By
From the * Amgen Institute and Ontario Cancer Institute, Department of Medical Biophysics and
Immunology, University of Toronto, M5G 2C1 Toronto, Ontario, Canada; Ontario Cancer Institute,
Department of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario, Canada; § Institute for Radiation and Cell Research, University of Wuerzburg, D-97078 Wuerzburg,
Germany;
Samuel Lunenfeld Research Institute and Department of Medical Genetics, University of
Toronto, Mount Sinai Hospital, Toronto, Ontario, Canada; and ¶ Institute for Experimental
Immunology, University of Zürich, 8091, Zürich, Switzerland
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.
Distinct and evolutionarily conserved signal transduction cascades mediate survival or death in response to
developmental and environmental cues. Multiple stimuli
for differentiation and cell growth activate the mitogen-activated protein kinases (MAPKs)1, also known as the extracellular signal-regulated kinases ERK1 and ERK2 (1),
which translocate to the nucleus and regulate the activity of
transcription factors (5). MAPKs are activated by the phosphorylation of a threonine and a tyrosine residue mediated by the dual specificity MAPK kinases MAPK/ERK kinase
(MEK)1 and MEK2, which relay Ras and Raf signal transduction to MAPK activation (6).
A second signaling cascade exists in all cells that leads to
the activation of stress-activated protein kinases (SAPKs) or Jun NH2 terminal kinase (JNKs; 9,10). The SAPK signaling
cascade is parallel and independent from MAPK activation
(11, 12). SAPKs/JNKs are activated in response to a variety
of cellular stresses such as changes in osmolarity and metabolism, DNA damage, heat shock, ischemia, inflammatory
cytokines, or ceramide (13). Activated SAPKs/JNKs
phosphorylate c-Jun, which leads to activation of the transcriptional complex AP-1 (19). SAPKs/JNKs are activated by the phosphorylation of tyrosine and threonine residues,
which is catalyzed by the dual specificity kinase SAPK/
ERK kinase (SEK)1 (also known as MAPK kinase [MKK4]
and JNK kinase; 20-22). In addition to SEK1, a novel
SAPK activator (SEK2 or MKK7) has been genetically identified but has not been cloned yet (23).
It has been proposed from transfection studies with dominant negative signaling mutants that the SEK1 To determine the role of SEK1 in B cell function and
CD28-mediated costimulation, we reconstructed T (23)
and B cell development in SEK1 gene-deficient chimeras
using recombination-activating gene (RAG)2 blastocyst complementation. We show that SEK1 is important for CD28-mediated costimulation for T cell proliferation and IL-2 production. B lymphocyte development was partially impaired. However, peripheral B cells displayed normal responses to IL-4 and to IgM and CD40 cross-linking, and
exhibited normal IgG class switching after vesicular stomatitis virus (VSV) infections. Moreover, we show that
CD28, but not SEK1, is crucial for VSV-specific germinal center formation. Interestingly, using the same activation
regimen, i.e., PMA plus Ca2+ ionophore which mimics
TCR-CD3- and CD28-mediated signal transduction (29),
SAPK activation was observed in peripheral T cells, but not
in thymocytes, from SEK1 Mice.
The generation of embryonic stem (ES) cells homozygous for the SEK1 mutation, SEK1 Immunocytometry.
Single cell suspensions from thymocytes,
spleen cells, mesenteric lymph node cells, and bone marrow cells
from SEK1 Cell Sorting.
Bone marrow cells were isolated from RAG2
Table 1.
T and B Cell Subpopulations in SEK1 SAPK/
JNK
c-Jun signaling cascade is a common intracellular
pathway required for the induction of apoptosis in response
to many types of cellular stresses (16, 24). However,
recent genetic evidence suggests that SEK1 and SEK1-mediated SAPK activation have no role in the induction of cell death in lymphocytes, but rather protect T cells from
CD95 (FAS) and CD3-mediated apotosis (23). The SAPK/
JNK signaling cascade is also triggered by certain growth stimulating factors and phorbol esters (9, 14, 29, 30). In B cells,
SEK1 and SAPK are activated in response to CD40 cross-linking (31, 32) and by the human STE20 homologue germinal center kinase (GCK) (33). The prominent expression of
GCK in germinal centers (34) suggested that the GCK/SAPK
pathway might be important for B cell differentiation or
activation. Moreover, biochemical studies in T cells indicated that SAPKs/JNKs are involved in the integration of
TCR-CD3 and CD28 costimulatory signals required for
proliferation and IL-2 production (29, 35). Failure to activate
SAPKs/JNKs in T cells may result in clonal anergy (36, 37).
