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Departments of
*
Medical Microbiology and
Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates; and
Section of Immunobiology and
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B transcription factors. Proximal steps in the
IL-1R pathway, however, remain poorly understood, and there is
conflicting evidence as to the importance of tyrosine phosphorylation
in IL-1R signaling. We have addressed this issue by examining the
ability of IL-1 to costimulate the activation of Lck-deficient Th2
cells. Our data demonstrate that, in the absence of Lck, the IL-1
costimulatory pathway is blocked despite the expression of normal
levels of IL-1RI. Moreover, the block is associated with a defective
degradation of IB-
and an incomplete activation of NF-B
heterodimeric complexes. Protein expression of NF-B monomers,
including p50, p65, and c-Rel, is equivalent in both wild-type and
Lck-deficient Th2 cell clones. Finally, we demonstrate that, in normal
Th2 cells, stimulation with IL-1 leads to a rapid induction in tyrosine
phosphorylation of several substrates including Lck itself. These
findings strongly suggest that Lck is required for signaling in the
IL-1 costimulatory pathway in Th2 lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by Th2 cells, which in turn act in an autocrine fashion to
facilitate their proliferation in response to IL-4. An alternative
mechanism was recently proposed whereby costimulation of Th2 cells via
TCR and IL-1 lead to the production of endogenous IL-1, which in
turn is used by Th2 cells as an autocrine growth factor in an
IL-4-independent fashion (6).
Major advances in the identification of the components of the IL-1R
signaling pathway have been made in the last few years (for reviews,
see Refs. 7, 8, 9, 10). The main effectors of this pathway are a
group of proteins that belong to the NF-B family of transcription
factors. In resting cells, NF-B is inactive and sequestered in the
cytoplasm as a heterodimer comprising two polypeptides of 50 kDa (p50)
and 65 kDa (p65), which are noncovalently associated with the
inhibitory IB proteins, including IB-. Triggering of the IL-1R
leads to the phosphorylation of IB- on two specific serine
residues, an event that signals its ubiqutination and degradation. The
released NF-B protein is then able to translocate to the nucleus and
bind to specific DNA regions within the promoter of many genes involved
in immune and inflammatory responses.
The serine-threonine kinase capable of phosphorylating IB- was
cloned and designated IB kinase
(IKK).4 This kinase comprises a large molecular
mass complex of 
700 kDa, the components of which are not all
yet identified (11, 12, 13). To date, the complex has been
shown to contain at least three subunits: IKK-, IKK-
, and IKK-
(14, 15, 16, 17). IKK- and IKK- act as the catalytic
subunits of the serine-threonine kinase, whereas IKK- is thought to
function as a regulatory subunit. Little is known about the way the
activity of the three subunits, or indeed of the larger complex as a
whole, is regulated. One aspect that has remained controversial is the
potential role of tyrosine phosphorylation in the activation of any of
the components of the IL-1R pathway. A previous study showed that
several substrates were tyrosine phosphorylated following IL-1R
triggering (18). Additionally, another study showed that
tyrosine phosphorylation of IB- can lead to NF-B activation
(19). Given that many of the components of the large
kinase complex are yet to be identified, it is possible that a tyrosine
kinase may be part of this complex. Alternatively, tyrosine
phosphorylation may be required for the activation of one or more
components of the kinase complex.
