Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) plays an important role in T and B lymphocyte signaling; however, the function of SHP-1 in Th cell differentiation, in particular, the Th1 response, has not been defined. In this study, we provide evidence that SHP-1 phosphatase negatively regulates Th1 cell development and IFN-γ production. Compared with the wild-type control, anti-CD3-activated mouse T lymphocytes carrying the motheaten viable mutation in the SHP-1 gene produced a significantly increased amount of IFN-γ in the presence of IL-12. This increase was also seen at the basal level without IL-12 addition. Similarly, Th1 cell differentiation and proliferation of anti-CD3-activated SHP-1 mutant lymph node cells in the presence or absence of IL-12 were markedly enhanced, indicating a negative role for SHP-1 phosphatase in such lymphocyte activities. Interestingly, IL-12-induced activation of Jak2 and STAT4, critical components for IL-12-mediated cellular responses, was shortened or attenuated in mutant T cells. Together these results suggest that SHP-1 negatively regulates Th1 cell development and functions through a mechanism that is not directly related to IL-12 signaling.
Naive T lymphocytes can differentiate to at least two functional/effector subsets during an immune response, i.e., Th1 cells, which secret IFN-γ to mediate cellular immunity, and Th2 cells, which produce IL-4, IL-5, and IL-13 to mediate allergic-type immunity. During these cellular processes, a number of cytokines can provide either instructive or selective signals to naive cells in the choice of their fates (1, 2). IL-12, a heterodimeric cytokine composed of a disulfide bond 40-kDa subunit and a 30-kDa subunit, has been shown to promote cell-mediated immunity against a variety of pathogens by inducing/favoring Th1 cell development, IFN-γ production, stimulation of the proliferation of activated T and NK cells, and enhancement of T and NK cell-mediated lymphocyte responses (3). IL-12 functions through binding to its cognate receptors on the cell surface, which consist of two noncovalently linked subunits, IL-12Rβ1 and IL-12Rβ2. The β1 subunit is the primary binding component in the mouse, conferring both high and low affinity binding sites to IL-12, while the β2 subunit plays a major role in triggering intracellular signal transduction, thereby mediating cellular responses to IL-12 (4). Previous studies have shown that IL-12Rβ2 is absent in naive T cells, but is induced during differentiation of naive cells along the Th1, but not the Th2 pathway, and is selectively expressed on the Th1, but not Th2 cells (5, 6). IL-12 functions through activation of several intracellular signaling cascades, particularly the Jak/STAT pathway. STAT4 has been shown to be important for the production of IFN-γ in response to IL-12 (7, 8, 9). Nevertheless, even though there is no doubt that IL-12 can significantly enhance Th1 cell response in the culture system, emerging evidence has indicated that IL-12 might function through selecting or clonally amplifying stochastic Th1 cells rather than inducing or instructing Th1 commitment (1, 2), and STAT4-independent Th1 cell development has also been described (10, 11, 12).
The precise regulation of Th1 cell development and function is not fully understood. As protein tyrosine phosphorylation and dephosphorylation are fundamental biochemical events in intracellular signal transduction, tyrosine phosphatases are presumably involved. Unfortunately, it is unclear what and how tyrosine phosphatases regulate these cellular processes. SHP-1,3 a Src homology 2 domain-containing protein tyrosine phosphatase, is predominantly expressed in hemopoietic and lymphoid cells. A putative negative role for this phosphatase in hemopoietic cell and lymphocyte regulation has been suggested by a number of studies using motheaten and motheaten viable mouse strains carrying spontaneous SHP-1 mutations (13, 14). Motheaten (me/me) and motheaten viable (mev/mev) mice contain a mutation in the coding region for the N-terminal Src homology 2 domain and the catalytic domain of SHP-1, respectively, which result in two aberrant loss-of-function 67- and 71-kDa proteins or SHP-1 deficiency (15, 16, 17). Homozygous mutant mice exhibit multiple hematological and immunological abnormalities in T and B lymphocytes, NK cells, granulocytes, and macrophages. Both me/me and mev/mev mice develop systemic autoimmune disease and die ∼3 and 9 wk after birth, respectively. High levels of Igs, particularly, autoantibodies in peripheral blood, overproduction of macrophages and CD5-positive (CD5+) B cells, and excessive erythropoiesis in the spleen suggest a primarily negative role for this phosphatase in hemopoietic cell development and lymphocyte function. Consistent with these phenotypes, it has been shown that SHP-1 attenuates signals emanating from receptors for erythropoietin (EPO), IL-3, GM-CSF, and M-CSF, and mediates inhibitory signals triggered by TCR, BCR, Igγ Fc domains (FcγRIIB1), NK cell inhibitory receptor, CD22, and CD72 (13, 14).