/
mice. These data provide
the first genetic evidence that SEK1-regulated stress signal
transduction has a role in CD28 costimulation for IL-2
production and proliferation. These results also show that signaling pathways for SAPK activation are developmentally regulated in T cells.
/
somatic chimeras using
RAG2
/
blastocyst complementation (23, 38), and CD28
/
mice (39) have been previously described. Since E14 ES cells are
derived from a 129/J mouse background, age-matched 129/J
mice were used as wild-type controls. T and B cells from SEK1
/
RAG2
/
mice were tested for the SEK1 mutation using PCR
(sense primer: 5
-ACAGCAAATTTTGGAAACAGC-3
; antisense primer: 5
-CTCCCCTACCCGGTAGAATTC-3
). All
data presented throughout this study were obtained from two independently derived SEK1
/
ES cell clones (No. 1-6 and No.
1-21), and all results were comparable between them. If not otherwise stated, all mice used for experiments were between 6 and
10 wk old. Mice were kept under pathogen-free conditions in accordance with guidelines of the Canadian Medical Research Council.
/
RAG2
/
chimeric, SEK1+/+RAG2
/
chimeric,
RAG2
/
, and 129/J mice were prepared as described (40), resuspended in immunofluorescence-staining buffer (PBS, 4% FCS,
0.1% NaN3) and incubated with appropriate Abs. The following
mAbs were used: anti-CD4 (FITC-, or PE-labeled), anti-CD8
(FITC-labeled, PE-labeled, or biotinylated), anti-TCR
/
(FITC-, or PE-labeled), anti-CD3-
(FITC-labeled), anti-B220
(FITC-labeled, PE-labeled, or biotinylated), anti-CD43 (FITC-labeled), anti-CD25/IL-2R-
(biotinylated), anti-H2Kb (FITC-labeled), anti-CD44 (PE-labeled), anti-FAS (PE-labeled, or biotinylated), anti-intercellular cell adhesion molecule 1 (ICAM-1;
biotinylated); anti-CD23 (PE-labeled), anti-CD28 (PE-labeled), anti-CTLA-4 (PE-labeled), anti-CD69 (FITC-labeled), anti-CD40L (gp39; biotinylated) (all above Abs were from PharMingen, San Diego, CA); anti-surface (s)IgM (clone B67; FITC-labeled, gift of C. Paige, Ontario Cancer Institute, Toronto, Canada), anti-sIgD (PE-labeled; gift of C. Paige), and anti-CD40 (FITC-
labeled; Serotec, Toronto, Canada). All staining combinations
were as indicated in the figure and table legends. Biotinylated Abs
were visualized using Streptavidin-RED670 (Life Technologies,
Burlington, Canada). Samples were analyzed using a FACScan®
(Becton Dickinson, Mountain View, CA).
/
,
SEK1
/
RAG2
/
chimeric, SEK1+/+RAG2
/
chimeric, and
129/J control mice and double stained for CD43 and B220 expression using anti-CD43-FITC and anti-B220-PE. CD43+B220+ and
B220+CD43
bone marrow B cell polulations (Fig. 2) were
sorted using a FACS® power sorter (FACS® Vantage). In all experiments, postsorting purity of CD43+B220+ and B220+CD43
populations was >98%. Sorted cells were analyzed for the SEK1 mutation using PCR (see above).
Fig. 2.
Immunocytometric analysis of B cell populations in the bone
marrow (left) and spleen (right) of 129/J, SEK1+/+ chimeric, SEK1/
chimeric, and RAG2
/
mice. Cells were isolated from 6-wk-old mice
and double stained using anti-B220 (PE) and anti-CD43 (FITC), or anti-B220 (PE) and anti-sIgM (FITC). Percentages of positive cells within a
quadrant are indicated. Note the partial block in the maturation from
CD43+B220+ pro-B cells to CD43
B220+ pre-B cells in the bone marrow and the reduced number of sIgM+ B cells in the spleen of SEK1
/
chimeric mice (see also Table 1). 10,000 viable cells were collected and
analyzed on a FACScan®. Total cell numbers were: 129/J bone marrow
(total lymphoid cells isolated from one femur), 7.9 × 106; 129/J spleen,
4.1 × 107; SEK1+/+ chimeric bone marrow, 8.3 × 106; SEK1+/+ chimeric spleen, 3.1 × 107; SEK1
/
bone marrow, 7.7 × 106; SEK1
/
chimeric spleen, 3.9 × 107; RAG2
/
bone marrow, 9.8 × 106;
RAG2
/
spleen, 1.3 × 107.
