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The Journal of Immunology, 2005, 174: 6122-6128.
Copyright © 2005 by The American Association of Immunologists

Impaired TGF-{beta} Responses in Peripheral T Cells of G{alpha}i2–/– Mice 1

Jim Y. Wu{dagger}, YongZhu Jin*, Robert A. Edwards{ddagger},§, Yujin Zhang{ddagger}, Milton J. Finegold{ddagger} and Mei X. Wu2,*,{ddagger}

* Wellman Center of Photomedicine, Massachusetts General Hospital, and Department of Dermatology, Harvard Medical School, Boston, MA 02114; {dagger} Synta Pharmaceuticals, Lexington, MA 02421; {ddagger} Department of Pathology, Baylor College of Medicine, Houston, TX 77030; and § Department of Pathology, University of California, Irvine, CA 92697


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Null mutation of heterotrimeric G protein {alpha}2 inhibitory subunit (G{alpha}i2) induces Th1-skewed hyperimmune responses in the colon, leading to chronic colitis and the development of colonic adenocarcinoma. However, the underlying molecular mechanisms and cellular basis, in particular, for the role of G{alpha}i2 in regulating immune responses, are poorly understood. We show here that peripheral T cells from G{alpha}i2-deficient mice do not respond normally to the inhibitory effects of TGF-{beta} on proliferation and cytokine production, revealing a previously unappreciated cross-talk between these two signaling pathways. Lack of G{alpha}i2 resulted in decreased phosphorylation of Smad2 and Smad3 in T cells at the basal levels as well as at the late but not early phase of TGF-{beta} stimulation, which appears to be ascribed to differential expression of neither cell surface TGF-{beta} receptors nor Smad7. The altered phosphorylation of Smad proteins involves phospholipase C-mediated signaling, a downstream signaling molecule of G{alpha}i2, because phospholipase C inhibitors could restore Smad2 and Smad3 phosphorylation in G{alpha}i2–/– T cells at levels comparable to that in wild-type T cells. Moreover, adoptive transfer of G{alpha}i2-deficient T cells into immunocompromised mice rendered an otherwise resistant mouse strain susceptible to trinitrobenzesulfonic acid-induced colitis, suggesting that an impaired response of G{alpha}i2-deficient T cells to TGF-{beta} may be one of the primary defects accounting for the observed colonic Th1-skewed hyperimmune responses. These findings shed new lights on the molecular and cellular basis of how G{alpha}i2 down-regulates immune responses, contributing to the maintenance of mucosal tolerance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Inflammatory bowel disease (IBD) 3 is a multifactorial disease, arising in the setting of an acquired imbalance of mucosal T cell immune responses, possibly to one or more normally occurring gut constituents. In accordance with this, mice with targeted disruptions in genes encoding IL-2, IL-10, or TCR {alpha}- or {beta}-chain, all develop colitis resembling IBD in humans, as these genes are known key players in T cell immune responses (1, 2, 3, 4). However, G protein {alpha}2 inhibitory subunit (G{alpha}i2), whose role in regulating T cell immune responses is poorly defined, has also been implicated in the pathogenesis of colitis, because mice with a targeted deletion of G{alpha}i2 developed colitis (5, 6). Moreover, genetic linkage studies in humans have mapped the G{alpha}i2 gene within an IBD susceptible locus at chromosome 3p21 (7), raising the possibility that G{alpha}i2 is one of the candidate genes contributing to IBD development in humans.

G{alpha}i2 is a member of the heterotrimeric G protein family that consists of multiple {alpha}, {beta} and {gamma} subunits. The GTP-binding proteins couple to seven transmembrane receptors, called G protein-coupled receptors, ligation of which elicits chemotaxis, migration, proliferation, and differentiation of leukocytes (8, 9, 10). There are three G{alpha}i isoforms, G{alpha}i1, G{alpha}i2, and G{alpha}i3, and all of them can be uncoupled from receptors by pertussis toxin (PTX), a potent Th1 adjuvant produced by Bordetella pertussis (11, 12). PTX has long been used to increase the severity of disease in animal models of Th1-mediated autoimmunity, such as experimental autoimmune encephalomyelitis (13), experimental autoimmune uveitis (14), as well as delayed-type hypersensitivity reactions (15). Consistent with the immune enhancing effect of PTX, T cells isolated from G{alpha}i2-deficient mice showed a hyperproliferative response and increased Th1-cytokine secretion following stimulation with engagement of the TCR/CD3 complexes (16). This in vitro hyperimmune response of T cells is in agreement with the abnormal immune response in the mouse that had increased numbers of activated CD4+ T cells with a memory phenotype and elevated levels of proinflammatory Th1-type cytokines in the intestinal mucosa (17). These observations argue strongly that G{alpha}i2 is a negative regulator for T cell immune responses.

