(Received for publication, August 24, 1995; and in revised form, October 31, 1995)
From the
Interleukin-4 (IL-4) and IL-13 are functionally similar
cytokines. The functional IL-4 receptor (IL-4R) consists of the
IL-4R chain (IL-4R
) and the IL-2R
chain
(
), which is shared by the IL-2, IL-7, IL-9, and IL-15
receptors. The functional IL-13R is thought to involve the IL-4R
but not
. In this study, we have analyzed activation
of members of the Janus tyrosine kinase (Jak) family and signal
transducers and activators of transcription (STAT) 6 induced by IL-4
and IL-13 in Epstein-Barr virus-transformed B cells derived from two
patients of X-linked severe combined immunodeficiency, who have
mutations of the
gene in the extracellular and
intracellular domains. In these B cells, IL-4 failed to induce tyrosine
phosphorylation of Jak3 and activation of STAT6, or activation of these
molecules was significantly decreased compared with Epstein-Barr
virus-transformed normal B cells. In contrast, IL-13 activated STAT6 in
these cells as well as normal B cells. However, Jak3 was not activated
by IL-13, even in normal B cells. These results clearly indicated that
is essential for activation of Jak3 and STAT6 in the
signal transduction pathway of IL-4 in human B cells and that IL-13
does not utilize
but activates STAT6 through an
alternative pathway, which is not impaired in B cells of X-linked
severe combined immunodeficiency patients.
Both IL-4 ()and IL-13 are pleiotropic cytokines that
are produced by activated T cells and manifest similar functions on
hematopoietic cells such as induction of immunoglobulin class switching
to IgG4 and IgE, expression of CD23 and major histocompatibility
complex class II on human B cells, and anti-inflammatory effects on
monocytes(1) . These functions are mediated by binding of
cytokines to their specific receptors. The functional IL-4 receptor
(IL-4R) has been shown to consist of at least two components, the
IL-4R
chain (IL-4R
) and the IL-2R
chain (2, 3) . Although the IL-2R
was originally
identified as the third chain of the IL-2R(4) , it has been
shown that the IL-2R
is shared by IL-2, IL-4, IL-7, IL-9, and
IL-15 receptors, and thus the IL-2R
was termed the common
chain
(
)(2, 3, 5, 6, 7, 8) .
The initial study of functional IL-13 receptor (IL-13R) suggested that
IL-4R and IL-13R share a common component(9) , and
has been proposed to be a common component (2, 3) . However, recent studies have revealed that
may not be a component of functional IL-13, because
it has been demonstrated that
was not chemically
cross-linked with radiolabeled IL-13 and that the functional IL-13R was
expressed on
-negative
cells(10, 11, 12, 13) . These
reports have also indicated that
is not absolutely
required for functional IL-4R. In contrast, the reports that both IL-4
and IL-13 induce tyrosine phosphorylation of the IL-4R
and that
anti-IL-4R
antibody inhibits proliferation induced by IL-13 and
binding of IL-13 on TF-1 cells suggest that functional IL-13R shares
IL-4R
rather than
(11, 14, 15)
Recently, involvement of the Janus tyrosine kinase (Jak) family and
downstream molecules, signal transducers and activators of
transcription (STAT), in many cytokine signal transduction pathways has
been extensively investigated(16) . IL-4 has been shown to
activate members of the Jak family, Jak1 and
Jak3(17, 18) , which associate with the IL-4R
chain and
,
respectively(7, 19, 20) . In contrast, IL-13
does not induce tyrosine phosphorylation of Jak3(21) , which is
consistent with the idea that
is not utilized in
functional IL-13R. Recently, STAT induced by IL-4 (IL-4 STAT/STAT6),
which is able to bind to the consensus sequence in the promoter of
IL-4-inducible genes (22, 23, 24) , has been
molecularly cloned(25) . IL-13 has also been demonstrated to
activate a DNA binding nuclear factor similar to that activated by
IL-4(11, 26) .
X-linked severe combined
immunodeficiency (XSCID) is an inherited immunodeficiency disease
characterized by absent or markedly reduced numbers of T cells and B
cells that function abnormally, resulting in recurrent infections in
early life and failure to thrive without bone marrow
transplantation(27, 28) . Recently, it has been
demonstrated that XSCID results in mutation of the gene (29, 30, 31) . Various mutations
of the
gene have been reported, such as one point
mutation at the extracellular domain or the cytoplasmic domain or the
exon/intron junction and deletion mutations at the cytoplasmic domain,
the second exon, or the transmembrane domain(32) . Cells from
XSCID patients provide an excellent assay system for cytokine signal
transduction where
is involved. It has been
demonstrated that IL-4R and IL-13R are able to transduce signals in
XSCID B cells(12) . However, the mechanism by which this is
done remains unclear. Here, we analyze the Jak-STAT pathway activated
by IL-4 and IL-13 in Epstein-Barr Virus (EBV)-transformed B cells
derived from two XSCID patients in order to evaluate the role of
in the signal pathways of IL-4 and IL-13.