[View Larger Version of this Image (51K GIF file)]
/
Chimeric Mice
Percentages ± SEM of positive cells per total cells
Cell subset
129/J
SEK1+/+
SEK1
/
RAG2
/
Thymus
CD4+CD8+
81.8 ± 2.2
84 ± 0.9
57.7 ± 4.2
0
CD4+CD8
11.6 ± 0.8
7.7 ± 0.4
27.2 ± 2.8
0
CD4
CD8+
3.2 ± 0.4
2.1 ± 0.7
7.5 ± 1.1
0
CD4
CD8
3.5 ± 0.6
6.1 ± 1.0
4.8 ± 0.4
100
Lymph node
CD4+CD8
48.2 ± 3.2
44.2 ± 4.3
55.1 ± 3.1
0
CD4
CD8+
19.0 ± 2.8
20.2 ± 3.7
22.1 ± 1.8
0
B220+slgM+
30.5 ± 1.6
26.6 ± 3.5
12.0 ± 3.0
0
Spleen
CD4+CD8
24.0 ± 5.3
22.6 ± 5.3
33.2 ± 2.8
0
CD4
CD8+
11.3 ± 2.0
8.9 ± 2.7
13.5 ± 1.5
0
B220+slgM+
33.5 ± 5.5
32.6 ± 3.5
16.7 ± 1.6
0
Bone marrow
B220+CD43+
11.9 ± 1.4
10.6 ± 2.5
13.4 ± 0.4
16.5 ± 3.5
B220+CD43
30.8 ± 2.5
34.5 ± 4.5
11.6 ± 1.6
<1
B220+CD25+
30.5 ± 2.1
32.0 ± 3.3
10.2 ± 1.5
<1
B220+CD25
10.5 ± 0.9
15.1 ± 2.7
12.6 ± 1.2
14.6 ± 4.1
Cells from thymi, mesenteric lymph nodes, spleens, and bone marrow (one femur) from 129/J (n = 6), SEK1+/+ chimeric (n = 3), SEK1 /
chimeric (n = 6), and RAG2
/
(n = 5) mice were stained with the indicated Abs and populations determined using a FACScan®. Total cell numbers
(× 106 ± SEM) were: 129J: thymus, 80 ± 6.3; LN, 24 ± 2.2; spleen, 21± 3.4; BM, 7.8 ± 0.7; SEK1+/+: thymus, 85 ± 10.1; LN, 22 ± 4.6; spleen,
32 ± 3.14; BM, 8.6 ± 2.5; SEK1
/
: thymus, 14 ± 4.1; LN, 29 ± 4.2; spleen 27 ± 5.3; BM, 10.6 ± 0.4; RAG2
/
: thymus, 1.1 ± 0.2; LN, 0.7±
0.2; spleen, 8.3 ± 1.6; BM 9.2 ± 0.9 (reference 23). Bold numbers indicate, statistically significant differences between 129/J or SEK1+/+ and
SEK1
/
subpopulations (Student's t test P <0.05).
B and T Cell Stimulation Assays.
Lymph node T cells were
negatively enriched from lymph nodes of SEK1/
RAG2
/
chimeric and SEK1+/+RAG2
/
chimeric mice using affinity
columns (R&D Sys. Inc., Minneapolis, MN) to avoid receptor
cross-linking during the purification process. Purified (>95%) T
cells (104) and freshly isolated thymocytes were placed into
round-bottom 96-well plates (Costar, Fisher Scientific, Unionville, Canada) in freshly prepared IMDM (10% FCS, 10
5 M
mercaptoethanol) and activated with PMA (12.5 ng/ml) plus Ca2+ ionophore A23617 (100 ng/ml), plate-bound anti-CD3-
(clone 145-2C11, hamster IgG; PharMingen), soluble anti-CD3-
(clone 145-2C11), and soluble anti-CD28 (clone 37.51, hamster
IgG; gift of Dr. J. Allison, University of California, Berkeley,
CA). PMA/Ca2+ ionophore and mAbs were added at optimal
concentrations determined in pilot studies. For CD3 cross-linking, plates were coated overnight (4°C) with 10 µg/well of rabbit
anti-hamster IgG (Jackson Labs., West Grove, PA), and subsequently with anti-CD3-
(37°C for 2 h, clone 145-2C11).
