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,
*
The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Melbourne, Victoria, Australia;
The Centenary Institute of Cancer Medicine and Cell Biology, Newtown, New South Wales, Australia,
Medical Foundation, University of Sydney, Sydney, Australia; and
§
The Ludwig Institute of Cancer Research, Royal Melbourne Hospital, Victoria, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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IL-4 signaling in B cells is mediated by a specific receptor, a
heterodimer consisting of 
and 
polypeptide chains
(7). Upon IL-4 binding, various signal transduction
molecules including the Janus activation kinases (8, 9)
and the transcription factor STAT-6 (10), are recruited to
the receptor and phosphorylated. Phosphorylated STAT-6 dimerizes,
enters the nucleus, and binds to IL-4 responsive elements, thereby
initiating transcription of downstream genes (10).
IL-4-responsive elements are found in the promoters of IL-4-inducible
genes such as CD23 (11), C1
(12), and C
(13). Indeed, transcription of
germline C1 and C is a prerequisite for switching to these
isotypes (14). Although little is known about the negative
regulation of IL-4 signaling, it is thought to be mediated by the
phosphatase SHP-1 (15). B cells from motheaten viable
(mev)3
mice, which have a mutation in the gene
encoding SHP-1 such that the protein retains only 10% of wild-type
catalytic activity (16), show enhanced activation of the
transcription factor STAT-6 upon IL-4 stimulation (15).
Through binding to the IL-4R, activated SHP-1 dampens or suppresses
positive IL-4 signaling (15). For SHP-1 to be recruited to
the IL-4R, its binding site needs to be phosphorylated. The kinase
responsible for this phosphorylation of the IL-4R and, consequently,
for initiating the subsequent negative regulation of the IL-4 signal is
currently undefined.
The src-related tyrosine kinase Lyn targets a number of cell
surface receptors for phosphorylation, including the B cell receptor
(BCR), CD22, and FcRIIb (17). At the BCR, Lyn
phosphorylates tyrosines within the Ig-Igß heterodimer,
facilitating recruitment of the kinase Syk and subsequent initiation of
positive downstream signaling events (17). This function
of Lyn, however, appears to be redundant, because positive signals are
transduced through the BCR in the absence of Lyn (17).
This is presumably due to the ability of other src family
members to associate with the BCR (18). A unique role for
Lyn in B cells is the initiation of negative signaling cascades upon Ag
stimulation. Upon BCR cross-linking, Lyn phosphorylates CD22 and
thereby induces recruitment of the phosphatase SHP-1, which suppresses
signals emanating from the BCR (19). In B cells, Lyn also
phosphorylates the IgG receptor FcRIIb (20), which
negatively regulates B cell proliferation (17). As for
CD22, the inhibitory function of FcRIIb is mediated by the
recruitment of a phosphatase, in this case SHIP (19). Lyn
knockout mice have several immunological abnormalities, including a
10-fold increase in both IgM serum titers and the frequency of IgM
Ab-forming cells (AFC), the production of autoantibodies and a
deficiency in mature B cells (21, 22). This phenotype
resembles that of the CD22 knockout (23) and
mev mice (24) and exemplifies
the role of Lyn in initiating this particular negative signaling
pathway.
The study reported here reveals a previously unsuspected role for Lyn as a negative regulator of IL-4 signaling in B cells. Mitogen-stimulated B cells from lyn-/- mice are hyper-responsive to Ig isotype switching induced by IL-4. As a consequence of this, adult lyn-/- mice have an increased frequency of IgG1 splenic B cells and a 10-fold increase in serum IgE. Our results show that in the absence of Lyn the threshold of IL-4 signaling is diminished, resulting in IL-4 hyper-responsive B cells. We hypothesize that Lyn acts by recruiting a phosphatase, possibly SHP-1, to the IL-4R. This work shows Lyn to be a potential regulator of the development of atopy and highlights that allergy predisposition may arise due to a defect in attenuating cytokine signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The generation and characterization of lyn-/- mice have been described (21). Mice were originally a C57BL/6 x 129/Sv intercross before an 11th generation C57BL/6 backcross line was established. Littermates of the lyn-/- mice were used as controls. Hemizygous transgenic mice of the Eµ-bcl-2-36 strain (25), backcrossed with inbred C57BL/6 mice for 20 generations, were provided by Dr. A. W. Harris (The Walter and Eliza Hall Institute, Melbourne, Australia). These mice were crossed with lyn-/- mice, and the F1 mice were intercrossed to select for bcl-2/lyn-/- and bcl-2/lyn+/+ mice. Ly5.1 congenic C57BL/6 mice were used to distinguish lyn-/- from lyn+/+ cell in in vitro coculture experiments.
