(Received for publication, October 29, 1996, and in revised form, December 17, 1996)
From the Interleukin-4 (IL-4) is an important cytokine for
B and T lymphocyte function and mediates its effects via a receptor
that contains Patients with X-linked severe combined immunodeficiency
(X-SCID)1 present with very few T cells and
absent mitogenic responses (1). Although B cells are present,
immunoglobulin levels are low, and specific antibody production is
lacking. The combination of these defects is fatal by 1-2 years of age
unless the immune system is reconstituted by allogeneic bone marrow
transplantation. These clinical manifestations are due to a wide range
of mutations in the common gamma chain ( IL-4 is thought to be important for mature B cell functions including
immunoglobulin class switching to IgG4 and IgE as well as expression of
CD23 and major histocompatibility complex class II genes (10). Because
IL-4 regulates B lymphocyte function, it is important to determine the
response of X-SCID B cells to this cytokine. The functional IL-4
receptor (IL-4R) consists of at least two components, IL-4R It might be predicted that in the absence of a functional
It was therefore important to analyze IL-4-signaling in a panel of
B-LCL derived from X-SCID patients with a wide range of Recombinant human IL-2 and IL-13
were the generous gifts of C. Paradise (Chiron Corp., Emeryville, CA)
and C. Reynolds (BRMP, Frederick, MD), respectively. Polyclonal rabbit
antisera against STAT6 was kindly provided by R. LaRochelle (National
Cancer Institute, Bethesda) or was obtained from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA). Anti-JAK1 antibodies were
purchased from Transduction Laboratories (Lexington, KY) and Santa Cruz
Biotechnology Inc. Anti-IRS-1 antibodies were obtained from J. Pierce
(National Institutes of Health, Bethesda). The 4G10 monoclonal
anti-phosphotyrosine antibody was purchased from Upstate Biotechnology
Inc. (Lake Placid, NY), and the pY72 anti-phosphotyrosine was the gift
of B. Sefton (Salk Institute, La Jolla, CA).
Male patients with SCID were initially diagnosed
with the X-linked form based on maternal X chromosome inactivation
patterns (3). Peripheral blood mononuclear cells from X-SCID patients and normal donors were obtained upon informed consent, and B cell lymphoblastoid cell lines (B-LCL) were established by Epstein-Barr virus immortalization. The mutations in the Cells
were stimulated essentially as described previously (15). Briefly,
cells were resuspended at 1 × 107 cells/ml and
incubated with either 103 units/ml IL-2, 100 ng/ml IL-4, or
250 ng/ml IL-13. After 10 min, cells were lysed in a 1% Nonidet P-40
lysis buffer. Supernatants were immunoprecipitated with the specified
antibody at 4 °C, and immunoprecipitates were collected on protein
A/G agarose (Santa Cruz Biotechnology Inc.) and separated on 7.5%
SDS-polyacrylamide gels. Membranes were blotted with the 4G10 and pY72
anti-phosphotyrosine mABs as described previously (31). Blots probed
with polyclonal JAK1, JAK3, STAT6, and IRS-1 antibodies were blocked in
TBS (150 mM NaCl, 20 mM Tris (pH 7.5))
containing 5% bovine serum albumin and 0.1% Tween 20. Blots were
incubated sequentially with the primary antibody and horseradish
peroxidase-conjugated goat anti-rabbit IgG (Amersham Corp.) and
visualized using the enhanced chemiluminescence detection system
(Amersham Corp.). For reblotting, filters were stripped as reported
(31).
B-LCLs were cultured
without serum for 4 h prior to a 15-min stimulation with IL-4 or
IL-13 (1000 units/ml). Cells were lysed in a buffer containing 0.5%
Nonidet P-40, 50 mM Tris-HCl (pH 8.0), 10% glycerol,
100 mM EDTA (pH 8.0), 50 mM NaF, 150 mM NaCl, 100 mM Na3VO4,
1 mM dithiothreitol, 400 mM
phenylmethylsulfonyl fluoride, and 1 mg/ml leupeptin and aprotinin.
