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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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Germinal center formation in SEK1βˆ’/βˆ’ and CD28βˆ’/βˆ’ mice. SEK1βˆ’/βˆ’, SEK1+/+, and CD28βˆ’/βˆ’ mice were immunized with VSV Indiana (2 Γ—  106 PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA+ cells localize to germinal centers and the marginal zone in (A) SEK1+/+ and (B) SEK1βˆ’/βˆ’ mice. (C) Absence of  germinal center formation and germinal center PNA+ B cells in VSV-infected CD28βˆ’/βˆ’ mice. Some PNA+ B cells are present in the marginal zone and  the red pulp of CD28βˆ’/βˆ’ mice. (D–F) CD4+ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and  the follicular mantle zone in (D) SEK1+/+ and (E) SEK1βˆ’/βˆ’ mice. (F) CD4+ T cells in the spleen of VSV-infected CD28βˆ’/βˆ’ mice. (G–I) VSV-specific B  cells in germinal centers of (G) SEK1+/+ and (H) SEK1βˆ’/βˆ’ mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28βˆ’/βˆ’ mice. (J) VSV-specific plasma cells in VSV-infected CD28βˆ’/βˆ’ mice.
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Figure 5: Germinal center formation in SEK1βˆ’/βˆ’ and CD28βˆ’/βˆ’ mice. SEK1βˆ’/βˆ’, SEK1+/+, and CD28βˆ’/βˆ’ mice were immunized with VSV Indiana (2 Γ— 106 PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA+ cells localize to germinal centers and the marginal zone in (A) SEK1+/+ and (B) SEK1βˆ’/βˆ’ mice. (C) Absence of germinal center formation and germinal center PNA+ B cells in VSV-infected CD28βˆ’/βˆ’ mice. Some PNA+ B cells are present in the marginal zone and the red pulp of CD28βˆ’/βˆ’ mice. (D–F) CD4+ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and the follicular mantle zone in (D) SEK1+/+ and (E) SEK1βˆ’/βˆ’ mice. (F) CD4+ T cells in the spleen of VSV-infected CD28βˆ’/βˆ’ mice. (G–I) VSV-specific B cells in germinal centers of (G) SEK1+/+ and (H) SEK1βˆ’/βˆ’ mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28βˆ’/βˆ’ mice. (J) VSV-specific plasma cells in VSV-infected CD28βˆ’/βˆ’ mice.

Mentions: The prominent expression of the GCK in follicular germinal centers (34) and activation of SAPK through GCK (33) suggested that the GCK/SAPK pathway might be important for B cell differentiation within germinal centers. Moreover, mice lacking CD28 (51) or CD40 (52, 53) do not develop germinal centers. Since all of these receptors can activate SAPKs/JNKs (10, 29, 31, 32), we tested whether virus-specific germinal center formation was normal in SEK1βˆ’/βˆ’ mice. Although VSV-specific germinal centers were completely absent in CD28βˆ’/βˆ’ mice after challenge with VSV, SEK1βˆ’/βˆ’RAG2βˆ’/βˆ’ chimeric mice developed germinal centers with normal morphology (Fig. 5) and at normal frequency (Table 3). Germinal center B cells were positive for PNA expression (Fig. 5, A–C). CD4+ T cells were mainly present in the T area, but were also observed within germinal centers (Fig. 5, D–F). Moreover, a light zone containing strongly VSV-binding germinal center B cells could be distinguished from a dark zone containing sIg-negative B lymphocytes (Fig. 5, G–I). It should be noted that VSV-specific plasma cells were detectable in the spleens of CD28βˆ’/βˆ’ mice (Fig. 5 J) and that CD28βˆ’/βˆ’ mice could still produce, albeit at low levels, neutralizing IgG Abs (Table 2). These data show that SEK1βˆ’/βˆ’RAG2βˆ’/βˆ’ mice can mount biologically relevant responses against VSV and that SEK1 has no apparent role in CD28-dependent, virus-specific germinal center formation.