Immunofluorescence staining of cells proved that neither
patient's B cells showed an appreciable fluorescent intensity,
whereas EBV-transformed B cells derived from a normal donor gave a
significant intensity by using antibody against ,
TUGh4.
In order to investigate involvement of in
activation of Jak1 and Jak3 in the B cells of XSCID, we analyzed
tyrosine phosphorylation of Jak1 and Jak3 induced by IL-4 and IL-13 in
two patients' B cells. As shown in Fig. 1A,
tyrosine phosphorylation of Jak1 induced by IL-4 in normal B cells was
much less than that in TF-1 cells. This observation is consistent with
the previous report in which levels of tyrosine phosphorylation of Jak1
upon IL-4 stimulation were varied and generally weak among different
cell types(35, 36) . Thus, we focused on analyzing
tyrosine phosphorylation of Jak3 by IL-4 in the patients' B
cells. Jak3 was tyrosine-phosphorylated in normal B cells upon IL-4
stimulation, whereas in B cells from patient 1, no tyrosine
phosphorylation of Jak3 was detected upon IL-4 stimulation, and
tyrosine phosphorylation of Jak3 was remarkably decreased in B cells
from patient 2 compared with that in normal B cells (Fig. 1B). The loaded amounts of Jak3 were not
significantly different in each sample (Fig. 1C),
indicating that tyrosine phosphorylation of Jak3 in the patients'
B cells is impaired. We assume that, although expression of
on B cells from patient 2 was not detected, mutated
, which anti-
antibody used in
staining of B cells was not able to recognize, is expressed on the cell
surface and transduces a reduced signal from the ligand-engaged
receptors. These results suggest that activation of Jak3 by IL-4
requires expression of
on the cell surface. In
contrast to IL-4, IL-13 did not induce tyrosine phosphorylation of Jak3 (Fig. 2A), which is consistent with the previous
report(21) . This could not be explained by the low
concentration of IL-13, because the concentration of IL-13 used in this
experiment was enough to activate STAT6 and induce synthesis of IgE in
human peripheral blood mononuclear cells (data not shown). Thus, these
results indicate that Jak3 is not involved in the signal transduction
pathway of IL-13.
Figure 1:
Tyrosine
phosphorylation of Jak1 and Jak3 by IL-4. A,
immunoprecipitates (Immuno. Pt.) with anti-Jak1 antibody (UBI)
from TF-1 and EBV-transformed normal B cells (3 10
cells) unstimulated or stimulated by 10 ng/ml IL-4 for 5 min were
probed by anti-phosphotyrosine (
PY) antibody (Ab). B and C, immunoprecipitates with
anti-Jak3 antibody (Santa Cruz Biochemistry) from EBV-transformed
normal B cells and patients' B cells (1
10
cells) unstimulated or stimulated by 10 ng/ml IL-4 for 5 min were
probed by anti-phosphotyrosine antibody (B) or anti-Jak3
antibody (C). N, P1, and P2 denote normal,
patient 1, and patient 2, respectively.
Figure 2:
Comparison of tyrosine phosphorylation of
Jak3 by IL-4 and IL-13. Immunoprecipitates (Immuno. Pt.) with
anti-Jak3 antibody (Santa Cruz Biochemistry) from EBV-transformed
normal B cells (1 10
cells) unstimulated or
stimulated by 10 ng/ml IL-4 or by 10 ng/ml IL-13 for 5 min were probed
by anti-phosphotyrosine (
PY) antibody (Ab) (A) or anti-Jak3 antibody (B).
It has already been shown that IL-4-responsive
genes, such as I, Fc
RIIa, and Fc
RI, have the consensus
sequence TTCNNN(N)GAA at the promoter region and that certain DNA
binding factors that associate with this portion are activated by
IL-4(22, 23, 24) . Recently, a member of the
STAT family activated by IL-4, which binds to this region, was
molecularly cloned and denoted as IL-4 STAT (STAT6)(25) , and
IL-13 has been shown to activate a STAT-like protein, which appeared to
be identical to the IL-4-induced STAT. However, it has not been
confirmed as STAT6 by anti-STAT6 antibody(11, 26) . In
order to assess the role of
in activation of the
STAT-like protein induced by IL-4 and IL-13, which was predicted to be
STAT6, we analyzed activation of the STAT-like protein using anti-STAT6
antibody in the patients' B cells. Activation of the DNA binding
factor induced by IL-4 and IL-13 was detected in normal B cells, and
addition of anti-STAT6 antibody to the DNA-protein complexes induced by
IL-4 and IL-13 supershifted the DNA-protein complexes (Fig. 3A), indicating that the DNA binding factor
induced by IL-4 and IL-13 is STAT6. Activation of STAT6 by IL-13
appears to be less than that by IL-4, and an even higher concentration
of IL-13 did not augment activation of STAT6 (data not shown). These
results suggest that in human B cells, the mechanism for activation of
STAT6 by IL-4 is distinct from that by IL-13.