Our current studies reveal a previously unappreciated cross-talk between G{alpha}i2- and TGF-{beta}-mediated signaling pathways in peripheral T cells that showed no response to the usual inhibitory effects of TGF-{beta} in the absence of G{alpha}i2. The lack of TGF-{beta} responses in G{alpha}i2-deficient T cells probably resulted from a shortened duration of Smad2 and Smad3 phosphorylation. This defect may contribute to colitis development in the animal, as suggested by an increase in colitis susceptibility of an otherwise resistant mouse strain following adoptive transfer of G{alpha}i2-deficient T cells. The findings explain hyperimmune responses with Th1 preference in G{alpha}i2-deficient mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

G{alpha}i2-knockout (KO) and wild-type (WT) control mice on the mixed 129Sv/C57BL/6 background were generated by gene targeting and backcrossed with C57BL/6 (B6) mice as described (5, 17). Both female and male mice were used at 4–6 wk of age in the present study unless otherwise indicated. The mice were housed in conventional cages at the animal facilities of the Baylor College of Medicine or Massachusetts General Hospital in accordance with institutional guidelines.

Cytokine production

Flat bottom 96-well plates (Costar) were precoated with 5 µg/ml goat anti-hamster IgG Ab (Ab) at 4°C for overnight, followed by washes. Then, 100 µl of 2 µg/ml hamster anti-CD3 mAb (specific for murine CD3{epsilon}) plus an equal amount of anti-CTLA-4 Ab (BD Pharmingen) or anti-CD3 mAb plus control Ab hamster Ig (2 µg/ml) were added in triplicate in the indicated wells, incubated for 1 h at 37°C, and then washed with PBS. T cells from G{alpha}i2-KO and WT control mice were seeded at 2 x 105 cells per well to the precoated 96-well plates. To test the inhibitory effects of IL-10 and TGF-{beta}, IL-10 at concentration of 20 ng/ml or TGF-{beta} at 4 ng/ml (both from R&D Systems) was added in triplicate to T cell cultures in plates precoated with anti-CD3 mAb. In some experiments, CD4+CD62LhighCD44low naive T cells were sorted from splenocytes by a Beckman Coulter Altra high speed cell sorter, after labeling of the cells with FITC-conjugated anti-CD62L and PE-conjugated anti-CD4 Abs, and biotin conjugated anti-CD44 Ab plus CyChrome-conjugated streptavidin (BD Pharmingen). The naive T cells (~96% purity) were stimulated with immobilized anti-CD3 mAb in the presence or absence of TGF-{beta} as above. Culture supernatants were collected after 48 h for IL-2 or 72 h for IFN-{gamma} assay and cytokine concentrations in the supernatants were assayed by a sandwich ELISA using specific Ab pairs from BD Pharmingen and recombinant murine IL-2 and IFN-{gamma} as standards. Culture supernatants collected after 48 h stimulation with immobilized anti-CD3 mAb alone were also assayed similarly for IL-10 and TGF-{beta} production using Abs specific for IL-10 and TGF-{beta} (BD Pharmingen).

Purification of lamina propia T cells

Lamina propia T cells were isolated from mouse colons as described (18), with some modifications. In brief, dissected colons were washed thoroughly with 40–50 ml of cold HBSS, opened longitudinally, cut laterally into 0.5- to 1-cm pieces, and incubated in HBSS containing EDTA (0.37 mg/ml) and DTT (0.145 mg/ml) at 37°C for 30 min in a shaking water bath. After two washes with HBSS, the resulting tissue was digested for 30 min in RPMI medium containing collagenase D (400 U/ml) and DNase I (0.1 mg/ml) in a shaking incubator at 37°C. Lamina propia cells were pelleted, layered on a 44/67.5% Percoll gradient, and centrifuged. Lymphocyte-enriched populations were collected at the 44/67.5% interface and passed through a mouse CD3+ T cell enrichment column (R&D Systems). The eluted lamina propia T cells were stimulated with immobilized anti-CD3 mAb in the presence or absence of TGF-{beta} as detailed in T cell proliferation assays.

T cell proliferation assays

Single cell suspensions prepared from lymph nodes (LNs) and spleens were treated with a mixture of rat anti-mouse mAbs against CD19, CD32, and CD16 followed by depletion of Ab-bound cells with BioMag goat anti-rat IgG (Polysciences) per the manufacturer’s instruction. The resulting T cells (~90% purity) were stimulated in triplicate in 96-well plates with indicated concentrations of immobilized anti-CD3 mAb (2C11) in the presence or absence of 4 ng/ml TGF-{beta}. [3H]Thymidine (Valeant Pharmaceuticals) of 0.5 µCi/well was added 5 days later. [3H]Thymidine incorporation was measured after 16 h on a Packard TopCount microplate scintillation counter. Lamina propia T cells prepared from the colon and naive T cells sorted from splenocytes as described above were stimulated similarly.