To directly assess the requirement for tyrosine phosphorylation in T
lymphocyte costimulatory pathways, we used stable T cell transfectants
of a Th2 clone in which Lck expression was inhibited by antisense RNA
(20, 21). The findings reported here demonstrate that in
normal Th2 cells, Lck is tyrosine phosphorylated in response to IL-1
stimulation. Consequently, Lck-deficient Th2 cells are unresponsive to
IL-1 stimulation. Moreover, this unresponsiveness appears to correlate
with the inability to degrade IB- and fully activate NF-B
heterodimeric complexes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The parental Th2 clone D10.G4.1 (hereafter referred to as D10)
expresses a TCR which specifically recognizes a peptide derived
from conalbumin in the context of the murine I-Ak
MHC class II molecule (22). Lck-deficient transfectants of
D10 were derived by transfecting a vector containing a 400-bp fragment
of murine Lck cloned in the antisense orientation. Expression of the
antisense RNA is under the control of SV40 promoter and human T cell
leukemia virus 1 long terminal repeat enhancer. One of the
transfectants, known as D8, which had been extensively characterized
previously (20), was used for the present studies (for the
sake of clarity, these cells will thereafter be referred to as
Lck-deficient T cells). Lck-deficient T cells were maintained by
biweekly stimulation with cognate Ag plus syngeneic feeder cells in the
presence of IL-1, IL-2, and IL-4 lymphokines. For all experiments,
parental as well as Lck-deficient T cells were used in the resting
phase at least 10–14 days after the last round of activation.
Recombinant human IL-1 was purchased from Genzyme (Cambridge, MA);
mouse rIL-2 and rIL-4 were obtained from BioSource International
(Camarillo, CA). The following mAbs were used: H57-597, anti-TCR
C (23); 11B11, anti-IL-4 (24); and
S4B6, anti-IL-2 (25). All mAb were affinity purified
from hybridoma culture supernatants on protein G-Sepharose (Pharmacia
Biotech, Piscataway, NJ).
Costimulation assay
To prepare Ab-coated plates, wells of flat-bottom microtiter
plates received 50 µl of H57-597 mAb diluted in PBS to a
concentration of 3 µg/ml. After incubation for 2 h at 22°C,
the plates were blocked for 30 min with 5% FCS-PBS and washed three
times before addition of T cells. Resting, viable, T cells were
isolated from maintenance cultures and activated at
104 cells/well in Ab-coated plates in the
presence or absence of graded concentrations of human rIL-1, as
indicated. In some assays, neutralizing Abs specific to IL-2 or IL-4
were also added to some cultures at a final concentration of 20
µg/ml. Cell proliferation was assessed after a 72 h-incubation period
following a pulse with 1 µCi of [3H]thymidine
for the last 18 h of culture.
Flow cytometry
The level of expression of lymphokine receptors for IL-1, IL-2,
and IL-4 was determined by flow cytometry using a three-step staining
procedure. Viable T cells (1 x 106
cells/sample) were stained with first-step mAbs on ice for 30 min. The
first-step mAbs used were PC61, rat IgG1 specific to IL-2R chain
(26), anti-CD121, rat IgG1 specific to mouse IL-1R
type I (BD PharMingen, San Diego, CA), or anti-CDw124, rat IgG2a
specific to mouse IL-4R (Genzyme). For positive control staining, cells
were stained with YCD3–1 mAb specific to CD3
(27). As
a negative control, TR310 mAb, rat IgG2b specific to TCR V7
(28) was also used (note that D10 cells are
V8.2+). For second-step staining,
biotin-conjugated goat anti-rat IgG Ab was used for 30 min on ice.
In the third step, cells were stained with PE-conjugated streptavidin
(Southern Biotechnology Associates, Birmingham, AL) also for 30 min on
ice. After extensive washing, cells were analyzed on a FACSort (BD
Biosciences, Mountain View, CA). Data collected from 15,000 cells were
analyzed using CellQuest software.
