Opposing roles of STAT1 and STAT3 in T cell–mediated hepatitis: regulation by SOCS (original) (raw)
Activation of multiple JAKs, STATs, SOCSs, and proapoptotic and antiapoptotic proteins in Con A–induced hepatitis. After injection of Con A, plasma levels of a wide variety of cytokines are dramatically elevated (13). To determine the interaction of these cytokines in Con A–induced hepatitis, activation of the JAK-STAT signaling pathway in the liver was examined. As shown in Figure 1a, Con A injection rapidly induced activation of JAK1 and JAK2, but not Tyk2. Consistent with activation of JAK1 and JAK2, multiple STATs were also activated. As shown in Figure 1a, STAT1, STAT3, STAT5, and STAT6 were activated in the liver with the peak effect occurring at 1–3 hours, whereas peak STAT4 activation was detected at 9 hours. Activation of STAT2 was not detected at any time point tested. Furthermore, injection of Con A significantly induced expression of both SOCS1 mRNA and SOCS3 mRNA, whereas it only slightly induced SOCS2 and CIS expression (Figure 1a).
Activation of multiple JAKs, STATs, SOCS, and apoptosis-associated proteins in the liver after injection of Con A. (a) and (b) C57BL/6J mice were injected with Con A (10 μg/g) at various time points. Total liver protein extracts and RNA were prepared and analyzed by Western blotting and RT-PCR (indicated by asterisks), respectively, using Ab’s and primers as indicated. Data are representative of three independent experiments with similar results. p, phosphorylated form.
The robust activation of STAT1 and STAT3 suggested that the downstream effectors of STAT1 and STAT3 may be important mediators of T cell–mediated hepatitis. STAT1 and STAT3 play complimentary, counterregulatory roles in apoptosis during Con A–induced hepatitis, because STAT1 activates the proapoptotic proteins IRF-1 and p21cip1/waf1 (30, 32), while STAT3 activates the antiapoptotic proteins Bcl-2 and Bcl-XL (33). As shown in Figure 1b, IRF-1 was activated in a biphasic response, with significant peak induction at 3 hours and again at 9 hours. Expression of Bcl-XL proteins was elevated with peak effect between 9 hours and 48 hours. Additionally, Bax, a proapoptotic protein, was significantly induced with peak effect at 9 hours. Caspase-3, an indicator of apoptosis, is generated as a 32-kDa precursor, which is enzymatically cleaved into p17/p12 mature subunits (34). Caspase-3 was activated 3–9 hours after Con A injection, as assessed by generation of the mature p17 subunit. On the contrary, expression of p21cip1/waf1, p53, and Bcl-2 proteins was not altered after injection of Con A.
Liver injury and STAT1 and SOCS1 activation are attenuated, but STAT3 and SOCS3 activation are enhanced and prolonged in Con A-induced hepatitis in STAT1–/– mice. To examine the potential role of STAT1 activation in T cell–mediated hepatitis, we compared Con A–induced hepatitis in STAT1–/– and STAT1+/+ mice. As shown in Figure 2, a and b, after injection of Con A, ALT activities were dramatically elevated in wild-type mice, but were not increased in STAT1–/– mice. Examination of liver pathology showed massive necrosis in STAT1+/+ mice, but not in STAT1–/– mice. A representative example of liver histology from STAT1+/+ and STAT1–/– mice obtained 9 hours after Con A injection is shown in Figure 2b.
Since IFN-γ has been shown to play an important role in Con A–induced hepatitis (14, 35), we compared the serum IFN-γ levels in both wild-type and STAT1–/– mice after injection of Con A. As shown in Figure 2c, Con A injection–induced elevation of IFN-γ was significantly attenuated in STAT1–/– mice compared with wild-type mice. Next, we compared activation of the JAK-STAT signaling pathway in the liver of Con A–induced hepatitis in STAT1+/+ and STAT1–/– mice. As shown in Figure 2d, JAK1 activation remained unchanged and JAK2 activation was decreased in STAT1–/– mice compared with STAT1+/+ mice. Activation and induction of STAT1, as expected, were absent in STAT1–/– mice, whereas activation of STAT3 was significantly enhanced and prolonged in these mice. Consistent with absent STAT1 activation and strong STAT3 activation in STAT1–/– mice, induction of IRF-1 protein was not detected, whereas induction of Bcl-XL protein was significantly enhanced in these mice (Figure 2d). Expression of SOCS in the liver of Con A–induced hepatitis was also examined. A 20- to 25-fold induction of SOCS1 mRNA expression was observed between 3 hours and 9 hours in STAT1+/+ mice, whereas five- to eightfold induction was detected in STAT1–/– mice (Figure 2d). In contrast with decreased SOCS1 induction, Con A injection–induced SOCS3 mRNA expression was significantly enhanced at 1 hour and 48 hours in STAT1–/– mice compared with STAT1+/+ mice. Similar weak activation of SOCS2 and CIS was detected in both STAT1+/+ and STAT1–/– mice.
