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Requirement for Caspase-8 in NF-κB Activation by Antigen Receptor

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Science  04 Mar 2005:
Vol. 307, Issue 5714, pp. 1465-1468
DOI: 10.1126/science.1104765

Abstract

Caspase-8, a proapoptotic protease, has an essential role in lymphocyte activation and protective immunity. We show that caspase-8 deficiency (CED) in humans and mice specifically abolishes activation of the transcription factor nuclear factor κB (NF-κB) after stimulation through antigen receptors, Fc receptors, or Toll-like receptor 4 in T, B, and natural killer cells. Caspase-8 also causes the αβ complex of the inhibitor of NF-κB kinase (IKK) to associate with the upstream Bcl10-MALT1 (mucosa-associated lymphatic tissue) adapter complex. Recruitment of the IKKα, β complex, its activation, and the nuclear translocation of NF-κB require enzyme activity of full-length caspase-8. These findings thus explain the paradoxical association of defective apoptosis and combined immunodeficiency in human CED.

The intracellular aspartate-specific cysteine protease caspase-8 initiates death receptor signaling for apoptosis (1). Recruitment into the death-signaling complex induces procaspase-8 oligomerization, followed by full processing into a highly active soluble tetramer (2, 3). However, caspase-8 is also essential for lymphocyte activation and protective immunity in mice and humans (4, 5). Patients with caspase-8 deficiency (CED) have defective apoptosis and immunodeficiency due to impaired activation of T, B, and natural killer (NK) lymphocytes (4).

Human peripheral blood leukocytes (PBLs) or mouse T cells treated with the pan-caspase inhibitor benzyloxycarbonylvalyl-alanyl–aspartic acid (O-methyl)–fluoro-methylketone (zVAD) have reduced antigen receptor–induced expression of interleukin-2 (IL-2) and its receptor subunit CD25 (4, 6, 7). Because nuclear factor κB (NF-κB) is required for IL-2 and CD25 gene transcription as well as lymphocyte activation (8, 9), we examined this transcription factor in cells exposed to caspase inhibitor. NF-κB family members—Rel (c-rel), RelA (p65), RelB, NF-κB1 (p105/50), and NF-κB2 (p100/52)—regulate gene transcription as dimers (8, 9). Lymphocyte stimulation causes phosphorylation and degradation of inhibitor of κB (IκB) proteins, leading to NF-κB nuclear translocation and transcriptional activation. Nuclear translocation of p65 or p50, which constitute the NF-κB heterodimer, was detected 5 min after T cell receptor (TCR) stimulation in control PBLs but not in cells treated with zVAD (Fig. 1A and fig. S1, A to C) (10). The difference occurred despite CD28 costimulation, indicating a defect in TCR signaling (Fig. 1A). Consistent with the zVAD effect, the caspase-8-deficient Jurkat T cell line I9.2 exhibited almost no NF-κB nuclear translocation after TCR stimulation compared to that in parental A3 cells (fig. S1, D to G, and fig. S2A), with a corresponding decrease in NF-κB nuclear binding activity but no effect on the binding activity of the transcription factors OCT1 or TFIID (Fig. 1B). TCR stimulation of I9.2 cells failed to induce transcription from an NF-κB promoter–driven luciferase reporter construct (Fig. 1C). NF-κB was also defective in A3 or primary human CD4+ cells in which caspase-8 was specifically knocked down using short hairpin RNAs (shRNAs) (fig. S3, A to C), A3 or mouse EL-4 T cells pretreated with zVAD (fig. S3, D to F, and fig. S4, A to D), and in caspase-8-deficient T cells from conditional knockout mice (fig. S4, E and F). These effects appeared to be selective for the TCR signaling pathway, because tumor necrosis factor–α (TNF-α)–induced NF-κB activation was not affected by zVAD or caspase-8 deficiency (Fig. 1 and figs. S1 to S4). Moreover, all other early activation events examined were essentially unaffected (fig. S5) (10). Together, these results suggest a selective role for caspase-8 in NF-κB activation.

Fig. 1.