Whole cell extracts were prepared by centrifugation, and
electrophoretic mobility shift assays were performed essentially as
reported (32), using a 32P random prime-labeled
double-stranded oligonucleotide corresponding to the GAS-like element
present in the CD23 promoter (5 For flow
cytometric analysis of surface markers, serum was removed from the
cells, and cells were cultured in RPMI 1640 with or without IL-4 for
48 h at a density of 5 × 105 per well in a
6-well plate. Cells were then incubated for 30 min at 4 °C in the
presence of 25 ng of phycoerythrin-conjugated-EBVCS-5 (anti-human CD23,
IgG1) or an isotype control. CD23 surface staining was measured using a
FACscan flow cytometer, and the data were analyzed with Cellquest
software (Becton-Dickinson, San Jose, CA). The mean fluorescence
intensities were calculated by deducting the corresponding isotype
control.
In order to investigate whether there
is any JAK3 associates with the shared
JAK1 constitutively associates with the IL-2R The large cytosolic docking molecule IRS-1 is also
tyrosine-phosphorylated in response to IL-4 and has been hypothesized
to be important for an IL-4-mediated proliferative response (35, 36).
We therefore examined the effects of IL-4 on IRS-1 phosphorylation in
X-SCID B cells. Following immunoprecipitation with specific antibodies,
we detected IL-4-induced tyrosine phosphorylation of IRS-1 in both
B-LCL from a normal donor and a patient with X-SCID (Fig.
1C). Thus, Stimulation of many cell types with IL-4 leads to
the phosphorylation of STAT6 (37). As JAK3 was not phosphorylated in
X-SCID B cells, it was important to determine whether IL-4-mediated
STAT6 phosphorylation would also be affected by the loss of
We also found that IL-4 stimulated DNA binding activity in control and
X-SCID B-LCL as assessed by an electrophoretic mobility shift assay
(Fig. 2B). Since IL-4 is known to induce the expression of
CD23 in B cells (41), we assessed DNA binding activity with a probe
corresponding to the interferon- In order to determine the role of
Our data indicate that JAK1,
STAT6, and IRS-1 can be phosphorylated by IL-4 in a
Stimulation of B cells with IL-4 results in proliferation,
immunoglobulin class switching, and regulation of cell surface proteins
such as major histocompatibility complex class II molecules and the
CD23 receptor. Previous reports have concluded that the JAK-STAT
pathway is not activated by IL-4 in the absence of The finding that JAK3 was not phosphorylated in X-SCID B-LCL stimulated
with either IL-2 or IL-4 was not surprising as a number of recent
studies have demonstrated a physical association between Matthews et al. (27) showed that functional responses such
as proliferation, IgE secretion, and CD23 expression could occur in
X-SCID B cells when co-stimulated in vitro by IL-4 or IL-13 together with CD40L or IgM. However, in our studies, we did not detect
an increase in CD23 expression in X-SCID B-LCL even though CD23
expression did increase in normal and Although the precise mechanism by which this
In contrast to other forms of SCID that present with a complete absence
of both T and B lymphocytes, X-SCID patients have normal to elevated
numbers of nonfunctional B cells. However, it is clear that the IL-4
signaling that we have detected is not sufficient for normal B cell
function. The findings presented here provide insight into IL-4 signal
transduction pathways and B cell function in X-SCID patients. The
physiological role of the described We are grateful to Drs. R. Callard, C. Rooney, W. Leonard, J. DiSanto, G. de Saint Basile, and A. Fischer for
their valuable suggestions. We thank M. Sitbon for his support and
critical comments on the manuscript. We appreciate the assistance of M. Dao.