Immunoprecipitation and immunoblotting
T cells (2 x 106/group) were treated
with 5 ng/ml rIL-1 for various times ranging from 15 min to 3
h. Whole-cell extracts were prepared in 1% Nonidet P-40 lysis buffer,
as previously described (20, 21), and protein content was
determined by the Bradford assay (Bio-Rad, Richmond, CA). Equal protein
amounts (50 µg/lane) were then run on 10% SDS-PAGE; the blots were
probed with a mAb specific to IB- (catalogue no. sc-1643; Santa
Cruz Biotechnology, Santa Cruz, CA) and developed using SuperSignal
substrate (Pierce, Rockford, IL). In other experiments, whole-cell
extracts were immunoblotted with Abs specific to p50, p65, or c-Rel
protein (Santa Cruz Biotechnology) or with 4G10
(anti-phosphotyrosine) mAb (Upstate Biotechnology, Lake Placid, NY)
and developed similarly. Immunoprecipitations were performed
essentially as previously described (20). Briefly, cell
lysates (prepared from 5 to 7 x 106
cells/group) were incubated for 2 h at 4°C with a mAb specific
to Src homology 3 domain of Lck (provided by Dr. A. Shaw, Washington
University, St. Louis, MO), followed by an overnight incubation with
protein A-Sepharose (Sigma-Aldrich, St. Louis, MO). After extensive
washing, the beads were resuspended in reducing sample buffer and
boiled for 5 min. The immunoprecipitates were resolved on 12%
SDS-PAGE, transferred to nitrocellulose membranes, immunoblotted with
4G10 Ab, and processed as described above.
EMSA
T cells were activated with IL-1 for 15 min, and whole-cell
extracts were prepared from 5 to 10 x 106
cells/group, as described previously (29). Clarified
extracts were incubated with 32P-labeled
double-stranded oligonucleotides comprising NF-B binding sites. The
Ig enhancer-derived sequence is 5'-AGTTGAGGGGACTTTCCCAGGC; the
mutant Ig sequence is 5'-CAGAGCAGACTTTCCGAGAGGC-3'; the IL-2
promoter-derived sequence is 5'-CCAAGAGGGATTTCACCTAAATCC-3'; and the
CD28 promoter- derived sequence is 5'-AAAGAAATTCCAAAGAGT-3'. For Ab
supershift assays, 1 µl of the indicated antisera was added to the
reaction before electrophoresis. Electrophoresis was conducted on
nondenaturing 5% polyacrylamide gels (acrylamide:bis = 39:1) in
0.45x Tris-borate-EDTA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-specific mAb, H57-597, in the
presence or absence of variable concentrations of rIL-1. As shown in
Fig. 1
A, IL-1 costimulated
a strong, dose-dependent proliferative response in wild-type D10 cells.
The fold increase in the proliferative response of T cells was 3.5x
and 9.3x in the presence of 20 and 200 pg/ml IL-1, respectively.
The addition of IL-1 alone to D10 cells failed to induce any
proliferation (Fig. 1A). The ability of IL-1 to
costimulate D10 cell proliferation was dependent on endogenously
produced IL-4 since the addition of anti-IL-4 mAb almost completely
abrogated IL-1 costimulatory activity. In sharp contrast, the
combination of immobilized anti-TCR mAb and rIL-1 failed to
induce any significant proliferation of Lck-deficient D10 cells (Fig. 1B). The response of both cell lines to stimulation by IL-2
was also evaluated. Normal D10 cells proliferated strongly when
stimulated with IL-2 alone, even when anti-IL-4 mAb was also
present in the culture (Fig. 1C). It is important to note
that the response was almost identical whether anti-IL-4 mAb was
present or absent (data not shown). Lck-deficient D10 cells also
proliferated in response to IL-2, but at a much reduced rate. When
stimulated with 1 or 10 U/ml IL-2, the response of Lck-deficient D10
cells represented only 52 or 28% of the response of normal D10 cells,
respectively (Fig. 1C). Moreover, increasing IL-2
concentrations (from 0.1 to 10 U/ml) led to a proportional increase in
the proliferative response of D10 cells. In contrast, the response of
Lck-deficient D10 cells reached a plateau at 1 U/ml, with no further
increase being observed upon stimulation with 10 U/ml IL-2 (Fig. 1C). A rather different picture emerged when the cell
proliferative response was evaluated upon stimulation with IL-4. In
this study, Lck-deficient cells consistently exhibited a stronger
proliferative response than normal D10 cells (Fig. 1D). In
this case, the response of wild-type D10 cells represented 24 or 48%
of Lck-deficient cells when stimulated with 20 or 200 U/ml IL-4,
respectively (Fig. 1D). In addition, Lck-deficient cells
appeared to have a lower threshold of activation than normal D10 cells,
as can be seen from their significant proliferative response when
stimulated with IL-4 at 2.0 U/ml. Taken together, these results suggest
that Lck-deficient cells are refractory to IL-1-mediated costimulation.