ELISPOT and ELISA assays
IgM and IgG1 AFCs were detected by ELISPOT as previously described (21). Briefly, cellulose ester-based plates (Millipore, Bedford, MA) were coated with sheep anti-mouse Ig (Silenus Laboratories, Hawthorn, Australia) as a capture reagent for 4 h and washed. Spleen cells (103–105) were cultured in triplicate wells for 20 h, after which plates were washed and developed with goat anti-mouse Ig conjugated to HRP (Southern Biotechnology Associates, Birmingham, AL). AFC were visualized by addition of 3-aminoethyl carbazole (Sigma, Sydney, Australia) and counted using a dissecting microscope. IgE titers were measured by ELISA, using rat anti-mouse IgE (clone 23G3, Southern Biotechnology Associates) as a plate coat and anti-IgE goat sera directly conjugated with HRP (Nordic Immunological Laboratories, Tilburg, The Netherlands) for developing. Results were calibrated against a purified mouse IgE mAb (clone SPE-7, ICM Biomedicals, Costa Mesa, CA).
B cell preparation and in vitro culture
B cells were enriched from pooled spleens of at least four control or lyn-/- mice by complement-mediated depletion of T cells using the Abs 30H12 (anti-Thy1), 3155 (anti-CD4), and RL172.4 (anti-CD8) and rabbit complement (C6 Diagnostics, Germantown, WI). The remaining cells were centrifuged through a Percoll density gradient, and the high density cells banding between the 65–72% layers were collected. This resulted in 85–90% B cell purity, of which <1% expressed surface IgG1. These cells were washed twice in serum-free RPMI and resuspended at 107 cells/ml in serum-free medium warmed to 37°C and containing CFSE (Molecular Probes, Eugene, OR) at a final concentration of 5 µM. Cells were incubated at 37°C for 10 min shielded from light, and the reaction was terminated by the addition of cold complete RPMI containing 5% FCS. Cells were washed twice and cultured at a density of 1 x 105 cells/ml with an optimal dilution of baculovirus-derived CD40L (gift from Dr. M. Kehry, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT) and purified rIL-4 (Immunex, Seattle, WA). At various time points, cells were harvested for staining with Abs and analysis by flow cytometry. For coculture experiments, purified B cells were not stained with CFSE before in vitro stimulation.
Immunostaining and flow cytometric analysis
Cells harvested from cell culture were stained with PE-conjugated RA3-6B2 (CD45R, otherwise known as B220; PharMingen, San Diego, CA) and Texas Red-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates) or biotinylated anti-IgE Ab R1E4, revealed with PE-streptavidin (Caltag, South San Francisco, CA), along with propidium iodide for dead cell exclusion and analyzed on a FACStarPlus (Becton Dickinson, San Jose, CA). Splenic IgG1 expressing B cells were identified as IgM-IgD-CD45R+ and IgG1+. After RBC removal, single-cell suspensions were stained with a mixture of Abs comprising PE conjugates of 331.12 (anti-IgM) and 11-26C (anti-IgD, Southern Biotechnology Associates), fluorescein-conjugated RA3-6B2 (CD45R), and Texas Red-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates). Analysis was performed on a FACStarPlus. Ly5.1 C57BL/6 cells were identified using a fluorescein conjugate of the mAb A20.1.7.
