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B Kinases 
and 
Have Distinct Roles in Regulating Murine T Cell Function1
* Division of Hematology-Oncology, Department of Medicine, Harold Simmons Cancer Center, and
Center for Immunology and Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B is a transcription factor that regulates a variety of genes
involved in the control of the immune and inflammatory responses.
Activation of NF-B is mediated by an inducible I-B kinase (IKK)
complex comprised of two catalytic subunits, IKK
and IKK
. In this
study, the role of these kinases in the development and function of T
lymphocytes was explored using transgenic mice expressing the
dominant-negative forms of one or both kinases under the control of a T
cell-specific promoter. Activation of the NF-B pathway in thymocytes
isolated from these transgenic mice following treatment with either PMA
and ionomycin or anti-CD3 was markedly inhibited. Although
inhibition of IKK and/or IKK function did not alter T cell
development in these transgenic mice, the proliferative response to
anti-CD3 was reduced in thymocytes isolated from mice expressing
dominant-negative IKK. However, inhibition of both IKK and IKK
was required to markedly reduce cytokine production in thymocytes
isolated from these transgenic mice. Finally, we demonstrated that
IKK and IKK have opposite roles on the regulation of
anti-CD3-induced apoptosis of double-positive thymocytes. These
results suggest that IKK and IKK have distinct roles in
regulating thymocyte function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B is a critical regulator of immune and
inflammatory responses (1, 2, 3). NF-B regulates the
expression of a variety of genes encoding cytokines, cytokine
receptors, chemokines, cell adhesion molecules, and cell surface
receptors that are critical for T and B lymphocyte function
(4). Targeted inactivation of genes in mice encoding
individual NF-B subunits has demonstrated the importance of these
proteins in regulating immune system functions (5). Gene
disruption of single NF-B subunits in mice, including p105/p50
(6, 7), p100/p52 (8, 9), c-Rel
(10), RelA (11), and RelB (12),
leads to a variety of defects in B and T cell proliferation and
associated immune function, but does not result in major defects in the
maturation of T and B cells. However, mice lacking multiple NF-B
subunits such as p105/p50 and p100/p52 (13), p105/p50 and
RelB (14, 15), RelA and c-Rel (16), and
p105/p50 and RelA (17) have more marked defects in B and T
cell development. These results indicate that the NF-B pathway is
critical for the development and function of both B and T
lymphocytes.
Members of the NF-B/Rel family, which include NF-B1 (p105/p50),
NF-B2 (p100/p52), RelA/p65, RelB, and c-Rel, are present
predominantly in the cytoplasm of resting cells, where they are bound
to a group of inhibitory proteins known as I-B (1, 2, 18, 19). In response to a variety of stimuli, including the
cytokines TNF- and IL-1, the I-B proteins are specifically
phosphorylated, leading to their ubiquitination and degradation by the
26S proteosome (19). This process results in nuclear
translocation of NF-B and the activation of a variety of genes
involved in the inflammatory and immune response.
Phosphorylation of the I-B proteins by the I-B kinases
(IKK)3 is a critical
step involved in the control of the NF-B pathway (3, 20, 21, 22, 23, 24). The IKK complex is composed of two catalytic subunits,
IKK and IKK, in addition to a regulatory subunit known as the
IKK
/NF-B essential modulator. Both IKK and IKK are able to
phosphorylate I-B (20, 21, 22, 23, 24), while the IKK/NF-B
essential modulator is a scaffold protein that is critical in
regulating IKK and IKK kinase activity (25, 26, 27, 28).
IKK and IKK have a high degree of amino acid homology and a
similar domain organization that includes an N-terminal kinase domain,
a leucine zipper that facilitates their heterodimerization and
homodimerization, and a C-terminal helix-loop-helix domain
(20, 21, 22, 23, 24, 29). IKK is a much more potent kinase for
I-B than is IKK, suggesting that IKK is the dominant kinase
involved in cytokine-mediated activation of the NF-B pathway
(22, 23, 30).
IKK and IKK have distinct functions in vivo. The function of
IKK and IKK has been investigated in IKK-deficient
(IKK-/-) and IKK-deficient
(IKK-/-) mice.
IKK-/- mice die of severe skin and skeletal
abnormalities shortly after birth (31, 32), whereas
IKK-/- embryos die of severe liver
degeneration due to massive hepatocyte apoptosis (29, 33, 34). These studies further indicate that IKK is the critical
kinase that phosphorylates I-B in response to proinflammatory
cytokines and, in addition, is important in activating genes that
prevent apoptosis (34, 35). In contrast, IKK has a more
important role in the development of the epidermis and skeletal system
(31, 32).
