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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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B cell activation and immunoglobulin production in SEK1−/−  mice. (A) Activation of splenic B cells. Purified splenic B cells (105/well)  from SEK1−/− (shaded bars) and SEK1+/+ (open bars) control mice were  seeded in medium containing no added stimulus (Control), soluble anti-Igμ Ab (10 μg/ml, clone B76), IL-4 (10 U/ml), soluble anti-CD40 (1  μg/ml), IL-4 (10 U/ml) plus soluble anti-CD40 (1 μg/ml), and 10 μg/ml  LPS (LPS). After 24 h, the cells were pulsed for 12 h with 1 μCi [3H]thymidine/well. The experiment shown is one of four experiments in which  conditions for stimulation varied (time, cell concentration, concentration  of activators). No significant differences (Student's t test; p > 0.05) were  observed in the [3H]thymidine uptake between SEK1−/− and SEK1+/+ B  cells in response to any of these conditions. [3H]thymidine uptake is  shown in cpm ± SD. (B) SEK1−/− mice produce normal levels of serum  immunoglobulin subclasses. Sera were collected from two individual  6-wk-old SEK1−/− (shaded bars) and two individual 6-wk-old SEK1+/+  (open bars) chimeric mice. The concentrations of Ig subclasses are shown  in μg/ml and were determined by ELISA. Standard deviations were <25  μg/ml.
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Figure 4: B cell activation and immunoglobulin production in SEK1−/− mice. (A) Activation of splenic B cells. Purified splenic B cells (105/well) from SEK1−/− (shaded bars) and SEK1+/+ (open bars) control mice were seeded in medium containing no added stimulus (Control), soluble anti-Igμ Ab (10 μg/ml, clone B76), IL-4 (10 U/ml), soluble anti-CD40 (1 μg/ml), IL-4 (10 U/ml) plus soluble anti-CD40 (1 μg/ml), and 10 μg/ml LPS (LPS). After 24 h, the cells were pulsed for 12 h with 1 μCi [3H]thymidine/well. The experiment shown is one of four experiments in which conditions for stimulation varied (time, cell concentration, concentration of activators). No significant differences (Student's t test; p > 0.05) were observed in the [3H]thymidine uptake between SEK1−/− and SEK1+/+ B cells in response to any of these conditions. [3H]thymidine uptake is shown in cpm ± SD. (B) SEK1−/− mice produce normal levels of serum immunoglobulin subclasses. Sera were collected from two individual 6-wk-old SEK1−/− (shaded bars) and two individual 6-wk-old SEK1+/+ (open bars) chimeric mice. The concentrations of Ig subclasses are shown in μg/ml and were determined by ELISA. Standard deviations were <25 μg/ml.

Mentions: Previously it has been shown that CD40 signaling in B cells leads to the induction of SAPK/JNK activity (31, 32). To determine the requirement of SEK1 for B cell activation, we measured proliferation of B cells in response to various stimuli. SEK1−/−RAG2−/− B cells responded normally to LPS, IL-4, anti-CD40, IL-4 plus anti-CD40, and Igμ cross-linking (Fig. 4 A). Moreover, SEK1−/−RAG2−/− B cells upregulated ICAM-1 and CD23 upon activation with anti-CD40 in the absence or presence of IL-4 (not shown; 42). The basal serum levels for the Ig subclasses IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA were also comparable between SEK1−/−RAG2−/− and SEK1+/+RAG2−/− chimeric mice (Fig. 4 B).


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)

B cell activation and immunoglobulin production in SEK1−/−  mice. (A) Activation of splenic B cells. Purified splenic B cells (105/well)  from SEK1−/− (shaded bars) and SEK1+/+ (open bars) control mice were  seeded in medium containing no added stimulus (Control), soluble anti-Igμ Ab (10 μg/ml, clone B76), IL-4 (10 U/ml), soluble anti-CD40 (1  μg/ml), IL-4 (10 U/ml) plus soluble anti-CD40 (1 μg/ml), and 10 μg/ml  LPS (LPS). After 24 h, the cells were pulsed for 12 h with 1 μCi [3H]thymidine/well. The experiment shown is one of four experiments in which  conditions for stimulation varied (time, cell concentration, concentration  of activators). No significant differences (Student's t test; p > 0.05) were  observed in the [3H]thymidine uptake between SEK1−/− and SEK1+/+ B  cells in response to any of these conditions. [3H]thymidine uptake is  shown in cpm ± SD. (B) SEK1−/− mice produce normal levels of serum  immunoglobulin subclasses. Sera were collected from two individual  6-wk-old SEK1−/− (shaded bars) and two individual 6-wk-old SEK1+/+  (open bars) chimeric mice. The concentrations of Ig subclasses are shown  in μg/ml and were determined by ELISA. Standard deviations were <25  μg/ml.
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Figure 4: B cell activation and immunoglobulin production in SEK1−/− mice. (A) Activation of splenic B cells. Purified splenic B cells (105/well) from SEK1−/− (shaded bars) and SEK1+/+ (open bars) control mice were seeded in medium containing no added stimulus (Control), soluble anti-Igμ Ab (10 μg/ml, clone B76), IL-4 (10 U/ml), soluble anti-CD40 (1 μg/ml), IL-4 (10 U/ml) plus soluble anti-CD40 (1 μg/ml), and 10 μg/ml LPS (LPS). After 24 h, the cells were pulsed for 12 h with 1 μCi [3H]thymidine/well. The experiment shown is one of four experiments in which conditions for stimulation varied (time, cell concentration, concentration of activators). No significant differences (Student's t test; p > 0.05) were observed in the [3H]thymidine uptake between SEK1−/− and SEK1+/+ B cells in response to any of these conditions. [3H]thymidine uptake is shown in cpm ± SD. (B) SEK1−/− mice produce normal levels of serum immunoglobulin subclasses. Sera were collected from two individual 6-wk-old SEK1−/− (shaded bars) and two individual 6-wk-old SEK1+/+ (open bars) chimeric mice. The concentrations of Ig subclasses are shown in μg/ml and were determined by ELISA. Standard deviations were <25 μg/ml.
Mentions: Previously it has been shown that CD40 signaling in B cells leads to the induction of SAPK/JNK activity (31, 32). To determine the requirement of SEK1 for B cell activation, we measured proliferation of B cells in response to various stimuli. SEK1−/−RAG2−/− B cells responded normally to LPS, IL-4, anti-CD40, IL-4 plus anti-CD40, and Igμ cross-linking (Fig. 4 A). Moreover, SEK1−/−RAG2−/− B cells upregulated ICAM-1 and CD23 upon activation with anti-CD40 in the absence or presence of IL-4 (not shown; 42). The basal serum levels for the Ig subclasses IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA were also comparable between SEK1−/−RAG2−/− and SEK1+/+RAG2−/− chimeric mice (Fig. 4 B).

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