The role of SHP-1 phosphatase in T lymphocyte differentiation has not been well characterized. SHP-1 has been shown to be required for IL-4 induction of IL-4Rα (18). It also negatively regulates IL-4-induced tyrosine phosphorylation of STAT6 (19) and the Th2 cell differentiation (20). However, the potential role for SHP-1 phosphatase in the Th1 cell development remains to be defined. Although this phosphatase has been demonstrated to play a negative role in the regulation of EPO-induced Jak/STAT signaling (21, 22), it is unclear whether it functions in the IL-12-induced Jak/STAT pathway in the same fashion. To address these issues, we examined Th1 responses in mev/mev lymphocytes mutant for SHP-1. The results have shown that Th1 cell development and IFN-γ production are significantly enhanced in SHP-1 mutant cells. Interestingly, IL-12Rβ2 signaling in the mutant cells is dampened, indicating that the negative role of SHP-1 phosphatase in Th1 cell differentiation and function is not via IL-12 signaling.
Materials and Methods
Mice and reagents
SHP-1 heterozygous mutant (mev/+) mice were purchased from The Jackson Laboratory and were housed in the American Red Cross vivarium. All animal procedures complied with the National Institutes of Health Guideline for the Care and Use of Laboratory Animals. Mice generated from the intercrosses of mev/+ mice were genotyped by PCR detection of wild-type (WT) and mutant SHP-1 loci from the tail genomic DNA, as we previously reported (23). Anti-IL-12Rβ2 and anti-STAT4 Abs were purchased from Santa Cruz Biotechnology. Anti-SHP-1, anti-Jak2, and anti-phosphotyrosine (PY) (4G10) Abs were obtained from Upstate Biotechnology. Anti-CD3, PE-labeled anti-CD4, anti-IL-4, FITC-labeled anti-IFN-γ Abs, the IFN-γ ELISA kit, and the IL-4 ELISA kit were supplied by BD Pharmingen. The CD4+ cell isolation kit was purchased from Miltenyi Biotec. Recombinant mouse IL-12 p40 and biotin-labeled anti-IL-18R α-chain (IL-18Rα) Ab were obtained from R&D Systems.
IFN-γ production and Th1 cell differentiation assays
Sex-matched 6- to 8-wk-old WT and mev/mev mice were euthanized. Lymph node cells or total CD4+ T cell population isolated from splenocytes by positive selection using the CD4+ cell enrichment kit were used for the IFN-γ production and the Th1 cell differentiation assays, as reported (7, 8). For the IFN-γ production assay, lymph node cells or CD4+ splenocytes were cultured at the density of 2 × 106 cells/ml in RPMI 1640 medium supplemented with 15% FCS and IL-12 (10 ng/ml) for 2 days in anti-CD3 (2 μg/ml)-coated tissue culture plates. Supernatants were then harvested, and the concentration of IFN-γ was determined by the IFN-γ ELISA. For the Th1 cell differentiation assay, lymph node cells or CD4+-enriched splenocytes were cultured in IL-12 and anti-IL-4 Ab (10 μg/ml)-containing medium in anti-CD3-coated plates for 6 days. The cells were then harvested and washed twice with prewarmed PBS, and then cultured in regular RPMI 1640 medium with 15% FCS at a concentration of 2 × 106 cells/ml in anti-CD3-coated plates for 24 h. Supernatants were collected for IFN-γ measurement by IFN-γ ELISA using the ELISA kit following the manufacturer’s instruction. Mouse rIFN-γ was used as a standard.
