We generated transgenic mice for STAT-4, a regulatory protein specifically associated with IL-12 signaling, under the control of a CMV promoter. These mice expressed strikingly increased nuclear STAT-4 levels in lamina propria CD4+ T lymphocytes upon systemic administration of dinitrophenyl-keyhole limpet hemocyanin and developed chronic transmural colitis characterized by infiltrates of mainly CD4+ T lymphocytes. The latter cells produced predominantly TNF and IFN-γ but not IL-4 upon activation with αCD3/CD28 or autologous bacterial Ags, consistent with a Th1-type cell response. Furthermore, chronic colitis in STAT-4 transgenic mice could be adoptively transferred to SCID mice by colonic and splenic CD4+ T cells that were activated with Ags from autologous bacterial flora. These data establish a critical molecular signaling pathway involving STAT-4 for the pathogenesis of chronic intestinal inflammation, and targeting of this pathway may be relevant for the treatment of colitis in humans.

The STAT family consists of regulatory proteins that deliver cytokine signals from the cell surface to the nucleus 1, 2 . Specificity of cytokine effects in T cells is maintained by recruitment of specific STAT proteins: whereas a STAT-1 (STAT-6)-containing DNA-binding complex is rapidly formed in response to IFN-γ (IL-4), STAT-3 and STAT-4 are strongly activated by IL-12 1, 2 . IL-12 is a heterodimeric cytokine produced mainly by activated follicular dendritic cells, B cells, and macrophages 3, 4, 5 . Binding of IL-12 to the high-affinity IL-12 receptor results in rapid activation of the Jak2 and Tyk2 signaling proteins and finally dimerization of STAT-4 and STAT-3 transcription factors 2 . In primary CD4+ T lymphocytes, STAT-4 is essential for IL-12-dependent IFN-γ promoter activity by binding to a specific recognition sequence in the IFN-γ promoter, thus providing a molecular explanation for the role of STAT-4 in Th1 T cell differentiation 3 . The critical role of the IL-12/STAT-4 pathway in driving Th1 T cell responses is further underlined by the finding that both IL-12 p40 4 and STAT-4 5 knockout mice manifest impaired Th1/IFN-γ cytokine responses.

Recently, various animal models of colitis caused by dysregulated T cell functions demonstrated the key role of Th1 T cells in the pathogenesis of chronic intestinal inflammation 6, 7, 8 . Because there is increasing evidence that IL-12/STAT-4-driven Th1 responses predominate in human Crohn’s disease 9 , we analyzed the effect of STAT-4 overexpression in a transgenic mouse model.

The murine STAT-4 cDNA 10 was cloned downstream of the CMV promoter into the pcDNA3.1 expression vector (Invitrogen, San Diego, CA) yielding the pcDNA3.1S4 vector. A 3.6-kp NruI/PvuII fragment of pcDNA3.1S4 containing the STAT-4 expression cassette was microinjected into pronuclei of fertilized eggs of FVB/NHSD mice. Mice were maintained under specific pathogen-free conditions in isolated cages.

Transgene-positive founder mice were identified by Southern blots and PCR amplification of genomic DNA isolated from the tail using the QiaAmp Tissue Kit (Qiagen, Hilden, Germany). Southern blots were probed with a radiolabeled 1.3-kb BamHI/EcoRI fragment of pcDNA3.1S4.

For immunization mice were i.p. injected with 100 μg dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH)3 (Calbiochem, Bad Soden, Germany) in CFA (Sigma, Deisenhofen, Germany) or alum. Control transgenic mice were injected with PBS.

Total RNA of spleen cells was prepared using Qiagen RNeasy columns (Qiagen). PCR was performed with a STAT-4-specific primer (5′-gctgaatgacggtgcaaacgg-3′) and a second transgene-specific primer (5′-gacagtgggagtggcacctt-3′) derived from sequences of the construct. PCR with β-actin primers (5′-tcctgtggcatccatgaa-3′ and 5′-cgcagctcagtaacagtc-3′) was made as control. Cycling conditions were as follows: 94°C for 30 s, 57°C for 30 s, and 72°C for 75 s for 30 cycles.