Western blot analysis

T cells that were purified by negative depletion as described above were treated with 5 ng/ml TGF-{beta} at varying times. To test effects of various inhibitors on the levels of Smad2 and Smad3 phosphorylation, T cells prepared as above were treated for 2 h with calyculin A (100 nM), D609 (20 and 100 nM), cAMP analog cAMP-8-CL (0.5 µM), ET-18-OCH3 (10 µM), or okadaic acid (100 nM). All the inhibitors were purchased from Calbiochem. The cells were then lysed in 50 mM Tris (pH 7.5) buffer containing 137 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, and 1% protease inhibitor mixture (Sigma-Aldrich). Approximately 100 µg of whole cell lysate proteins were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted, either immediately or after stripping, with an indicated Ab. Abs specific for phospho-Smad2 (Ser465/467) and Smad2 protein were obtained from Cell Signaling Technology, and Abs against Smad3, Smad7, or G3PDH proteins were purchased from Upstate, Santa Cruz Biotechnology, or Sigma-Aldrich, respectively. The rabbit anti-phospho-Smad3 Ab was a generous gift from Dr. E. Leof, generated against the peptide COOH-GSPSIRCSpSVpS of Smad3 protein (19). Ab-specific proteins were detected by incubation of the membrane with HRP-linked corresponding secondary Ab and developed using a SuperSignal West Pico Kit (Pierce).

Cell surface expression of TGF-{beta} receptors

Expression of TGF-{beta} receptors on cell surface was detected by covalently cross-linking 125I-labeled TGF-{beta} to TGF-{beta} receptors on T cell surface. Briefly, T cells prepared as above were stimulated with or without 5 ng/ml TGF-{beta} for 2 h and then were washed four times on ice, each for 30 min, with binding buffer (0.9 mM CaCl2, 0.49 mM MgC12, and 1 mg/ml BSA in PBS). The stimulated and unstimulated cells (1 x 107) were incubated on ice for 3 h with 1 µCi 125I-labeled TGF-{beta} (Amersham Pharmacia Biotech) in binding buffer. For specificity controls, unstimulated cells were incubated with 125I-labeled TGF-{beta} in the presence of 10 ng of unlabeled TGF-{beta}. After the incubation, the cells were washed in BSA-free binding buffer and cross-linked by disuccinimidyl suberate (Pierce) per the manufacturer’s instructions. The labeled cells were washed, lysed by SDS-PAGE sample buffer, and subjected to SDS-PAGE analysis followed by autoradiography.

Induction of colitis

To address a possible role for T cells in colitis development in G{alpha}i2–/– mice, WT littermate mice at 4–5 wk of age were irradiated at a dose of 7.5 Gy in a Cobalt-60 gamma irradiator and i.p. administrated the following day with WT or G{alpha}i2-deficient T cells isolated from littermate mice at a dose of 1 x 107 T cells per mouse. T cells (CD3+) were sorted from the spleen of healthy G{alpha}i2–/– mice and littermate control mice on a Beckman Coulter Altra cell sorter. The purity of the resulting T cells was >99%. The irradiated mice receiving either WT or G{alpha}i2-KO T cells were monitored for 5–8 mo for spontaneous colitis development. Alternatively, colitis was induced in the mice 10 days after adoptive transfer of T cells. In brief, 1 mg of the haptenating reagent trinitrobenzesulfonic acid (TNBS) (Sigma-Aldrich) in 100 µl of 50% ethanol or 50% ethanol alone was administered per rectum via a catheter fitted onto a l-ml syringe into the rectum of the mice that were previously irradiated and transferred with either WT or G{alpha}i2-KO T cells. The animals were weighed every other day for 12 days. The animals that died within 3 days following TNBS injection due to chemical toxicity were excluded in the study.

Histological examination

Colons from experimental and control mice were removed within 24 h of any overt signs of terminal illness or 12 days after TNBS injection, fixed with 10% formalin, embedded in paraffin, and sectioned at 4 µm. Sections were then stained with H&E by standard methods and subjected to histological examination. The degree of inflammation was graded on the basis of the following criteria: 0, no signs of inflammation; 1, low level of leukocyte infiltration (<10%) but no structural changes; 2, moderate leukocyte infiltration but no ulceration; 3, high level of leukocyte infiltration, thickening of the colon wall, and superficial ulceration; and 4, transmural infiltration, extensive ulceration, and thickening of the colon wall.

Statistical analysis

The Student’s two-tailed t test was used to analyze the significance of experimental group and relevant controls.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Peripheral T cells from G{alpha}i2-deficient mice are unresponsive to TGF-{beta}

The inhibitory cytokines TGF-{beta} and IL-10 and cell surface molecule CTLA-4 are critical in the maintenance of mucosal tolerance. To address whether exaggerated immune responses of G{alpha}i2–/– T cells could be ascribed to aberrant expression and/or function of these inhibitory molecules, we first examined the expression and responses of CTLA-4. Flow cytometry analysis revealed comparable or even slightly higher levels of CTLA-4 on G{alpha}i2-deficient T cells relative to WT, after 40 h of stimulation via ligation of the TCR/CD3 complex (data not shown). In agreement with the well-described inhibitory effect of CTLA-4, cross-linked anti-CTLA-4 Ab blocked IL-2 and IFN-{gamma} secretion similarly by both WT and G{alpha}i2–/– T cells when compared with anti-CD3 mAb stimulation alone, suggesting a nearly normal CTLA-4 response in T cells in the absence of G{alpha}i2 (Fig. 1, left panel).