IFN-γ intracellular staining
For examining IFN-γ production on a per cell basis, differentiated cell populations were washed and recultured in anti-CD3-bound dishes for 6 h in the presence of monensin (2 μM), which inhibits intracellular protein transport and thereby the secretion of newly produced proteins. Cells were harvested and fixed with 2% paraformaldehyde. Fixed cells were permeabilized in PBS containing 2% FCS and 0.1% saponin and then stained with FITC-labeled anti-IFN-γ Ab, followed by FACS analyses.
Immunoprecipitation and immunoblotting
Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 2 mM Na3VO4, 1 mM PMSF, and 1 μg/ml leupeptin/aprotinin). Whole cell lysates (500 μg) were immunoprecipitated with 1–2 μg of purified Abs. Immunoprecipitates were washed three times with HNTG buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1% glycerol, 0.1% Triton X-100, and 1 mM Na3VO4) and resolved by SDS-PAGE, followed by immunoblottings with the indicated Abs.
Anti-CD3-activated lymph node cells (1 × 104 cells/well) were cultured in 96-well round-bottom plates with IL-12-containing RPMI 1640 medium supplemented with 15% FCS. The cells were pulsed for the last 18 h of 48-h incubation with 1 μCi/well [3H]TdR (New England Nuclear) and harvested onto glass-fiber filters. [3H]TdR incorporation was analyzed by liquid scintillation counting, and results were expressed as mean cpm of triplicate cultures.
The role of SHP-1 tyrosine phosphatase in regulating Th1 cell development and function has not been characterized. To address this issue, mev/mev mice that are defective in SHP-1 function (15, 16, 17) were used. The mev/mev mice exhibit a dramatic change in cell composition in the spleen due to the shift of hemopoiesis from the bone marrow to the spleen (24, 25). As a result, a majority of splenocytes are myeloid and erythroid cells, but not lymphocytes. Therefore, we decided to use lymph nodes to examine Th1 responses. As shown in Fig. 1,A, lymph nodes dissected from mev/mev mice are 3–5 times larger than those from WT littermates. This phenotype is very likely to result from loss of inhibitory roles of SHP-1 phosphatase in T lymphocyte signaling. The cell composition in mutant lymph nodes, however, appears not to be significantly changed. The percentages of CD4+ cells in WT and mev/mev lymph nodes are comparable (Fig. 1 B).
Two assays were next performed to determine Th1 cell differentiation, i.e., the IFN-γ production assay and the Th1 differentiation assay (see details in Materials and Methods). IFN-γ production by anti-CD3-activated mev/mev lymphocytes in the presence of IL-12 was significantly increased compared with WT cells (Fig. 2,A). The IFN-γ production experiment was also repeated with CD4+-enriched splenocytes (see Materials and Methods); similar results were obtained (data not shown). To rule out the possibility that the different efficiencies of IFN-γ production might be attributed to a difference in the induction of the IL-12R, expression of the IL-12R on activated WT and mev/mev lymphocytes was examined. Both immunoblotting (Fig. 2,B) and flow cytometric (Fig. 2 C) analyses showed that IL-12Rβ2, the signaling component of the IL-12R, was equally induced in the anti-CD3-activated lymphocytes of both types. Thus, the results indicated that SHP-1 negatively regulated Th1 response.
To further define the function of SHP-1 phosphatase in lymphocyte processes, we conducted the Th1 differentiation assay. In the presence of anti-CD3, IL-12, and anti-IL-4 Ab, WT and mev/mev lymph node cells were induced to differentiate toward Th1 cells. Six days later, Th1 cells differentiated from naive lymphocytes were determined by examining the IFN-γ-producing capacities of the differentiated cell populations after restimulation with anti-CD3. As shown in Fig. 3 A, Th1 cell differentiation defined by IFN-γ production was dramatically enhanced in the mev/mev culture. It is worth mentioning that we also examined production of IL-4, one of the Th2 cytokines, in the Th1 cell differentiation systems using ELISA. The results showed that the IL-4 produced by the same cell populations was minimal in either WT or mutant cultures (data not shown), suggesting that no significant Th2 cells were generated in our Th1 differentiation procedures.