CD40 Cross-linking. For CD40-mediated upregulation of ICAM-1 and CD23 (42), purified B cells were activated with anti-CD40 (2 µg/ml; Serotec) in the absence or presence of IL-4 (50 U/ml) in IMDM (10% FCS, 37°C). After 24 h of activation, cells were harvested and triple stained with Abs reactive against B220 (PE), sIgM (FITC), and ICAM-1 (biotin) or CD23 (biotin). Biotinylated Abs were visualized using Streptavidin-RED670 and staining of cells was analyzed using a FACScan®.
Detection of Ig-subclasses.
Sera were collected from 6-wk-old
individual SEK1/
RAG2
/
and SEK1+/+RAG2
/
chimeric
mice. The concentrations of Ig subclasses were determined by
ELISA with isotype-specific, alkaline phosphatase-conjugated Abs (Southern Biotechnology Assoc. Birmingham, AL). Serum Ig
concentrations were determined by fivefold serial dilutions and
calculated according to standard charts as described previously (39).
VSV Infections and Detection of VSV-neutralizing Abs.
Mice were
immunized with VSV-Indiana (2 × 106 PFU, intravenously). After 4, 8, and 12 d, sera were collected and neutralizing IgM and
IgG Ab titers determined as described (43). In brief, 1:2 dilutions
of 40-fold prediluted serum were incubated with VSV for 90 min. The presence of remaining infectious virus was determined
by incubating the VSV serum samples with fibroblasts for another
24 h. Serum dilutions that reduced the number of viral plaques by
50% were taken as specific titers. IgG titers were determined after
preincubation of sera with 2 mercaptoethanol, a procedure that
eliminates IgM (43).
Germinal Center Formation and Immunohistochemistry.
To determine formation of germinal centers, 6 wk old SEK1/
RAG2
/
and SEK1+/+RAG2
/
chimeric mice and CD28
/
mice were
infected with VSV-Indiana as described above. Spleens from
VSV-infected animals were harvested 12 d after the intial infection, frozen in liquid nitrogen, and processed for cryosections. Cryostat sections (5 µm) were fixed in acetone (10 min). Sections were incubated with PNA (diluted 1:200) and bound PNA was
detected by rabbit anti-PNA Abs (diluted 1:300; DAKO, Glostrup, Denmark). CD4 was detected by the rat mAb YTS191.
Binding of primary Abs was detected by alkaline phosphatase-labeled goat Abs to rabbit or rat Ig (1:80 dilution; Jackson Labs.)
followed by alkaline phosphatase-labeled donkey Abs against goat
Ig (1:80 dilution; DAKO). Alkaline phosphatase was visualized using Napthol AS-BI phosphatase and New Fuchsin as a substrate,
which yields a red precipitate. Endogenous alkaline phosphatase
activity was blocked by levamisole (44).
SAPK/JNK Activities.
Thymocytes and purified lymph node
T cells (5 × 106) were activated with PMA (50 ng/ml) and the
Ca2+ ionophore A23617 (1 µg/ml) as previously described (23,
29). Cells were lysed in ice-cold lysis buffer (10 mM NaCl, 20 mM Pipes, pH 7.0, 0.5% NP-40, 5 mM EDTA, 0.05 mercaptoethanol, 100 µM Na3VO4, 50 mM NaF, 20 µg/ml leupeptin,
and 1 mM benzamidine). Cleared lysates were adjusted to equal
protein concentrations (BioRad Protein Assay; Bio Rad Labs.,
Hercules, CA). SAPKs/JNKs were immunoprecipitated (1 h,
4°C) using polyclonal rabbit anti-SAPK/JNK Abs reactive against
all SAPK/JNK isoforms (10). Immune complexes were harvested
on protein A-Sepharose beads. For kinase assays, immune complexes were washed three times with PBS-Triton buffer (150 mM
NaCl, Na2HPO4, 4 mM NaH2PO4, 0.1% Triton X-100, 100 mM Na3VO4, 50 mM NaF, 20 µg/ml leupeptin, and 1 mM benzamidine). SAPK/JNK kinase assays were performed in 20 µl of
kinase buffer (10 mM MgCl2, 50 mM Tris-Cl, pH 7.5, 1 mM
EGTA, pH 7.5) in the presence of 1.2 µCi [32P]
-ATP and 5 µg
glutathione-S-transferase-c-Jun as in vitro substrate (30°C, 30 min). The reaction was stopped by the addition of 2× SDS sample buffer. Phosphoproteins were separated by SDS-PAGE and
visualized by autoradiography as described (10). The levels of expression of SAPK/JNKs in thymocytes and lymph node cells were determined by immunoblotting using goat anti-JNK1 and
rabbit anti-JNK2 polyclonal Abs (both from Santa Cruz Biotechnology Inc., Santa Cruz, CA; 23).