Furthermore, however, Lck-deficient cells are perfectly capable of
responding to lymphokine stimulatory signals, in particular IL-4,
demonstrating that these cells do not have a generalized growth
defect.
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shows the results of staining normal
(A–D) and Lck-deficient (E–H) D10 cell lines
with mAbs specific to IL-1RI (B and F), IL-4R
(C and G), IL-2R (D and H),
or, as a control, CD3 (A and E). The results
demonstrate that all three lymphokine receptors are expressed on
Lck-deficient cells at either comparable (IL-1R), or increased (IL-2R
and IL-4R), levels compared with the parent D10 line. The higher
expression of IL-2R and IL-4R on Lck-deficient cells is most likely due
to the continuous culture of the cells in the presence of these two
lymphokines (20). Thus, the refractory response of
Lck-deficient cells to IL-1 is not a consequence of altered expression
of IL-1RI.
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B-, thereby allowing NF-B complexes to
translocate to the nucleus. To determine the integrity of the IL-1
pathway, normal as well as Lck-deficient T cells were activated with
IL-1 and whole-cell extracts were then analyzed for the expression of
IB-. As can be seen (Fig. 3),
significant levels of IB- were observed in resting cells (both
parental and Lck-deficient cells). After 30 min of IL-1 stimulation,
IB- was degraded in D10 cells to an almost undetectable level.
The IB- protein returned to normal levels by 60 min, presumably
due to de novo protein synthesis, as has been described elsewhere
(30). This pattern of IB- degradation in response to
appropriate stimuli is entirely consistent with previous studies
(31). Surprisingly, however, no evidence of IB-
degradation was observed in Lck-deficient T cells (Fig. 3). The lack of
IL-1-induced IB degradation is not due to a change in kinetics
since no alteration in IB- level was seen up 3.5 h following
IL-1 treatment in Lck-deficient T cells (Fig. 3 and data not
shown).
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B complexes.
Stimulation of D10 cells with IL-1 leads to the activation of two main
NF-B complexes (Fig. 4A,
complexes N1 and N2). Moreover, activation of these complexes appears
to occur with slightly different kinetics. Thus, whereas the
DNA-binding activity of the N2 complex is apparent at 30 min and
increases in strength after 2 h of IL-1 stimulation, the activity
of the N1 complex is maximal at 30 min of stimulation and is weaker at
2 h. Surprisingly, IL-1 treatment of Lck-deficient cells led to
the specific activation of the N1 complex only. No evidence for the
activation of the N2 complex was observed in Lck-deficient T cells
(Fig. 4, A and B). To determine the composition
of the NF-B complexes, EMSA was conducted in the presence of Abs
specific to p65 and p50 proteins (Fig. 4B). As shown,
anti-p65 Abs supershifted the N1 complex completely but had no
effect on the N2 complex. In contrast, addition of anti-p50 Ab
decreased the binding activity of both complexes and led to the
appearance of a new supershifted band (Fig. 4B). Based on
these studies, we conclude that the N1 complex corresponds to the
p65/p50 heterodimer, while the N2 complex contains p50/p50 homodimer.
Interestingly, however, the N2 complex was not supershifted completely
by the anti-p50 Ab, suggesting the presence of other NF-B dimers
in that complex. Attempts to supershift the complex with anti-c-Rel
Ab have so far been unsuccessful (data not shown).