To determine whether the increased IFN-γ production in the mutant cell population is attributed to the enhanced Th1 differentiation or to the increased IFN-γ production on a per cell basis, we performed intracellular cytokine staining for IFN-γ. The results showed that both the percentage of IFN-γ-producing cells and the expression level per cell determined by the mean value of staining density were increased in the SHP-1 mutant population (Fig. 3,B). Thus, it is clear that loss of SHP-1 function not only increases Th1 cell differentiation, but also enhances IFN-γ production on a per cell basis. We also examined differentiated cell populations for expression of the IL-18R, one of the reported Th1 cell surface markers. The percentages of the IL-18R-positive cells were comparable in both WT and mutant populations. However, the expression level of the IL-18R determined by the mean value of staining density was significantly increased in SHP-1 mutant cells (Fig. 3 C). Because IL-18 also plays an important role in Th1 cell function (2), this result suggests that differentiated SHP-1 mutant cells may receive enhanced IL-18 stimulation, which may also be in part associated with the elevated IFN-γ production in mutant cells. The observation that there is no significant difference in the percentages of IL-18Rα-positive cells between the two cell populations would indicate that the IL-18R is not as specific as IFN-γ production to Th1 cells. Collectively, the Th1 differentiation results further confirm a negative role for SHP-1 phosphatase in Th1 cell development.
We also assessed proliferation of WT and mev/mev cells in response to IL-12. Anti-CD3-activated lymph node cells were cultured in IL-12-containing medium for 48 h. [3H]TdR incorporation results showed that mev/mev cells proliferated much faster than WT cells at all concentrations of IL-12 tested (Fig. 4). Intriguingly, while we were analyzing the data from this experiment and those described above, it always came to our attention that the basal levels (in the absence of IL-12) of IFN-γ production or DNA synthesis activity were also markedly increased in SHP-1 mutant cells. This observation prompted us to determine the role of SHP-1 phosphatase in IL-12-induced signal transduction.
We thus examined the potential involvement of SHP-1 phosphatase in IL-12 signaling. Tyrosine phosphorylation of SHP-1 in response to IL-12 stimulation was analyzed. Lymph node T cells from WT C57BL/6 mice were activated with anti-CD3 for 48 h. Activated cells were cultured in serum-free medium in regular tissue culture plates for 5 h and then stimulated with IL-12 (10 ng/ml) for various periods of time. As shown in Fig. 5, phosphorylation of SHP-1 was nicely induced by IL-12 stimulation. As tyrosine phosphorylation is important for SHP-1 catalytic activation and protein-protein interactions, this result indicates that SHP-1 phosphatase is involved in the signal transduction of IL-12. Additionally, Jak2 was found greatly activated following IL-12 stimulation, as evidenced by its tyrosine phosphorylation response. STAT4, the downstream substrate of Jak2, was also markedly tyrosine phosphorylated, consistent with its important role in IL-12-induced cellular response (7).
We next analyzed IL-12-induced signal transduction in SHP-1 mutant lymphocytes. Lymph node cells isolated from WT and mev/mev mice were activated with anti-CD3 for 48 h. As expected, tyrosine phosphorylation levels of cellular proteins were greatly increased in mutant lymphocytes (data not shown). After being transferred to serum-free medium in regular tissue culture plates for 5 h (to decrease the basal level of tyrosine phosphorylation), the cells were then stimulated with IL-12, as described above. Anti-PY immunoblotting analysis of whole cell lysates showed that tyrosine phosphorylation of cellular proteins in activated WT cells was induced by IL-12. However, this response in SHP-1 mutant cells was diminished (Fig. 6,A). Compared with that in WT cells, Jak2 activation, defined by its tyrosine phosphorylation, was slightly elevated in mev/mev cells at the early time point (10 min) (Fig. 6,B). This is consistent with previous observations that SHP-1 functions as a negative regulator on Jak2 in the signal transduction of EPO (21, 22). Interestingly, activation of Jak2 could not be sustained; its tyrosine phosphorylation in mev/mev cells was significantly decreased after 40 min (Fig. 6,B). Activation (tyrosine phosphorylation) of STAT4, the key component in IL-12-mediated cellular response, was also reduced in mev/mev cells at all time points examined (Fig. 6 B). This experiment was repeated several times; similar results were produced each time. Altogether, the biochemical results suggest that SHP-1 phosphatase is required for the maintenance of the IL-12-induced Jak2/STAT4 pathway.