Division of Research Immunology and Bone
Marrow Transplantation, Childrens Hospital Los Angeles,
Los Angeles, California 90027, § Institut de
Génétique Moléculaire de Montpellier,
34033 Montpellier, France,
Clinical Gene Therapy Branch,
Lymphocyte Cell Biology Section,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
c. B cells derived from patients with
X-linked severe combined immunodeficiency (X-SCID) are deficient in
c and provide a useful model in which to dissect the
role of this subunit in IL-4-mediated signaling. We found that although
IL-4 stimulation of X-SCID B cells did not result in Janus tyrosine
kinase-3 (JAK3) phosphorylation, other IL-4 substrates including JAK1
and IRS-1 were phosphorylated. Additionally, we detected signal
transducers and activators of transcription 6 (STAT6) tyrosine
phosphorylation and DNA binding activity in X-SCID B cells with a wide
range of
c mutations. However, reconstitution of these
X-SCID B cells with
c enhanced IL-4-mediated responses
including STAT6 phosphorylation and DNA binding activity and resulted
in increased CD23 expression. Thus,
c is not necessary
to trigger IL-4-mediated responses in B cells, but its presence is
important for optimal IL-4-signaling. These results suggest that two
distinct IL-4 signaling pathways exist.
c) gene that
result in either a lack of
c message, unstable
c proteins that are poorly expressed, or defective
c receptor subunits that are expressed but nonfunctional (2-4).
c was originally identified as a component of
the IL-2 cytokine receptor (IL-2R
), but as it has been shown to be
shared by receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, it is now designated
c (5-9).
and
c subunits (6, 11). Signal transduction through the
IL-4R, as well as through other hematopoietic receptors, is initiated
by activation of Janus family tyrosine kinases (JAKs) (12, 13). IL-4
elicits tyrosine phosphorylation of the JAK family members JAK1 and
JAK3, which interact with the IL-4R
and
c subunits,
respectively (14-17). The current model of cytokine signaling proposes
that upon cytokine binding, members of the JAK family are rapidly
activated and subsequently tyrosine-phosphorylate the receptor, forming
a docking site for signal transducers and activators of transcription
(STATs) that are also phosphorylated by JAKs (12, 13). The STAT
proteins then dimerize and translocate to the nucleus where they bind
DNA sequences on target genes. One important STAT that is activated in
response to IL-4, STAT6 (IL-4 STAT), has been shown to bind to promoter
sequences of IL-4-inducible genes (18-21). As STAT6 knockout animals
parallel the IL-4 null phenotype and exhibit defects in Th2 helper T
cell differentiation and immunoglobulin class switching (22-24), STAT6
appears to be essential for many IL-4-mediated effects.
c chain, X-SCID B cells would not be able to respond to
IL-4. Indeed, two groups have reported that neither JAK1
phosphorylation nor STAT6 DNA binding activity is induced upon IL-4
stimulation of Epstein-Barr virus-transformed B cells (B-LCL) derived
from X-SCID patients (25, 26). However, Matthews and colleagues (27) have recently demonstrated that although X-SCID B cells cannot undergo
immunoglobulin class switching, they can proliferate in vitro in response to IL-4 when co-stimulated with CD40 ligand or
anti-IgM. Although the underlying biochemical mechanisms are not clear,
these data suggest that IL-4-mediated signaling in X-SCID B cells can
occur.
c mutations in order to clarify these discrepancies.
IL-4 failed to stimulate JAK3 tyrosine phosphorylation in X-SCID B
cells, but we found that JAK1 and IRS-1 were phosphorylated. IL-4 also induced STAT6 phosphorylation as well as DNA binding activity in these
cells. However, STAT6 activation and CD23 expression were significantly
enhanced in X-SCID B-LCL reconstituted with wild type
c.
Thus, we have demonstrated a
c-JAK3-independent pathway
through which IL-4 activates JAK1 and STAT6 in B cells derived from
X-SCID patients. These results have important implications for the
understanding of IL-4 signal transduction and the lack of mature B cell
function in patients with X-SCID.