Cells were incubated for 20 min with 10 U/ml rIL-12 (obtained from Genzyme, Germany), and, subsequently, nuclear proteins were extracted. EMSA studies were made as described 3 . For supershift assays, polyclonal anti-STAT-4 (L-18; Santa Cruz Biotechnology, Heidelberg, Germany) Abs were used.

Frozen sections were fixed in 4% paraformaldehyde and incubated overnight with the primary Ab (polyclonal rabbit anti-STAT-4 Ab, C20; Santa Cruz; monoclonal anti-mouse IFN-γ and TNF; PharMingen, San Diego, CA). Sections were then incubated with a biotinylated secondary IgG Ab (Vector, Burlingame, CA) followed by incubation with streptavidin-conjugated Cy2 (Dianova, Hamburg, Germany). A second cycle of staining was made using anti-CD4 as primary Ab (rat anti-mouse CD4; PharMingen) and streptavidin-conjugated Cy3. Three mice per group were analyzed.

The degree of inflammation on microscopic cross-sections of the colon was graded semiquantitatively from 0 to 4 (0, no signs of inflammation; 1, very low level; 2, low level of leukocytic infiltration; 3, high level of leukocytic infiltration, thickening of the colon wall; 4, transmural inflammation, loss of goblet cells, thickening of the colon wall), as previously described 7 . Grading was done in a blinded fashion by the same observer on at least three colon cross-sections in each animal per group.

Spleen CD4+ T cells (>95% purity) were isolated using immunomagnetic beads (Dynal, Oslo, Norway) with subsequent bead detachment 3 . CD4-enriched LPMC were isolated from resected large bowel specimens as described 7 .

A total of 1 × 106 cells from three mice per group were cultured in 1 ml complete RPMI 1640 7 in triplicate wells. Cells were activated with precoated anti-CD3 mAb (10 μg/ml) and soluble anti-CD28 Abs (1 μg/ml; PharMingen). Supernatants were removed after 48 h and assayed for cytokine concentration by ELISA (PharMingen).

Colonic bacterial Ags were prepared as described 11 . For generation of APC, 2 × 107 freshly isolated spleen cells were incubated overnight with 100 μg/ml bacterial sonicates and subsequently irradiated with 3000 rad. For proliferation assays, 5 × 104 cells in triplicate wells were incubated with 5 × 104 Ag-pulsed APC for 5 days in complete media. 0.25 μCi/well [3H]thymidine (DuPont, Les Ulis, France) were added for 18 h. The incorporation of [3H]thymidine was determined by β scintillation counting.

Splenic CD4+ T cells and LPMC from STAT-4 transgenic mice and control mice were isolated as described above and incubated over a period of 4 days with APC that were loaded with autologous bacterial Ags. Finally, 1 × 106 cells were i.p. injected in CB.17 SCID mice, IL-12 p40 knockout mice (4; kindly donated by Dr. E. Schmitt, Department of Immunology, University of Mainz, Mainz, Germany), or wild-type control mice.

Data were compared by the student’s t test for independent events (Statworks for Macintosh).

Because previous studies suggested a key role for IL-12 in the pathogenesis of colitis 7, 8 , we generated STAT-4 transgenic mice using a vector containing the murine STAT-4 cDNA under control of a human CMV-promoter (Fig. 1 a). Southern blot analysis (not shown) of the offspring mice demonstrated chromosomal integration of the construct in 12 mice, and two independent founders (no. 6 and 15) were used for additional experiments.

