IL-7 is a growth factor for both murine and human, T cell lineage cells. In the mouse, abundant IL-7 mRNA expression has been demonstrated in bone marrow stroma, thymus, spleen, liver, kidney, and keratinocytes (1
). Recent studies have shown that IL-7 is expressed in human thymus, spleen, and keratinocytes (7
), although a potential role of IL-7 in peripheral lymphoid tissues remains unclear. We have demonstrated that human intestinal epithelial cells produce IL-7 that serves as a regulatory factor for proliferation of intestinal mucosal lymphocytes expressing functional IL-7 receptor (11
). In this study, we demonstrated the presence of epithelial cell–derived IL-7 in the normal murine intestine.
To clarify the mechanism by which intestinal epithelial cell-derived IL-7 regulates the mucosal lymphocytes, we investigated transgenic mice carrying murine IL-7 cDNA and SRα promoter. The SRα expression system is shown to be one or two orders of magnitude more active than SV40 early promoter in a wide variety of cell types including lymphoid cells and epithelial cells, and promotes a high level of expression of various lymphokine cDNAs (17
). We established transgenic mice carrying murine IL-7 cDNAs by three promoters, SRα, SV40 early, and metallothionein I (16
). SRα/IL-7 transgenic mice developed severe dermatitis in 7 of 30 founder mice at 4–9 wk of age. However, SV40 early and metallothionein I/IL-7 transgenic mice never developed dermatitis in 9-mo observation periods. Interestingly, some SRα/IL-7 transgenic lines, but not SV40 early/IL-7, or metallothionein I/IL-7 transgenic mice developed anal bleeding and rectal prolapse. Therefore, we used the SRα/ IL-7 transgenic mice in this study. The fact that plural SRα/IL-7 transgenic lines developed rectal prolapse means that this phenomenon was caused by the transgene itself and not by the positional effect of the transgene insertion on chromosome.
In this study, we provide the evidence that SRα/IL-7 transgenic mice developed chronic colitis with perianal bleeding and rectal prolapse. Histological analysis showed erosions, redness, wall thickening with goblet cell-depleted epithelium, and mononuclear cell infiltration. In the gastrointestinal tract, inflammation was limited to the colon and ileum, and the most severe inflammation occurred in the rectum. The inflammatory cells then consisted of lymphocytes, plasma cells, and macrophages in the IL-7 transgenic mice. These mimicked histopathological characteristics of ulcerative colitis in humans (20
In the IL-7 transgenic mice, the SRα/IL-7 transgene was constitutively expressed in the thymus, spleen, kidney, brain, lung, and skin, but not in the liver and muscle as previously reported (16
). Interestingly, development of colitis in the IL-7 transgenic mice was parallel with the SRα/IL-7 transgene expression in the colonic mucosa. Transgenic mice with SRα/IL-7 transgene expression in the colonic mucosa developed colitis, but mice without transgene in the colonic mucosa did not. This result indicated that SRα/IL-7 transgene expression in the colonic mucosa was directly related to chronic inflammation in the colon of transgenic mice. It would be interesting to clarify whether the increased expression of the transgene in the colonic mucosa induces chronic colitis or whether chronic inflammation in the colonic mucosa induces the activation of the transgene. However, conclusive evidence has not been easily obtained. The final cumulative frequencies of colitis in the line 34 IL-7 transgenic mice were not 100%, but 60%. It was quite difficult or impossible to assess which appears earlier in the same mice, the development of colitis or SRα/ IL-7 transgene mRNA expression in the colonic mucosa. However, we found that SRα/IL-7 transgene mRNA expression was not changed in the thymus, spleen, and skin without dermatitis between mice with colitis and mice without colitis (Watanabe, M., unpublished observation). Moreover, we found that 15–20% of transgenic mice with the SRα/IL-7 transgene mRNA expression in the colonic mucosa did not develop chronic colitis. The preliminary histopathological observation of colonic mucosa of these mice demonstrated that they have little infiltration of mononuclear cells in the lamina propria. On the basis of these findings, we suggest that the expression of detectable SRα/ IL-7 transgene mRNA in the colonic mucosa is a sign of developing colitis. However, we cannot deny that after initiation of colitis by the transgene expression, inflammation in the colonic mucosa also induces the activation of the transgene and eventually transgene mRNA expression is increased.