Cytokines and Antibodies
c gene in
each X-SCID B-LCL have been previously described (3, 28, 29). Each X-SCID B-LCL is named by the position and identity of the base pair or
amino acid substitution within the
c cDNA or
protein, respectively. M1I has an M to I substitution at amino acid 1, and other substitutions include R224W, R226C, R226H, and F227C. R289*
prematurely terminates the
c protein at R289, dup235-7 has a duplication of amino acids 235-237, 924delC has a C deleted at
cDNA position 924 resulting in a missense amino acid after amino
acid 308. The X-SCID B-LCL identified as cDNA468+1 has a mutation
which disrupts the splice donor site after exon 3 and results in no
detectable
c mRNA (3). The X-SCID B-LCL
cDNA468+1 (boy 5) into which the wild type
c gene
was introduced by retroviral mediated transduction has previously been
reported (30) and is noted as cDNA468+1/
c.
Expression of
c protein on the cell surface of control
and X-SCID B-LCL was assessed using a
c-specific rat
monoclonal antibody, TUGH4 (Pharmigen, San Diego, CA).
Immunofluoresence analyses revealed either normal, trace, or absent
levels of
c protein on the cell surface (data not
shown). The HUT78 T cell line was obtained from the ATCC (Rockville,
MD). All cell lines were cultured in RPMI 1640 with 10% fetal calf
serum, 2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin.
-AAGACCATTTCTAAGAAATCTATC-3
) (33).
Briefly, whole cell extracts were incubated with labeled probe in
binding buffer for 15 min at 4 °C prior to electrophoresis on 6%
polyacrylamide gels and autoradiography. When used, anti-STAT6 antibodies were incubated with cell extracts for 15 min following the
addition of probe.
JAK1 and IRS-1, but Not JAK3, Are Phosphorylated following IL-4
Stimulation of X-SCID B-LCL
c-independent cytokine-mediated signal
transduction in X-SCID B cells, we assessed the phosphorylation of the
major intermediates determined to be involved in IL-4 signaling.
c subunit of the IL-2
and IL-4 receptors and is tyrosine-phosphorylated upon addition of either cytokine (14-17). We therefore analyzed JAK3 tyrosine
phosphorylation in response to IL-2 and IL-4 stimulation in
Epstein-Barr virus- transformed X-SCID B-LCL with heterogeneous
c mutations. Although JAK3 was tyrosine-phosphorylated
upon both IL-2 and IL-4 stimulation of a control B-LCL and the HUT78 T
cell line, we did not detect tyrosine phosphorylation of JAK3 following
IL-2 or IL-4 stimulation of any of the X-SCID B-LCLs (Fig.
1A, upper panel). Equivalent levels of immunoprecipitated JAK3 could be demonstrated in each lane by
immunoblotting the same filter with a polyclonal anti-JAK3 antibody
(Fig. 1A, lower panel). These data indicate that
JAK3 is not phosphorylated following IL-2 or IL-4 stimulation of X-SCID B-LCL with a diversity of
c mutations (28, 29, Fig. 1).
Fig. 1.
Tyrosine phosphorylation of JAK3, JAK1, and
IRS-1 following IL-2 and IL-4 stimulation. Normal
(control) B-LCL, B-LCL derived from X-SCID patients with
various c mutations, and the HUT-78 T cell line were
stimulated with IL-2 (1000 units/ml) or IL-4 (100 ng/ml) for 10 min at
37 °C. X-SCID B-LCL are labeled by the amino acid position of their
mutation within the
c protein, except for the B-LCL
cDNA468+1 where there is a 1-base pair insertion at position 468 of
the
c cDNA. The ability to detect
c
protein in each cell line was assessed by immunofluorescence with an
anti-
c monoclonal antibody (
c IF) and is
indicated as either normal (+), absent (
), or trace (Tr)
levels. Lysates were immunoprecipitated (IP) with either a
rabbit polyclonal anti-JAK3 antibody (A), rabbit polyclonal
anti-JAK1 antibody (B), or a rabbit polyclonal anti-IRS-1 antibody (C), resolved on polyacrylamide gels, and
immunoblotted with an anti-phosphotyrosine monoclonal antibody
(
PY) (upper panels). Blots were then stripped
and reprobed with either anti-JAK3 or anti-JAK1 antibodies to verify
equivalent levels of protein in each lane (lower
panels).