Previous studies using transgenic mice overexpressing I-B
(36) or expressing dominant-negative (DN) forms of
IB in T cells (37, 38) indicated that the NF-B
pathway is critical in regulating T cell proliferation and cytokine
production (37, 38). The NF-B pathway is also important
for the development of CD8-positive T cells (36, 38) and
for the regulation of T cell survival (37, 38). Recently,
several groups used IKK-/- and
IKK-/- radiation chimeras to investigate the
roles of these kinases in the development and function of the immune
system (35, 39, 40). These studies demonstrated that
IKK is important in B cell maturation and the formation of secondary
lymphoid organs through its ability to phosphorylate and induce
processing of p100, whereas IKK is critical in preventing
TNF--induced apoptosis in developing lymphocytes. However, NF-B
could promote apoptosis in T cells under some circumstances (37, 41). Although it was shown that mature B cells lacking IKK
have an increased turnover rate and increased spontaneous apoptosis in
vitro (39, 40), the role of IKK in regulating apoptosis
in T cells is unclear, as is the role of IKK and IKK in
regulating T cell proliferation and cytokine production.
A direct comparison of the role of IKK and IKK on the development
and function of T lymphocytes has not previously been reported. In this
study, we use transgenic mice expressing dominant-negative IKK
(DNIKK) and dominant-negative IKK (DNIKK) either individually
or in combination specifically in T cells. We demonstrate that IKK
and IKK have distinct roles in T cell function. Both DNIKK and
DNIKK inhibited NF-B activation in T cells following treatment
with PMA and ionomycin. DNIKK was a stronger inhibitor of
anti-CD3-induced NF-B activation than was DNIKK. Accordingly,
DNIKK, but not DNIKK, markedly reduced the proliferative response
of T cells following TCR cross-linking by inhibiting cell cycle
progression. In addition, thymocytes from mice expressing both DNIKK
and DNIKK exhibited severe defects in cytokine production. Finally,
we assayed the effect of DNIKK and DNIKK on the apoptosis of
thymocytes. Surprisingly, following the in vivo administration of
anti-CD3, DNIKK mice exhibited increased apoptosis of
double-positive thymocytes, while DNIKK mice exhibited decreased
apoptosis. These results provide the first direct comparison of the
roles of IKK and IKK on the development and function of murine T
lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and CD2/DNIKK transgenic mice
An influenza hemagglutinin (HA)-tagged human IKK cDNA
containing substitution of serine residue 176 with alanine
(HA-IKKA176) or a Flag-tagged human IKK cDNA containing
substitutions of serine residues 177 and 181 with alanine
(FL-IKKA177/181) were inserted into the VA hCD2 cassette
(42) to obtain T cell-specific expression of these genes.
The linearized transgenes CD2/DNIKK and CD2/DNIKK were
microinjected into the pronuclei of F1 zygotes of
the C57BL/6 and DBA2 strains. Transgenic founders were bred and
maintained in a specific pathogen-free colony. PCR using primer pairs
hybridizing to the 5' and 3' portions of the HA-IKKA176 or
FL-IKKA177/181 cDNA were used to genotype transgenic progeny and
their littermates in the colony. Southern blot analysis was used to
confirm the presence of the transgenes in the founders. Progeny of
different founders were tested for transgene protein expression, and
those with highest levels of expression were crossed to generate
transgenic mice that exhibited T cell-specific expression of both
DNIKK and DNIKK.
Flow cytometry analysis
Thymocytes and splenocytes from either wild-type or transgenic littermates were prepared in RPMI medium and stained with fluorescent Abs against cell surface markers. The Abs and reagents used for cell surface staining were: FITC-conjugated anti-CD4, PE-conjugated anti-CD3, and PerCP-conjugated anti-CD8 (BD PharMingen, San Diego, CA). Fluorescence analysis was performed using a FACSCaliber flow cytometer (BD Biosciences, San Diego, CA).
In vivo response to T-dependent Ags
Immunization of mice with T-dependent Ag trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) and quantification of TNP-specific IgM, IgG1, and IgG2a were performed, as described previously (43). TNP-KLH and TNP-BSA were obtained from Biosearch Technologies (Novato, CA).
Immunoprecipitation and Western blot analysis
Thymocytes were lysed in TNE buffer (1% Triton X-100, 10
mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA) containing a mixture of
protease inhibitors (Roche, Somerville, NJ). Cell lysates were then
incubated overnight with an M2 mAb against the Flag epitope
(Sigma-Aldrich, St. Louis, MO) or a polyclonal Ab against the HA
epitope (sc-805; Santa Cruz Biotechnology, Santa Cruz, CA), followed by
incubation with protein G-Sepharose beads (Sigma-Aldrich) for 1 h.