IFN-γ and IL-6 induce prolonged STAT3 activation in the STAT1–/– mouse hepatocytes. The above data indicate that Con A injection-induced STAT3 activation in the liver is prolonged in STAT1–/– mice, which could be due to either increased levels of STAT3-activating cytokines or increased response of STAT1–/– hepatocytes to STAT3 activation. Our observation that Con A injection–induced activation of JAKs was not enhanced in STAT1–/– mice (Figure 2d) suggested that Con A–induced prolonged activation of STAT3 in STAT1–/– mice is not due to increased levels of STAT3 activating cytokines such as IL-6. Indeed, Con A injection–induced elevation of serum IL-6 levels was decreased in STAT1–/– mice compared with wild-type mice (data not shown). To examine whether STAT1–/– hepatocytes were more susceptible to STAT3 activation, mouse hepatocytes from STAT1+/+ and STAT1–/– mice were stimulated with IFN-γ and IL-6, which are the two major cytokines responsible for STAT1 and STAT3 activation, respectively, in the livers of mice with Con A–induced hepatitis (see Figures 4 and 5). As shown in Figure 3a, IFN-γ treatment induced STAT1 activation and IRF-1 expression in STAT1+/+ mouse hepatocytes, but not in STAT1–/– mouse hepatocytes. On the contrary, the same IFN-γ treatment caused prolonged STAT3 activation and enhanced Bcl-XL expression in STAT1–/– mouse hepatocytes (Figure 3a). IFN-γ–induced SOCS1 mRNA expression was completely abolished in STAT1–/– mouse hepatocytes, whereas induction of SOCS3 mRNA expression was enhanced and prolonged in these cells, compared with wild-type cells (Figure 3a).
The effects of IL-6 on STAT activation and SOCS induction in STAT1+/+ and STAT1–/– mouse hepatocytes are shown in Figure 3b. IL-6 treatment caused weak STAT1 activation and IRF-1 induction in STAT1+/+, but not in STAT1–/– mouse hepatocytes. On the contrary, the same IL-6 treatment slightly enhanced STAT3 activation and Bcl-XL protein expression in STAT1–/– mouse hepatocytes compared with STAT1+/+ mouse hepatocytes. IL-6 treatment induced enhanced and prolonged SOCS3 mRNA expression in STAT1–/– mouse compared with STAT1+/+ mouse hepatocytes, but did not affect SOCS1 mRNA expression in either cell type. Taken together, these findings suggest that Con A–induced prolonged activation of STAT3 in STAT1–/– mice is caused by increased response of STAT1–/– hepatocytes to STAT3 activation.
Liver injury and STAT1 and SOCS1 activation are attenuated, whereas activation of STAT3 is slightly enhanced in Con A–induced hepatitis in IFN-γ–/– mice. IFN-γ, which is elevated in the serum after administration of Con A (Figure 2c), is an important activator of STAT1 signaling. To explore the role of IFN-γ/STAT1 signaling in T cell–mediated hepatitis, we injected IFN-γ–/– mice with Con A and assessed the subsequent molecular and pathological changes in the liver. Con A injection–induced STAT1 activation and induction were almost completely abolished in the liver of IFN-γ–/– mice, whereas activation of STAT3 was slightly but not significantly enhanced in IFN-γ–/– mice compared with IFN-γ+/+ mice; JAK1 and JAK2 activation were decreased in IFN-γ–/– mice (Figure 4a). Consistent with weak STAT1 activation, IRF-1 gene expression was not upregulated in IFN-γ–/– mice. Bcl-XL protein expression was slightly reduced in IFN-γ–/– mice compared with IFN-γ+/+ mice. Additionally, a 13- to 24-fold induction of SOCS1 mRNA was observed between 3 hours and 9 hours after injection of Con A in IFN-γ+/+ mice, whereas only a eight- to tenfold increase was detected in IFN-γ–/– mice (Figure 4a). Furthermore, induction of SOCS3 expression was slightly but not significantly enhanced in IFN-γ–/– mice compared with IFN-γ+/+ mice. Weak activation of SOCS2 and CIS was seen in both IFN-γ+/+ and IFN-γ–/– mice.
Next we compared Con A–induced liver injury in IFN-γ+/+ and IFN-γ–/– mice. As shown in Figure 4b, injection of Con A significantly induced serum ALT levels in IFN-γ+/+ mice, with a peak effect at 9 hours; in comparison, serum ALT levels were not significantly increased in IFN-γ–/– mice. Examination of liver histology showed massive necrosis in IFN-γ+/+ mice, whereas IFN-γ–/– mice appeared to be largely protected from hepatic injury. A representative example of liver histology from 9-hour Con A–injected IFN-γ+/+ mice and IFN-γ–/– mice is shown in Figure 4c.