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)

Germinal center formation in SEK1βˆ’/βˆ’ and CD28βˆ’/βˆ’ mice. SEK1βˆ’/βˆ’, SEK1+/+, and CD28βˆ’/βˆ’ mice were immunized with VSV Indiana (2 Γ—  106 PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA+ cells localize to germinal centers and the marginal zone in (A) SEK1+/+ and (B) SEK1βˆ’/βˆ’ mice. (C) Absence of  germinal center formation and germinal center PNA+ B cells in VSV-infected CD28βˆ’/βˆ’ mice. Some PNA+ B cells are present in the marginal zone and  the red pulp of CD28βˆ’/βˆ’ mice. (D–F) CD4+ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and  the follicular mantle zone in (D) SEK1+/+ and (E) SEK1βˆ’/βˆ’ mice. (F) CD4+ T cells in the spleen of VSV-infected CD28βˆ’/βˆ’ mice. (G–I) VSV-specific B  cells in germinal centers of (G) SEK1+/+ and (H) SEK1βˆ’/βˆ’ mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28βˆ’/βˆ’ mice. (J) VSV-specific plasma cells in VSV-infected CD28βˆ’/βˆ’ mice.
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Figure 5: Germinal center formation in SEK1βˆ’/βˆ’ and CD28βˆ’/βˆ’ mice. SEK1βˆ’/βˆ’, SEK1+/+, and CD28βˆ’/βˆ’ mice were immunized with VSV Indiana (2 Γ— 106 PFU). Serial spleen sections were processed for immunostaining 12 d after immunization as described in Materials and Methods. Original magnifications: (A–I) 200; (J) 400. (A–C) PNA+ cells localize to germinal centers and the marginal zone in (A) SEK1+/+ and (B) SEK1βˆ’/βˆ’ mice. (C) Absence of germinal center formation and germinal center PNA+ B cells in VSV-infected CD28βˆ’/βˆ’ mice. Some PNA+ B cells are present in the marginal zone and the red pulp of CD28βˆ’/βˆ’ mice. (D–F) CD4+ T cells localize mainly to the periarteriolar lymphatic sheaths, but are also present in germinal centers and the follicular mantle zone in (D) SEK1+/+ and (E) SEK1βˆ’/βˆ’ mice. (F) CD4+ T cells in the spleen of VSV-infected CD28βˆ’/βˆ’ mice. (G–I) VSV-specific B cells in germinal centers of (G) SEK1+/+ and (H) SEK1βˆ’/βˆ’ mice. Note the presence of VSV-specific B cells outside the germinal centers that show cytoplasmic staining. These cells are VSV-specific plasma cells (44). (I) VSV-specific germinal centers are absent in VSV-infected CD28βˆ’/βˆ’ mice. (J) VSV-specific plasma cells in VSV-infected CD28βˆ’/βˆ’ mice.
Mentions: The prominent expression of the GCK in follicular germinal centers (34) and activation of SAPK through GCK (33) suggested that the GCK/SAPK pathway might be important for B cell differentiation within germinal centers. Moreover, mice lacking CD28 (51) or CD40 (52, 53) do not develop germinal centers. Since all of these receptors can activate SAPKs/JNKs (10, 29, 31, 32), we tested whether virus-specific germinal center formation was normal in SEK1βˆ’/βˆ’ mice. Although VSV-specific germinal centers were completely absent in CD28βˆ’/βˆ’ mice after challenge with VSV, SEK1βˆ’/βˆ’RAG2βˆ’/βˆ’ chimeric mice developed germinal centers with normal morphology (Fig. 5) and at normal frequency (Table 3). Germinal center B cells were positive for PNA expression (Fig. 5, A–C). CD4+ T cells were mainly present in the T area, but were also observed within germinal centers (Fig. 5, D–F). Moreover, a light zone containing strongly VSV-binding germinal center B cells could be distinguished from a dark zone containing sIg-negative B lymphocytes (Fig. 5, G–I). It should be noted that VSV-specific plasma cells were detectable in the spleens of CD28βˆ’/βˆ’ mice (Fig. 5 J) and that CD28βˆ’/βˆ’ mice could still produce, albeit at low levels, neutralizing IgG Abs (Table 2). These data show that SEK1βˆ’/βˆ’RAG2βˆ’/βˆ’ mice can mount biologically relevant responses against VSV and that SEK1 has no apparent role in CD28-dependent, virus-specific germinal center formation.

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