The immunoprecipitates were then subjected to Western blot analysis
using Abs directed against either IKK (sc-7218) or IKK (sc-7330)
obtained from Santa Cruz Biotechnology.
RT-PCR analysis of IKK or IKK mRNA isolated from thymocytes
of wild-type and transgenic mice
Total RNA was extracted from thymocytes using RNeasy
mini-columns (Qiagen, Chatsworth, CA) and subjected to RT-PCR analysis.
The oligonucleotide primers used to amplify GAPDH have been described
(44). Primers used to amplify a 411-bp fragment of both
the mouse and human IKK cDNA included the 5' primer,
5'-ctgaggttggtgtcattgg-3', and the 3' primer,
5'-cagaactctgtgtacaggc-3'. Primers used to amplify a 341-bp fragment of
both mouse and human IKK were the 5' primer,
5'-gtgtcagctgtatccttc-3', and the 3' primer,
5'-gctccacagcctgctcc-3'. The sense primers were end labeled with
[-32P]ATP. The PCR products were analyzed by
digestion with BstEII or EcoRI for IKK and
IKK, respectively. BstEII cuts the cDNA fragment
amplified from endogenous mouse IKK, but not DNIKK (human) to
generate two fragments of 247 and 164 bp, whereas EcoRI cuts
the cDNA fragment amplified from DNIKK (human), but not the mouse
IKK, to generate fragments of 176 and 165 bp. Following gel
electrophoresis and autoradiography, the intensity of the radioactive
species was measured by phosphor imager analysis (Cyclone; Packard
Instrument, Meriden, CT).
Stimulation of thymocytes and EMSA
Thymocytes from wild-type and transgenic mice were incubated
either in complete RPMI alone or with PMA (50 ng/ml) and ionomycin (200
ng/ml) for 15 min or with immobilized anti-CD3 (145-2C11, 10
µg/ml) alone or in combination with anti-CD28 (16 µg/ml;
Southern Biotechnology Associates, Birmingham, AL) for 4 h at
37°C. Nuclear extracts were then prepared from the cells according to
published methods (45, 46). To test NF-B binding, a
32P-labeled oligonucleotide probe containing the
MHC class I-B site (47) or the NF-Y binding site (Santa
Cruz Biotechnology) was incubated with the nuclear extracts. The
binding reaction contained 60,000 cpm of the radiolabeled probe, 4 µg
nuclear protein, 500 ng poly(dI-dC) (Amersham Biosciences, Piscataway,
NJ), 10 µg BSA, 20 mM HEPES (pH 7.9), 1 mM EDTA, 1% Nonidet P-40,
5% glycerol, and 5 mM DTT in a final volume of 20 µl.
Reactions were incubated at room temperature for 30 min and subjected
to electrophoresis on a 5% polyacrylamide gel in 0.5x Tris-buffered
EDTA buffer. For supershift assays, 5 µg rabbit polyclonal Ab
directed against p65 (sc-7151X), p50 (sc-1190X), p52 (sc-298X), c-Rel
(sc-272X), or normal rabbit IgG (sc-2027) obtained from Santa Cruz
Biotechnology was added to the binding reactions and incubated for 30
min on ice before the samples were subjected to gel electrophoresis.
The gels were dried and exposed to x-ray film and quantified by
phosphor imager analysis.
Cell culture and proliferation assay
Freshly isolated thymocytes and splenocytes were cultured in RPMI containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 25 mM HEPES, and 50 mM 2-ME in 96-well plates at 37°C, 5% CO2. Anti-CD3 Ab was purified from the culture supernatant of 145-2C11 hybridoma using HiTrap rProtein A column (Amersham Biosciences) and used at 10 µg/ml alone or in combination with anti-CD28 (16 µg/ml; Southern Biotechnology Associates) to coat the plates overnight at 4°C. Mouse rIL-2 (Endogen, Woburn, MA) was added to the cultures at a concentration of 1–10 ng/ml. The cells were cultured for 48 h before they were pulsed with [3H]thymidine (NEN, Boston, MA) for 14–18 h and harvested onto glass fiber filter paper. The amount of [3H]thymidine incorporated was quantified by a beta scintillation counter.