[View Larger Version of this Image (22K GIF file)]
and IL-4R
subunits
of the IL-2 and IL-4 receptors, respectively, and is phosphorylated upon receptor stimulation (14, 16, 34). It might be predicted that in
the absence of JAK3 phosphorylation, JAK1 would not be phosphorylated
in response to IL-2 or IL-4 in X-SCID B-LCL. In fact, JAK1 was not
phosphorylated in any of the X-SCID B-LCLs tested following the
addition of IL-2 (Fig. 1B, upper panel). However,
treatment with IL-4 resulted in JAK1 phosphorylation in all X-SCID
B-LCLs. It is of interest to note that the phosphorylation of JAK1 was
observed irrespective of the presence of a
c subunit on
the surface of the X-SCID B-LCL. These results demonstrate that JAK1 is
activated by an IL-4-responsive pathway in X-SCID B-LCL which is
independent of
c and JAK3.
c expression is not required for
IL-4-mediated IRS-1 phosphorylation.
c. Previous data indicated that IL-13, which shares many
structural and functional characteristics with IL-4 (38-40), but not
IL-4 itself, induced STAT6 activation in X-SCID B-LCL (25). However, we
found that STAT6 was tyrosine-phosphorylated upon both IL-4 and IL-13
stimulation in every X-SCID B-LCL (Fig. 2A).
Nevertheless, the IL-4-stimulated phosphorylation of STAT6 was
significantly greater in control B-LCL than in all X-SCID B-LCL tested
(Figs. 2A and 3). In contrast, the IL-13-mediated
stimulation of STAT6 was roughly equivalent among the control and
X-SCID B-LCLs.
Fig. 2.
Analysis of STAT6 tyrosine phosphorylation
and DNA binding activity upon IL-4 and IL-13 stimulation of X-SCID
B-LCL. A, immunoprecipitation with a polyclonal anti-STAT6
antibody was performed on lysates from control and X-SCID B-LCL
following either no stimulation () or stimulation with IL-4 (100 ng/ml) or IL-13 (250 ng/ml) for 10 min at 37 °C and immunoblotted
with anti-phosphotyrosine monoclonal antibodies (upper
panel). Blots were then stripped and reprobed with the anti-STAT6
antibody to monitor the level of STAT6 (lower panel).
B, whole cell extracts were incubated with
32P-labeled GAS oligonucleotide and then subjected to
polyacrylamide gel electrophoresis. For supershift reactions, cells
were preincubated with either an anti-STAT6 antibody or a control
antibody prior to addition of probe and were either untreated (
) or
stimulated with IL-4 (+).
[View Larger Version of this Image (38K GIF file)]
Fig. 3.
STAT6 phosphorylation is enhanced following
c reconstitution of an X-SCID B-LCL.
Immunoprecipitation with a polyclonal anti-STAT6 antibody was
performed on lysates from a control (normal), X-SCID B-LCL
(cDNA468+1), and the same X-SCID B-LCL reconstituted with
c (XSCID+
c) following
either no stimulation (
) or stimulation with IL-4 (+).
Immunoprecipitates were blotted with anti-phosphotyrosine monoclonal
antibodies (upper panel) and reprobed with an anti-STAT6 antibody (lower panel).
