Lineage-restricted function of nuclear factor kappaB-inducing kinase (NIK) in transducing signals via CD40 - PubMed (original) (raw)

Lineage-restricted function of nuclear factor kappaB-inducing kinase (NIK) in transducing signals via CD40

N Garceau et al. J Exp Med. 2000.

Abstract

CD40 signaling in B cells and dendritic cells (DCs) is critical for the development of humoral and cell-mediated immunity, respectively. Nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK) has been implicated as a central transducing kinase in CD40-dependent activation. Here, we show that although NIK is essential for B cell activation, it is dispensable for activation of DCs. Such data provide compelling evidence that different intermediary kinases are used by different cellular lineages to trigger NF-kappaB activation via CD40.

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Figures

Figure 1

Figure 1

Role of NIK in activation of B cells. aly/aly and aly/+ splenic B cells were cultured in vitro and assayed for their ability to proliferate (A), produce Ig (B), and upregulate cell surface markers (C) in response to CD40 stimulation. (A) Induction of proliferation by anti-CD40 (10 μg/ml), FGK115, LPS (50 μg/ml), and anti-IgM (goat anti–mouse IgM) was measured by the incorporation of [3H]thymidine from 66 to 72 h after initiation of culture. All cultures contained 10 ng/ml of IL-4. (B) Induction of Ig secretion was performed using sCD154 or LPS in combination with IL-4 (10 ng/ml) and IL-5 (10 ng/ml). Ig secretion was measured using an isotype-specific ELISA. (C) Expression of cell surface molecules on splenic B cells from _aly/_+ (top) or aly/aly (bottom) mice after culture with (purple histogram) or without (green outline histogram) sCD154 for 48 h was measured by flow cytometry.

Figure 1

Figure 1

Role of NIK in activation of B cells. aly/aly and aly/+ splenic B cells were cultured in vitro and assayed for their ability to proliferate (A), produce Ig (B), and upregulate cell surface markers (C) in response to CD40 stimulation. (A) Induction of proliferation by anti-CD40 (10 μg/ml), FGK115, LPS (50 μg/ml), and anti-IgM (goat anti–mouse IgM) was measured by the incorporation of [3H]thymidine from 66 to 72 h after initiation of culture. All cultures contained 10 ng/ml of IL-4. (B) Induction of Ig secretion was performed using sCD154 or LPS in combination with IL-4 (10 ng/ml) and IL-5 (10 ng/ml). Ig secretion was measured using an isotype-specific ELISA. (C) Expression of cell surface molecules on splenic B cells from _aly/_+ (top) or aly/aly (bottom) mice after culture with (purple histogram) or without (green outline histogram) sCD154 for 48 h was measured by flow cytometry.

Figure 1

Figure 1

Role of NIK in activation of B cells. aly/aly and aly/+ splenic B cells were cultured in vitro and assayed for their ability to proliferate (A), produce Ig (B), and upregulate cell surface markers (C) in response to CD40 stimulation. (A) Induction of proliferation by anti-CD40 (10 μg/ml), FGK115, LPS (50 μg/ml), and anti-IgM (goat anti–mouse IgM) was measured by the incorporation of [3H]thymidine from 66 to 72 h after initiation of culture. All cultures contained 10 ng/ml of IL-4. (B) Induction of Ig secretion was performed using sCD154 or LPS in combination with IL-4 (10 ng/ml) and IL-5 (10 ng/ml). Ig secretion was measured using an isotype-specific ELISA. (C) Expression of cell surface molecules on splenic B cells from _aly/_+ (top) or aly/aly (bottom) mice after culture with (purple histogram) or without (green outline histogram) sCD154 for 48 h was measured by flow cytometry.

Figure 3

Figure 3

Responses of DCs in aly/aly and aly/+ mice. (A) The NIK mutation does not affect CD40-induced IL-12 production by DCs. _aly/_+ or aly/aly DCs were cultured at 2 × 106 cells/ml in cRPMI with GM-CSF/IL-4 (both at 10 ng/ml) with or without anti-CD40 (10 μg/ml). Culture supernatant was assayed for IL-12 on day 3 by ELISA. (B) The NIK mutation does not affect the antigen-presentation capacity of DCs. DCs were purified as in A and were pulsed with OVA peptide (323–339) for 90 min, washed extensively, and then plated with 105 OTII cells (OVA-specific Tg T cells) at various DC densities as indicated. At 48 h, culture supernatants were assayed for the presence of IL-2 by ELISA.

Figure 3

Figure 3

Responses of DCs in aly/aly and aly/+ mice. (A) The NIK mutation does not affect CD40-induced IL-12 production by DCs. _aly/_+ or aly/aly DCs were cultured at 2 × 106 cells/ml in cRPMI with GM-CSF/IL-4 (both at 10 ng/ml) with or without anti-CD40 (10 μg/ml). Culture supernatant was assayed for IL-12 on day 3 by ELISA. (B) The NIK mutation does not affect the antigen-presentation capacity of DCs. DCs were purified as in A and were pulsed with OVA peptide (323–339) for 90 min, washed extensively, and then plated with 105 OTII cells (OVA-specific Tg T cells) at various DC densities as indicated. At 48 h, culture supernatants were assayed for the presence of IL-2 by ELISA.

Figure 2

Figure 2

Activation of NF-κB by NIK and aly NIK. Effect of aly NIK expression on IκBα phosphorylation in primary B cells. B cells from aly/aly and aly/+ mice were stimulated in vitro for 2, 5, or 15 min with sCD154, or for 5 min with LPS (50 μg/ml) or PMA (10 ng/ml) plus ionomycin (25 nM), and were assayed for phosphorylation of IκBα by Western blot with a phospho-specific anti-IκBα.

Figure 4

Figure 4

Phosphorylation of IκBα in response to CD40 engagement is normal in DCs from aly/aly mice. DCs were stimulated in vitro with sCD154 or TNF-α as indicated, and were assayed for phosphorylation of IκBα by Western blot with a phospho-specific IκBα antibody.

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