exposure. (a–d) Before DSS exposure no histological differences of the colonic mucosa could be observed between the IL-2^+/+ and IL-2^+/− mice, both displaying a well defined crypt structure, intact epithelia and normal cellularity of the lamina propria. (e–h) After 3 days DSS exposure no significant differences could be observed in the colonic mucosa when comparing the IL-2^+/+ and IL-2^+/− mice. The cellularity of lamina propria was not significantly increased and the crypt structure was still intact. However, some signs of epithelial erosion and thinning could be seen. (i–l) After 6 days DSS exposure the histological differences between the IL-2^+/+ and IL-2^+/− mice were evident. The lamina propria of the IL-2^+/+ mice shows a massive increase in cellularity (arrows in (i)and (j)), the crypt structure is almost destroyed (arrowheads in (j)) and the epithelial erosion evident. In the IL-2^+/− mice increase of cellularity (arrows in (k)) and epithelial erosion can also be observed but is less marked than in the IL-2^+/+ mice. In the IL-2^+/− mice the crypt structure is still quite well preserved (arrowheads in (l)).Original magnification: 40× in (a), (c), (e), (g), (i), (k), and 220× in (b), (d), (f), (h), (j), (l).

Figure 4

Total histological score of IL-2^+/+ and IL-2^+/− mice after DSS exposure. The histological score of the DSS colitis was determined by grading the extent and level of inflammation, the crypt damage and the area affected. Before DSS exposure the histological score was 0 for both groups and is therefore not included in the figure. By day 3 of DSS exposure (black bars) both groups displayed somewhat increased histological scores with no significant difference between the IL-2^+/+ (WT) and IL-2^+/− (HZ) groups. At day 6 of DSS exposure (grey bars) the IL-2^+/+ mice had significantly higher histological scores when compared to the IL-2^+/− mice. Bars indicate mean + 1SD of histological scores of six individual mice. **P < 0·01 and ***P < 0·001.

Figure 5

DSS colitis is associated with significantly increased frequencies of both myeloid and lymphoid cells in lamina propria of IL-2^+/+ mice but with an increase of only myeloid cell numbers in IL-2^+/− mice. Markers for different immune cell types were used in immunomorphometric analysis of cells of the lamina propria before and at day 6 of DSS exposure (n=6 mice per group). Macrophages (F4/80⁺) and granulocytes (Ly-6G⁺) were significantly increased in frequency in the lamina propria of both DSS-treated IL-2^+/+ (WT) and DSS treated IL-2^+/− (HZ) mice. In the IL-2^+/+ mice T cells (CD3⁺) and B cells (CD19⁺) were also significantly increased upon DSS exposure but not in DSS-treated IL-2^+/− mice. Analysis of the two major subtypes of T cells (CD4⁺ and CD8⁺) showed that none of these increased in number in the DSS-treated IL-2^+/− mice while both subtypes were increased in the IL-2^+/+ mice after DSS exposure. CD25⁺ cells were present normally and increased in frequency after DSS exposure in both IL-2^+/+ and IL-2^+/− mice. Bars indicate mean + 1SD. *P < 0·05, **P < 0·01 and ***P < 0·001.

Figure 6

Influx of macrophages and granulocytes but not T cells in IL-2^+/− mice during DSS colitis. (a–c). Before DSS exposure moderate levels of F4/80⁺ macrophages (arrow in (a)) and few granulocytes (data not shown) were observed in the lamina propria of the colon in both IL-2^+/+ and IL-2^+/− mice ((a) and data not shown). After DSS exposure and the development of colitis an influx of macrophages and granulocytes was observed in both IL-2^+/+ and IL-2^+/− mice (arrows in (b) and (c) and data not shown). (d–f) Before DSS-induced colitis CD3⁺ T cells were found scattered within the lamina propria of both IL-2^+/+ and IL-2^+/− mice (arrow in (d) and data not shown). After DSS exposure and the development of colitis, T cells increased in number in the lamina propria of IL-2^+/+ mice (arrows in (e)). However, no increase was seen in the lamina propria of the IL-2^+/− mice and only few CD3⁺ T cells were found (arrows in (f)).Original magnification in (a–f) is 220×.

Figure 7

IL-2^+/+ but not IL-2^+/− mice show increased expression levels of cytokine mRNAs in colonic T cells after DSS exposure. Expression levels of IL-2 (a), IFN-γ (b), IL-10 (c), and IL-4 (d) mRNAs were determined by real-time quantitative RT–PCR in RNA extracted from CD3⁺ cells freshly isolated from large intestine of IL-2^+/+ and IL-2^+/− mice before and after 6 days DSS exposure. Cytokine mRNA levels were normalized to the content of the house-keeping gene 18S rRNA in the respective sample. (a) At day 0 the IL-2 mRNA level in T cells from large intestine of IL2^+/− (HZ) was 1/3–1/2 of that in T cells isolated from IL-2^+/+ (WT) mice. After DSS treatment, IL-2 mRNA expression levels increased significantly in large intestinal T cells of IL-2^+/+ mice. In contrast, DSS-exposed IL-2^+/− mice showed a tendency for decreased expression levels of IL-2 mRNA. (b)No significant differences were observed for expression levels of IFN-γ mRNA in large intestinal T cells of IL-2^+/+ or IL-2^+/− mice either before or after DSS exposure. (c) There was a tendency for increased expression levels of IL-10 mRNA in both IL-2^+/+ and IL-2^+/− mice upon DSS exposure. (d) Expression levels of IL-4 mRNA were similar in untreated IL-2^+/+ and IL-2^+/− mice. DSS exposure caused significant increases in IL-4 mRNA expression levels in colonic T cells of IL-2^+/+ mice, whereas no increase was seen in IL-2^+/− mice. Results are displayed as the mean value + 1SD of three RNA pools, each pool containing the combined RNA extracted from CD3⁺ cells from six mice. *P < 0·05.