T cell proliferation in response to interleukins 2 and 7 requires p38MAP kinase activation.

Interleukin-2 (IL-2) is a potent T cell mitogen. However, the signaling pathways by which IL-2 mediates its mitogenic effect are not fully understood. One of the members of the mitogen-activated protein kinase (MAPK) family, p42/44MAPK (ERK2/1), is known to be activated by IL-2. We have now investigated the response to IL-2 of two other members of the MAP kinase family, p54MAP kinase (stress-activated protein kinase (SAPK)/Jun-N-terminal kinase (JNK)) and p38MAP kinase (p38/Mpk2/CSBP/RK), which respond primarily to stressful and inflammatory stimuli (e.g. tumor necrosis factor-alpha, IL-1, and lipopolysaccharide). Here we show that IL-2, and another T cell growth factor, IL-7, activate both SAPK/JNK and p38MAP kinase. Furthermore, inhibition of p38MAP kinase activity with a specific pyrinidyl imidazole inhibitor SB203580 that prevents activation of its downstream effector, MAPK-activating protein kinase-2, correlated with suppression of IL-2- and IL-7-driven T cell proliferation. These data indicate that in T cells p38MAP kinase has a role in transducing the mitogenic signal.

Interleukin-2 (IL-2) is a potent T cell mitogen. However, the signaling pathways by which IL-2 mediates its mitogenic effect are not fully understood. One of the members of the mitogen-activated protein kinase (MAPK) family, p42/44MAPK (ERK2/1), is known to be activated by IL-2. We have now investigated the response to IL-2 of two other members of the MAP kinase family, p54MAP kinase (stress-activated protein kinase (SAPK)/Jun-N-terminal kinase (JNK)) and p38MAP kinase (p38/Mpk2/CSBP/RK), which respond primarily to stressful and inflammatory stimuli (e.g. tumor necrosis factor-␣, IL-1, and lipopolysaccharide). Here we show that IL-2, and another T cell growth factor, IL-7, activate both SAPK/JNK and p38MAP kinase. Furthermore, inhibition of p38MAP kinase activity with a specific pyrinidyl imidazole inhibitor SB203580 that prevents activation of its downstream effector, MAPK-activating protein kinase-2, correlated with suppression of IL-2and IL-7-driven T cell proliferation. These data indicate that in T cells p38MAP kinase has a role in transducing the mitogenic signal.
Interleukin-2 (IL-2) 1 is a key factor in driving the proliferation of activated T lymphocytes; a crucial event in mounting an effective immune response (1). The high affinity IL-2 receptor is a heterotrimeric complex composed of ␣, ␤, and ␥c subunits, the latter being shared with the receptors for IL-4, IL-7, IL-9, and IL-15, other T cell growth factor cytokines (2).
The apparent redundancy of ERK activation in T cell proliferation is in contrast to other cellular systems, where these kinases have been implicated in mitogenic responses to growth factors (33). This functional link is supported by the transforming potential of the proximal activators of this pathway, i.e. oncogenic Ras (reviewed in Ref. 34) and Raf (35) and a constitutively active form of MEK1 (36), as well as by studies with dominant-negative and antisense cDNA (37). The inhibition of fibroblast proliferation by a specific inhibitor of MEK1, PD098059 (38), further supports a role for this pathway in proliferation.
Recently two other subgroups of the MAP kinase family have been characterized, SAPK/JNK and p38MAP kinase. These kinases respond to a variety of physicochemical stresses (e.g. UV light, translational inhibitors, hyperosmolarity), lipopolysaccharide, and the pro-inflammatory cytokines TNF-␣ and IL-1 (39 -43).
Unlike ERK, these "stress kinases" have not been implicated previously in mitogenesis. The response of these enzymes to IL-2, other T cell growth factors, or other cytokines barring those mentioned above has not been investigated previously, although SAPK/JNK has been shown to be activated in T cells by co-stimulation through CD3 and CD28 (44). This study shows that both SAPK/JNK and p38MAP kinase are activated by IL-2. In addition (which is not the case for ERK) they are also activated by IL-7. The inhibition of p38MAP kinase activity by the specific inhibitor SB203580 resulted in suppression of T cell proliferation in response to IL-2 and IL-7, suggesting that rather than being solely involved in stress responses, in T cells at least, p38MAP kinase is required to transduce the mitogenic signal.
(Sigma) incorporated into the cell membrane of live gated cells (47).
Human peripheral blood T cell isolation and proliferation were as described previously (48). SB203580 was added to the cells 15 min prior to the addition of cytokine where indicated.
In Vitro Kinase Assays-In vitro kinase assays for SAPK/JNK were performed on immunoprecipitates resuspended in 50 l of assay buffer and 30 l of 0.1 mg/ml GST-ATF2- (19 -96)  For the in vitro kinase assays for p38MAP kinase, Hsp27 kinase (which is similar, if not identical to MAPKAP kinase-2) and Hsp27 were purified from KB cells, and recombinant Hsp27 was prepared as described previously (41). To the immunoprecipitates were added 35 l of kinase assay buffer, 30 l of Hsp27 kinase (MAPKAP kinase-2), which had been previously inactivated by treatment with protein phosphatase 2A, and Hsp27 (1.5 g/track); the reaction was initiated and terminated as for the SAPK/JNK assays. All products were separated by SDSpolyacrylamide gel electrophoresis, and dried gels were autoradiographed at Ϫ70°C.