Quantification of IL-4R expression
Splenic B cells were purified by FACS sorting, and 2.5 x
106 cells were cultured with CD40L and IL-4 for
24 h (described above) before RNA extraction using the RNeasy Mini
Kit (Qiagen, Melbourne, Australia). For cells that were unstimulated,
including the positive control cell line CTLL-2, RNA extraction from
2.5 x 106 cells immediately followed
purification. One-tenth of the RNA product was converted to cDNA using
Superscript II reverse transcriptase (Life Technologies, Grand Island,
NY) in a mix containing random hexamer primers (Pharmacia, Piscataway,
NJ) for 30 min at 37°C. The extracellular domain of the IL-4R was
amplified by PCR from 1/50th of the cDNA using the oligonucleotides
5'-ATCCGCACTTCCACGTGTGAGTGG-3' (forward) and 5'-CAGCTGGAAGTTGTA
CCACGT-3' (reverse), resulting in an 
550-bp product. cDNA was
amplified for ß-actin using oligonucleotides from Clontech (Palo
Alto, CA). For amplification of the IL-4R and ß-actin, 25 cycles
were used, which was previously determined to be in the linear range
for product amplification and not saturating. PCR products were run
through a 1.5% agarose gel before transfer to nylon membrane
(
-Probe, Bio-Rad, Hercules, CA). The filter was hybridized with
32P end-labeled internal oligonucleotide probes
for ß-actin (Clontech) and IL-4R of the sequence
5'-TTCATAGTCTATAATGTGACCTAC-3'. The results were visualized and
quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale,
CA).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Naive B cells from lyn-/- mice showed
division-linked isotype switching to IgG1 over a range of IL-4
concentrations, starting at 1 U/ml. This is apparent in contour plots
(Fig. 1
A) and as the
percentage of cells within each division that have switched (Fig. 1B). No wild-type B cells switched to IgG1 at 1 U/ml of IL-4
over eight cell divisions within 5 days. Control B cells commence
switching to IgG1 at 10 U/ml IL-4, with the level increased at 100
U/ml. Thus, in contrast to control B cells,
lyn-/- B cells switch to IgG1 at earlier
divisions and have a higher proportion of switched cells when a plateau
is reached at the later divisions.
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). Both
lyn-/- and control B cells only initiated
switching to IgE after seven cell divisions; however, at both 10 and
100 U/ml IL-4, the percentage of lyn-/- B
cells expressing IgE was increased compared with the control value.
These results were not due to the differential death of
isotype-switched B cells, because cell numbers in both cultures
remained comparable throughout the experiment (data not shown). These
results show that Lyn is involved in setting both the threshold of IL-4
responsiveness and the strength of that signal, at least where this is
reflected in isotype switch.
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A). After 4 days of culture
the proportion of each B cell type that had switched to IgG1 was
determined by flow cytometry. Control and
lyn-/- cells were resolved by different
Ly5 alleles. The heightened responsiveness of
lyn-/- B cells was again apparent as was
the fact that the presence of lyn-/-
cells in the culture did not enhance switching of
lyn+/+ B cells. Thus, the increased
sensitivity of lyn-/- B cells to IL-4 is
intrinsic to the cells themselves.
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-chain and ß-actin (to normalize
for RNA content) were amplified using subsaturating cycles of PCR, and
the products were quantified after hybridization with internal
oligonucleotide probes. The results showed that IL-4R expression is
not enhanced in lyn-/- B cells relative
to that in wild-type cells (Fig. 3
B). In resting B cells the
levels of IL-4R in lyn+/+ and
lyn-/- cells were similar, being 0.4 and
0.3 the level of ß-actin, respectively. Upon activation, IL-4R
levels remained essentially unchanged relative to actin levels at 0.2
in lyn-/- and 0.3 in
lyn+/+ B cells. These results indicate that
differences in IL-4R levels are unlikely to contribute to the enhanced
responsiveness of lyn-/- B cells to IL-4
stimulation.
IL-4 is known to enhance mitogen-induced B cell proliferation
(3). To determine whether this facet of the IL-4 response
was also affected by Lyn, we examined the proliferation of
lyn-/- and control B cells cultured with
a constant amount of CD40L and varying doses of IL-4 (Fig. 4). This measurement involved calculating
the fraction of the B cell population within each cell division peak as
revealed by the CFSE profile (for example Fig. 1A). Doing
this revealed no significant difference in the proliferation of
lyn-/- B cells in response to CD40L plus
IL-4 compared with controls. In the absence of IL-4,
lyn-/- and control B cells proliferated
to an equal extent in response to CD40L. Addition of IL-4 enhanced the
proliferation of both B cell types to an equal extent. This enhancement
was apparent as a shift in the peak of the proliferation profile to a
higher division number. That the absence of Lyn affects isotype
switching and not proliferation suggests that these aspects of IL-4
signaling may be controlled by distinct mechanisms
(30).
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A). In contrast, control mice
had no detectable IgG1 AFC until 8 wk of age. As opposed to the
lifelong elevation of IgM AFC in lyn-/-
mice (data not shown), the elevated frequency of IgG1 AFC was not
maintained but remained relatively normal throughout adulthood (Fig. 5A). The transient nature of the increased frequency of IgG1
AFC in lyn-/- mice may be due to either
the 10-fold increase in the serum IgM concentration in adult
lyn-/- mice affecting their ability to
mount T-dependent responses (21) or to isotype switching
to IgG1 not necessarily being an end point of differentiation, as such
cells can continue to isotype switch to IgE (see below).