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B
complexes appears to be independent of protein levels of the various
monomers. Western blotting of cell extracts using Abs specific to p50,
p65, or c-Rel demonstrated that both cell lines express equivalent
levels of proteins (Fig. 4
C; data not shown). To further
confirm the specificity of the observed DNA-binding complexes, an
oligonucleotide competition experiment was undertaken. The results
demonstrate that both N1 and N2 complexes can be competed specifically
by known NF-B-binding oligonucleotides (Fig. 4D,
lanes 3, 4, 10, and 11) but
not by unrelated (lanes 8 and 9) or
mutated (lanes 5 and 6) oligonucleotides.
Interestingly, the fastest migrating band, which is prominent in
resting (Fig. 4D, lane 1) as well as
IL-1-stimulated (lanes 2 and 7) D10 cells,
can be competed by oligonucleotides containing a mutation in the
NF-B motif (lanes 5 and 6), suggesting
that this band is most likely in a different family of transcription
factors. Taken together, these data strongly indicate that Lck is
required for a full activation of IL-1-stimulated NF-B dimeric
complexes.
The above data demonstrate that IL-1 signaling is defective in
Lck-deficient Th2 cells. However, how and where in the pathway Lck is
involved remains unknown. To shed some light on this, wild-type D10
cells were stimulated with IL-1 and cell extracts were analyzed for any
evidence of IL-1-induced tyrosine phosphorylation. As shown in Fig. 5A, IL-1 triggering of Th2
cells led to a significant increase in the tyrosine phosphorylation of
at least four protein bands running at a molecular mass of 38, 56, 59,
and 116 kDa. The magnitude and kinetics of tyrosine phosphorylation of
these bands appear to vary significantly. The most prominent of these
bands was the 56-kDa protein which was of great interest to us since
this could potentially represent Lck. To further identify this protein,
cell extracts were prepared following IL-1 stimulation of D10 cells and
immunoprecipitated with a mAb to Lck. The precipitated protein was then
run on a 12% gel and blotted with anti-phosphotyrosine mAb. The
results show that Lck is indeed tyrosine phosphorylated in response to
stimulation by IL-1 (Fig. 5B, middle lane). No
Lck band was seen in untreated cells (Fig. 5B,
left lane) or in cells treated with a 10x lower
concentration of IL-1 (right lane). We conclude that
stimulation of cloned Th2 cells with IL-1 can induce the tyrosine
phosphorylation of several protein substrates, including Lck. Attempts
to determine the IL-1-induced tyrosine phosphorylation pattern in
Lck-deficient cells were unsuccessful. This is mainly due to the fact
that, in our hands, IL-1-induced phosphorylation could readily be
observed when T cells were rested by an overnight incubation in the
absence of any growth factors. Such treatment of Lck-deficient cells
routinely resulted in a significant degree of death due to their
heightened susceptibility to apoptosis, as previously reported
(21). Whether Lck is responsible for the observed tyrosine
phosphorylation, however, remains to be demonstrated. Whatever the
mechanism, our data support a role for Lck in the IL-1 signaling
pathway and demonstrate an IL-1-dependent increase in tyrosine
phosphorylation in treated Th2 cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B- and, consequently, inhibition of NF-B activation.
Moreover, our study demonstrates that Lck itself is tyrosine
phosphorylated in response to IL-1 stimulation in Th2 cells, strongly
indicating that Lck may be one of the components of the IL-1RI
signaling pathway.
NF-B is a master transcription factor with key involvement in the
control of cellular response to proinflammatory stimuli. The main
pathway through which NF-B is activated involves the induced
phosphorylation of two serine residues in the N-terminal region of
IB proteins. In the case of IB-, this event is catalyzed by
the serine/threonine IKK enzyme complex (11, 12, 13).
Alternatively, recent evidence had shown that NF-B can be activated
in response to hypoxia and reoxygenation in Jurkat T cells by the
phosphorylation of IB- on tyrosine 42 (19). This
modification, which has been shown to involve Lck and phosphoinositide
3-kinase, does not lead to ubiquitination and degradation of IB-
but rather induces the dissociation of IB- from NF-B, allowing
the latter to translocate into the nucleus (19, 32).