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  		FIGURE 1. IL-2 and IFN-{gamma} production induced by ligation of the TCR/CD3 complex in the presence of inhibitory cytokines or Ab. Splenic T cells from G{alpha}i2–/– (KO) and WT mice were cultured at 2 x 105 cells per well and stimulated with immobilized anti-CD3 mAb (2 µg/ml) in the presence or absence of cross-linked anti-CTLA-4 Ab (2 µg/ml), IL-10 (20 ng/ml), or TGF-{beta} (4 ng/ml) as detailed in Materials and Methods (left panel). CD4+CD62LhighCD44low naive T cells were sorted and stimulated with anti-CD3 mAb in the presence or absence of TGF-{beta} as above (right panel). Culture supernatants were harvested 48 h later for IL-2 (top panel) and 72 h for IFN-{gamma} (bottom panel) assays. The levels of cytokines were determined with commercially available ELISA kits. Data are expressed as mean ± SD of four independent experiments, each performed in triplicate, for splenic T cells or two for naive T cells. The percentages of inhibition relative to absence of an inhibitory Ab or cytokine are shown on related bars.

 
We next assessed levels of IL-10 and TGF-{beta} production following ligation of the TCR/CD3 complex. Again, our data showed no obvious differences in IL-10 secretion in the presence or absence of G{alpha}i2 (data not shown). Contrary to a hyperimmune phenotype, G{alpha}i2-deficient T cells actually released slightly higher levels of TGF-{beta} than did WT T cells (data not shown). When their inhibitory effects were tested, IL-10 inhibited the production of IL-2 and IFN-{gamma} in T cells from either WT or KO mice after ligation of the TCR/CD3 complex, but to a slightly greater extent in WT T cells than G{alpha}i2-deficient T cells (Fig. 1, left panel). A lack of significant defects in IL-10 and CTLA-4 responses in G{alpha}i2-deficient T cells suggests that their contribution to the colitis phenotype of the mouse may be minimal.

In sharp contrast, TGF-{beta} was almost entirely ineffective as an inhibitor of anti-CD3-stimulated production of either IL-2 or IFN-{gamma} in G{alpha}i2-deficient T cells, suppressing only 11% of IL-2 and 16% of IFN-{gamma} production. This was remarkably less than the 84% inhibition for IL-2 and 75% for IFN-{gamma} observed in WT T cells (Fig. 1, left panel). Furthermore, TGF-{beta} failed to suppress proliferation of peripheral T cells in G{alpha}i2-deficient mice. As can be seen in Fig. 2A, LN T cells from WT mice proliferated vigorously when stimulated with increasing concentrations of anti-CD3 mAb, which was completely abrogated in the presence of TGF-{beta}. Strikingly, under similar conditions, TGF-{beta} was not only unable to inhibit proliferation of T cells; it augmented proliferation at low concentrations (< 0.1 µg/well) of anti-CD3 mAb in the absence of G{alpha}i2. This result was reproducibly obtained in at least 20 mice tested. Similar impairment in TGF-{beta} responses was observed in T cells from the spleen and lamina propria T cells (Fig. 2, C and D), but not in thymocytes (data not shown).



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  		FIGURE 2. Proliferation of peripheral T cells in the presence of TGF-{beta}. T cells from G{alpha}i2–/– (KO) or WT mice were stimulated with increasing concentrations (A and B) or 0.05 µg/well (C and D) of anti-CD3 mAb for 5 days in the presence or absence of TGF-{beta}. Means ± SD of [3H]thymidine uptake at final 16 h of the culture in triplicate is shown. A, LN T cells; B, CD4+CD62LhighCD44low naive T cells; C, T cells from the spleen; and D, lamina propia (LP) T cells. A representative result of at least six experiments for T cells from LNs and the spleen, and two for naive T cells or LP T cells is shown.

 
Because G{alpha}i2-deficient T cells do not respond to TGF-{beta}-mediated growth inhibition at all anti-CD3 mAb concentrations tested, it is unlikely that the threshold for the maximal T cell activation accounts for a failure of TGF-{beta} to suppress T cell proliferation in the absence of G{alpha}i2. In particular, while proliferation of G{alpha}i2–/– T cells was similar to that of WT T cells at anti-CD3 mAb concentration higher than 0.2 µg/well, TGF-{beta} efficiently inhibited WT T cell proliferation, but was without effect on G{alpha}i2-deficient T cells. Moreover, the lack of a response to TGF-{beta} could not be ascribed either to the presence of a relatively high proportion of activated T cells in G{alpha}i2–/– mice (17). In support, we showed that CD4+CD62LhighCD44low naive T cells isolated from G{alpha}i2-deficient mice were as refractory to TGF-{beta}-mediated suppression of cytokine secretion and proliferation induced by TCR/CD3 engagement as total LN T cells (Fig. 1, right panel, and Fig. 2B). The observation leads to a conclusion that TGF-{beta} depends on the presence of G{alpha}i2 for executing its inhibitory effects on cytokine production and proliferation of peripheral T cells. The findings are highly relevant to the hyperimmune responses observed in the mouse, as TGF-{beta} is one of the major cytokines in down-regulating immune responses and maintaining mucosal immune tolerance.