Recent biochemical studies implied that the
SAPK/JNK signaling pathway is operating in T cells, and
that cell proliferation and IL-2 production induced by CD28
costimulation may be mediated via SAPK/JNK (29, 36,
37). SEK1/
RAG2
/
chimeric mice have a smaller thymus, but normal numbers of peripheral T cells (Table 1; reference 23). To test the role of SEK1 in CD28 costimulation,
lymph node T cells were cultured in anti-CD3-
Ab-coated
plates in the absence or presence of various concentrations of
soluble anti-CD28 Abs. Whereas SEK1
/
RAG2
/
and
SEK1+/+RAG2
/
T cells reponded in the same way to
CD3-
activation alone, CD28-mediated upregulation of
proliferation and IL-2 production were significantly reduced in SEK1
/
T cells (Fig. 1, A and B). Reduced proliferation and IL-2 production were also observed in
SEK1
/
T cells after stimulation with PMA/Ca2+ ionophore (Fig. 1, A and B), which mimic TCR-CD3- and
CD28-mediated signal transduction (29, 45).
Since the proliferative response to plate-bound anti-
CD3- Abs alone was still very vigorous (Fig. 1 A), we analyzed T cell activation in response to suboptimal concentrations of soluble anti-CD3-
Abs. As shown in Fig. 1, C and
D, proliferation and IL-2 production of SEK1
/
RAG2
/
and SEK1+/+RAG2
/
chimeric T cells were minimal after stimulation with soluble anti-CD3-
alone. Although
the addition of anti-CD28 greatly enhanced the proliferation and IL-2 production of SEK1+/+RAG2
/
T cells,
SEK1
/
RAG2
/
T cells were significantly impaired in
proliferation and IL-2 production (Fig. 1, C and D). It
should be noted that freshly isolated T cells from SEK1
/
mice displayed upregulated expression of CD28, but normal surface expression of the TCR-
/
-CD3-
complex,
IL-2R-
chain (CD25), CD69, and adhesion molecules
such as ICAM-1 (not shown). These data provide the first
genetic evidence that SEK1 plays an important role in T
cell proliferation and IL-2 production in transmitting CD28 signals to downstream effector molecules.
To determine the effect of the SEK1 mutation on B cell development, single
cell suspensions from spleen, lymph nodes, and bone marrow of SEK1/
RAG2
/
chimeric, SEK1+/+RAG2
/
chimeric, RAG2
/
, and control 129/J mice were stained
with mAbs against B lineage-specific markers (Fig. 2, Table
1). The bone marrow of 129/J mice contained a relatively
low number (12%) of B220+CD43+ pro-B cells and a
larger population (30%) of B220+CD43
pre-B cell precursors, and mature B cells in peripheral lymphatic organs
expressed both IgM (Fig. 2) and IgD (not shown) on the cell surface (46). By contrast, B cell differentiation in the bone marrow of RAG2
/
mice was blocked at the pro-B
cell stage (B220+CD43+IgM
) and RAG2
/
mice did
not have any mature sIgM+ B cells (Fig. 2, Table 1; 47, 48). B-cell development and expression of sIgM and sIgD
were restored in chimeras derived from injection with parental SEK1+/+ ES cells. In contrast, the relative and total
numbers of B220+CD43
bone marrow cells and B220+
sIgM+ peripheral B cells were significantly reduced in
SEK1
/
RAG2
/
chimeric mice (Fig. 2, Table 1). Peripheral B cells from SEK1
/
mice expressed normal levels of CD23, CD40, CD44, ICAM-1, CD95 (FAS), and
H2Kb on the cell surface (not shown). The partial block in
the development from B220+CD43+ pro-B cells to more
mature B220+CD43
pre-B cells was also observed by alterations in IL-2R-
chain (CD25) expression, an early B
cell maturation marker that is expressed before sIgM expression (46). Although ~75% of 129/J or SEK1+/+ chimeric B220+ bone marrow B cells expressed CD25 on the
cell surface, expression of CD25 was significantly reduced
in SEK1
/
bone marrow B cells (Table 1).