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B). This revealed a
significantly higher fraction of such cells in the bcl-2
transgenic lyn-/- mice compared with
bcl-2 control mice. Quantification of these data showed the
difference to be significant both as a fraction of spleen and as a
fraction of B cells (Table I). No surface
IgE+ cells were observed in either strain;
however, serum analysis revealed 10-fold higher IgE titers in
nontransgenic spleens of lyn-/- mice at
both 8 wk and 10 mo of age (Fig. 5C). Thus
lyn-/- mice display attributes consistent
with the behavior revealed by our in vitro studies (Figs. 1 and 2).
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RIIb
(20), and PIR-B (35), respectively. The
cytoplasmic domain of the IL-4 -chain contains an ITIM motif
(I/VXYXXL) at aa 707–712 of the human and mouse sequences. Taking into
account the function of Lyn at other surface receptors, we hypothesize
that Lyn is responsible for phosphorylating a site on the IL-4
-chain that allows for the subsequent binding of a phosphatase to
the receptor. Macrophages from mev mice
are hyper-responsive to GM-CSF (36), the receptor for
which is a target for Lyn phosphorylation (37), supporting
the argument that Lyn and SHP-1 may cooperate in the negative control
of cytokine signaling. Recently, a new family of signaling molecules, known variously as the suppressors of cytokine signaling (SOCS) (38), STAT-induced STAT inhibitor (39), and Janus kinase binding (40) proteins have been shown to be part of negative feedback loops for several cytokine receptors. They act at various sites in the signaling pathway; for example, SOCS-1 binds to and inhibits the activity of the Janus activation kinase proteins (38), whereas CIS (cytokine inducible SH2 protein) competes for STAT binding sites on the cytokine receptor (41). As yet no SOCS protein involved in IL-4 signaling has been defined. It is possible that Lyn influences the activity of such a SOCS protein at the IL-4R. Given, however, the mechanism by which Lyn initiates negative signaling pathways at other cell-surface receptors, this mode of action appears to us the less likely of the two discussed.
The abnormally high levels of IgE in the serum of
lyn-/- mice indicates that setting the
signaling thresholds for cytokines such as IL-4 could play a role in
predisposing individuals to allergic responses. Predisposition to
allergies have been extensively studied with a focus on genetic
influences and the balance between Th1 and Th2 responses
(5). Particular attention has been given to the levels of
IL-4 and IFN- production during immune responses, with the logic
that aberrant levels of IL-4 may lead to excessive IgE production
(5) (42). Although this reasoning is valid,
recent studies into the internal processing of cytokine signals provide
new avenues for research into atopy (43). Although the
underlying mechanism by which Lyn acts remains to be fully defined,
this report is the first describing a nonreceptor protein being
responsible for setting the threshold of IL-4 responsiveness and
provides a means by which this issue can be pursued.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David Tarlinton, The Walter and Eliza Hall Institute for Medical Research, P.O. The Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia. E-mail address:
3 Abbreviations used in this paper: mev, motheaten viable; AFC, Ab-forming cells; BCR, B cell receptor; CFSE, carboxy fluorescein succinimidyl ester; ITIM, immunoreceptor tyrosine-based inhibitory motif; SHP, SH2-containing protein tyrosine phosphatase; ELISPOT, enzyme-linked immunospot; CD40L, CD40 ligand; SOCS, suppressors of cytokine signaling.