Defective NF-κB translocation and transcriptional activation in CED T cells. (A) Quantitation of the fraction of cells by immunofluorescence confocal microscopy showing NF-κB p65 nuclear translocation. Results are shown as the mean ± SD for PBLs from three normal donors, with or without zVAD, and then stimulated with antibodies to CD3ϵ (anti-CD3ϵ) and anti-CD28 (1 μg/ml each), anti-CD3ϵ (1 μg/ml) alone, or TNF-α (10 ng/ml) as indicated. (Inset) Representative merged views of p65 (green), Hoechst (red), overlay (yellow) from unstimulated (a and b), or cells stimulated with anti-CD3ϵ and anti-CD28 (c and d), pretreated with dimethyl sulfoxide (DMSO) (a and c), or zVAD (b and d). (B) NF-κB gel shift of nuclear extracts prepared from A3 and I9.2 cells after 20 min of stimulation with anti-CD3ϵ and anti-CD28 (1 μg/ml each) (+), control (–), or TNF-α (10 ng/ml). OCT1 or TFIID gel shifts of the same nuclear extracts are shown. (C) Relative light units (RLU) of luciferase activity for an NF-κB luciferase reporter construct transfected into A3 or I9.2 cells and stimulated for 16 hours with anti-CD3ϵ, anti-CD28 (2 μg/ml each), and goat antibody to mouse immunoglobulin G (IgG) (5 μg per ml) or TNF-α (30 ng per ml) one day after transfection. Asterisk indicates P < 0.05 by the unpaired Student's t test for experimental compared to corresponding control.

We next examined NF-κB activation in PBLs from NIH CED family 66 (4). Early activation events in T cells were essentially normal, but NF-κB did not translocate to the nucleus after TCR stimulation in PBLs that were homozygous for CED (fig. S6, Fig. 2A, and fig. S7A). Notably, the heterozygous mother showed no such impairment, indicating that a single functional copy of the gene sufficed for NF-κB signaling.

Fig. 2.

Impaired NF-κB in lymphocytes from caspase-8-deficient humans. N, normal donor; M, healthy mother (+/m); P1, proband (m/m); P2, affected sibling (m/m). Genotype: +, normal; m, mutant. (A) Quantitation of the fraction of cells by immunofluorescence confocal microscopy showing NF-κB p65 nuclear translocation. T lymphocytes were stimulated with anti-CD3ϵ and anti-CD28 (1 μg/ml each) or anti-CD3ϵ (1 μg/ml) alone. (B) Lysates from stimulated T cells, immunoblotted for phosphorylated-IκBα (P-IκBα) and IκBα. (C) NF-κB p65 nuclear translocation in B cells stimulated with biotinylated anti-IgG (20 μg/ml) and streptavidin (20 μg/ml), lipopolysaccharide (LPS, 50 μg/ml), or CD40 ligand (CD40L, 2.5 μg/ml). (D) NF-κB p65 nuclear translocation in NK cells stimulated with anti-CD16 (20 μg/ml) or 2B4 (10 μg/ml) and goat antibody to mouse IgG (20 μg/ml).

Phosphorylation activates the IκB kinase (IKK) complex, composed of the two catalytic subunits IKKα and IKKβ and a regulatory subunit IKKγ (NEMO) (8, 9). Active IKK phosphorylates IκBα, which marks IκBα for degradation, thereby liberating NF-κB for nuclear translocation and transcriptional activity. However, we saw no IκBα phosphorylation or degradation in TCR-stimulated PBLs from a CED patient (Fig. 2B and fig. S7I). Hence, caspase-8 was necessary for NF-κB activation after TCR stimulation.