FIGURE 1.

a, Map of the vector cassette of the linearized STAT-4 construct derived from the the pcDNA3.1S4 plasmid that was used for microinjection. Restriction sites and location of the primers for transgene specific PCR are indicated. b, Transgene-specific PCR analysis of spleen and colon mRNA of immunized and unimmunized STAT-4 transgenic mice and control wild-type mice. Only spleen and colon cells from DNP-KLH-treated STAT-4 transgenic mice expressed transgenic STAT-4 mRNA. c, High expression of STAT-4 in splenic T cells from immunized STAT-4 transgenic mice compared with unimmunized STAT-4 transgenic control mice. Gel-retardation analysis of spleen nuclear extracts from STAT-4 transgenic mice was performed using a radiolabeled STAT-4 consensus-binding site. The location of the STAT-4 signal is indicated. Addition of a STAT-4-specific Ab strongly reduced intensity of the STAT-4 protein-DNA complex. The free probe in all lanes had comparable intensity (not shown).

FIGURE 1.

a, Map of the vector cassette of the linearized STAT-4 construct derived from the the pcDNA3.1S4 plasmid that was used for microinjection. Restriction sites and location of the primers for transgene specific PCR are indicated. b, Transgene-specific PCR analysis of spleen and colon mRNA of immunized and unimmunized STAT-4 transgenic mice and control wild-type mice. Only spleen and colon cells from DNP-KLH-treated STAT-4 transgenic mice expressed transgenic STAT-4 mRNA. c, High expression of STAT-4 in splenic T cells from immunized STAT-4 transgenic mice compared with unimmunized STAT-4 transgenic control mice. Gel-retardation analysis of spleen nuclear extracts from STAT-4 transgenic mice was performed using a radiolabeled STAT-4 consensus-binding site. The location of the STAT-4 signal is indicated. Addition of a STAT-4-specific Ab strongly reduced intensity of the STAT-4 protein-DNA complex. The free probe in all lanes had comparable intensity (not shown).

Close modal

Further analysis of the transgenic mice showed no detectable levels of transgenic STAT-4 mRNA in spleen, colon, and liver cells as determined by RT-PCR. Because the CMV promoter is constitutively silenced in many tissues of transgenic mice but inducible by cellular stimulation in vivo 12 , we analyzed if activation of the immune system with DNP-KLH would up-regulate transgene expression. It was found that colon and spleen cells from DNP-KLH-treated STAT-4 transgenic mice but not from PBS-treated transgenic control mice expressed transgene-specific mRNA after 7 days, suggesting induction of the CMV promoter in vivo (Fig. 1,b). This finding was associated with increased STAT-4 protein levels in splenic T cells of immunized transgenic mice compared with control mice (Fig. 1 c).

Within 7–14 days after i.p. injection of DNP-KLH plus CFA STAT-4 transgenic mice developed macroscopic and histologic signs of severe transmural colitis (Table I), which was observed as long as the animals were followed (3 mo). The use of alum instead of CFA for immunization resulted in the development of a milder colitis in STAT-4 transgenic mice compared with DNP-KLH plus CFA. In contrast, CFA-treated STAT-4 transgenic mice and DNP-KLH plus CFA-treated nontransgenic control mice failed to develop colitis (Table I). DNP-KLH-induced colitis was accompanied by diarrhea, weight loss, and massive thickening of the bowel wall (Figs. 2, a and g). Histopathological analysis of the ileum and large bowel of DNP-KLH-treated animals showed severe mucosal inflammation in STAT-4 transgenic mice (Fig. 2, b–f; Table I). This inflammatory process was characterized by destruction of the crypt architecture and dense infiltrates of granulocytes, macrophages, and lymphocytes in the lamina propria.

Table I.

Histologic scoring of colon cross-sections in immunized and unimmunized STAT-4 transgenic mice, nontransgenic control mice, and mice reconstituted with spleen CD4+ T cells from STAT-4 transgenic mice after 7–8 wka

