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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: 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.

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 IL-2 production (B) of SEK1−/−  (shaded bars) and SEK1+/+ chimeric (open bars) thymocytes. Thymocytes  (105 T cells/well) were activated with plate-bound anti–CD3-ε (1 μg/ ml) and soluble anti-CD28 Abs (100 ng/ml; CD3-ε + CD28) or PMA  (12.5 ng/ml) plus Ca2+ ionophore (100 ng/ml) (PMA + Ca). Rabbit  anti–hamster Ig-coated plates without addition of anti–CD3-ε/CD28 Abs  are shown as controls. After 48 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. The low IL-2 production of thymocytes after PMA/Ca2+ ionophore stimulation is due the  fact that PMA/Ca2+ ionophore also induces proliferation of  CD4−CD8−TCR− thymocytes, the majority of which does not produce  IL-2. One result representative of three independent experiments is  shown.
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Figure 7: Proliferation (A) and IL-2 production (B) of SEK1−/− (shaded bars) and SEK1+/+ chimeric (open bars) thymocytes. Thymocytes (105 T cells/well) were activated with plate-bound anti–CD3-ε (1 μg/ ml) and soluble anti-CD28 Abs (100 ng/ml; CD3-ε + CD28) or PMA (12.5 ng/ml) plus Ca2+ ionophore (100 ng/ml) (PMA + Ca). Rabbit anti–hamster Ig-coated plates without addition of anti–CD3-ε/CD28 Abs are shown as controls. After 48 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. The low IL-2 production of thymocytes after PMA/Ca2+ ionophore stimulation is due the fact that PMA/Ca2+ ionophore also induces proliferation of CD4−CD8−TCR− thymocytes, the majority of which does not produce IL-2. One result representative of three independent experiments is shown.

Mentions: The results in SEK1−/− lymph node T cells indicated that SEK1 has a role in CD28-mediated costimulation for proliferation and IL-2 production in peripheral T cells and that lymph node T cells use a second signaling pathway for SAPK activation that is independent of SEK1. Since this second signaling pathway is not operational in SEK1−/− thymocytes, we tested proliferation and IL-2 production of SEK1−/− thymocytes in response to PMA/Ca2+ ionophore and CD3/ CD28 activation. Surprisingly, 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 (Fig. 7). These data further confirm that SEK1 relays CD28 costimulatory signals to IL-2 production in T cells. However, these results in thymocytes also indicate that CD28 costimulation and PMA/Ca2+ ionophore–triggered signaling pathways exist that can mediate proliferation and IL-2 production independently of SAPK activation.


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 IL-2 production (B) of SEK1−/−  (shaded bars) and SEK1+/+ chimeric (open bars) thymocytes. Thymocytes  (105 T cells/well) were activated with plate-bound anti–CD3-ε (1 μg/ ml) and soluble anti-CD28 Abs (100 ng/ml; CD3-ε + CD28) or PMA  (12.5 ng/ml) plus Ca2+ ionophore (100 ng/ml) (PMA + Ca). Rabbit  anti–hamster Ig-coated plates without addition of anti–CD3-ε/CD28 Abs  are shown as controls. After 48 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. The low IL-2 production of thymocytes after PMA/Ca2+ ionophore stimulation is due the  fact that PMA/Ca2+ ionophore also induces proliferation of  CD4−CD8−TCR− thymocytes, the majority of which does not produce  IL-2. One result representative of three independent experiments is  shown.
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Related In: Results  -  Collection

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Figure 7: Proliferation (A) and IL-2 production (B) of SEK1−/− (shaded bars) and SEK1+/+ chimeric (open bars) thymocytes. Thymocytes (105 T cells/well) were activated with plate-bound anti–CD3-ε (1 μg/ ml) and soluble anti-CD28 Abs (100 ng/ml; CD3-ε + CD28) or PMA (12.5 ng/ml) plus Ca2+ ionophore (100 ng/ml) (PMA + Ca). Rabbit anti–hamster Ig-coated plates without addition of anti–CD3-ε/CD28 Abs are shown as controls. After 48 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. The low IL-2 production of thymocytes after PMA/Ca2+ ionophore stimulation is due the fact that PMA/Ca2+ ionophore also induces proliferation of CD4−CD8−TCR− thymocytes, the majority of which does not produce IL-2. One result representative of three independent experiments is shown.
Mentions: The results in SEK1−/− lymph node T cells indicated that SEK1 has a role in CD28-mediated costimulation for proliferation and IL-2 production in peripheral T cells and that lymph node T cells use a second signaling pathway for SAPK activation that is independent of SEK1. Since this second signaling pathway is not operational in SEK1−/− thymocytes, we tested proliferation and IL-2 production of SEK1−/− thymocytes in response to PMA/Ca2+ ionophore and CD3/ CD28 activation. Surprisingly, 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 (Fig. 7). These data further confirm that SEK1 relays CD28 costimulatory signals to IL-2 production in T cells. However, these results in thymocytes also indicate that CD28 costimulation and PMA/Ca2+ ionophore–triggered signaling pathways exist that can mediate proliferation and IL-2 production independently of SAPK activation.

Bottom Line: 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.

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