T cell-mediated pathology in two models of experimental colitis depends predominantly on the interleukin 12/Signal transducer and activator of transcription (Stat)-4 pathway, but is not conditional on interferon gamma expression by T cells

Abstract

The requirements for interleukin (IL)-12/signal transducer and activator of transcription (Stat)-4 signaling and induction of T cell-specific interferon (IFN)-gamma expression in the development of T helper cell (Th)1-type pathology were examined in two different models of experimental colitis. In each model, abnormal reconstitution of the T cell compartment in immunodeficient mice by adoptive cell transfer leads to a wasting syndrome and inflammation of the colon, induced by IFN-gamma and tumor necrosis factor (TNF)-alpha-producing T cells. We show here that treatment with anti-IL-12 antibodies in one of the models, or reconstitution with T cells from Stat-4-deficient (Stat-4(null)) mice in both models resulted in a milder disease in the majority of recipient animals, compared with those that were left untreated or that had been reconstituted with wt cells. Protected mice in each group also harbored lower frequencies of IFN-gamma-producing T cells than did diseased mice, suggesting that effects on wasting and colitis resulted from the attenuation of IFN-gamma expression by T cells. To test whether the development of pathogenic T cells in the two colitis models was directly dependent on T cell-specific IFN-gamma expression, IFN-gammanull donors were used for T cell reconstitution in each system. Surprisingly, large numbers of IFN-gammanull-reconstituted mice developed wasting and colitis, which in many cases was of comparable severity to that seen in animals reconstituted with wt cells. Furthermore, T cells from these animals expressed TNF-alpha, demonstrating that they had retained the ability to produce another proinflammatory cytokine. Taken together, these results demonstrate that in some forms of chronic experimental colitis the development of pathogenic T cells is influenced predominantly, though not exclusively, by IL-12 via the actions of Stat-4 proteins. Furthermore, our data suggest that in the models of colitis studied here the effects of IL-12/Stat-4 or other Th1 promoting pathways are not limited to the induction of IFN-gamma gene expression in T lymphocytes.

Figure 1

Disease is reduced in the absence of the IL-12/Stat-4 pathway. Disease activity scores of Tgε26 mice that were transplanted with (C57BL/6 × CBA/J)F1 BMCs (F1→ Tgε26) and received a weekly dose of anti–IL-12 antibody are compared with PBS-treated F1→ Tgε26 mice (untreated). Tgε26 and RAG^null mice were reconstituted with Stat-4^null BMCs and CD45RB^hi CD4⁺ T cells, respectively, and compared with animals that received 129/SvEv/wt cells. For comparison, results are also presented from RAG^null mice that received CD45RB^lo CD4⁺ T cells from wt and Stat-4^null donors. Clinical disease activity scores were determined according to parameters of weight loss, colon thickening, and stool consistency (described in Materials and Methods). The means of collective individual scores, obtained from three separate experiments, are plotted ± SD (BM→ Tgε26 and CD45RB^hi transfer groups, n = 10–15/ group; CD45RB^lo, n = 3–4/group).

Figure 2

Wasting and colitis are both affected by the absence of IL-12/Stat-4 signaling. The divisible components of disease, weight loss and histological colitis, are represented. (A) Weight loss. The body weights of animals measured at the end of each experiment was divided by the starting body weight (on the day of cell transfer), to calculate the percentage of starting body weight. Each point represents data from an individual animal. (B) The extent of mucosal inflammation was examined histologically and graded (see Materials and Methods). Represented are the combined scores for cell infiltration (0–3) and colon thickening (0–3) on a scale of 1–6. Each point represents the score assigned to an individual animal.

Figure 2

Wasting and colitis are both affected by the absence of IL-12/Stat-4 signaling. The divisible components of disease, weight loss and histological colitis, are represented. (A) Weight loss. The body weights of animals measured at the end of each experiment was divided by the starting body weight (on the day of cell transfer), to calculate the percentage of starting body weight. Each point represents data from an individual animal. (B) The extent of mucosal inflammation was examined histologically and graded (see Materials and Methods). Represented are the combined scores for cell infiltration (0–3) and colon thickening (0–3) on a scale of 1–6. Each point represents the score assigned to an individual animal.