[View Larger Version of this Image (44K GIF file)]
activation sequence (GAS) element
in the CD23 promoter (42). As shown in Fig. 2B, gel-retarded
CD23 GAS element-binding complexes were formed following IL-4
stimulation of both a control B-LCL as well as an X-SCID B-LCL
(cDNA468+1). However, as might be expected from our finding that
STAT6 was phosphorylated at lower levels in IL-4-stimulated X-SCID
B-LCL as compared with control B-LCL (Fig. 2A), IL-4-induced DNA binding activity was significantly lower in all X-SCID B-LCL than
in control B-LCL (Fig. 2B and data not shown). To ascertain whether the DNA binding complexes contained STAT6, we performed supershift analyses using STAT6 antisera. In each of the IL-4-induced DNA binding complexes, a supershift was detected using STAT6 antisera but not with a control rabbit antisera (Fig. 2B). This
demonstration of IL-4-stimulated STAT6 DNA binding activity in cells
that do not express
c further supports the presence of
an IL-4 signaling pathway independent of
c and JAK3.
c
c in IL-4-mediated signaling, we assessed STAT6
phosphorylation and DNA binding activity in an X-SCID B-LCL in which
c expression was reconstituted using retroviral mediated
gene transduction (30). The data presented in Fig. 3
demonstrate that IL-4-mediated STAT6 phosphorylation was significantly
enhanced in the
c reconstituted X-SCID B-LCL, approaching the level observed in the control B cell line. Moreover, anti-STAT6 supershifted DNA binding complexes induced by IL-4 were more
abundant in
c reconstituted X-SCID B-LCL than in the X-SCID B-LCL (data not shown). Therefore, wild type levels of STAT6
phosphorylation and DNA binding activity in control B-LCL are likely
due to significant transduction through the
c-dependent IL-4 pathway.
c Reconstituted B-LCL
c-independent fashion. However, it was not clear whether
these IL-4-mediated biochemical responses in B cells derived from
X-SCID patients would result in biological outcomes, such as changes in
gene expression in X-SCID B cells. It has been shown that IL-4 induces
CD23 (Fc
RII) antigen expression in normal mature B cells (10). In
order to determine whether
c expression had a
significant effect on IL-4 functional responses, IL-4-induced regulation of CD23 in normal, X-SCID, and
c
reconstituted X-SCID B-LCL was examined. A 48-h incubation of control B
cells with IL-4 resulted in an almost 2-fold increase in CD23 levels
(Fig. 4), whereas no changes in CD23 expression were
detected in B-LCL derived from a number of patients. However, in
X-SCID cells reconstituted with
c, there was an almost
2-fold increase of CD23 levels when compared with the same cells
incubated without IL-4. This was a consistent finding in four
replicate experiments. These results suggest that
c is important for transducing some of the functional responses to IL-4.
Fig. 4.
Modulation of CD23 cell surface expression by
IL-4. Normal, X-SCID, and
c-reconstituted B cells were treated with or
without IL-4 in serum-free conditions for 48 h. CD23 expression was measured by fluorescence-activated cell sorting as described. Expression in untreated and IL-4-stimulated cells are shown as open and
closed histograms, respectively. The mean fluorescence intensity of
CD23 on the cell surface, with or without IL-4 stimulation, was
analyzed with Cellquest software (Becton-Dickinson). The mean fluorescence intensity in the normal, X-SCID, and
c
reconstituted X-SCID B-LCL increased by 1.93 (170.36 to 330.09), 1.00 (183.45 to 181.76), and 1.48 (232.56 to 344.91)-fold, respectively.
Results are representative of data from four independent
experiments.
[View Larger Version of this Image (24K GIF file)]
c (25, 26). However, IL-4 can induce functional, although suboptimal, responses in X-SCID B cells (27) suggesting that the IL-4 signal transduction pathway is conserved in these cells. X-SCID B cells, therefore, provide an important model in which to examine the generation of
c-independent IL-4 responses. In this
study, we determined that JAK1, IRS-1, and STAT6 were
tyrosine-phosphorylated in response to IL-4 in B cell lines derived
from X-SCID patients with a wide diversity of
c
mutations and that IL-4 induced detectable levels of STAT6 DNA binding
activity in these cells.