Figure 3:
Activation of STAT6 by IL-4 and IL-13 in
EBV-transformed normal B cells and XSCID patients' B cells.
Nuclear extracts from EBV-transformed normal B cells and XSCID
patients' B cells unstimulated or stimulated by 10 ng/ml IL-4 (A and B) or by 10 ng/ml IL-13 (A and C) for 30 min were incubated with P-labeled probe
from the I
promoter and analyzed in EMSA. Anti-STAT6 antibody (Ab) was added in the DNA-protein complex (A). N, P1, and P2 denote normal, patient 1, and
patient 2, respectively.
In B cells from
patient 1, activation of STAT6 by IL-4 was completely abolished,
whereas IL-4 induced significantly reduced activation of STAT6 in B
cells from patient 2, compared with normal B cells (Fig. 3B). Activation levels of STAT6 correlated well
with the intensities of tyrosine phosphorylation of Jak3, suggesting
that activation of Jak3 is required for activation of STAT6 in the IL-4
signal transduction pathway. In contrast to IL-4, IL-13 activated STAT6
in one patient's B cells to the same level as normal B cells, and
higher activation of STAT6 was observed in another patient's B
cells (Fig. 3C). Thus far, we don't know the
reason why activation of STAT6 by IL-13 is higher in patient 2 than in
normal B cells. These results suggest that the signal transduction
pathway arising from via Jak3 is essential for
IL-4-induced activation of STAT6 and that IL-13 utilizes an alternative
pathway to activate STAT6.
In this study, we demonstrated that activation of Jak3 and
STAT6 by IL-4 was impaired in EBV-transformed B cells established from
XSCID patients who have mutations in the gene encoding
, while activation of STAT6 induced by IL-13 was
intact without activation of Jak3 in the same cells. These results
clearly show that IL-13 does not require
for
activation of STAT6. Although both Jak1 and Jak3 are
tyrosine-phosphorylated by IL-4, recent reports have demonstrated that
the degree of tyrosine phosphorylation of Jak1 is much less than that
of Jak3 in TF-1 cells and that Jak1 is not tyrosine-phosphorylated by
IL-4 in human B cells(35, 36) . We also found that
tyrosine phosphorylation of Jak1 in EBV-transformed B cells was much
less than that in TF-1 cells. These results suggest that Jak1 plays
different roles in IL-4-mediated signal transduction in different cell
types.
Matthews et al.(12) have shown that B cells
derived from a -negative XSCID patient, which were
costimulated with IL-4 and soluble CD40 ligand, were able to generate
IgE synthesis to the same level as normal B cells. Furthermore,
germ-line
transcript is also found to be induced by IL-4 in the
patients' B cells used in this study. (
)However, our
results demonstrated that the existence of
is
essential for activation of STAT6 by IL-4 in EBV-transformed B cells.
These observations suggest that the Jak-STAT pathway is not necessary
for IL-4-induced immunoglobulin class switching. However, the study of
-deficient mice demonstrated that the sera from
-deficient mice do not contain detectable levels of
IgE(37) . Although the reason for these discrepancies is not
clear, it is possible that human B cells, but not mouse B cells, have
an alternative IL-4-mediated signal pathway for IgE synthesis, which
does not require activation of the known Jak-STAT pathway. In addition,
Lin et al.(11) have demonstrated that IL-4 activates
a STAT protein in COS7 cells expressing IL-4R
without expression
of
, providing evidence that
is not
essential for activation of STAT6 by IL-4. Taken together, these
findings raise the possibility that there are three distinct signal
transductin pathways of IL-4. The first one is the Jak-STAT pathway,
which is transduced through IL-4R
and
, as seen
in normal human B cells. The second one is the Jak-STAT pathway, which
does not require
, as seen in COS7 cells. The third
one is the signal pathway, which induces IgE synthesis without
activating the known Jak-STAT pathway, as seen in human XSCID B cells.
The third signal pathway seems to be defective in mouse B cells.
Molecular cloning of a IL-13 receptor will clarify this possibility.
Hou et al.(25) have suggested that phosphorylated
tyrosine residues of IL-4R at 578 and 606 amino acids may be
important for activation and dimerization of STAT6. Recently, Quelle et al.(38) reported that activation of STAT6 by IL-4
in a mouse myeloid cell line 32D requires the distal portion of the
intracellular domain of IL-4R
. In our studies, we have
demonstrated that the portion in the intracellular domain of IL-4R
between amino acids 353 and 393, numbering from the methionine start of
the signal peptide, is essential for the synthesis of germ-line
transcript induced by IL-4 (39) , which is also essential for
IL-4-mediated mitogenic signal(40) . These findings also
suggest that the Jak-STAT pathway in IL-4-mediated signal transduction
is not essential for induction of IgE synthesis and proliferation.
Further investigation is awaited to establish the physiological role of
the Jak-STAT pathway in the signal transduction of IL-4 and IL-13.