To analyze whether the observed block in B cell maturation was due to the SEK1 mutation and not due to low
chimerism and contribution of RAG2/
cells to pro-B cells,
we FACS® sorted B220+CD43+ and B220+CD43
bone
marrow cells and analyzed the genotype of sorted cells by PCR (Fig. 3). Both B220+CD43+ pro-B cells and the
more mature B220+CD43
B cell populations in SEK1
/
chimeras contained mutant, but not wild-type SEK1 alleles
indicating that both populations were derived from SEK1
/
ES cells. These data imply that SEK1-mediated signaling
plays a role at the transition from B220+CD25
CD43+
pro-B cells to B220+CD25+CD43
pre-B cells in the bone
marrow.
B Cell Activation.
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).
VSV Infections and IgG Class Switching.
VSV infections
are exclusively controlled by neutralizing Abs (49). All
neutralizing Abs are directed against the VSV glycoprotein which is present in a highly repetitive form in the viral envelope. Due to this repetitiveness, neutralizing IgM Abs are
induced in complete absence of T cell help (49). However,
the isotype switch from IgM to IgG is strictly T cell dependent (50). Recently, it has also been shown that the production of VSV-neutralizing IgG Abs is decreased in
CD28/
mice (39). Since, SEK1
/
T cells had reduced
proliferation and IL-2 production in response to CD28 costimulation (Fig. 1), but SEK1
/
B cells could be activated
normally and produced normal levels of Ig subclasses (Fig.
4), we examined T cell help and IgG class switching in
SEK1
/
RAG2
/
and SEK1+/+RAG2
/
mice after infection with VSV (Table 2). Neutralizing serum IgM was
assessed 4 d and neutralizing serum IgG levels were measured 4, 8, and 12 d after VSV infection. VSV infection induced rapid, T cell-independent IgM production, followed
by a T helper cell and CD28 costimulation-dependent IgG
response (Table 2). Surprisingly, both early IgM production and IgG class switching were comparable between
SEK1
/
RAG2
/
and SEK1+/+ RAG2
/
mice (Table
2). Moreover, SEK1
/
mice survived for more than 4 wk
after infection, indicating that the B cell response was protective.
|
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 SEK1/
mice. Although VSV-specific germinal centers were completely absent in CD28
/
mice after challenge with VSV,
SEK1
/
RAG2
/
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
CD28
/
mice (Fig. 5 J) and that CD28
/
mice could still
produce, albeit at low levels, neutralizing IgG Abs (Table
2). These data show that SEK1
/
RAG2
/
mice can
mount biologically relevant responses against VSV and that
SEK1 has no apparent role in CD28-dependent, virus-specific germinal center formation.
|
Although SEK1/
peripheral T cells displayed reduced proliferation and IL-2 production in response to
CD28 in vitro, SEK1 expression was not an absolute requirement for T cell activation in vivo. Thus, similar to our
data that shows that SEK1-dependent and SEK1-independent signaling pathways for SAPKs/JNKs activation exist in
ES cells (23), it was possible that a SEK1-independent
pathway for SAPK/JNK activation was operational in T
cells. To test this hypothesis, we examined SAPKs/JNKs
activation in peripheral T cells and thymocytes in response
to PMA/Ca2+ ionophore (Fig. 6 A), which mimic TCR-
CD3 and CD28-mediated signal transduction (29,45).
SAPK activation, i.e., SAPK-mediated c-Jun phosphorylation,
was observed in SEK1+/+, but not in SEK1
/
, thymocytes,
indicating that SEK1 is the crucial genetic regulator of
PMA/Ca2+ ionophore-triggered activation of SAPKs/JNKs
in thymocytes (23; Fig. 6 A). Surprisingly, using the same
PMA/Ca2+ ionophore activation regimen, SAPK activation was observed in peripheral lymph node T cells from
both SEK1
/
and SEK1+/+ mice (Fig. 6 A). The levels of
SAPKs (JNK1 and JNK2) expression were comparable among
SEK1
/
RAG2
/
and SEK1+/+RAG2
/
thymocytes and
peripheral T cells (Fig. 6 B), suggesting that the observed
differences in PMA/Ca2+ ionophore-mediated SAPK activation in SEK1
/
thymocytes versus SEK1
/
lymph node
T cells were not the result of alterations in SAPK expression. Our data that SAPKs are activated in peripheral T cells, but not in thymocytes, from SEK1
/
chimeras in response to the same stimulus PMA/Ca2+ ionophore indicate
that signaling pathways for SAPK/JNK activation are developmentally regulated.