Decreases in phosphorylation levels of both Smad2 and Smad3 in G{alpha}i2–/– T cells

To investigate the mechanism underlying impaired TGF-{beta} responses in absence of G{alpha}i2, phosphorylation levels of Smad2 and Smad3 were evaluated in the presence vs absence of G{alpha}i2. Smad2 and Smad3 are immediate downstream signaling molecules of TGF-{beta}. Phosphorylation on Ser465/467 of Smad2 and a corresponding position (Ser423/425) on Smad3 at the C terminus is necessary for translocation of cytosolic Smad2/4 and Smad3/4 complexes to the nucleus following activation of TGF-{beta} receptors (20, 21). As can be seen in Fig. 3A, when Smad2 and Smad3 phosphorylation levels were compared in T cells isolated from WT and G{alpha}i2–/– mice following treatment with TGF-{beta} for varying times, we observed an ~70% decrease in the basal levels of both phosphorylated Smad2 and Smad3 but not their unphosphorylated counterparts in G{alpha}i2–/– T cells compared with WT T cells (Fig. 3A, lane 1 vs 2), as determined by densitometry analysis with Glykobandscan software. However, after 30 min of stimulation with TGF-{beta}, although phosphorylation of both Smad2 and Smad3 was augmented in T cells from either mouse, the increments were much greater in G{alpha}i2–/– T cells than WT T cells, bringing about similar levels of Smad2 and Smad3 phosphorylation in T cells from these two groups of mice (Fig. 3A, lanes 3 vs 4). The result suggests that a lack of G{alpha}i2 does not impede the signal propagating from TGF-{beta} receptors to Smad2 and Smad3 phosphorylation in the early phase of TGF-{beta} stimulation. Interestingly, the phosphorylation of Smad2 and Smad3 declined slightly at 1 h, profoundly at 2 h after TGF-{beta} stimulation in the absence of G{alpha}i2, but no such a decrease was observed in the presence of G{alpha}i2. The levels of Smad2 and Smad3 phosphorylation decreased by 64 and 52%, respectively, at 2 h of stimulation (Fig. 3A, lanes 7 vs 8), and a similar reduction could be extended to 6 h or overnight (data not shown). The decreases in phosphorylation levels of Smad2 and Smad3 were not accompanied by similar decreases in the levels of Smad2 and Smad3 proteins, suggesting that decreases in the phosphorylation rather than protein degradation account for the observed difference in the phosphorylation of Smad2 and Smad3, which may be one of the primary reasons behind defective TGF-{beta}-mediated inhibition of immune responses in G{alpha}i2-deficient T cells.



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  		FIGURE 3. Decreased phosphorylation of Smad2 and Smad3 in T cells in the absence of G{alpha}i2. A, Western blot analysis of Smad2 and Smad3 phosphorylation and protein levels in T cells from G{alpha}i2–/– (even lanes) and WT (odd lanes) littermates. Splenic and LN T cells were stimulated with 5 ng/ml TGF-{beta} at the indicated times. The same membrane was stripped and blotted with Abs specific for phospho-Smad2, Smad2 protein, phospho-Smad3, or Smad3 protein, respectively. One representative result of three experiments performed is shown. B, Comparable expression levels of TGF-{beta} receptors I and II on T cells in the presence or absence of G{alpha}i2. T cells prepared from G{alpha}i2–/– (even lanes) and WT (odd lanes) littermates were either left unstimulated (lanes 1–4) or stimulated with 5 ng/ml TGF-{beta} for 2 h, followed by extensive wash. The cells were then incubated with 125I-labeled TGF-{beta}, cross-linked by disuccinimidyl suberate, lysed, and subjected to SDS-PAGE analysis followed by autoradiography. As a specificity control, unstimulated T cells were incubated with 125I-labeled TGF-{beta} in the presence of 200-fold excess unlabeled TGF-{beta} (lanes 1 and 2). One representative result of two experiments performed is shown. C, Western blot analysis of Smad7 expression in T cells from G{alpha}i2–/– (odd lanes) and WT (even lanes) littermates. Splenic and LN T cells were stimulated with 5 ng/ml TGF-{beta} at the indicated times and subjected to Western blot analysis using anti-Smad7 Ab (top panel). The same membrane was stripped and blotted with anti-G3PDH Ab (bottom panel). One representative result of three experiments performed is shown.

