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Impaired CD28-mediated interleukin 2 production and proliferation in stress kinase SAPK/ERK1 kinase (SEK1)/mitogen-activated protein kinase kinase 4 (MKK4)-deficient T lymphocytes.

Nishina H, Bachmann M, Oliveira-dos-Santos AJ, Kozieradzki I, Fischer KD, Odermatt B, Wakeham A, Shahinian A, Takimoto H, Bernstein A, Mak TW, Woodgett JR, Ohashi PS, Penninger JM - J. Exp. Med. (1997)

Bottom Line: These results show that signaling pathways for SAPK activation are developmentally regulated in T cells.Although SEK1(-/-) thymocytes failed to induce SAPK/JNK in response to PMA/Ca2+ ionophore, SEK1(-/-)RAG2(-/-) thymocytes proliferated and made IL-2 after PMA/Ca2+ ionophore and CD3/CD28 stimulation, albeit at significantly lower levels compared to SEK1(+/+)RAG2(-/-) thymocytes, implying that CD28 costimulation and PMA/Ca2+ ionophore-triggered signaling pathways exist that can mediate proliferation and IL-2 production independently of SAPK activation.Our data provide the first genetic evidence that SEK1 is an important effector molecule that relays CD28 signaling to IL-2 production and T cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Amgen Institute and Ontario Cancer Institute, Department of Medical Biophysics and Immunology, University of Toronto, M5G 2C1 Toronto, Ontario, Canada.

ABSTRACT
The dual specific kinase SAPK/ERK1 kinase (SEK1; mitogen-activated protein kinase kinase 4/Jun NH2 terminal kinase [ JNK] kinase) is a direct activator of stress-activated protein kinases ([SAPKs]/JNKs) in response to CD28 costimulation, CD40 signaling, or activation of the germinal center kinase. Here we show that SEK1(-/-) recombination-activating gene (RAG)2(-/-) chimeric mice have a partial block in B cell maturation. However, peripheral B cells displayed normal responses to IL-4, IgM, and CD40 cross-linking. SEK1(-/-) peripheral T cells showed decreased proliferation and IL-2 production after CD28 costimulation and PMA/Ca2+ ionophore activation. Although CD28 expression was absolutely crucial to generate vesicular stomatitis virus (VSV)-specific germinal centers, SEK1(-/-)RAG2(-/-) chimeras mounted a protective antiviral B cell response, exhibited normal IgG class switching, and made germinal centers in response to VSV. Interestingly, PMA/Ca2+ ionophore stimulation, which mimics TCR-CD3 and CD28-mediated signal transduction, induced SAPK/JNK activation in peripheral T cells, but not in thymocytes, from SEK1(-/-) mice. These results show that signaling pathways for SAPK activation are developmentally regulated in T cells. Although SEK1(-/-) thymocytes failed to induce SAPK/JNK in response to PMA/Ca2+ ionophore, SEK1(-/-)RAG2(-/-) thymocytes proliferated and made IL-2 after PMA/Ca2+ ionophore and CD3/CD28 stimulation, albeit at significantly lower levels compared to SEK1(+/+)RAG2(-/-) thymocytes, implying that CD28 costimulation and PMA/Ca2+ ionophore-triggered signaling pathways exist that can mediate proliferation and IL-2 production independently of SAPK activation. Our data provide the first genetic evidence that SEK1 is an important effector molecule that relays CD28 signaling to IL-2 production and T cell proliferation.