For the in vitro MAPKAP kinase-2 assays, the activated enzyme was immunoprecipitated from lysates of 5-10 ϫ 10 6 cell equivalents for 3 h at 4°C. The immunoprecipitates washed as for the p38MAP kinase and SAPK/JNK assays, resuspended in 50 l of kinase assay buffer containing 30 M final concentration of Hsp27 peptide (sequence KKLN-RTLSVA (49)). Reactions were initiated with 10 l of 180 M ATP containing 0.5 Ci of [␥-32 P]ATP. Following 20 min at 25°C, reactions were terminated by spotting the supernatants onto squares of P81 paper and placing into 0.75% orthophosphoric acid. Following three washes in acid and one in acetone, the squares were subjected to scintillation counting.

RESULTS
IL-2 and IL-7 Activate SAPK/JNK-SAPK/JNK activation was measured by phosphorylation of GST Jun-(2-89) (43) or GST-ATF-2-(19 -96) (50) as substrates, after immunoprecipitation from human (Kit-225) and murine (CT6) T cells. The enzyme was activated when precipitated from IL-2-treated cells, as compared with that from unstimulated cells (Fig. 1, a  and b). Treatment of the responsive CT6 cells with IL-7 also activated this kinase (Fig. 1b). SAPK/JNK activation was dosedependent for both cytokines (Fig. 1, c and d) and was maximal at 2 ng/ml (100 pM). This correlates with the proliferative response of CT6 to either cytokine (46). As expected, activated SAPK/JNK was also immunoprecipitated from T cells stressed with anisomycin (Fig. 1a) or exposed to TNF-␣ (Fig. 1b). No kinase activity was precipitated with non-immune rabbit serum (Fig. 1, a and b), which confirmed the specificity of the assay.
IL-2 and IL-7 Cause the Activation of p38MAP Kinase-Generally, stressful and inflammatory stimuli that activate SAPK/JNK also activate a related enzyme, p38MAP kinase. We therefore investigated whether this second stress kinase would respond to T cell mitogens. The activity of p38MAP kinase immunoprecipitated from CT6 cells was assayed by a kinase cascade involving Hsp27 kinase (which is similar, if not identical to, MAPKAP kinase-2 (51)) and its substrate (41). A substantial elevation in Hsp27 phosphorylation was observed with p38MAP kinase immunoprecipitates from cytokine-treated cells (Fig. 2a). Kinetic studies showed maximal activation by IL-7 was delayed when compared with IL-2 (Fig. 2b), although this has not been apparent in all experiments. TNF-␣ also activated p38MAP kinase as expected, although it should be noted that CT6 cells possess only the p75 TNF receptor (p75 TNFR) (52), while previous studies on the TNF-␣ response have used cells that also express the p55 TNFR. No kinase activity was precipitated by non-immune rabbit serum (Fig. 2  a). Unlike SAPK/JNK, p38MAP kinase has not been previously shown to be activated in T cells. . 3a) with the specific p38MAP kinase inhibitor, SB203580, equally inhibited proliferation induced by either IL-2 or IL-7 (IC 50 ϳ3 M). A similar IC 50 is observed for inhibition of TNF-␣ production by monocytes 2 and for collageninduced platelet aggregation (45). We next examined the effect of this inhibitor on primary T cells isolated from human blood (Fig. 3, b and c). These were treated with anti-CD3 and then induced to proliferate to IL-2 and IL-7. SB203580 inhibited the proliferation over a concentration range similar to that observed in the T cell lines. The compound was not toxic at the maximum concentrations used as judged by merocyanine staining in conjunction with fluorescence-activated cell sorter analysis (47) (not shown), nor did it inhibit all responses to the cytokines, since expression of c-Myc induced by IL-2 or IL-7 (14) was unaffected by 10 M SB203580 (Fig. 3d).

A p38MAP Kinase Inhibitor, SB203580, Suppresses IL-2 and IL-7-driven Proliferation of CT6 Cells and Activated PBMC but Not Cytokine-induced c-Myc Expression-Preincubation (15 min) of CT6 cells (Fig
IL-2 Activation of MAPKAP Kinase-2 Is Sensitive to SB203580 -The effect of SB203580 on T cell proliferation led us to question how closely this correlated with inhibition of one known function of p38MAP kinase. MAPKAP kinase-2 is phosphorylated and activated by p38MAP kinase in a number of cell types (41,51), and this is inhibited by SB203580 (53). As expected MAPKAP kinase-2 was activated by IL-2 (Fig. 4a) in a dose-dependent manner as judged by assays of the enzyme immunoprecipitated from CT6 cells. Activity was maximal at 20 ng/ml IL-2; the proliferative response of the cells displayed a similar dose dependence (not shown). The activation of the enzyme was inhibited by SB203580 (Fig. 4b) in the concentration range 0.1-1 M, in agreement with the in vitro inhibition of p38MAP kinase (53) and previous studies on MAPKAP kinase-2 (54). There was no MAPKAP kinase-2 activity in control immunoprecipitations with nonspecific antisera (not shown). In contrast, studies of ERK phosphorylation by gel retardation assay showed that the IL-2-induced phosphorylation was unaffected by 10 M SB203580 (Fig. 4c), as was the SAPK/JNK activation measured by GST-ATF-2 phosphorylation in specific immunoprecipitates (Fig. 4d), indicating the specificity of the compound. DISCUSSION This study shows that the previously termed "stress-activated kinases," p38MAP kinase and SAPK/JNK are activated in T cells by the mitogenic cytokines IL-2 and IL-7. Moreover evidence is presented that p38MAP kinase is involved in transducing these mitogenic responses. This is in contrast to ERK, which has been shown previously not to be necessary for T cell proliferation (14), opposing the paradigm in other cells that it is ERK that is required for proliferative effects and that p38MAP kinase is involved in stress responses.