 
Diminished phosphorylation of Smad2 and Smad3 at the basal levels or at the late phase of TGF-{beta} stimulation did not appear to result from the amount of TGF-{beta} receptors on T cell surface, because TGF-{beta} receptors I and II were expressed at comparable levels on T cells from either mouse when the cells were treated in the same conditions (Fig. 3B, odd vs even lanes). We also detected a significant decline in TGF-{beta}-induced expression of Smad7, an inhibitor for TGF-{beta} responses, in agreement with an impaired TGF-{beta} signaling in T cells after null mutation of G{alpha}i2 (Fig. 3C). The result also suggests that Smad7 is unlikely to play a significant role in lack of TGF-{beta} responses in G{alpha}i2–/– T cells.

We next tested the potential signaling pathways linking G{alpha}i2 with TGF-{beta}. To this end, various phosphatase and kinase inhibitors that may block down-stream signaling of G{alpha}i2 were used to treat T cells. As shown in Fig. 4A, treatment of the cells with a phospholipase C (PLC) inhibitor D609 at a concentration of 100 nM, but not at 20 nM, enhanced phosphorylation of Smad2 and Smad3 in G{alpha}i2-deficient T cells to levels comparable to those seen in WT T cells (22). Consistent with this, another PLC inhibitor named ET-18-OCH3, which selectively inhibits phosphatidylinositol-specific PLC (PI-PLC), also restored phosphorylation of these two Smad proteins in G{alpha}i2–/– T cells (Fig. 4B, lanes 5 vs 6). The results implicate involvement of PLC, a down-stream signaling molecule of G{alpha}i2, in altered levels of Smad2 and Smad3 phosphorylation in lack of G{alpha}i2. In contrast, inhibitors including calyculin A (100 nM) for phosphatases I and II and cAMP analog cAMP-8-CL (0.5 µM) for PKA (data not shown) showed little effects on the levels of phosphorylated Smad2 and Smad3, suggesting that phosphatases I and II and PKA may not be essential in the reduced phosphorylation of these two proteins (Fig. 4B, lanes 3 vs 4). No effect of calyculin A on the levels of phosphorylated Smad2 and Smad3 was not due to a loss of activity of this compound, because its activity was validated by its ability to sufficiently block T cell proliferation (data not shown). Unlike calyculin A, okadaic acid at 100 nM, another phosphatase inhibitor for broad serine/threonine phosphatases, augmented phosphorylation of Smad2 and Smad3 similarly as PLC inhibitors (Fig. 4B, lanes 7 vs 8) (23). How this phosphatase inhibitor affects phosphorylation levels of Smad2 and Smad3 requires further investigation.



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  		FIGURE 4. Restoration of Smad2 and Smad3 phosphorylation by PLC inhibitors or okadaic acid. A, Increased phosphorylation of Smad2 and Smad3 in G{alpha}i2–/– T cells in the presence of a PLC inhibitor. T cells isolated from G{alpha}i2–/– (even lanes) and WT (odd lanes) mice were treated for 2 h with or without PLC inhibitor D609 at indicated concentrations and then subjected to Western blot analysis. One representative result of three experiments performed is shown. B, Effects of various inhibitors on Smad2 and Smad3 phosphorylation in the absence of G{alpha}i2. T cells isolated from G{alpha}i2–/– (odd lanes) and WT (even lanes) mice were treated for 2 h with or without calyculin A (Caly A, 100 nM), ET-18-OCH3 (ET-18, 10 µM), or okadaic acid (OA, 100 nM), followed by Western analysis. One representative result of three experiments performed is shown.

 
TNBS-induced colitis in mice receiving G{alpha}i2–/– T cells

Because the abrogation of TGF-{beta} signaling in T cells alone can induce colitis in mice (24), impaired responses of G{alpha}i2-deficient T cells to TGF-{beta} may be a primary defect leading to colitis in G{alpha}i2-deficient mice. To test this possibility, splenic T cells isolated from healthy G{alpha}i2–/– and control WT mice were injected i.p. into gamma-irradiated cognate WT mice. Colitis development was monitored in the recipient mice for 5–8 mo, but no overt clinical signs of colitis were seen in the recipients (data not shown). These observations are in agreement with the study showing that adoptive transfer of G{alpha}i2-deficient T cells into RAG2–/–129Sv mice failed to induce colitis in the receipt, unless the G{alpha}i2–/– T cells were first activated in vitro with Ags prepared from Helicobacter pylori (25). To activate T cells, the haptenating reagent TNBS, which induces colitis in susceptible mouse strains, was instilled into the distal colon of irradiated mice that were previously infused with either G{alpha}i2-deficient T cells or WT T cells. As shown by the weight curves in Fig. 5A, mice transferred with WT T cells did not develop any clinical signs of colitis, as this mouse strain is resistant to TNBS-induced colitis (26). They gained weight steadily following a brief initial decline. In contrast, mice receiving G{alpha}i2–/– T cells experienced a progressive weight loss that was more significant in the second week than in the first week following TNBS instillation. The mice developed diarrhea, wasting, a scruffy coat, and a hunched over habitus; overall an 18% weight loss was measured 12 days after instillation. The colons of diseased mice revealed striking hyperemia, with shortening and thickening. The average colitis score in two experiments evaluated by histologic examination was 2.89 (4 being the most severe score) in mice transferred with G{alpha}i2–/– T cells, significantly higher than the 0.6 score obtained with mice receiving WT T cells (p < 0.01) (Fig. 5B). Control mice receiving 50% ethanol alone developed negligible inflammation in both mice. In severe cases, we observed dense lymphocytic infiltrates in the lamina propria, with increased numbers of intraepithelial lymphocytes (Fig. 6D). Multifocal mucosal ulceration was present, in association with thickening of the colon wall and distortion of the crypt architecture (Fig. 6B). These results underscore a critical role for T cells in colitis development in G{alpha}i2–/– mice.