Received for publication April 9, 1999. Accepted for publication August 2, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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chain. Immunity 4:113.[Medline]
1 RNA: analysis of the promoter/enhancer. EMBO J. 11:145.[Medline]
germline transcript: identification of a novel IL-4 responsive element. J. Immunol. 150:5408.[Abstract]
RII) phosphorylation. Mol. Cell. Biol. 16:4735.[Abstract]
subunit of the interleukin-4 receptor. N. Engl. J. Med. 337:1720.This article has been cited by other articles:
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  V. Parravicini, A.-C. Field, P. D. Tomlinson, M. Albert Basson, and R. Zamoyska Itch-/-{alpha}{beta} and {gamma}{delta} T cells independently contribute to autoimmunity in Itchy mice Blood, April 15, 2008; 111(8): 4273 - 7282. [Abstract] [Full Text] [PDF]
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 B. Guo, D. Blair, T. C. Chiles, C. A. Lowell, and T. L. Rothstein Cutting Edge: B Cell Receptor (BCR) Cross-Talk: The IL-4-Induced Alternate Pathway for BCR Signaling Operates in Parallel with the Classical Pathway, Is Sensitive to Rottlerin, and Depends on Lyn J. Immunol., April 15, 2007; 178(8): 4726 - 4730. [Abstract] [Full Text] [PDF]
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J. Kitaura, Y. Kawakami, M. Maeda-Yamamoto, V. Horejsi, and T. Kawakami Dysregulation of Src Family Kinases in Mast Cells from Epilepsy-Resistant ASK versus Epilepsy-Prone EL Mice J. Immunol., January 1, 2007; 178(1): 455 - 462. [Abstract] [Full Text] [PDF]
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C.-L. Chu and C. A. Lowell The Lyn Tyrosine Kinase Differentially Regulates Dendritic Cell Generation and Maturation J. Immunol., September 1, 2005; 175(5): 2880 - 2889. [Abstract] [Full Text] [PDF]
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S.-J. E. Beavitt, K. W. Harder, J. M. Kemp, J. Jones, C. Quilici, F. Casagranda, E. Lam, D. Turner, S. Brennan, P. D. Sly, et al. Lyn-Deficient Mice Develop Severe, Persistent Asthma: Lyn Is a Critical Negative Regulator of Th2 Immunity J. Immunol., August 1, 2005; 175(3): 1867 - 1875. [Abstract] [Full Text] [PDF]
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K. W. Harder, C. Quilici, E. Naik, M. Inglese, N. Kountouri, A. Turner, K. Zlatic, D. M. Tarlinton, and M. L. Hibbs Perturbed myelo/erythropoiesis in Lyn-deficient mice is similar to that in mice lacking the inhibitory phosphatases SHP-1 and SHIP-1 Blood, December 15, 2004; 104(13): 3901 - 3910. [Abstract] [Full Text] [PDF]
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 S. Odom, G. Gomez, M. Kovarova, Y. Furumoto, J. J. Ryan, H. V. Wright, C. Gonzalez-Espinosa, M. L. Hibbs, K. W. Harder, and J. Rivera Negative Regulation of Immunoglobulin E-dependent Allergic Responses by Lyn Kinase J. Exp. Med., June 7, 2004; 199(11): 1491 - 1502. [Abstract] [Full Text] [PDF]
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M. L. Hibbs, K. W. Harder, J. Armes, N. Kountouri, C. Quilici, F. Casagranda, A. R. Dunn, and D. M. Tarlinton Sustained Activation of Lyn Tyrosine Kinase In Vivo Leads to Autoimmunity J. Exp. Med., December 16, 2002; 196(12): 1593 - 1604. [Abstract] [Full Text] [PDF]
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Y. Xu, S.-J. E. Beavitt, K. W. Harder, M. L. Hibbs, and D. M. Tarlinton The Activation and Subsequent Regulatory Roles of Lyn and CD19 After B Cell Receptor Ligation are Independent J. Immunol., December 15, 2002; 169(12): 6910 - 6918. [Abstract] [Full Text] [PDF]
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B. A. Croker, D. M. Tarlinton, L. A. Cluse, A. J. Tuxen, A. Light, F.-C. Yang, D. A. Williams, and A. W. Roberts The Rac2 Guanosine Triphosphatase Regulates B Lymphocyte Antigen Receptor Responses and Chemotaxis and Is Required for Establishment of B-1a and Marginal Zone B Lymphocytes J. Immunol., April 1, 2002; 168(7): 3376 - 3386. [Abstract] [Full Text] [PDF]
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B. O'Laughlin-Bunner, N. Radosevic, M. L. Taylor, Shivakrupa, C. DeBerry, D. D. Metcalfe, M. Zhou, C. Lowell, and D. Linnekin Lyn is required for normal stem cell factor-induced proliferation and chemotaxis of primary hematopoietic cells Blood, July 15, 2001; 98(2): 343 - 350. [Abstract] [Full Text] [PDF]
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) with
lyn-/- (
) B cells in the presence of
CD40L and the indicated concentrations of IL-4 for 4 days. B cells were
then analyzed for surface IgG1, with the two B cell types in the
mixture being distinguished by expression of different Ly5 alleles.
These results are representative of three experiments.
B, Quantification of IL-4R
) and
lyn-/- (