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Proliferation (A and C) and IL-2 production (B and D) of SEK1−/− chimeric (shaded bars) and SEK1+/+ chimeric (open bars) T cells. Purified  lymph node responder T cells (105 T cells/well) were activated with (A and B) plate-bound anti–CD3-ε (1 μg/ml) and different concentrations of soluble anti-CD28 Ab (10, 100, and 200 ng/ml) or PMA (12.5 ng/ml) plus Ca2+ ionophore (100 ng/ml) (PMA + Ca); and (C and D) soluble anti–CD3-ε  and soluble anti-CD28 Abs at the indicated concentrations. Rabbit anti–hamster Ig-coated plates without addition of anti–CD3-ε (−) or CD28 (0) Abs  are shown as controls in (A and B). (C and D) data from two individual SEK1−/− and SEK1+/+ chimeric mice are shown. After 24 h of stimulation, proliferation was determined by [3H]thymidine uptake, and IL-2 production was determined by ELISA. Data of triplicate cultures ± SD are shown. Similar  results were obtained after 48 and 72 h of culture (not shown). One result representative of seven independent experiments is shown.
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Figure 1: Proliferation (A and C) and IL-2 production (B and D) of SEK1−/− chimeric (shaded bars) and SEK1+/+ chimeric (open bars) T cells. Purified lymph node responder T cells (105 T cells/well) were activated with (A and B) plate-bound anti–CD3-ε (1 μg/ml) and different concentrations of soluble anti-CD28 Ab (10, 100, and 200 ng/ml) or PMA (12.5 ng/ml) plus Ca2+ ionophore (100 ng/ml) (PMA + Ca); and (C and D) soluble anti–CD3-ε and soluble anti-CD28 Abs at the indicated concentrations. Rabbit anti–hamster Ig-coated plates without addition of anti–CD3-ε (−) or CD28 (0) Abs are shown as controls in (A and B). (C and D) data from two individual SEK1−/− and SEK1+/+ chimeric mice are shown. After 24 h of stimulation, proliferation was determined by [3H]thymidine uptake, and IL-2 production was determined by ELISA. Data of triplicate cultures ± SD are shown. Similar results were obtained after 48 and 72 h of culture (not shown). One result representative of seven independent experiments is shown.

Mentions: Recent biochemical studies implied that the SAPK/JNK signaling pathway is operating in T cells, and that cell proliferation and IL-2 production induced by CD28 costimulation may be mediated via SAPK/JNK (29, 36, 37). SEK1−/−RAG2−/− chimeric mice have a smaller thymus, but normal numbers of peripheral T cells (Table 1; reference 23). To test the role of SEK1 in CD28 costimulation, lymph node T cells were cultured in anti–CD3-ε Ab-coated plates in the absence or presence of various concentrations of soluble anti-CD28 Abs. Whereas SEK1−/−RAG2−/− and SEK1+/+RAG2−/− T cells reponded in the same way to CD3-ε activation alone, CD28-mediated upregulation of proliferation and IL-2 production were significantly reduced in SEK1−/− T cells (Fig. 1, A and B). Reduced proliferation and IL-2 production were also observed in SEK1−/− T cells after stimulation with PMA/Ca2+ ionophore (Fig. 1, A and B), which mimic TCR–CD3- and CD28-mediated signal transduction (29, 45).


Impaired CD28-mediated interleukin 2 production and proliferation in stress kinase SAPK/ERK1 kinase (SEK1)/mitogen-activated protein kinase kinase 4 (MKK4)-deficient T lymphocytes.

Nishina H, Bachmann M, Oliveira-dos-Santos AJ, Kozieradzki I, Fischer KD, Odermatt B, Wakeham A, Shahinian A, Takimoto H, Bernstein A, Mak TW, Woodgett JR, Ohashi PS, Penninger JM - J. Exp. Med. (1997)

Proliferation (A and C) and IL-2 production (B and D) of SEK1−/− chimeric (shaded bars) and SEK1+/+ chimeric (open bars) T cells. Purified  lymph node responder T cells (105 T cells/well) were activated with (A and B) plate-bound anti–CD3-ε (1 μg/ml) and different concentrations of soluble anti-CD28 Ab (10, 100, and 200 ng/ml) or PMA (12.5 ng/ml) plus Ca2+ ionophore (100 ng/ml) (PMA + Ca); and (C and D) soluble anti–CD3-ε  and soluble anti-CD28 Abs at the indicated concentrations. Rabbit anti–hamster Ig-coated plates without addition of anti–CD3-ε (−) or CD28 (0) Abs  are shown as controls in (A and B). (C and D) data from two individual SEK1−/− and SEK1+/+ chimeric mice are shown. After 24 h of stimulation, proliferation was determined by [3H]thymidine uptake, and IL-2 production was determined by ELISA. Data of triplicate cultures ± SD are shown. Similar  results were obtained after 48 and 72 h of culture (not shown). One result representative of seven independent experiments is shown.
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Related In: Results  -  Collection