Interestingly, this pathway can also be readily activated by treatment
with pervanadate, a potent inhibitor of tyrosine phosphatases
(19). The present study demonstrates that degradation of
IB- in response to IL-1 stimulation is also inhibited in
Lck-deficient cells. The tyrosine phosphorylation status of IB-
following IL-1 treatment in our cell lines has not yet been examined.
It would also be interesting to test the response of our cell lines to
pervanadate treatment. Taken together, experimental evidence suggests
the involvement of Lck in both pathways of NF-B activation in T
cells.
The molecular mechanism for the role of Lck in the IL-1 signaling
pathway is not understood. We have shown that IB- is not properly
degraded in Lck-deficient cells following IL-1 stimulation. This points
to a dysfunction in the IKK kinase complex, influencing either the
activity of IKK itself or one of the hitherto uncharacterized
constituents of the large complex. Experimental evidence for the former
possibility was recently provided by You et al. (33) who
demonstrated that Shp-2, an Src homology 2-containing phosphotyrosine
phosphatase, is associated with the IKK complex. A functional Shp-2 was
shown to be required for the efficient serine phosphorylation of
IB- by the IKK complex in response to stimulation by
proinflammatory cytokines such as IL-1 and TNF-. Together with our
present findings, these results suggest that tyrosine
phosphorylation/dephosphorylation events are essential for proper
signaling through the IL-1RI complex.
Interestingly, the defect in Lck expression resulted in a differential
effect on the activation of the various NF-B complexes. The most
pronounced effect, observed in Lck-deficient cells, was on the
defective formation of N2 complexes following stimulation with IL-1.
The N2 complexes most likely consist of a mixture of p50/p50 homodimers
as well as other yet to be identified p50-containing heterodimers. In
contrast, the prototypic p50/p65 heterodimers could still be activated,
albeit at moderately decreased levels, in Lck-deficient cells. The fact
that this was observed in the absence of efficient IB-
degradation suggests that 1) a small degree of IB- degradation is
taking place, which accounts for the observed p50/p65 activity; or 2)
other IB inhibitor proteins may be selectively degraded in the
absence of Lck, allowing for the activation of p50/p65 heterodimers.
For example, a specific and nonoverlapping role has been previously
described for IB- in the activation of NF-B (31).
In that study, IB- was shown to be specifically activated by a
select group of inducers, including IL-1 and LPS, and resulted in
persistent activation of NF-B (31). In contrast, other
proinflammatory stimuli, such as TNF-, induced the degradation of
IB-, but not IB-, leading to the commonly observed
transient activation of NF-B dimers. Thus, it is reasonable to
propose that IL-1 could induce the observed p50/p65 dimers via the
degradation of IB-. Implicit in this argument is the suggestion
that the activities of IB- and IB- may be regulated by
slightly different mechanisms, insomuch as Lck is required for the
latter but not the former. At present, no data are available in support
of this contention. Previous studies demonstrated that IB-
regulated NF-B activity via an autoregulatory feedback loop
(reviewed in Ref. 34). NF-B stimulatory signals achieve
their effect through the induced degradation of IB-. The active
NF-B dimers can then up-regulate the expression of de novo IB-
mRNA due to the presence of NF-B sites in the IB- promoter.
Replenishment of the IB- pool prevents further translocation of
NF-B dimers, effectively shutting down the NF-B response and
ensuring that the responsive genes are activated only transiently. This
negative feedback loop is not operative in the control of IB-
activation since IB- is unique in being the only species of
IBs whose expression is not regulated by NF-B. Indeed, the
importance of IB- activation was highlighted by studies that
demonstrated that activation of T cells via the CD28 costimulatory
molecule led to a rapid, and selective, degradation of IB- and
persistent activation of NF-B (35).