Because the enhancement in Th1 differentiation of SHP-1 mutant cells appears to be unrelated to the function of SHP-1 in IL-12 signaling, we next performed Th1 cell differentiation for anti-CD3-activated WT and SHP-1 mutant lymphocytes in the absence of IL-12. To block the Th2 differentiation, anti-IL-4 Ab was still added to the culture systems. As shown in Fig. 7,A, without extrinsic signal (IL-12), both WT and SHP-1 mutant cells can still differentiate to IFN-γ-producing cells, albeit at lower levels. This is consistent with the selective model of Th1 development, i.e., IL-12 is not absolutely required for Th1 cell differentiation (2, 11). Interestingly, in the absence of IL-12, Th1 cell differentiation efficiency (the percentage of IFN-γ-producing cells) as well as the IFN-γ production level per cell (the mean value of staining density) were also greatly increased by loss of SHP-1 function (Fig. 7 B). These results further confirm that SHP-1 phosphatase negatively modulates Th1 response via other mechanisms that are not directly linked to IL-12 signaling.
Our results presented in this work suggest that SHP-1 phosphatase negatively regulates Th1 cell differentiation and IFN-γ production. This function of SHP-1 appears to be unrelated to its role in the IL-12-induced Jak/STAT pathway. Even though activation of Jak2 and STAT4 is shortened and attenuated (but not blocked) by the motheaten viable mutation of SHP-1 (Fig. 6), Th1 cell development and IFN-γ production of anti-CD3-activated SHP-1 mutant T lymphocytes (in the presence of IL-12) are markedly enhanced (Figs. 2,A and 3). Moreover, in the absence of IL-12, Th1 cell differentiation was also significantly increased in SHP-1 mutant cells (Fig. 7).
Although IL-12 has been shown to promote Th1 cell differentiation and there is no doubt that in the presence of IL-12, efficient Th1 type responses are obtained, several lines of evidence have indicated that IL-12 might not be an absolute requirement and it might have a role more in the fixation and amplification of Th1 cells than in their commitment (2, 11). IL-12 might also function by synergy with many other activating or instructive stimuli such as the TCR-CD3 complex, the CD28 receptor, IL-2, and IL-27 (2). In T cells, two different pathways for the induction of IFN-γ production exist: one pathway is induced by stimulation through TCRs, CD3, and Fc receptors. The other pathway is induced by stimulation with IL-12 alone, or in synergy with IL-18 or the ligand for CD28 (2). Thus, the IL-12-IFN-γ axis might amplify the commitment of Th1 cell differentiation in conjunction with signaling through the TCR or other receptors, such as IL-18R.
The detailed mechanisms by which Th1 cell differentiation and IFN-γ production of SHP-1 mutant cells are enhanced remain to be defined. Several possibilities may exist. First, TCR signaling has been shown to induce early Th1 polarization of naive T cells independent of IL-12 (11, 26), and strength and quality of TCR signaling are important for Th1 cell commitment. The negative role of SHP-1 phosphatase in TCR signaling has been well established (13, 14). In this study, we also found that even without IL-12 stimulation, the DNA-synthesizing activity of anti-CD3-activated SHP-1 mutant lymphocytes is markedly elevated (Fig. 4) and tyrosine phosphorylation levels of cellular proteins were greatly increased in mutant lymphocytes cultured in anti-CD3-bound dishes (data not shown). Therefore, increased effects of anti-CD3 activation in SHP-1 mutant cells might contribute to the enhancement of their subsequent Th1 differentiation. Second, a number of cytokines produced by activated or differentiated lymphocytes synergize with IL-12 in priming Th1 cell differentiation (1, 2). These activating or instructive cytokines also play important roles in polarizing Th1 cells. SHP-1 might negatively regulate the signal transduction of such stimuli. Third, the stress-activated MAPK, p38, functions in both IL-12 and TCR signaling. p38 kinase has been shown to promote Th1 responses in CD4+ T cells by up-regulation of IFN-γ transcription (8, 9, 10, 27). It is also possible that SHP-1 negatively regulates Th1 cell development and function by suppression of p38 activity. Finally, recent studies have shown that SHP-1 is localized in both the cytoplasm and the nucleus (28). It is possible that the as yet unknown nuclear function of SHP-1 phosphatase might be involved in the regulation of lymphocyte activities. Loss of the nuclear function of SHP-1 enhances the basic status of cell life, and this in turn increases cellular responses to instructive or selective signals for Th1 development. This idea is supported by our recent cell cycle analysis data, i.e., after depletion of extrinsic signals (growth factors), G1 arrest of catalytically inactive SHP-1 C455S-overexpressing BaF/3 cells was attenuated (our unpublished data). It is expected that further characterization of the nuclear function of SHP-1 would help better understand molecular mechanisms of this phosphatase in the regulation of lymphocyte function.