Scorep Values
Nontransgenic mice   
Untreated 0.1 ± 0.3  
CFA plus DNP-KLH-treated 0.3 ± 0.5 NS 
Adoptive transfer: STAT-4 > IL−12 KO mice   
IL-12 p40 wild-type mice 0.7 ± 0.8  
IL-12 p40 knockout mice 0.3 ± 0.4 NS 
STAT-4 transgenic mice   
Untreated 0.3 ± 0.4  
CFA-treated 0.2 ± 0.4 NS 
CFA plus DNP-KLH-treated 3.1 ± 1.1 < 0.05 
Alum instead of CFA 1.3 ± 0.5 < 0.05 
Adoptive transfer: STAT-4 > SCID mice   
T cells STAT-4 transgenic mice 2.7 ± 0.9  
T cells control mice 0.4 ± 0.6 < 0.05 
Scorep Values
Nontransgenic mice   
Untreated 0.1 ± 0.3  
CFA plus DNP-KLH-treated 0.3 ± 0.5 NS 
Adoptive transfer: STAT-4 > IL−12 KO mice   
IL-12 p40 wild-type mice 0.7 ± 0.8  
IL-12 p40 knockout mice 0.3 ± 0.4 NS 
STAT-4 transgenic mice   
Untreated 0.3 ± 0.4  
CFA-treated 0.2 ± 0.4 NS 
CFA plus DNP-KLH-treated 3.1 ± 1.1 < 0.05 
Alum instead of CFA 1.3 ± 0.5 < 0.05 
Adoptive transfer: STAT-4 > SCID mice   
T cells STAT-4 transgenic mice 2.7 ± 0.9  
T cells control mice 0.4 ± 0.6 < 0.05 
a

Grading was done in a blinded fashion on at least three colon cross-sections in each animal (n = 3–5) per group as specified in Materials and Methods. Data represent mean values ± SD. Significant differences are indicated.

FIGURE 2.

Analysis of colitis in immunized STAT-4 transgenic animals. a, Macroscopic appearance of the colon in unimmunized (top) and immunized (bottom) STAT-4 transgenic mice (founder 15) 3 wk after treatment with DNP-KLH. The colon of immunized STAT-4 transgenic mice was enlarged and strikingly thickened. b–f, High- and low-power histologic analysis of the colon from unimmunized (e and f) and immunized (b–d) STAT-4 transgenic mice 4 wk after treatment with DNP-KLH. Colon paraffin sections were stained with hematoxylin and eosin. Note the transmural inflammation, edema, focal destruction of the mucosa in immunized STAT-4 transgenic mice, and absence of inflammation in unimmunized STAT-4 transgenic control mice. Magnification: b, ×25; e, ×50; c and f, ×100; d, ×150. g, Weight curves of STAT-4 transgenic mice and nontransgenic control mice at indicated time points after immunization with DNP-KLH plus CFA. Data represent mean values ± SD from at least three mice per group.

FIGURE 2.

Analysis of colitis in immunized STAT-4 transgenic animals. a, Macroscopic appearance of the colon in unimmunized (top) and immunized (bottom) STAT-4 transgenic mice (founder 15) 3 wk after treatment with DNP-KLH. The colon of immunized STAT-4 transgenic mice was enlarged and strikingly thickened. b–f, High- and low-power histologic analysis of the colon from unimmunized (e and f) and immunized (b–d) STAT-4 transgenic mice 4 wk after treatment with DNP-KLH. Colon paraffin sections were stained with hematoxylin and eosin. Note the transmural inflammation, edema, focal destruction of the mucosa in immunized STAT-4 transgenic mice, and absence of inflammation in unimmunized STAT-4 transgenic control mice. Magnification: b, ×25; e, ×50; c and f, ×100; d, ×150. g, Weight curves of STAT-4 transgenic mice and nontransgenic control mice at indicated time points after immunization with DNP-KLH plus CFA. Data represent mean values ± SD from at least three mice per group.

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To characterize colitis in STAT-4 transgenic mice, we performed immunohistochemical studies. It was found that STAT-4 colitic mice showed dense infiltrates of CD4+ T cells in the colon compared with DNP-KLH-treated control mice (Fig. 3). To further determine STAT-4 protein levels in transgenic T cells, we performed double-staining analysis. We observed that colonic CD4+ T cells from STAT-4 transgenic mice express higher amounts of STAT-4 compared with control cells (Fig. 3).

FIGURE 3.