We next evaluated NF-κB activation in B cells and NK cells. B cells from a CED patient failed to translocate NF-κB into the nucleus after B cell receptor (BCR) stimulation (Fig. 2C and fig. S7C). Ramos B cells, in which caspase-8 was knocked down using specific shRNAs, displayed a similar defect (fig. S7, F and G). NF-κB activation induced by lipopolysaccharide (LPS) requires signaling through Toll-like receptor 4 (TLR4). In B cells from a CED patient, but not an autoimmune lymphoproliferative syndrome patient bearing a Fas mutation (ALPS, Type Ia), NF-κB nuclear translocation was abrogated after LPS stimulation (Fig. 2C and fig. S7, C to E). By contrast, NF-κB could be normally activated in CED B cells through CD40 (Fig. 2C and fig. S7C) (11). NK cells from CED patients failed to translocate NF-κB into the nucleus after FcγRIII or 2B4 (NK cell–activating receptor) stimulation (Fig. 2D and fig. S7H). NF-κB activation proceeds normally in caspase-8-deficient mouse embryo fibroblasts after TNF-α or Fas stimulation (12). Thus, CED impairs both adaptive immunoreceptor signaling and certain pathways of innate immunity that activate NF-κB.

Early TCR activation events link to NF-κB activation through protein kinase Cθ (PKCθ) activation (13, 14). PKCθ phosphorylation recruits the CARMA1-Bcl10-MALT1 (CBM) complex to the immunological synapse (15, 16). This complex of adapter molecules causes activation of the IKK complex. However, the CBM and IKK complexes do not appear to interact directly (17), and how the signal for NF-κB activation is conveyed between the two is not well understood (15, 16, 18). The amounts of active phosphorylated PKCθ were not altered by caspase-8 deficiency (fig. S5C). In normal Jurkat cells, IKKα phosphorylation occurred at 5 min after TCR stimulation, and IKKα, β and IκBα phosphorylation occurred maximally after 10 min, followed by IκBα degradation (Fig. 3A and fig. S8A). IKK phosphorylation coincided with the appearance of in vitro kinase activity using glutathione S-transferase (GST)–IκBα as a substrate (Fig. 3B). IKK phosphorylation, IκBα phosphorylation and degradation, and in vitro kinase activity were reduced in I9.2 cells (Fig. 3, A and B). Thus, caspase-8 acts between PKCθ and IKK in the NF-κB pathway.

Fig. 3.

Requirement for active caspase-8 in linking the CARMA1-Bcl10-MALT1 (CBM) complex with the IκB kinase (IKK) complex for TCR-induced NF-κB activation. (A to E) Lysates (WCL) from Jurkat A3 or I9.2 cells at various times after stimulation with anti-CD3ϵ and anti-CD28 (1 to 2 μg/ml each) or TNF-α (30 ng/ml for 5 min). Immunoblotting (A), or immunoprecipitations (IP) of caspase-8 (C), Bcl10 (D), or FADD (E) followed by immunoblotting are shown using indicated antibodies against phosphorylated IκBα (P-IκBα), IκBα, the α and/or β subunits of phosphorylated IKK (P-IKK), Bcl10, MALT1, caspase-8, or FADD. Solid arrowhead indicates full length (p54 and 52). Open arrowhead indicates partly processed (p43 and 41) forms of caspase-8. Asterisks indicate Ig heavy chain in immunoprecipitates. For (B), IKK in vitro kinase activity was assessed in coimmunoprecipitates of IKKγ by the ability to phosphorylate GST-IκBα. (F) Activity of an NF-κB luciferase reporter in I9.2 cells transfected with the indicated caspase-8 expression constructs. GFP, green fluorescence protein–expressing vector; FL, full-length wild-type caspase-8. Cells were stimulated for 24 hours with anti-CD3ϵ and anti-CD28 (2 μg/ml each) and goat antibody to mouse IgG (5 μg/ml) 2 days after transfection. Asterisks indicate P < 0.001 by the Student's t test compared to the GFP control. (G) Biotinylated-VAD (b-VAD) was incubated with protein lysates after TCR stimulation as in (F), then precipitated with streptavidin, and IKK, caspase-8, and Bcl10 proteins were detected by immunoblotting. (H) Lysates before (input) and after (output) precipitation of b-VAD–bound proteins, shown immunoblotted for full-length caspase-8 and β-actin. (I and J) Lysates from human PBLs were prepared at various times after stimulation with anti-CD3ϵ and anti-CD28 (1 μg/ml each). IP of caspase-8 (I) or using b-VAD (J) were followed by immunoblotting to detect IKKα, β, Bcl10, and caspase-8 proteins.