IL-2 Production in SEK1
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.
SAPKs/JNKs are activated in response to many cellular
stresses such as osmolarity changes, metabolic poisons, DNA-damaging agents, heat shock, ischemia/reperfusion injury, UV-,
or -irradiation (9, 10, 13, 14, 18, 25). The dual specificity kinase SEK1 (JNK kinase/MKK4) has been identified as a potent
and direct activator of SAPKs/JNKs in vitro and in cell lines
in vivo (20). Although it has been shown genetically that a
second SAPK activator, SEK2, exists (23), SEK1 is the only
cloned kinase that can directly activate SAPKs/JNKs (11, 12).
In addition to the induction of SAPK/JNK activity by
many types of cellular stresses (10), SAPKs/JNKs are activated in response to certain growth factors, heterotrimeric
G proteins, phorbol esters, CD40 cross-linking, and CD28-mediated costimulation in T cells (9, 10, 14, 29, 54).
Moreover, activation of SAPKs/JNKs leads to phosphorylation of c-Jun and activation of Jun/Fos heterodimeric
AP-1 complexes, which are involved in the coordinate activation of IL-2 transcription (10, 19, 20, 55). In T cells, ligation of the TCR results in rapid activation of the
Ras Raf
MEK
MAPK signaling cascade (11, 56).
However, activation of the MAPK cascade is not sufficient
for effective IL-2 production and cell proliferation for
which T cells require a second signal (35). Recently, it has
been shown that coordinate stimulation of the TCR-CD3
complex and the costimulatory receptor CD28 correlates
with the activation of SAPKs/JNKs, phosphorylation of
c-Jun, and induction of AP-1 activity (29). These biochemical data indicated that T cells use two distinct signaling cascades for antigen-specific activation, TCR-triggered
MAPK activation and TCR-CD28-induced activation of
SAPKs/JNKs. Importantly, it has been suggested that failure to activate SAPKs/JNKs in T cells might result in
clonal anergy and the induction of immunological tolerance (36, 37).
Our demonstration of defective IL-2 production and
proliferation in SEK1/
T cells in response to CD28 costimulation and PMA/Ca2+ ionophore provides the first
genetic evidence that the stress signaling kinase SEK1 is a
downstream effector involved in TCR and CD28 coreceptor signaling. However, the impairments of proliferation and IL-2 production were not complete, and a strong activation signal via the TCR-CD3 complex alone triggered
normal proliferation of SEK1
/
T cells. Thus, although
SEK1 appears to be necessary for adequate IL-2 production
and proliferation in T cells, another activator(s) can compensate for the SEK1 deficiency in peripheral T cells. This
hypothesis is in line with our biochemical data on SEK1-independent activation of SAPKs/JNKs in lymph node T
cells in response to PMA/Ca2+ ionophore stimulation. Interestingly, this second pathway for SAPK/JNK activation
is only operational in peripheral T cells but not in thymocytes, indicating that signaling pathways for SAPK/JNK
activation are developmentally regulated in T cells. It has
also been shown that proliferation and IL-2 production are
normal in c-Jun
/
RAG2
/
T cells, suggesting that not
only c-Jun, but also other Jun family members, i.e., JunD
and JunB, may have a role in T cell activation (57). The
exact role of distinct SAPK/JNK activators, SEK1 versus SEK2, and of different Jun family transcription factors in
CD28-mediated IL-2 production and T cell activation
needs to be determined.
SEK1/
RAG2
/
thymocytes failed to induce SAPK/
JNK in response to PMA/Ca2+ ionophore. Interestingly,
SEK1
/
RAG2
/
thymocytes still proliferated and produced IL-2 after PMA/Ca2+ ionophore and CD3/CD28
stimulation, albeit at significantly lower levels compared to
SEK1+/+RAG2
/
thymocytes. These data further confirmed that SEK1 relays CD28 costimulatory signals to IL-2
production in T cells. However, these results also indicate
that, at least in thymocytes, CD28 and PMA/Ca2+ ionophore-triggered signaling pathways exist that can mediate proliferation and IL-2 production independently of SAPK
activation. Besides activation of SEK1 and SAPKs/JNKs,
additional downstream effectors for CD28 signaling have
been identified including PI3
K, PLC
1, Raf-1, and Vav
(see review in reference 58). In particular, it has been
shown that Vav, Ras, and the Vav-associated tyrosine phosphoprotein SLP76 can cooperate to induce nuclear
factor of activated T cells activity and IL-2 secretion after
activation of the TCR (59). Nevertheless, a growing
body of evidence suggests that Vav functions as a guanine
nucleotide (GDP/GTP)-exchange factor for members of the
Rho family of small GTPases that regulate activation of the
SAPK pathway (62). Whether Vav can relay TCR-mediated signals to proliferation and IL-2 production independently of SAPK activation needs to be determined.