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  		FIGURE 5. Induction of colitis by colonic instillation of TNBS in mice adoptively transferred with G{alpha}i2-deficient T cells. A, Weight curve of adoptively transferred mice following TNBS instillation. WT littermate mice were irradiated and adoptively transferred with either WT or G{alpha}i2-deficient (KO) T cells. The mice were instilled with TNBS into the colon lumen 10 days later. Mean ± SD (n = 7) of weight changes in the mice is shown over a 12-day period. * and **, Statistical significance (p < 0.05) or high significance (p < 0.01), respectively. B, Colitis score of TNBS-treated mice adoptively transferred with G{alpha}i2-deficeint T cells. Colitis was induced by instillation of TNBS as in A. The colons were fixed, prepared for histological examination, and graded using a disease activity index as described in Materials and Methods. Data collected from mice infused with G{alpha}i2–/– T cells (n = 16) and WT T cells (n = 11) for each treatment were shown.

 


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  		FIGURE 6. Histological examination of diseased colons. Shown are H&E-stained cross-sections of the colon removed from 10 days after TNBS instillation. A and C, Sections (x100) of cecum or colon, respectively, of control mice infused with WT T cells and instilled with TNBS in 50% ethanol. B and D, Sections from mice infused with G{alpha}i2–/– T cells and instilled with TNBS in 50% ethanol. Note: colitis is present in B and D, but not in A and C.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our study shows for the first time that TGF-{beta} relies, at least in part, on the presence of G{alpha}i2 for its inhibitory effects on proliferation and cytokine production in mouse peripheral T cells. Lack of G{alpha}i2 abrogated the inhibitory effects of TGF-{beta} on cytokine production and proliferation of T cells, which may be one of the primary defects underlying the Th1-skewed hyperimmune responses in the mice. Numerous studies have demonstrated an important role for TGF-{beta} in mucosal homeostasis (27, 28, 29, 30). For instance, expression of TGF-{beta} by intranasal administration of a TGF-{beta} encoding plasmid suppresses the Th1 cytokine response seen in TNBS-induced colitis by down-regulation of IFN-{gamma} and IL-12 expression and ameliorates the disease (27, 28, 31). The importance of TGF-{beta} in balancing mucosal immunity is also underscored by the development of IBD in mice transgenic expressing a dominant-negative TGF-{beta} receptor type II under a T cell-specific promoter that abrogates TGF-{beta} signaling specifically in T cells (24).

In view of the importance of TGF-{beta} in controlling colonic Th1 immune responses, we would expect that adoptive transfer of G{alpha}i2-deficient T cells into immunocompromised mice alone would be sufficient for induction of colitis in the recipient mice. This is apparently not the case in our study. Consistent with this is the development of neither clinic signs of colitis, nor abnormality in T cell homeostasis in Smad3-deficient mice that also displayed an impaired response to TGF-{beta}-mediated inhibition of anti-CD3-stimulated T cell proliferation and cytokine production (32). These studies emphasize distinct roles for G{alpha}i2 and Smad3 molecules in the etiology of colon diseases. A lack of spontaneously developing colitis in mice adoptively transferred with G{alpha}i2–/–T cells suggests that B cells, macrophages, dendritic cells, and other leukocytes may also play roles in the initiation of colitis in the mice. In this regard, G{alpha}i2 appears to be required for development of IL-10-producing B cells that are predominantly activated by LPS that is highly expressed in enteric microflora (33). B cells in G{alpha}i2–/– mice are deficient in LPS-induced proliferation and IL-10 production. However, the major source of IL-10 production seems to be macrophages and Th2 and T regulatory cells (34). Macrophages were constitutively activated in G{alpha}i2-deficient mice before the onset of colitis and yet there were no differences in the levels of IL-10 production in homogenate of either the colon or small intestine (our unpublished data). These observations challenge a role for a B cell abnormality in the onset of disease in the mice.