We used coimmunoprecipitation and immunoblotting to show that TCR stimulation induced caspase-8 to associate with the Bcl10-MALT1 complex followed by recruitment of IKK (Fig. 3C). Formation of this complex coincided with IKK phosphorylation and activation at 10 min after stimulation. Absence of caspase-8 abrogated IKKα, β recruitment and activation by the CBM complex (Fig. 3, A and D). The adapter protein FADD associated with caspase-8 and Bcl10-MALT1 at an earlier time (5 min) but disappeared before the recruitment of IKK to form the holocomplex (Fig. 3E). Caspase-8 appears to act before IKK, because reconstituting I9.2 cells with constitutively active IKK restored NF-κB activation (fig. S8, B and C). Thus, caspase-8 is integral to the assembly and activation of the CBM-IKK complex in response to antigen receptors.

Our observation that zVAD blocks NF-κB activation after TCR stimulation suggests that caspase-8 enzymatic activity may be essential. Indeed, we found that wild-type caspase-8, but not the catalytically inactive C360S mutant (where Cys360 is replaced by Ser), enhanced NF-κB responses to TCR stimulation in transfected I9.2 cells (Fig. 3F). We next tested caspase-8 autoprocessing mutants (D210A, D374A, and D384A) by substituting alanines for aspartic acid residues (10). These mutants increased NF-κB activity in I9.2 cells after TCR stimulation, indicating that enzymatic activity, but not autoprocessing, was required (Fig. 3F and fig. S8F). Consistent with this conclusion, only full-length forms of caspase-8, which are known to be enzymatically active (2, 3), were detected in the CBM complex (Fig. 3E). We therefore used a more sensitive probe, biotinylated-VAD (b-VAD), which bonds covalently to the catalytic cysteine of active caspases. Precipitation of b-VAD detected a small amount of enzymatically active full-length caspase-8 in lysates from unstimulated T cells (Fig. 3G). TCR stimulation caused activation of full-length caspase-8 and induced physical interaction with Bcl10 at times when IKK was recruited, phosphorylated, and active (Fig. 3G). Depletion of the b-VAD–bound species revealed that only a minor fraction (10 to 15%) of the total caspase-8 became enzymatically active after TCR stimulation (Fig. 3H and fig. S8G). Hence, NF-κB activation by antigen receptors requires enzyme activity of full-length caspase-8. In the NF-κB–activating holocomplex, caspase-8 appears to be bound, unprocessed, and only weakly activated, by contrast to caspase-8 in a death-inducing complex (2, 3).

Caspase-8 now emerges both as a pivotal molecule for death-receptor signaling and as a selective signal transducer for NF-κB during the early genetic response to an antigen. This explains the requirement for caspase activity and caspase-8 for lymphocyte activation and c-rel responses after antigen receptor stimuli (47, 19). Full-length, unprocessed, but active caspase-8 serves as a crucial link for the CBM and IKK complexes leading to NF-κB activation not only in lymphoid cell lines, but also in freshly isolated human lymphocytes (Fig. 3, I and J). After antigen receptor stimulation, MALT1-dependent recruitment of the ubiquitin ligase TRAF6 to the CBM complex may enhance regulatory polyubiquitination of IKKγ (20, 21). IKKγ ubiquitination, but not phosphorylation of IKKα, β, occurred in the absence of caspase-8, indicating that ubiquitination may be necessary but not sufficient for IKK activation (fig. S8H).

CED patients manifest certain diagnostic criteria for ALPS, most notably impaired lymphocyte apoptosis (22). However, the combined T, B, and NK cell immunodeficiency is not seen in ALPS patients with Fas, Fas ligand, or caspase-10 mutations. Our findings reveal how a single protease regulates both lymphocyte proliferation and programmed death through different molecular forms. The molecular mechanism we have unveiled may be useful in understanding and treating other varieties of immunodeficiency and disordered lymphocyte homeostasis.

Supporting Online Material

www.sciencemag.org/cgi/content/full/307/5714/1465/DC1

Materials and Methods

SOM Text

Figs. S1 to S8

References

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