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Figure 1: Proliferation (A and C) and IL-2 production (B and D) of SEK1−/− chimeric (shaded bars) and SEK1+/+ chimeric (open bars) T cells. Purified lymph node responder T cells (105 T cells/well) were activated with (A and B) plate-bound anti–CD3-ε (1 μg/ml) and different concentrations of soluble anti-CD28 Ab (10, 100, and 200 ng/ml) or PMA (12.5 ng/ml) plus Ca2+ ionophore (100 ng/ml) (PMA + Ca); and (C and D) soluble anti–CD3-ε and soluble anti-CD28 Abs at the indicated concentrations. Rabbit anti–hamster Ig-coated plates without addition of anti–CD3-ε (−) or CD28 (0) Abs are shown as controls in (A and B). (C and D) data from two individual SEK1−/− and SEK1+/+ chimeric mice are shown. After 24 h of stimulation, proliferation was determined by [3H]thymidine uptake, and IL-2 production was determined by ELISA. Data of triplicate cultures ± SD are shown. Similar results were obtained after 48 and 72 h of culture (not shown). One result representative of seven independent experiments is shown.
Mentions: Recent biochemical studies implied that the SAPK/JNK signaling pathway is operating in T cells, and that cell proliferation and IL-2 production induced by CD28 costimulation may be mediated via SAPK/JNK (29, 36, 37). SEK1−/−RAG2−/− chimeric mice have a smaller thymus, but normal numbers of peripheral T cells (Table 1; reference 23). To test the role of SEK1 in CD28 costimulation, lymph node T cells were cultured in anti–CD3-ε Ab-coated plates in the absence or presence of various concentrations of soluble anti-CD28 Abs. Whereas SEK1−/−RAG2−/− and SEK1+/+RAG2−/− T cells reponded in the same way to CD3-ε activation alone, CD28-mediated upregulation of proliferation and IL-2 production were significantly reduced in SEK1−/− T cells (Fig. 1, A and B). Reduced proliferation and IL-2 production were also observed in SEK1−/− T cells after stimulation with PMA/Ca2+ ionophore (Fig. 1, A and B), which mimic TCR–CD3- and CD28-mediated signal transduction (29, 45).

Bottom Line: These results show that signaling pathways for SAPK activation are developmentally regulated in T cells.Although SEK1(-/-) thymocytes failed to induce SAPK/JNK in response to PMA/Ca2+ ionophore, SEK1(-/-)RAG2(-/-) thymocytes proliferated and made IL-2 after PMA/Ca2+ ionophore and CD3/CD28 stimulation, albeit at significantly lower levels compared to SEK1(+/+)RAG2(-/-) thymocytes, implying that CD28 costimulation and PMA/Ca2+ ionophore-triggered signaling pathways exist that can mediate proliferation and IL-2 production independently of SAPK activation.Our data provide the first genetic evidence that SEK1 is an important effector molecule that relays CD28 signaling to IL-2 production and T cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Amgen Institute and Ontario Cancer Institute, Department of Medical Biophysics and Immunology, University of Toronto, M5G 2C1 Toronto, Ontario, Canada.

ABSTRACT
The dual specific kinase SAPK/ERK1 kinase (SEK1; mitogen-activated protein kinase kinase 4/Jun NH2 terminal kinase [ JNK] kinase) is a direct activator of stress-activated protein kinases ([SAPKs]/JNKs) in response to CD28 costimulation, CD40 signaling, or activation of the germinal center kinase. Here we show that SEK1(-/-) recombination-activating gene (RAG)2(-/-) chimeric mice have a partial block in B cell maturation. However, peripheral B cells displayed normal responses to IL-4, IgM, and CD40 cross-linking. SEK1(-/-) peripheral T cells showed decreased proliferation and IL-2 production after CD28 costimulation and PMA/Ca2+ ionophore activation. Although CD28 expression was absolutely crucial to generate vesicular stomatitis virus (VSV)-specific germinal centers, SEK1(-/-)RAG2(-/-) chimeras mounted a protective antiviral B cell response, exhibited normal IgG class switching, and made germinal centers in response to VSV. Interestingly, PMA/Ca2+ ionophore stimulation, which mimics TCR-CD3 and CD28-mediated signal transduction, induced SAPK/JNK activation in peripheral T cells, but not in thymocytes, from SEK1(-/-) mice. These results show that signaling pathways for SAPK activation are developmentally regulated in T cells. Although SEK1(-/-) thymocytes failed to induce SAPK/JNK in response to PMA/Ca2+ ionophore, SEK1(-/-)RAG2(-/-) thymocytes proliferated and made IL-2 after PMA/Ca2+ ionophore and CD3/CD28 stimulation, albeit at significantly lower levels compared to SEK1(+/+)RAG2(-/-) thymocytes, implying that CD28 costimulation and PMA/Ca2+ ionophore-triggered signaling pathways exist that can mediate proliferation and IL-2 production independently of SAPK activation. Our data provide the first genetic evidence that SEK1 is an important effector molecule that relays CD28 signaling to IL-2 production and T cell proliferation.

Show MeSH
Related in: MedlinePlus