The present data provide strong direct evidence as to the importance of
Lck in the IL-2R signaling pathway. Lck-deficient T cells exhibited a
profoundly lower capacity to proliferate in response to IL-2. This was
seen despite a demonstrated 5-fold increase in IL-2R expression on
Lck-deficient cells when compared with parental D10 cells. The
reduction in IL-2-induced cellular proliferation of Lck-deficient cells
is most likely a consequence of the noncovalent association of Lck with
the -chain of the IL-2R (36, 37). Furthermore, the role
of tyrosine kinases, such as Lck, in the phosphorylation and activation
of Janus kinase 1 and Janus kinase 3 kinases, integral components of
the IL-2 signaling pathway, is incompletely understood
(38). In sharp contrast, IL-4 signaling is unaffected in
Lck-deficient D10 cells, demonstrating that the observed effect is
specific to the IL-2R pathway. In fact, IL-4-induced proliferation was
significantly stronger in Lck-deficient in comparison to normal cells,
presumably a consequence of increased surface expression of IL-4R on
the former cells. It is interesting to note that, unlike IL-2R, the
IL-4R complex consists of only two chains (- and common -chain)
and lacks a -chain. Furthermore, no association between any of the T
cell-expressed Src-tyrosine kinases and the IL-4R complex has been
reported (39). Given that Lck deficiency led to a partial
reduction in IL-2-induced cellular proliferation, our findings indicate
that the association between the IL-2R -chain and Lck is of
physiological importance and plays a complementary role in
IL-2R-mediated signaling.
In conclusion, our findings demonstrate that a deficiency in the
expression of the Src-protein tyrosine kinase Lck in Th2 cells has a
profound effect on the IL-1 signaling pathway. The delivery of IL-1
costimulatory signal to Lck-deficient Th2 cells was completely blocked.
This was associated with (1) lack of IB- degradation
and (2) differential activation of NF-B complexes.
Moreover, evidence is presented that Lck itself is tyrosine
phosphorylated in response to IL-1 stimulation. Our data provide
evidence for an important role for Lck in the IL-1R signaling pathway
in T lymphocytes. Whether Lck kinase activity is required for this
pathway, or Lck is acting in a kinase-independent manner
(40), remains to determined. Finally, the data suggest
that activation of p50-p50 complexes is dependent on the specific
degradation of IB-, whereas p50-p65 complexes can be activated
independently of IB-, perhaps through the degradation of other
inhibitory proteins, such as IB- (31).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Basel K. al-Ramadi, Department of Medical Microbiology, Faculty of Medicine and Health Sciences, UAE University, P. O. Box 17666, Al Ain, United Arab Emirates. E-mail address: ramadi.b{at}uaeu.ac.ae
3 Current address: Blood Research Institute, Blood Center of South East Wisconsin, Milwaukee, WI 53201.
4 Abbreviation used in this paper: IKK, IB kinase.
Received for publication May 4, 2001. Accepted for publication October 12, 2001.
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S. Nakae, Y. Komiyama, H. Yokoyama, A. Nambu, M. Umeda, M. Iwase, I. Homma, K. Sudo, R. Horai, M. Asano, et al. IL-1 is required for allergen-specific Th2 cell activation and the development of airway hypersensitivity response Int. Immunol., April 1, 2003; 15(4): 483 - 490. [Abstract] [Full Text] [PDF]
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). Parallel sets of cultures
were set up containing 20 µg/ml of a neutralizing anti-IL-4 mAb
(
). The dose-response curves of cells stimulated with IL-1 only (in
the absence of anti-TCR mAb) are also shown (
). As controls,
parental and Lck-deficient D10 cells were stimulated with the indicated
concentrations of IL-2 in the presence of a fixed concentration (20
µg/ml) of anti-IL-4 mAb (C) or with IL-4 alone
(D). All data points represent the means ± SEM.
The results are representative of at least five independent
experiments.