Another interesting finding in this study is that SHP-1 dynamically regulates IL-12 signal transduction. Even though SHP-1 negatively modulates Th1 cell development in the presence or absence of IL-12, this phosphatase is indeed involved in the IL-12 signaling pathway, as evidenced by its phosphorylation response (Fig. 5). SHP-1 appears to play a complex role in IL-12 signal transduction. It temporarily inhibits Jak2 activation, and then turns to promote the Jak/STAT pathway. Jak2 activation was slightly elevated early following IL-12 stimulation in mev/mev cells, but its activity could not be sustained. Forty minutes after IL-12 stimulation, tyrosine phosphorylation of Jak2 was markedly decreased in mutant cells compared with that in WT cells. As a result, STAT4 activation was also attenuated (Fig. 6 B).
Protein tyrosine phosphatases function in signal transduction by dephosphorylating other signaling molecules. However, they may not always play a negative role in cell signaling. Some tyrosine phosphatases do function to enhance signal transduction. For example, SHP-2, a SHP-1-related tyrosine phosphatase, has been demonstrated to positively modulate a number of receptor or cytosolic tyrosine kinase-initiated signaling pathways (29, 30). Analyses of B cells pharmacologically or genetically ablated for the receptor protein tyrosine phosphatase CD45 indicate that CD45 acts primarily to promote BCR-evoked lymphocyte activation (31, 32). A negative role of SHP-1 phosphatase in T and B cell Ag receptor signaling has been extensively characterized (13, 14). SHP-1 also interacts and dephosphorylates ITIM motifs on the coreceptor of BCR such as CD22, CD72, and FcγRIIB (14). In the cytokine-mediated signaling pathways, SHP-1 has been shown to negatively modulate Jak2 and the Erk pathways (21, 22, 33). However, a positive role of SHP-1 phosphatase in FcεRI-mediated activation of JNK kinase and TNF-α production and the IFN-γ-induced Jak/STAT pathway has also been reported (34, 35). It is unclear at this point how SHP-1 phosphatase functions kinetically in IL-12 signaling. Clearly, further investigations are required to define why SHP-1 phosphatase is required for a sustained activation of the Jak2/STAT4 pathway induced by IL-12. It would be interesting to determine whether SHP-1 directly or indirectly interacts with the negative regulators of the IL-12-induced signaling pathways, such as suppressor of cytokine signaling and protein inhibitor of activated STAT family members, because these negative regulators have recently been shown to play important inhibitory roles in IL-12-induced signaling and cellular responses in lymphocytes (36, 37, 38).
We thank Drs. Ueli Gubler, Zhizhuang J. Zhao, John J. O’Shea, and Christine Couldrey for the reagents, helpful discussions, and critical reading of the manuscript.
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.
This work was supported by National Institutes of Health Grant R01 HL68212 (to C.-K.Q.).
Abbreviations used in this paper: SHP, Src homology 2 domain-containing protein tyrosine phosphatase; EPO, erythropoietin; PY, phosphotyrosine; WT, wild type.