In every murine model so studied, spontaneous colitis does not occur if an animal is reared in a germfree environment. This suggests that stimulation by one or more Ags derived from the bacterial flora is necessary for the initiation and/or maintenance of mucosal inflammation. The bacterial flora varies from one mouse strain to another and may change with environmental factors (35). Lack of an appropriate T cell stimulation may account for the absence of disease in our adoptive transfer mice. TNBS, which haptenates autologous colonic proteins with a TNP moiety and elicits an immune response against modified colonic self-Ags, has been commonly used for induction of chronic colitis in BALB/c and SJL/J mice, characterized by severe diarrhea, weight loss, and rectal prolapse, an illness that mimics some characteristics of Crohn’s disease in humans. TGF-{beta} has been shown to be a primary mechanism of counterregulation of Th1 cell-mediated mucosal inflammation induced by this haptenating agent (36). Therefore, given defective G{alpha}i2–/– T cell responses to TGF-{beta}, the autoimmune response induced by TNBS would be persistent, giving rise to colitis. Indeed, mice infused with G{alpha}i2–/– T cells developed colitis following instillation of TNBS into the colon lumen, whereas the same treatment failed to induce colitis in mice with adoptive transfer of WT T cells, as this strain of mice are resistant to TNBS-induced colitis (26). The result clearly suggests that T cell dysfunction in G{alpha}i2-deficient mice renders the mice susceptible to colitis development.

Our study showed that a lack of G{alpha}i2 resulted in decreased levels of Smad2 and Smad3 phosphorylation and consequent expression of Smad7, but it did not affect cell surface expression of TGF-{beta} receptors. The findings suggest that TGF-{beta} receptors or Smad7 may not be the key in the impaired TGF-{beta} responses in G{alpha}i2–/– T cells. A potential interplay between G{alpha}i2 and TGF-{beta} signaling pathways may be through either reduced activity of a Smad2 and Smad3 kinase or increased activity of a Smad phosphatase or Smad-binding partner that protects Smad2 and Smad3 proteins from phosphorylation, leading to reduced phosphorylation of Smad2 and Smad3 proteins in the absence of G{alpha}i2, which is under current investigation. Significance of increasing phosphorylation levels of Smad2 and Smad3 by okadaic acid in the absence of G{alpha}i2 remains to be determined, because treatment of cells with this phosphatase inhibitor would affect phosphorylation/dephosphorylation of many signaling proteins. Although the specific phosphatase or Smad-binding partner responsible for the decreased phosphorylation of Smad2 and Smad3 is unknown at present, restoration of phosphorylation of Smad2 and Smad3 by PLC inhibitors in absence of G{alpha}i2 raises an intriguing possibility that enhanced activity of PLC in the absence of G{alpha}i2 may play a role in the reduction of Smad2 and Smad3 phosphorylation in G{alpha}i2–/– mice. In this regard, Watkins et al. (37) showed that G{alpha}i2–/– cells had increased basal and hormone-stimulated PLC activity that subsequently augmented the levels of phosphatidyl inositol-3,4,5-triphosphate and Ca2+ flux (37, 38). These two important second messenger molecules function signal upstream of a variety of responses such as activation of p70S6K, Art/protein kinase B, Ca2+-calmodulin-dependent protein kinase II (Cam Kinase II), protein kinase C and others (3, 4, 5, 6, 7, 8, 9), thus opening a variety of avenues for G{alpha}i2 and TGF-{beta} signaling pathways to interplay. Further characterization of the Smad-binding partner or a specific phosphatase that directly regulates Smad2 and Smad3 phosphorylation will help us to understand the interplay of G{alpha}i2 and TGF-{beta} signalings and identify potential targets for the selective manipulation of Th1 immune responses.


   Acknowledgments
 
We thank Drs. Edwards B. Leof for anti-phospho-Smad3 antibody, X. H. Feng for stimulating discussion, Thomas Flotte and his pathology group for help in preparation and examination of the murine colitis samples, and James Versalovic for reading the manuscript. We are grateful to Lu Zhang for her technical assistance and animal husbandry.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work is supported by the National Institutes of Health Grant AI050822, Research Scholar Grant RSG-01-178-01-MGO from the American Cancer Society, and a Moran foundation award (PRJ 00-114) from the Baylor College Medicine (to M.X.W.), pilot grants (to M.X.W.) from National Institutes of Health Project Grant DK43351 and Public Health Service Grant DK56338, which fund the Center for the Study of Inflammatory Bowel Disease at the Massachusetts General Hospital and the Texas Gulf Coast Digestive Diseases Center, respectively. Back

2 Address correspondence and reprint requests to Dr. Mei X. Wu, Wellman Center for Photomedicine, Wel 204, Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114. E-mail address: mwu2{at}partners.org Back

3 Abbreviations used in this paper: IBD, inflammatory bowel disease; TNBS, trinitrobenzesulfonic acid; G{alpha}i2, G protein {alpha}2 inhibitory subunit; KO, knockout; WT, wild type; PLC, phospholipase C; LN, lymph node; PTX, pertussis toxin. Back

Received for publication November 10, 2004. Accepted for publication March 2, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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