Southern blot hybridization and competitive PCR demonstrated that expression of IL-7 mRNA was increased in the inflamed colonic mucosa of the IL-7 transgenic mice. The distribution of IL-7 mRNA in the colonic tissues was quite interesting. Analysis of differential expression of IL-7 mRNA showed that colonic mucosal lymphocytes expressed a significant amount of IL-7 mRNA, but IL-7 mRNA expression in the colonic epithelial cells was unchanged in the inflamed mucosa. Therefore, increased IL-7 mRNA expression in the inflamed colonic mucosa of IL-7 transgenic mice was derived from the colonic mucosal lymphocytes. Interestingly, immunohistochemical analysis demonstrated that IL-7 protein expression in the colonic mucosa and epithelial cells seemed to be decreased at colitis lesion in the IL-7 transgenic mice. Recent studies demonstrated that intestinal epithelial cells express IL-7 receptor and IL-7 regulates the growth and function of epithelial cells by autocrine or paracrine mechanism (22
). Overexpression of IL-7 in mucosal lymphocytes may lead to the downregulation of IL-7 protein production in the colonic epithelial cells. However, our previous study indicated that IL-7 protein expression by immunohistochemical technique was demonstrated only in the tissue where secreted IL-7 protein was stored and accumulated (11
). Therefore, we assessed IL-7 protein production by isolated cells. Isolated epithelial cells, IELs, and LPLs form the inflamed colonic mucosa of IL-7 transgenic mice produced an increased amount of IL-7. These results indicated that IL-7 production and secretion were increased in the inflamed colonic mucosa of IL-7 transgenic mice in parallel with the increase of IL-7 mRNA, but accumulation of IL-7 protein in the colonic mucosa was decreased in the colitis mucosa. We have shown that intestinal epithelial goblet cells are the major source of mucosal IL-7 in the human intestinal mucosa (11
). In this study, goblet cells in the murine colon also produce IL-7. In the inflamed colonic mucosa of IL-7 transgenic mice, histopathological examination revealed the depletion of goblet cells. This result is consistent with the findings that IL-7 protein accumulation was decreased in the inflamed colonic mucosa of the transgenic mice.
How does mucosal lymphocyte–produced IL-7 in the colonic mucosa of IL-7 transgenic mice affect the proliferation of mucosal lymphocytes? We have demonstrated IL-7 mRNA expression and protein production by human colonic epithelial cell line HT29-18-N2 cells after they differentiated into the goblet cell phenotype (Watanabe, M., unpublished observation). However, these cell lines produced marginal amounts of IL-7 in the culture supernatants though these high levels of expressed IL-7 mRNA. The puzzle regarding the low IL-7 protein production is not specific to the HT29 sublines. As other investigators discussed (9
), almost all of the cells and cell lines that have been found to produce IL-7 release IL-7 protein only at low levels. The case is the most extreme in the human cells, since it was difficult to detect IL-7 protein production even in the SK-HEP cell line that was used to clone the cDNA for the human IL-7 (24
). These observations suggested that IL-7 protein may be rapidly degraded. Locally produced IL-7 from IELs and LPLs in the colonic mucosa may be also degraded or internalized when it does not accumulate in the colonic mucin of the goblet cells. Therefore, we suggest that accumulation of IL-7 protein in the mucus of the goblet cells in the colonic mucosa is crucial for regulating the proliferation of mucosal lymphocytes.
The importance of IL-7 as a mediator of local inflammatory responses remains unclear. However, a possible role for IL-7 in mucosal inflammation has been recently suggested by the finding of decreased IL-7 mRNA expression and IL-7 protein accumulation in the colonic epithelium in inflamed mucosa of patients with ulcerative colitis (Watanabe, M., unpublished observation). We have also demonstrated that IL-7 mRNA expression and IL-7 protein accumulation was significantly increased in the colonic mucosa of patients with acute Salmonella infectious colitis, where goblet cell depletion was not observed. In the IL-7 transgenic mice, decreased IL-7 protein accumulation in the colonic epithelial cells was demonstrated after the onset of colitis. These results favor the idea that the decrease of IL-7 protein accumulation due to the deletion of goblet cells involves the chronic inflammation of the intestinal mucosa.
Lymphoid infiltrates in the lamina propria at the colitis lesion of IL-7 transgenic mice were dominated by CD4+
T cells. Analysis of cytokine production by anti-CD3 and anti-CD28 mAb-stimulated lamina propria CD4+
T cells from chronic inflamed colonic mucosa of IL-7 transgenic mice showed elevated levels of IL-2 and IFN-γ production. In contrast, IL-4 secreted by those lamina propria T cells were decreased in IL-7 transgenic mice. These results indicate that Th1 type CD4+
T cells were dominated in the infiltrating lamina propria T cells in the chronic inflamed colonic mucosa of IL-7 transgenic mice. This Th1 pattern of cytokine production from lamina propria CD4+
T cells was also observed in the inflamed colonic mucosa of mice with the hapten reagent 2, 4, 6-trinitrobenzene sulfonic acid–induced colitis (19
). Flow cytometric analysis of isolated cells from intraepithelium demonstrated that CD4+
T cells, but not CD8+
T cells were increased at the colitis lesions. Interestingly, infiltrating CD8+
T cells mainly expressed the α/β heterodimer, indicating that these cells were thymus dependent. TCR-γ/δ intraepithelial T cells were not increased, although Uehira et al. reported that transgenic mice constitutively expressing IL-7 developed severe dermatitis and double negative γ/δ T cells were the main components of the infiltrating mononuclear cells at the dermatitis lesion (16
). Recent evidence of an extrathymic pool of TCR-α/β mucosal lymphocytes in the gut suggests that intestinal epithelial cells may share some differentiation-inducing capacities with thymic epithelial cells, leading to in situ TCR rearrangement of extrathymically derived T cells (25
). We have recently demonstrated the presence of a novel lymphoid tissue, cryptopatches (CP), in the murine intestinal mucosa where clusters of IL-7 receptor+
lympho-hematopoietic progenitor developed in IL-7–dependent fashion (26
). These results indicated that IL-7 may mediate an extrathymic pathway of mucosal T cell proliferation. Therefore, overexpression of IL-7 protein in the colitis mucosa of acute stage and decrease of IL-7 protein accumulation in goblet cell–depleted colonic mucosa of the chronic colitis may disturb IL-7–dependent proliferation of lymphocytes and progenitors in the mucosal tissues.