Immunohistologic analysis of colonic cross-sections from DNP-KLH-treated nontransgenic control mice (lower panel) and immunized (upper panel) STAT-4 transgenic mice. Double staining for CD4+ T lymphocytes plus STAT-4 (A and B, G and H)-, CD4 plus IFN-γ (C and D, I and J)-, and CD4 plus TNF (E and F, K and L)-expressing T lymphocytes in the lamina propria of STAT-4 transgenic mice and controls. There was a striking increase of STAT-4-, IFN-γ-, and TNF-expressing CD4+ T lymphocytes in immunized STAT-4 transgenic mice. Colocalization of cytokines and T cells is indicated by arrows. Magnification: ×400

FIGURE 3.

Immunohistologic analysis of colonic cross-sections from DNP-KLH-treated nontransgenic control mice (lower panel) and immunized (upper panel) STAT-4 transgenic mice. Double staining for CD4+ T lymphocytes plus STAT-4 (A and B, G and H)-, CD4 plus IFN-γ (C and D, I and J)-, and CD4 plus TNF (E and F, K and L)-expressing T lymphocytes in the lamina propria of STAT-4 transgenic mice and controls. There was a striking increase of STAT-4-, IFN-γ-, and TNF-expressing CD4+ T lymphocytes in immunized STAT-4 transgenic mice. Colocalization of cytokines and T cells is indicated by arrows. Magnification: ×400

Close modal

Next, we analyzed cytokine production by spleen T cells from STAT-4 transgenic mice. We observed that anti-CD3/CD28-activated T cells from immunized STAT-4 transgenic mice produced on average more TNF (7 ng/ml) and IFN-γ (32 ng/ml) than T cells from untreated control mice (TNF, 2 ng/ml; IFN-γ, 13 ng/ml) and DNP-KLH-treated control mice (TNF, 3 ng/ml; IFN-γ, 18 ng/ml). In contrast, there was a low production of the Th2 cytokine IL-4 in both immunized STAT-4 transgenic mice (2 ng/ml) and immunized control mice (2 ng/ml) compared with untreated control mice (26 ng/ml). Furthermore, no significant differences in IL-10 production were found (immunized STAT-4 transgenic mice, 7 ng/ml; immunized control mice, 5 ng/ml; unimmunized mice, 8 ng/ml), suggesting a predominant Th1 cytokine profile in immunized STAT-4 transgenic mice.

In additional studies, we focused on the expression of TNF and IFN-γ in situ. In these studies, CD4+ T lymphocytes were analyzed by double-staining analysis using specific Abs to CD4, IFN-γ, and TNF. As shown in Fig. 3, the number of IFN-γ- and TNF-expressing CD4+ T lymphocytes was strikingly higher in the inflamed colon of STAT-4 transgenic mice compared with control mice, suggesting an activation of Th1 T cells in the lamina propria.

Previous studies suggested an important role of the intestinal microflora in T cell-dependent enterocolitis 11, 13, 14 . Because intestinal T cells are tolerant to autologous bacterial Ags but reactive to heterologous Ags 11 , we analyzed if colitis in STAT-4 transgenic mice is associated with a loss of tolerance toward autologous bacterial Ags. Accordingly, we stimulated lymphocytes from STAT-4 transgenic mice and control mice with sonicates from autologous and heterologous bacterial flora 11 . It was found that the latter cells proliferated when stimulated with Ags from the heterologous flora (DNP-KLH, 11,016 ± 1,129 CPM; untreated control mice, 12,489 ± 2,080 CPM), as previously described 11 . However, lymphocytes from STAT-4 transgenic mice proliferated even stronger (28,499 ± 3,558 CPM) when stimulated with Ags from the autologous flora and produced increased amounts of IFN-γ (STAT-4 flora, 2900 pg/ml vs. flora untreated mice, 0 pg/ml), suggesting increased reactivity to autologous bacterial Ags in STAT-4 transgenic mice.