Similar to the reported reduction in CD4+CD8+ thymocyte
numbers (23), SEK1/
RAG2
/
chimeric mice had a
partial block in the transition from B220+CD25
CD43+
pro-B cells to B220+CD25+CD43
pre-B cells in the
bone marrow. This effect was due to the SEK1 mutation
and not due to a low chimerism and contribution of
RAG2
/
cells to the CD43+ population (Fig. 3). However, splenic B cells from SEK1
/
RAG2
/
chimeras displayed normal proliferation in reponse to Igµ, CD40, IL-4,
or LPS activation. Moreover, basal Ig levels of all subclasses and IgG class switching after viral challenge were comparable between SEK1
/
and SEK1+/+ B cells. These results
imply that SEK1 has a role in early differentiation of B cell
precursors, but SEK1 is not necessary for proliferation and
Ig secretion of peripheral B cells.
The prominent expression of the GCK within 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. Both CD28- and CD40-triggered signaling cascades lead to the activation of SAPKs/
JNKs (10, 29, 31, 32). Although SEK1 is an important component of CD28-mediated IL-2 production and T cell
proliferation and VSV-specific germinal center formation is
dependent on CD28 expression (Fig. 5), our results clearly
indicate that SEK1 is not involved in germinal center formation in response to VSV infection. SEK1-independent
activation of peripheral T cells might explain the fact that
SEK1/
chimeric mice can mount CD28 costimulation
dependent responses against VSV infections. Since multiple
downstream effectors for CD28 signaling have been identified including SEK1/SAPK, PI3
K, PLC
1, Vav, or Raf-1
kinase (see reviewed in reference 58), it is possible that
CD28-mediated signaling for IL-2 production and CD28-dependent signaling for germinal center formation are biochemically different.
SEK1 (MKK4, JNK kinase) is a direct activator of stress-activated protein kinases (SAPK/JNK) in response to CD28 costimulation, CD40 ligation, and activation of the GCK. SEK1/
RAG2
/
chimeric mice have a
partial block in B cell maturation, but peripheral B cells
displayed normal responses to IL-4, IgM, and CD40 cross-linking and normal IgG class switching of neutralizing Abs
after viral challenge. T cells from chimeric mice showed
decreased proliferation and IL-2 production in response to
CD28 costimulation and PMA/Ca2+ ionophore activation.
Although CD28 was absolutely crucial to generate VSV-specific germinal centers, SEK1
/
chimeras made normal
germinal centers in response to VSV. Interestingly, PMA/
Ca2+ ionophores' stimulation, which mimic TCR-CD3 and
CD28-mediated signal transduction, induced SAPK/JNK
activation in peripheral T cells, but not in thymocytes,
from SEK1
/
mice. These results demonstrate 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+ ionophores and CD3/CD28 stimulation, albeit at significantly lower levels compared to SEK1+/+
RAG2
/
thymocytes, indicating that CD28 costimulation
and PMA/Ca2+ ionophore-triggered signaling pathways
exist that can mediate proliferation and IL-2 production independently of SAPK activation. These 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.
Address correspondence to Josef Penninger, Amgen Institute and Ontario Cancer Institute, Department of Medical Biophysics and Immunology, University of Toronto, 620 University Ave., Suite 706, M5G 2C1 Toronto, Ontario, Canada. Phone: 416-204-2241; FAX: 416-204-2278; E-mail: jpenning{at}amgen.com
Received for publication 2 April 1997 and in revised form 9 July 1997.
James R. Woodgett is supported by a grant from the Medical Research Council of Canada. Klaus-Dieter Fischer and Alan Bernstein are supported by the Medical Research Council and National Cancer Institute of Canada.We thank J. Allison and C. Paige for reagents; R. Hakem and S. Gardener for technical support; and H.W. Mittrücker, K. Bachmaier, A. Hakem, D. Bouchard, C. Sirard, M. Saunders, S. Nishina, L. Zhang, and S. Bagby for critical comments.
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