We have demonstrated that mucosal T cells in the normal murine as well as human colonic mucosa constitutively express the receptor for IL-7. In the present study, IL-7 receptor mRNA expression was strongly detected in the inflamed colonic mucosa of IL-7 transgenic mice, but not readily detectable in the colonic mucosa from normal littermate mice. Receptor expression was not strong in the colonic mucosa of transgenic mice without obvious inflammation. These findings indicated that the infiltrating mucosal lymphocytes in the colonic lesion of IL-7 transgenic mice highly expressed the IL-7 receptor. Interestingly, IL-7 receptor expression was quite marked in the mucosal lymphocytes of severely inflamed colonic mucosa from patients with ulcerative colitis (Watanabe, M., unpublished observation). These results raise the possibility that decreased mucosal IL-7 protein accumulation observed in the colonic mucosa with chronic inflammation of IL-7 transgenic mice may result in the increase of infiltrating mucosal lymphocytes expressing the IL-7 receptor. Previous studies have shown that IL-7 stimulates the proliferation of human mature T cells after exogenous stimulation in short time culture, suggesting that IL-7 receptor is not expressed by resting PBLs, but expressed after activation. Therefore, our results indicate that mucosal lymphocytes are activated in the transgenic mice. These activated T cells may produce regulatory factors such as cytokines that augment the inflammation process.
It has been reported that IL-7 acts synergistically with anti-CD3 Ab to stimulate proliferation of human T cells in the thymus and peripheral blood. However, our previous studies demonstrated that IL-7 inhibited anti-CD3–induced proliferation of mucosal T cells in the normal colonic mucosa. Moreover, we showed that IL-7 alone inhibited the growth of mucosal lymphocytes isolated from the inflamed colonic mucosa (Watanabe, M., unpublished observation). These suggested that IL-7 may inhibit the proliferation of activated and IL-7 receptor–expressing mucosal T cells in the intestinal mucosa.
We are now doing the precise time course studies concerning development of colitis, IL-7 protein expression in the colonic mucosa, and phenotype of infiltration lymphocytes in the IL-7 transgenic mice. IL-7 transgenic mice developed acute colitis with infiltrating neutrophils and lymphocytes at 1–3 wk of age. At that time, IL-7 protein was significantly expressed in the inflamed colonic mucosa of IL-7 transgenic mice, but then decreased with depletion of goblet cells. Accumulation of IL-7 protein in the colonic mucosa was significantly decreased at 8–12 wk of age. We also demonstrated that at 1–3 wk of age, infiltrating T cells in the acute colitis lesion were γ/δ T cells. This result is consistent with the findings that γ/δ T cells were accumulated at the acute dermatitis lesion of IL-7 transgenic mice (16
), and is contrasted with the our findings that α/β T cells were infiltrated in the chronic colitis lesion of the IL-7 transgenic mice. Therefore, we supposed that at 1–3 wk, IL-7 transgenic mice developed γ/δ T cell–mediated acute colitis due to the overexpression of IL-7 protein, but with the inflammation IL-7 protein–producing goblet cells decreased and accumulation of IL-7 protein in the colonic mucosa decreased in the goblet cell–depleted epithelial cells. Since we demonstrated that mucosal IL-7 regulates the proliferation of mucosal lymphocytes by apoptotic mechanism (Watanabe, M., unpublished observation), decreased accumulation of IL-7 protein may lead to the chronic inflammation of colon in the IL-7 transgenic mice. These results are consistent with our recent findings that IL-7 protein expression was increased in the inflamed colonic mucosa of acute Salmonella enterocolitis
and initial attack of ulcerative colitis, but significantly decreased at the chronic colitis lesion in the relapsing-remission type and chronic continuous type of ulcerative colitis (Watanabe, M., unpublished observation).
In this paper, we demonstrate that the dysregulation of mucosal IL-7–mediated immune regulation results in the acute and chronic colitis in IL-7 transgenic mice. This colitis mimicked several histopathological characteristics of ulcerative colitis in humans. A major limitation in understanding the pathogenesis responsible for the mucosal injury observed in human inflammatory bowel disease has been due to the lack of animal models that possess the histopathological features of human disease. A series of valuable new models that spontaneously arise in gene knockout mice gives a major impact in our understanding of the mechanisms that lead to chronic intestinal inflammation (27
). Although the mechanism by which these mice developed chronic colitis is unclear, series of the study using these mice and our IL-7 transgenic mice may lead to the understanding of the pathogenesis of human inflammatory bowel disease.