The activation of p38MAP kinase by IL-2 and IL-7 is the first evidence of the activation of this kinase in T cells. The recent identification of highly specific pyrinidyl imidazole inhibitors of p38MAP kinase allowed us to investigate its role in T cells responding to IL-2 or IL-7. These inhibitors abrogate the synthesis of TNF-␣ and IL-1 by monocytes stimulated with lipopolysaccharide (42), and one, termed SB203580, has been shown to be highly specific for p38MAP kinase (53). The concentrations of drug that inhibited proliferation were approximately one log higher than those required to inhibit the activation of the immediate substrate MAPKAP kinase-2. This difference in dose might be partly accounted for by the nature of the two responses: one is a complex response and is measured 24 h after stimulation, the other a single enzyme assayed after 20 min. The possibility that SB203580 inhibits another protein kinase responsive to IL-2 is unlikely, as it has no activity when tested on a large number of other kinases at high concentration (53). However, the possible existence of non-kinase targets cannot be absolutely discounted. These results imply that p38MAP kinase has a novel role in mediating the proliferative response of T cells to cytokines.
The nature of the signaling pathways leading to the activation of p38MAP kinase by IL-2 and IL-7 is an open question. The activation of MKK3/6 (55, 56), the G-proteins Rac and Cdc42 and p21-activated kinase have been shown or implicated in the activation of p38MAP kinase in a variety of cells (57,58), however the response of these to IL-2 is unknown. p21 ras , which is activated by IL-2 (16,17), has also been suggested as a proximal activator for the kinase (59). However IL-7 does not appear to stimulate the Shc/p21 ras /ERK1/2 cascade (14). As IL-2 and IL-7 both activate tyrosine kinases Jak1, Jak3, and p56 lck (3,4,6,29,32), it is possible that these may be involved in initiating the signaling cascade to these kinases, and this is currently being investigated. Moreover, the events distal to the kinase are similarly unknown and warrant investigation. Our data have shown that the induction of c-Myc was not affected and that the pathway required for this event is still largely unknown. In other cells p38MAP kinase has been implicated in the translational control of TNF synthesis by unknown mechanisms (42). p38MAP kinase has also been shown to phosphorylate and activate the transcription factors ATF-1 and CREB (54), Elk-1 (60), CHOP (61), and ATF-2 (62). None of these are known to be activated by IL-2, and although Elk-1 is involved in promoting fos transcription induced by the cytokine, it is not required for IL-2-driven cell proliferation (27).
The role played by SAPK/JNK in IL-2 and IL-7 function could not be easily approached, as no specific inhibitor exists for this kinase. However, IL-2 does induce the synthesis of c-Jun, in which SAPK/JNK has been implicated (43). The activation of SAPK/JNK has been demonstrated previously in T cells (44) in response to antibody cross-linking of CD3 and CD28. This involves a calcium signal, a requirement unique to T lymphocytes. However, neither IL-2 nor IL-7 elevate intracellular calcium (2), and thus the mechanism of activation of SAPK/JNK in T cells by cytokines is distinct from that described previously. Nothing is known of the response of MKK4  (63,64) or MEKK (59), which are proximal activators of SAPK/ JNK, to IL-2 and IL-7. Furthermore despite its activation by some growth factors, e.g. epidermal growth factor on HeLa cells (58), cholecystokinin on pancreatic cells (65), and both endothelin and thrombin on airway smooth muscle cells (66), no role for SAPK/JNK in T cell mitogenesis has been defined.
In summary, we have shown that the previously termed stress-activated kinases p38MAP kinase and SAPK/JNK are activated in T cells by the mitogenic cytokines IL-2 and IL-7. Furthermore, using the specific inhibitor SB203580 a previously undescribed role for p38MAP kinase in mitogenesis has been observed. Moreover, p38MAP kinase is one of the few elements of IL-2 and IL-7 signaling pathways that can be ascribed a role in T cell proliferation. The involvement of these kinases in inflammatory responses has identified them as likely therapeutic targets. The observation that these kinases are also activated by T cell mitogenic cytokines and that at least p38MAP kinase has a role in transducing the proliferative response could have important consequences for any pharmacological modulation of these enzymes.