Finally, we analyzed if colitis could be adoptively transferred by LPMC and CD4+ T cells from immunized STAT-4 transgenic animals to SCID mice. Accordingly, purified splenic CD4+ T cells and LPMC from wild-type and STAT-4 transgenic animals were incubated with APC loaded with bacterial Ags from the autologous flora and finally injected in SCID mice. Reconstitution of SCID mice with STAT-4 transgenic spleen T cells (histologic score, 2.5 ± 0.9) or LPMC (score, 2.3 ± 0.8) that were activated with Ags from the autologous flora led to severe transmural colitis that was indistinguishable from that of DNP-KLH-treated STAT-4 mice (Table I). In contrast, no severe colitis was found in SCID mice reconstituted with T cells from DNP-KLH-treated nontransgenic control mice (Table I). Furthermore, no severe colitis was seen in T cell-reconstituted IL-12-knockout mice or wild-type control mice (Table I), possibly due to an intact or even activated counter-regulatory Th2 and Th3 cytokine response in the lamina propria of these mice.

Here, we demonstrate that inducible overexpression of the IL-12-associated STAT-4 signaling protein in transgenic mice is associated with the development of severe chronic intestinal inflammation. Thus, activation of the IL-12/STAT-4 pathway emerges as a key regulatory mechanism in the pathogenesis of chronic intestinal inflammation.

Because Abs to IL-12 have been shown to abrogate established Th1-dependent colitis 7, 8 , we focused in the present study on the functional role of the STAT-4 pathway by using a transgenic mouse system. Interestingly, we could not detect transgenic STAT-4 mRNA in the spleen, liver, and colon of several STAT-4 founder lines, suggesting that the CMV promoter was silenced at least in these tissues. However, activation of the immune system by systemic administration of DNP-KLH resulted in expression of transgenic STAT-4 mRNA and increased STAT-4 protein levels in lamina propria T cells of STAT-4 transgenic mice but not nontransgenic control mice. Overexpression of STAT-4 in immunized mice was associated with macroscopic and histologic signs of colitis that was characterized by a transmural inflammation of T cells expressing high levels of nuclear STAT-4. These data demonstrate a predominant pathogenic role of STAT-4 in chronic colitis that is further underlined by recent studies showing the absence of colitis in CD45RBhigh T cell-reconstituted SCID mice in which the STAT-4 protein had been inactivated by homologous recombination 8 . The presence of activated nuclear STAT-4 in splenic and lamina propria T cells of STAT-4 transgenic mice correlated with high expression of the proinflammatory cytokines TNF and IFN-γ. Together with the decreased production of IL-4, these data suggest an important pathogenic role of Th1-type T cells for onset and maintenance of colitis.

Studies in germfree mice suggested an important role of the intestinal microflora in the pathogenesis of colitis 6, 13 . This is supported by studies showing that T cells from C3H/HejBir mice that respond to coecal bacterial Ags are capable to adoptively transfer colitis 14 . Interestingly, mucosal inflammation in STAT-4 transgenic mice was restricted to sites with the highest bacterial load (terminal ileum, colon), suggesting that mucosal Ags could play a role in the pathogenesis of colitis. In support of this hypothesis, we found that spleen cells of colitic STAT-4 transgenic mice proliferate to autologous bacterial Ags presented on APCs and consecutively produce proinflammatory Th1 cytokines. Finally, colitis in STAT-4 transgenic mice could be adoptively transferred to SCID mice by LPMC and splenic T cells that were activated via APC loaded with autologous bacterial Ags. These data directly indicate a key role of T cells that respond to luminal Ags in the pathogenesis of colitis in STAT-4 transgenic mice, and a similar mechanism has been recently suggested for the pathogenesis of Crohn’s disease in humans 15 .

In summary, the present data provide further insights into the pathogenic role of the STAT-4 signaling protein in the pathogenesis of colitis and define a critical molecular signaling pathway for the development of colonic inflammation. Taken together with recent data on the role of IL-12 and STAT-4 in human Crohn’s disease, these data suggest that activation of the IL-12/STAT-4 pathway is a key regulatory mechanism in the pathogenesis of chronic intestinal inflammation.

We thank Drs. Timothy Hoey (Tularik, San Francisco, CA), Sven Pettersson (Karolinska Institute, Stockholm, U.K.), and Warren Strober (National Institutes of Health, Bethesda, MD) for helpful discussions and critical reading of the manuscript.