Cell cycle analysis
Cell cycle analysis of nonstimulated and stimulated thymocytes was performed using a 5-bromo-2'-deoxyuridine (BrdU) flow kit (BD PharMingen). Thymocytes were cultured in RPMI in 24-well plates with or without anti-CD3 (10 µg/ml) and IL-2 (2 ng/ml) for 60 h before they were pulsed with BrdU for 30 min. According to the manufacturer’s instructions, the cells were then processed and stained with FITC-conjugated anti-BrdU and 7-amino actinomycin D (7-AAD) to determine the amount of BrdU incorporated and the total DNA content, respectively. Flow cytometry analysis was performed using FACSCaliber (BD Biosciences).
RNase protection assay
To measure the mRNA levels of multiple cytokines in thymocytes, freshly isolated thymocytes were stimulated with immobilized anti-CD3 (10 µg/ml) and anti-CD28 Abs (Southern Biotechnology Associates; 16 µg/ml) for 4–6 h. Total RNA was prepared from 2 x 107 stimulated cells using TRIzol (Life Technologies, Rockville, MD) in combination with the RNeasy Kit (Qiagen). Briefly, the stimulated cells were immediately homogenized with TRIzol reagent and extracted with chloroform. Total RNA in the cell extract was then bound to a silica gel-based mini-column and eluted with diethyl pyrocarbonate water. Approximately 2 µg of the RNA samples was subjected to electrophoresis on a 1.2% denaturing formaldehyde agarose gel in MOPS buffer to confirm the integrity of the RNA. Following DNase I treatment, 4 µg RNA from each sample was used to hybridize with labeled mCK-1b probe that contains fragments of multiple cytokine mRNA using the Riboquant RNase protection assay kit (BD PharMingen). The hybridized samples were treated with RNase, followed by proteinase K, and fractionated on a 5% denaturing polyacrylamide gel. The radioactive species on the gel were identified according to their mobility, and their intensities were quantified by ChemiImager 4400 (Alpha Innotech Corporation, San Leandro, CA) after autoradiography.
In vivo apoptosis analysis and TUNEL assay
Wild-type and transgenic littermates that were 5–7 wk old were injected i.p. with 100 µl PBS or PBS containing 25 or 50 µg anti-CD3 (145-2C11) (37). At 48 h after anti-CD3 administration, the thymocytes of treated and control mice were counted and analyzed by flow cytometry for the surface expression of CD4 and CD8. The absolute numbers of thymocyte subsets were calculated. Cryosections of thymi isolated from control and anti-CD3-treated mice were subjected to TUNEL assay using an in situ cell death detection kit (Roche), according to the manufacturer’s instructions. The stained sections were photographed using a Zeiss microscope system (Thornwood, NY).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and DNIKK
mutants in T lymphocytes
In an attempt to inhibit the function of endogenous IKK and
IKK specifically in T cells, we generated transgenic mice that
expressed DN forms of human IKK (A176) (48) or human
IKK (A177/181) (23, 49) by inserting these DN genes
into a modified human CD2 promoter cassette. This cassette confers
position-independent and transgene copy number-dependent expression of
these genes in the T cell lineage (42). The DNIKK and
DNIKK cDNAs contained amino-terminal HA and Flag epitopes,
respectively, to facilitate their detection in murine T cells. The
IKK protein in which serine residues 177 and 181 in the
mitogen-activated protein 3 kinase activation loop were substituted
with alanine has a DN phenotype that inhibits NF-B activation in
response to treatment with proinflammatory cytokines such as TNF-
and IL-1 (20, 21, 23, 49). The IKK protein in which
serine residue 176 was substituted with alanine could not be
phosphorylated or activated by the upstream kinase NF-B-inducing
kinase and inhibited endogenous IKK function
(48). Because mouse and human IKK or IKK have
greater than 90% amino acid identity, we expected that these DN forms
of IKK and IKK would inhibit the function of endogenous mouse
IKK and IKK, and thus alter NF-B activation in T
lymphocytes.
Constructs containing either the DNIKK or DNIKK cDNAs were
microinjected into the pronuclei of C57BL/6XDBA/2 zygotes (Fig. 1
). Southern blot analysis indicated that
four founders designated A, B, C, and D for the CD2/DNIKK construct
and three founders designated A, B, and C for the CD2/DNIKK
construct contained integrated transgenes (Fig. 1B). As
expected, progeny from all of these founders were able to express the
DNIKK proteins in T cells in a copy number-dependent manner (data not
shown). Progeny of the B founders of DNIKK and DNIKK that
expressed high levels of DNIKK and DNIKK, respectively, were
crossed to generate DNIKK mice that expressed both
DNIKK and DNIKK. As shown by immunoprecipitation and Western blot
assays of protein extracts prepared with thymocytes from wild-type and
DNIKK mice (Fig. 1C), DNIKK protein was expressed at
similar levels in the thymocytes of DNIKK and DNIKK mice, and
DNIKK protein was expressed at similar levels in the thymocytes from
DNIKK and DNIKK mice.