c and JAK3 (14, 16). This interaction is thought to be
critical for IL-2, IL-4, IL-7, IL-9, and IL-15 signaling in lymphocytes (14, 16, 43). The data described here show that IL-4-induced JAK1 and
IRS-1 phosphorylation as well as STAT6 activation can occur via
mechanisms that are independent of
c and JAK3. IRS-1 is
thought to be important for mediating IL-4 proliferative effects, but
the mechanism by which IL-4 stimulates IRS-1 phosphorylation is unclear
(44). Janus kinases likely play an important role (45), and our data
suggest that in the absence of JAK3, JAK1 may mediate IRS-1 tyrosine
phosphorylation in response to IL-4. It remains to be determined
whether other tyrosine kinases known to be induced by IL-4, including
JAK2, Tyk2, and Fes (46-48), play a role in IL-4-mediated responses in
X-SCID B cells.
c/reconstituted
X-SCID B-LCL following stimulation with IL-4. Because of the already high level of CD23 expression on Epstein-Barr virus-transformed B
cells, slight increases in CD23 expression may be difficult to detect.
Nevertheless, even in the studies performed by Matthews et
al. (27) in primary B cells, the increase in CD23 in X-SCID B
cells was significantly lower than that detected in control B cells
(27). Suboptimal CD23 activation in response to IL-4 suggests that the
observed humoral deficiency in X-SCID patients may result, at least in
part, from the inability to transduce the full set of IL-4-induced
signals through non-
c containing receptors. Our finding
that IL-4-induced STAT6 phosphorylation and CD23 expression were
restored to wild type levels following introduction of
c
into these cells indicates that
c potentiates the IL-4
signal transduction pathway. The importance of JAK3 in this
c-dependent pathway is further supported by
the recent observation that IL-4-induced CD23 expression and STAT6
activation are also suboptimal in B cells that express
c
but are deficient in JAK3 (49).
c-independent signaling pathway is initiated is not
clear, several groups have previously shown IL-4-induced proliferation
and protein phosphorylation in endothelial, colon carcinoma, and
plasmocytoma cell lines that lack
c (27, 46, 47,
50-52). The
c-independent pathway in these cells has
been suggested to function through a receptor that is shared by IL-4
and IL-13 since antibodies to the IL-4R
chain can block the binding
and function of IL-4 as well as IL-13 (53-57). Accordingly, IL-4 and
IL-13 stimulate the phosphorylation of many of the same downstream
substrates (47, 51, 55, 58-60). Moreover, IL-13 receptor subunits that
have varied capacities to bind IL-4 have recently been identified (61,
62). Our data support common signaling mechanisms as IL-4-activated
JAK1 and STAT6 but not JAK3 in X-SCID B cells, and this concurs with
what has been reported for IL-13 (47, 51 58, 60). Thus, an
IL-4-mediated signal transduction pathway in
c-deficient
X-SCID cells may occur via the same mechanisms utilized by IL-13.
c/JAK3 independent
IL-4R pathway in normal individuals as well as in patients with X-SCID
awaits further investigation.
*
This work was supported in part by a Howard Hughes Medical
Institute Postdoctoral Physician Grant (to N. T.), the Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program (to S. A. O.), the Fondation pour la Recherche Medicale, Philippe Foundation, and National Institutes of Health Grant AI25071.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Institut de
Génétique Moléculaire de Montpellier, 1919 Route de
Mende, 34033 Montpellier, Cedex 1 France. Tel.: 33-467 61 36 28; Fax:
33-467 04 02 31; E-mail: taylor{at}igm.cnrs-mop.fr.
**
Present address: Dept. of Pediatrics, University of Brescia,
Spedali Civili, 25123 Brescia, Italy.
¶¶
Present address: DNAX Research Institute, Palo Alto, CA
94304.
1
The abbreviations used are: X-SCID, X-linked
severe combined immunodeficiency; IL, interleukin; JAK, Janus tyrosine
kinase; STAT, signal transducers and activators of transcription;
B-LCL, B cell lymphoblastoid cell lines; GAS, interferon- activation site; R, receptor; IRS, insulin receptor substrate.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.