1

The research of M.F.N. was supported by grants from the Deutsche Forschungsgemeinschaft (Ne 490/1-1, Ne 490/2-1) and the Gerhard Hess program of the Deutsche Forschungsgemeinschaft (Ne 490/3-1).

3

Abbreviations used in this paper: DNP-KLH, dinitrophenyl-keyhole limpet hemocyanin; LPMC, lamina propria mononuclear cell; EMSA, electrophoretic mobility shift assay.

1
O’Shea, J. J..
1997
. Jaks, STATs, cytokine signal transduction, and immunoregulation: are we there yet?.
Immunity
7
:
1
2
Jacobson, N. G., S. J. Szabo, R. M. Weber-Nordt, Z. Zhong, R. D. Schreiber, J. E. Darnell, Jr, K. M. Murphy.
1995
. Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4.
J. Exp. Med.
181
:
1755
3
Barbulescu, K., C. Becker, J. F. Schlaak, E. Schmitt, K.-H. Meyer-zum Büschenfelde, M. F. Neurath.
1998
. Cutting Edge: IL-12 and IL-18 differentially regulate the transcriptional activity of the human IFN-gamma promoter in primary CD4+ T lymphocytes.
J. Immunol.
160
:
3642
4
Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, M. K. Gately.
1996
. IL-12-deficient mice are defective in IFN γ production and type 1 cytokine responses.
Immunity
4
:
471
5
Kaplan, M. H., Y. L. Sun, T. Hoey, M. J. Grusby.
1996
. Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice.
Nature
382
:
174
6
Elson, C. O., R. B. Sartor, G. S. Tennyson, R. H. Riddell.
1995
. Experimental models of inflammatory bowel disease.
Gastroenterology
109
:
1344
7
Neurath, M. F., I. Fuss, B. L. Kelsall, E. Stuber, W. Strober.
1995
. Antibodies to interleukin 12 abrogate established experimental colitis in mice.
J. Exp. Med.
182
:
1281
8
Simpson, S. J., S. Shah, M. Comiskey, Y. P. de Jong, B. Wang, E. Mizoguchi, A. K. Bhan, C. Terhorst.
1998
. 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 γ expression by T cells.
J. Exp. Med.
187
:
1225
9
Monteleone, G., L. Biancone, R. Marasco, G. Morrone, O. Marasco, F. Luzza, F. Pallone.
1997
. Interleukin 12 is expressed and actively released by Crohn’s disease intestinal lamina propria mononuclear cells.
Gastroenterology
112
:
1169
10
Zhong, Z., Z. Wen, J. E. Darnell, Jr.
1994
. Stat3 and Stat4: members of the family of signal transducers and activators of transcription.
Proc. Natl. Acad. Sci. USA
91
:
4806
11
Duchmann, R., E. Schmitt, P. Knolle, B. K. Meyer-zum Büschenfelde, M. Neurath.
1996
. Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12.
Eur. J. Immunol.
26
:
934
12
Loser, P., G. S. Jennings, M. Strauss, V. Sandig.
1998
. Reactivation of the previously silenced cytomegalovirus major immediate-early promoter in the mouse liver: involvement of NFκB.
J. Virol.
72
:
180
13
Rath, H. C., H. H. Herfarth, J. S. Ikeda, W. B. Grenther, J. T. E. Hamm, Jr, E. Balish, J. D. Taurog, R. E. Hammer, K. H. Wilson, R. B. Sartor.
1996
. Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human β2 microglobulin transgenic rats.
J. Clin. Invest.
98
:
945
14
Cong, Y., S. L. Brandwein, R. P. McCabe, A. Lazenby, E. H. Birkenmeier, J. P. Sundberg, C. O. Elson.
1998
. CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell type 1 response and ability to transfer disease.
J. Exp. Med.
187
:
855
15
Sartor, R. B..
1997
. Pathogenesis and immune mechanisms of chronic inflammatory bowel diseases.
Am. J. Gastroenterol.
92
:
5S