STAT3 and SOCS3 activation are attenuated, but liver injury, STAT1 activation, and STAT1-controlled IRF-1 are enhanced, in Con A–induced hepatitis in IL-6–/– mice. STAT3 and its downstream antiapoptotic factors such as Bcl-2 and Bcl-XL are largely activated by the cytokine IL-6. To determine the role of IL-6/STAT3 signaling in hepatitis, IL-6–/– mice were injected with Con A, and the subsequent molecular and pathological changes in the liver were assessed. As shown in Figure 5a, Con A injection–mediated activation of STAT3 was markedly attenuated at 1 and 3 hours, whereas STAT1 activation was significantly potentiated at 3, 6, and 9 hours in IL-6–/– mice compared with IL-6+/+ mice; JAK1 and JAK2 activation were downregulated in IL-6–/– mice. Consistent with upregulation of STAT1 activation, induction of IRF-1 gene expression was enhanced in IL-6–/– mice. On the contrary, induction of Bcl-XL protein was diminished in IL-6–/– mice, which correlated with downregulation of STAT3 activation in these mice. Con A injection–induced SOCS mRNA expression was also examined. As shown in Figure 5a, Con A injection–induced SOCS1 mRNA expression was enhanced and prolonged, whereas SOCS3 mRNA induction was attenuated in IL-6–/– mice compared with IL-6+/+ mice. Furthermore, weak induction of CIS observed in IL-6+/+ mice was barely detected in IL-6–/– mice. Similar activation of SOCS2 was seen in both IL-6+/+ and IL-6–/– mice.
Liver injury was also compared in IL-6+/+ and IL-6–/– mice. As shown in Figure 5b, Con A injection–induced elevation of ALT levels was markedly increased and prolonged in IL-6–/– mice compared with IL-6+/+ control mice. Likewise, histological examination of liver revealed much more sever necrosis in IL-6–/– mice compared with IL-6+/+ control mice (Figure 5c). Finally, the levels of serum IFN-γ in wild-type and IL-6–/– mice were determined. As shown in Figure 5d, elevation of serum IFN-γ levels at 3 and 9 hours after administration of Con A is significantly enhanced in IL-6–/– mice compared with wild-type mice.
The above data indicate that Con A–induced liver injury is exacerbated in IL-6–/– mice, suggesting that IL-6 imparts protective effects against hepatic injury. To explore this hypothesis, we examined whether administration of IL-6 protects against Con A–induced liver injury. As shown in Figure 5e, injection of IL-6 markedly suppressed Con A–induced liver injury, consistent with a previous report (36).
Con A injection–mediated activation of CD4+ and NKT Cells is abolished in STAT1–/– and IFN-γ –/– mice but remains unchanged in IL-6–/– mice. Activation of CD4+ and NK T cells, which have been shown to play an important role in Con A–induced hepatitis (11, 37), was examined in the IFN-γ, STAT1, and IL-6 knockout mice. Liver lymphocyte activation was determined by FACS analysis of early activation marker CD69+. As shown in Figures 6, a, b, and c, administration of Con A caused a significant increase in CD69+ positive cells (lower- and upper-right quadrant in CD4+/CD69+ panel) in three strains of wild-type mice and IL-6–/– mice, but such activation was almost completely abolished in STAT1–/– mice and IFN-γ–/– mice. These findings suggest that activation of total liver lymphocytes including CD4+ and NK T cells is suppressed in STAT1–/– mice and IFN-γ–/– mice. Furthermore, Con A injection caused a significant increase in CD4+CD69+ double-positive cells (upper-right quadrant in CD4+/CD69+ panel) in wild-type mice and IL-6–/– mice, but not in STAT1–/– mice and IFN-γ–/– mice (Figure 6, a, b, and c), suggesting that activation of CD4+ cells is inhibited in STAT1–/– mice and IFN-γ–/– mice.
Con A injection–mediated activation of CD4+ and NK T cells is abolished in STAT1–/– and IFN-γ–/– but not in IL-6–/– mice. Wild-type and knockout mice were injected with Con A for 3, 6, and 9 hours. Hepatic lymphocytes were isolated. The surface of CD4+CD69+ or NK1.1+CD3+ was analyzed by flow cytometry. The flow cytometric analysis is representative of three independent experiments. The upper-right quadrant in each panel shows CD3+CD69+ or NK1.1+CD3+ double-positive cells (percentage of the total hepatic lymphocytes). Values are shown in d as means ± SEM from three mice at each time point. *P < 0.001 and #P < 0.01 vs. corresponding Con A–treated wild-type groups at the same time points.
It has been shown that NK T cells are rapidly activated and consequently depleted after administration of Con A (11, 37), suggesting that NK T cell activation results in depletion. As shown in Figure 6, injection of Con A led to significant depletion of NK T cells (NK1.1+CD3+ double-positive cells in upper-right quadrant) in wild-type mice, but not in STAT1–/– and IFN-γ–/– mice. On the contrary, similar NK T cell depletion was observed in IL-6–/– mice compared with wild-type mice (Figure 6, c and d). These findings suggest that NK T cell activation is impaired in STAT1–/– and IFN-γ–/– mice, but not in IL-6–/– mice in Con A–mediated hepatitis.