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and DNIKK mRNA
levels with that of the endogenous IKK and IKK mRNA (Fig. 2). To distinguish the expression of the
mouse and human IKK RNAs, we chose to amplify either a 411-bp fragment
with a BstEII site in the mouse, but not the human IKK
(Fig. 2A), or a 341-bp fragment with an EcoRI
site in the human, but not the mouse IKK (Fig. 2B). The
level of DNIKK mRNA, represented by the 411-bp fragment, was
>20-fold higher than that of the endogenous mouse IKK mRNA
represented by the 247-bp fragment in the DNIKK and DNIKK mice
(Fig. 2A). The level of DNIKK mRNA, represented by the
176-bp fragment, was 10-fold higher than that of the endogenous mouse
IKK represented by the 341-bp fragment in the DNIKK and
DNIKK mice (Fig. 2B). These results suggest that both
DNIKK and DNIKK were expressed at significantly higher levels
than the endogenous kinases.
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B DNA binding in the thymocytes from the DNIKK
transgenic mice
Next, we addressed whether DNIKK expression altered NF-B
activation in T cells. We performed EMSAs using nuclear extracts
prepared from both nonstimulated and PMA/ionomycin or
anti-CD3-stimulated thymocytes isolated from wild-type and
different DNIKK transgenic mice. NF-B DNA-binding activity was
strongly induced by both PMA/ionomycin and anti-CD3 stimulation in
thymocytes isolated from wild-type mice (Fig. 3, A and B).
PMA/ionomycin-induced NF-B DNA-binding activity was reduced in
thymocytes isolated from DNIKK and DNIKK mice, and was further
reduced in the thymocytes from DNIKK mice (Fig. 3A).
There was only a moderate decrease in anti-CD3-induced NF-B
DNA-binding activity in DNIKK thymocytes as compared with the
wild-type thymocytes, whereas this activity was markedly reduced in
DNIKK and DNIKK thymocytes (Fig. 3B). In the
presence of anti-CD3 and anti-CD28, which provides a
costimulatory signal for T cell activation, the defects in NF-B
activation seen in thymocytes from DNIKK and DNIKK mice were
partially overcome. There was comparable DNA-binding activity to a
constitutively active transcription factor, NF-Y, in these extracts
(Fig. 3, A and B). Next, a variety of Abs
directed against multiple NF-B subunits was used in a supershift
assay to analyze the components of NF-B complex induced by
anti-CD3 alone or in combination with anti-CD28 (Fig. 3C). This analysis indicated that under both conditions, the
activated NF-B DNA-binding complex in wild-type and DNIKK (data not
shown) thymocytes contained the p65 and p50 NF-B subunits (Fig. 3C).
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B activation
was inhibited in thymocytes from all three lines of transgenic mice,
whereas anti-CD3-induced NF-B activation was blocked
predominantly in thymocytes isolated from DNIKK and DNIKK
transgenic mice, but only to a lesser degree in thymocytes from
DNIKK mice, suggesting that IKK plays a more important role in
anti-CD3-mediated NF-B activation. More importantly,
anti-CD28 costimulation partially overcame the inhibition of
NF-B activation mediated by TCR signaling. Normal T cell development and T-dependent Ab response in DNIKK transgenic mice
Because both DN forms of IKK and IKK inhibited NF-B
activation in the thymocytes isolated from the transgenic mice, we next
asked whether the expression of these DN kinases altered T cell
development. The thymus and spleen isolated from the DNIKK,
DNIKK, and DNIKK mice were of normal size and structure, as
determined by pathological examination with H&E staining (data not
shown). Thymocytes and splenocytes (data not shown) isolated from these
transgenic mice have normal surface expression of CD4 and CD8 (Fig. 4A). These results suggest
that the overall T cell development in the DNIKK transgenic mice is not
altered by the expression of DNIKK or DNIKK. Moreover, these
mutant mice appeared to have normal IgM, IgG1, and IgG2a responses to
the T-dependent Ag TNP-KLH (Fig. 4B), indicating that T
cells from these transgenic mice are capable of providing cognate help
to B cells during specific Ab response. Thus, the overall T cell
development and function appear to be normal.