Figure 3

Histology of colitis. Sections taken from the distal region of colon are compared after hematoxylin and eosin staining. Objectives are all ×10 and scoring is as described in Materials and Methods and Fig. 2. (A) Normal colon taken from an untransplanted Scid mouse. (B) Severe disease (6+) in a RAG^null mouse transplanted with CD45RB^hi T cells from a 129/SvEv/wt donor showing extensive cellular infiltration and crypt destruction. (C) Mild disease (2+) in a RAG^null mouse transplanted with CD45RB^hi T cells from a Stat-4^null donor. The basic structure of the mucosa is intact, but reveals some infiltration and crypt distortion. (D) Mild disease (2+) and presence of a granuloma in a RAG^null mouse transplanted with CD45RB^hi T cells from a Stat-4^null donor. (E) Severe disease (5+) in a Tgε26 mouse transplanted with wt (129/SvEv) bone marrow. Evident is extensive crypt elongation, and a pronounced cellular infiltration. (F) Mild disease (2+) in a BM→ Tgε26 mouse treated with a weekly dose of anti–IL-12 antibody. This section reveals a low level of cellular infiltrate, moderate crypt elongation, and reduced thickening of the bowel wall.

Figure 4

The effect of the IL-12/Stat-4 pathway on IFN-γ production by T cells. T cells from the MLNs from each group of BM→ Tgε26 and CD45RB^hi→ RAG^null mice were compared for production of IFN-γ. T cells were stimulated overnight using anti-CD3 antibody and stained for cytoplasmic expression of IFN-γ, and the frequency of cytokine positive cells was assessed by flow cytometry. (A) Dot plots comparing the frequency IFN-γ production by CD4⁺ MLN T cells from BM→ Tgε26 mice treated with anti–IL-12 antibody or PBS, Stat-4^null→ Tgε26 and wt→ Tgε26 controls, and CD45RB^hi→ RAG^null mice that had received Stat-4^null and 129/SvEv/wt (129/wt) cells. Plots show staining of cytoplasmic IFN-γ after analytical gating on CD4⁺ T cells. Gates are drawn after determination of background staining after preblocking with anti–IFN-γ antibodies and staining with isotype control antibodies of irrelevant specificity. The percentages of positive cells falling in the designated gates are shown. Frequencies of T cells from healthy wt mice expressing IFN-γ after identical CD3 stimulation or T cells from each of the represented groups without CD3 staining fall below 1% (data not shown) (B) The percentages of IFN-γ–positive T cells from the MLNs of different groups of mice were determined. Shown are the mean values ± SD calculated for each group (n = 5–9/group).

Figure 5

T cells deficient in IFN-γ production can cause disease in each of the colitis models. Tgε26 mice and C57BL/6/Scid or RAG^null mice received CD45RB^hi CD4⁺ T cells and BMCs, respectively, from C57BL/6/IFN-γ^null donors, and were monitored for disease. These animals were compared with mice that received cells from C57BL/6/wt (B6/wt) donors. Plotted are the mean clinical disease activity scores ± 1 SD for each group of animals (n = 4–12 mice/group).

Figure 6

Wasting and colitis in the absence of IFN-γ expression in T cells. (A) Change in body weights and (B) histological colitis are represented as described in Fig. 2 in animals reconstituted with IFN-γ^null T cells. In the CD45RB^hi transfer model, closed symbols represent RAG^null, rather than Scid recipients mice.

Figure 6

Wasting and colitis in the absence of IFN-γ expression in T cells. (A) Change in body weights and (B) histological colitis are represented as described in Fig. 2 in animals reconstituted with IFN-γ^null T cells. In the CD45RB^hi transfer model, closed symbols represent RAG^null, rather than Scid recipients mice.

Figure 7

Histological colitis in IFN-γ^null T cell–reconstituted mice. Sections taken from distal colon are shown from (A) an IFN-γ^null→ Tgε26 mouse with mild colitis (2+ histological score), (B) IFN-γ^null→ Tgε26 mouse severe colitis (4+ histological score), and (C) IFN-γ^null→ Scid mouse with severe colitis (5+ histological score).

Figure 8

T cells from IFN-γ^null–recipient mice produce TNF-α. MLN T cells were examined for expression of IFN-γ (hatched bars) and TNF-α (closed bars) using flow cytometry after overnight in vitro stimulation with an anti-CD3 antibody. The mean percentages of cytokine-positive cells in each group are shown ± SD (n = 5–10/group).