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is the dominant kinase regulating the proliferation of
thymic T cells in response to anti-CD3 treatment
NF-B is important in mediating TCR signaling (50, 51). Activation of T cells by TCR engagement results in their
proliferation and cytokine production. First, we addressed whether the
expression of DNIKK and DNIKK altered the proliferation of T
cells in response to anti-CD3 stimulation. Thymocytes and
splenocytes from wild-type and transgenic mice were stimulated with
either immobilized anti-CD3 Ab alone or in combination with
anti-CD28 or IL-2. As shown by the amount of
[3H]thymidine incorporation, the proliferative
response of thymocytes from DNIKK and DNIKK mice in response
to anti-CD3 alone was significantly reduced compared with that seen
with thymocytes isolated from DNIKK and wild-type mice (Fig. 5A). However, this defect
could be substantially rescued by the addition of IL-2 or
anti-CD28, which provide costimulatory signals (Fig. 5A). These results indicated that IKK was more important
than was IKK in regulating the T cell proliferative response induced
by TCR cross-linking, and that costimulatory signals such as
anti-CD28 or IL-2 could overcome this defect.
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thymocytes
To investigate the mechanisms involved in the proliferative
defects seen in T cells isolated from transgenic mice containing
DNIKK, the cell cycle progression of thymocytes stimulated with
anti-CD3 was analyzed by BrdU labeling, followed by staining with
anti-BrdU and 7-AAD. Flow cytometry analysis was then performed to
determine the incorporation of BrdU and the total DNA content (Fig. 6). Only cells in the S phase incorporate
significant amounts of BrdU during the 30-min labeling period, while
cells in the G0/G1 and
G2/M phase can be distinguished by differences in
their DNA content.
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, 
20% of the thymocytes from wild-type and
DNIKK mice were in the S phase after anti-CD3 treatment.
However, only 5–10% of thymocytes isolated from DNIKK and
DNIKK mice were in S phase. The decrease in the percentage of the
S phase cells of DNIKK and DNIKK thymocytes was associated
with a corresponding increase in the percentage of cells in the
G0/G1 phase of the cell
cycle. These results indicated that the progression from the
G0/G1 phase to the S phase
in the thymocytes from DNIKK and DNIKK mice was reduced. In
contrast, the expression of DNIKK did not significantly alter cell
cycle progression of thymocytes. The defect in cell cycle progression
seen with the DNIKK and DNIKK thymocytes could be rescued by
treatment with IL-2, as reflected in the similar percentages of S phase
cells seen in wild-type and transgenic thymocytes. These results are
consistent with the reduced NF-B DNA-binding activity and the
reduced [3H]thymidine incorporation in
thymocytes isolated from mice expressing DNIKK following stimulation
with anti-CD3. Differential cytokine expression in thymocytes isolated from DNIKK transgenic mice
To determine whether the cytokine production by T cells is
affected by inhibition of IKK or IKK, we analyzed the cytokine
profile of both untreated and anti-CD3/anti-CD28-stimulated
thymocytes isolated from wild-type and DNIKK transgenic mice (Fig. 7). The mRNA levels of multiple cytokines
were analyzed using RNase protection assays performed with a mouse
cytokine multiprobe template set. Similar amounts of RNA were analyzed
from each of these mice, as demonstrated by the detection of similar
levels of the housekeeping genes L32 and
GAPDH (Fig. 7A). Thymocytes isolated from
DNIKK and DNIKK transgenic mice produced relatively comparable
levels of multiple cytokines, including IL-4, IL-5, IL-10, IL-13, IL-2,
IL-3, and IFN-, when compared with thymocytes isolated from
wild-type mice. However, there were some differences in the levels of
IL-4, IL-5, IL-13, and IFN- noted in the DNIKK and DNIKK
transgenic mice. In contrast, thymocytes isolated from DNIKK
transgenic mice produced markedly reduced levels of the analyzed
cytokines (Fig. 7, A and B), indicating
that inhibition of both IKK and IKK can block the production of
multiple cytokines in thymocytes.
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and DNIKK on
anti-CD3-induced apoptosis in double-positive T cells
CD4+CD8+ thymocytes
undergo apoptotic cell death when activated through the TCR complex by
systemic administration of anti-CD3 Ab, a phenomenon that is
probably related to autoAg-induced negative selection during T cell
maturation (52, 53, 54, 55, 56, 57). Previously, it was noted that the
CD4+CD8+ thymocytes
isolated from the transgenic mice that expressed a DNI-B protein
in T cells were protected from anti-CD3-induced apoptosis
(37). Because the DNI-B protein inhibited nuclear
translocation of NF-B proteins, these results suggested that
activation of the NF-B pathway leads to proapoptotic effects in
immature thymocytes stimulated with anti-CD3.
Thus, we addressed whether inhibition of IKK and/or IKK altered
the survival of CD4+CD8+ T
cells following the in vivo administration of anti-CD3 (Fig. 8). In both wild-type and transgenic
mice, 50 µg anti-CD3 depleted more thymocytes than 25 µg
anti-CD3, indicating a dose-dependent effect of anti-CD3 Ab on
thymocyte depletion (Fig. 8, A and B). A decrease
in the size (data not shown) of the thymus and fewer numbers of total
thymocytes were noted in mice following anti-CD3 treatment (Fig. 8B). This depletion of thymocytes was associated with a
selective decrease in the number of the double-positive, but not
single-positive or double-negative, thymocytes (Fig. 8B).
Flow cytometry analysis indicated that the double-positive thymocytes
from DNIKK transgenic mice were significantly protected against
anti-CD3-induced apoptosis (Fig. 8A). This result is
similar to that obtained with DNI-B transgenic mice
(37). In contrast, thymocytes isolated from the DNIKK
transgenic mice had the opposite phenotype with increased
susceptibility to apoptosis as compared with thymocytes isolated from
either wild-type or DNIKK mice (Fig. 8A). Finally, there
was increased protection against anti-CD3-induced apoptosis in
thymocytes expressing both DNIKK and DNIKK as compared with
thymocytes obtained from wild-type mice (Fig. 8A). Analysis
of thymic tissue from these mice using an in situ TUNEL assay
demonstrated that anti-CD3 administration induced massive apoptosis
in thymus isolated from normal and DNIKK mice (Fig. 8C),
while the number of apoptotic cells in the thymus isolated from
DNIKK and DNIKK mice was significantly reduced (Fig. 8C). These results suggest that IKK has an antiapoptotic
effect on anti-CD3-activated double-positive T cells, while IKK
has a proapoptotic effect on these cells. In thymocytes containing both
DNIKK and DNIKK, the proapoptotic effect of IKK was dominant
over the antiapoptotic effect of IKK.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IKK in the ontogeny and regulation of T cell function. Because
disruption of IKK (31, 32) and
IKK (29, 33, 34) genes results in early
lethality of mice, we studied the effects of DN mutants of IKK and
IKK on both the development and function of T lymphocytes. Because
the DN mutants were expressed at higher levels than the endogenous
kinases, the endogenous kinases were most likely substantially
inhibited and the function of these kinases in T cells could be
addressed.
NF-B DNA-binding activity was substantially inhibited in thymocytes
from both DNIKK and DNIKK mice following treatment with PMA and
ionomycin. There was even greater inhibition noted in thymocytes from
DNIKK double-mutant mice. Although IKK is not as critical as
IKK in the cytokine-mediated activation of NF-B pathway
(29, 31, 32), there is significant evidence demonstrating
that IKK contributes to the activation of the conventional NF-B
pathway (48, 58, 59, 60). Our results suggest that IKK is
involved in the activation of NF-B pathway in T cells following
treatment with PMA and ionomycin. However, IKK appeared to be
dispensable for the NF-B activation induced by anti-CD3
treatment in thymocytes. Consistent with previous results demonstrating
that PKC-
mediates NF-B activation upon TCR engagement (61, 62) by activating IKK (63), our data suggest
that IKK is the dominant kinase mediating TCR-induced NF-B
activation.
The impaired proliferative response induced by anti-CD3-mediated
TCR cross-linking in thymocytes isolated from DNIKK and DNIKK
transgenic mice suggests that IKK is the dominant kinase involved in
this process. This defect was associated with marked alterations in
cell cycle progression. The relatively normal cell cycle progression
and proliferative response induced by anti-CD3 in thymocytes from
DNIKK mice may be attributed to their relatively normal NF-B
activation. Unlike the data from mice expressing a DNI-B molecule
(38), IL-2 was able to largely correct the proliferative
defects of thymocytes from DNIKK and DNIKK transgenic mice in
response to anti-CD3 treatment. Furthermore, these results suggest
that the IL-2 signaling pathway in these cells is intact.
Our results suggest that IKK and IKK are critical for the
production of cytokines in thymocytes. Inhibition of both IKK and
IKK in thymocytes from DNIKK transgenic mice reduced the
expression of both Th1 and Th2 cytokines following stimulation with
anti-CD3 and anti-CD28. However, thymocytes isolated from
DNIKK or DNIKK transgenic mice expressed significant levels of
these cytokines. These results suggest that IKK and IKK most
likely provide redundant functions required for production of Th1 and
Th2 cytokines. Interestingly, thymocytes from DNIKK mice expressed
somewhat increased levels of mRNA encoding the Th1 cytokine IFN-,
while DNIKK thymocytes tended to express somewhat higher levels of
IL-4, IL-5, and IL-13 as compared with thymocytes obtained from
wild-type mice. Thus, our data demonstrate that subtle changes in the
cytokine profiles of DNIKK and DNIKK thymocytes most likely
reflect distinct effects of IKK and IKK on NF-B induction of
Th1 and Th2 cytokines. It was surprising that the Ab response to the
T-dependent Ag TNP-KLH in DNIKK transgenic mice appeared normal,
despite the cytokine defects noted in thymocytes from these mice. This
may reflect the possibility that peripheral T cells are not defective
in cytokine production during T-dependent Ab response and/or that
additional signals provided by other types of cells in vivo, as
compared with the response seen in vitro, can rescue the function of T
cells lacking normal IKK and IKK.
Anti-CD28 costimulation has been shown to result in enhanced NF-B
activation in T cells (64, 65, 66, 67). Our results demonstrated
that anti-CD28 costimulation could significantly correct the
defects in NF-B activation induced by anti-CD3 in thymocytes
expressing DNIKK. Consistent with this result, the proliferative
defects in these thymocytes could also be largely rescued in the
presence of anti-CD28. However, anti-CD28 costimulation was not
able to rescue the cytokine defects in thymocytes from DNIKK
mice. These results indicate that either the overall amount and/or the
duration of NF-B activation may be critical in mediating certain T
cell responses.
NF-B has been demonstrated to have both antiapoptotic
(38) and proapoptotic (37, 41) effects on the
survival of lymphocytes. Hettmann et al. (37) demonstrated
that CD4+CD8+ thymocytes
isolated from transgenic mice expressing a DNI-B mutant were more
resistant to anti-CD3-induced apoptosis than were wild-type cells.
This protective effect of blocking the NF-B pathway in
double-positive thymocytes correlated with the high levels of the
antiapoptotic protein Bcl-xL (68).
In agreement with these results, we found that there was reduced
apoptosis in double-positive thymocytes isolated from the DNIKK mice
following systematic administration of anti-CD3. However,
peripheral CD4+ and CD8+
single-positive T cells isolated from transgenic mice expressing
DNI-B exhibited increased apoptosis upon anti-TCR stimulation
(38). Thus, NF-B is likely to regulate the expression
of distinct proapoptotic and antiapoptotic genes in cells at different
developmental stages and in response to different stimuli.
Although IKK-mediated NF-B activation may prevent apoptosis
induced by TNF- treatment during the physiological development of
lymphocytes and hepatocytes (33, 34, 35), our results suggest
that it may promote apoptosis induced by TCR activation in
double-positive thymocytes. In contrast to the results with IKK,
IKK has an antiapoptotic role in double-positive thymocytes
following treatment with -CD3. IKK has also been demonstrated to
prevent apoptosis in B cells (39, 40). Although the
mechanism by which IKK prevents apoptosis remains to be elucidated,
it is possible that it mediates these effects through p52, which has
been demonstrated to be important in preventing apoptosis of T cells
(69). Our results indicate that the proapoptotic effects
of DNIKK are dominant over the antiapoptotic effects of
DNIKK, as reflected by the decreased amounts of apoptosis seen in
double-positive T cells isolated from DNIKK transgenic
mice.
In this study, transgenic mice in which IKK and IKK function is
partially inhibited have allowed us to gain additional insights into
the distinct physiological roles of these kinases in the development
and function of T cells. Normal levels of IKK and IKK activity
are not required for the development of T lymphocytes. However, IKK,
but not IKK, is critical in mediating the proliferative response of
thymic T cells upon TCR stimulation, while both of these kinases are
critical in regulating cytokine production. Finally, IKK and IKK
have important and distinct roles in regulating apoptosis of immature
thymocytes. These studies will help to address the distinct roles of
IKK and IKK in regulating immune function.
|
Acknowledgments |
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and
CD2/DNIKK constructs, Noelle Williams for helpful discussions, and
Alex Herrera for assistance with the figures. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Richard B. Gaynor, Division of Hematology-Oncology, Department of Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8594. E-mail address: gaynor{at}utsw.swmed.edu
3 Abbreviations used in this paper: IKK, I-B kinase; 7-AAD, 7-amino actinomycin D; BrdU, 5-bromo-2'-deoxyuridine; DN, dominant-negative; HA, hemagglutinin; KLH, keyhole limpet hemocyanin; TNP, trinitrophenyl.
Received for publication October 11, 2001. Accepted for publication February 6, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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