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(Journal of Leukocyte Biology. 2001;69:825-830.)
© 2001 by Society for Leukocyte Biology

Sulfhydryl-2 domain-containing protein tyrosine phosphatase-1 is not a negative regulator of interleukin-4 signaling in murine mast cells

Erik D. White, Ryan P. Andrews and Gurjit K. Khurana Hershey

Division of Pulmonary Medicine, Allergy, and Clinical Immunology, Department of Pediatrics, Children’s Hospital Medical Center, Cincinnati, Ohio

Correspondence: Gurjit K. Khurana Hershey, M.D., Ph.D., Division of Pulmonary Medicine, Allergy, and Clinical Immunology, Children’s Hospital Medical Center, 3333 Burnet Ave., Cincinnati OH 45229. E-mail: Gurjit.Hershey{at}chmcc.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sulfhydryl-2 domain-containing tyrosine phosphatase-1 (SHP-1) has an important role in the negative regulation of many receptors including the interleukin (IL)-4 receptor. Motheaten mice (me/me) have a homozygous mutation in SHP-1 and do not possess functional SHP-1. Pre-B-cell lines derived from me/me mice have been reported to display prolonged IL-4-dependent activation of signal transducer and activator of transcription-6 (Stat6). We evaluated IL-4-dependent Stat6 activation and Fc{varepsilon} receptor 1 (Fc{varepsilon}RI) modulation in bone marrow-derived mast cells (BMMCs) from me/me and wild-type mice. IL-4 down-regulated Fc{varepsilon}RI expression in wild-type BMMCs but had no effect on Fc{varepsilon}RI expression in me/me BMMCs. Furthermore, me/me mast cells did not exhibit enhanced or prolonged IL-4-induced Stat6 activation compared with wild-type cells, indicating that mast cells possess alternative tyrosine phosphatases that are responsible for down-regulating Stat6 or can substitute for SHP-1. Thus, SHP-1 is not a negative regulator of IL-4 signaling in BMMCs. These results demonstrate the complexity and cellular specificity of these signaling pathways and indicate a previously unrecognized role for SHP-1 in murine mast cells.

Key Words: mast cells/basophils • protein kinases/phosphatases • signal transduction • cytokine receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-4, a pleiotropic T helper cell type 2-derived cytokine, plays a critical role in promoting allergic inflammation. IL-4 induces transcriptional activation of many genes including the {varepsilon} heavy-chain locus [1 ] and genes encoding vascular cell adhesion molecule-1 [2 ], CD23 [3 ] and major histocompatibility complex class II [4 5 ].Furthermore, studies have demonstrated that IL-4 is critical for the development of T helper cell type 2 cells [6 7 ]. IL-4 is also a potent regulator of mast cell phenotype, growth, and differentiation [8 9 10 11 ]. Mast cells have a pivotal role in atopic reactions through the release of preformed and newly synthesized mediators. Degranulation of mast cells occurs through antigen-mediated cross-linking of the surface high-affinity immunoglobulin (Ig) E Fc{varepsilon} receptor 1 (Fc{varepsilon}RI). The molecular events that regulate Fc{varepsilon}RI expression are poorly understood. IgE has been shown to be a potent inducer of surface Fc{varepsilon}RI expression in human and murine mast cells [12 13 ]. Recently, IL-4 has been shown to inhibit murine mast cell Fc{varepsilon}RI expression [14 ]. Furthermore, IL-4 promotes intercellular adhesion molecule-1 expression [15 ] but inhibits c-kit expression in mast cells [16 17 ]. Thus, IL-4 may function as both a positive and negative regulator of mast cells.

Biologic responses to IL-4 are mediated via two types of receptor complexes. The type I receptor consists of an IL-4-binding 140-kDa {alpha} chain (IL-4R{alpha}) [18 19 20 ] and the {gamma}c chain, which is common to multiple receptor systems [21 22 ]. Upon the binding of IL-4 to the {alpha} chain, the {gamma}c chain heterodimerizes with the {alpha} chain, and activation of associated Janus kinases JAK3 and JAK1, respectively, occurs [23 24 25 26 ]. JAK activation leads to the phosphorylation of signal transducer and activator of transcription-6 (Stat6), which mediates gene induction [27 28 ]. In nonhematopoietic cells, IL-4 can signal in the absence of the {gamma}c chain through the type II receptor, which is composed of IL-4R{alpha} and IL-13R{alpha}1 [29 30 ]. Signaling via this receptor also leads to activation of the JAK-Stat pathway.

Although much is known about the IL-4 signaling pathway, the molecular mechanisms responsible for the dephosphorylation of key signaling intermediates and for negative modulation of the IL-4 cascade remain to be elucidated. Sulfhydryl 2 domain-containing tyrosine phosphatase-1 (SHP-1) dephosphorylates regulatory phosphotyrosine residues and has been implicated in termination of signaling via many cytokine receptors including erythropoietin, IL-2, IL-3, colony-stimulating factor, and IL-4 receptor {alpha} (IL-4R{alpha}) [31 32 33 34 35 36 37 ]. Recent studies have shown that SHP-1 may also have a positive regulatory role in some pathways including the mitogen-activated protein kinase pathway [38 39 ] and epidermal growth factor-induced Stat activation [40 ]. Motheaten mice (me/me) are homozygous for a spontaneous frameshift mutation in the SHP-1 gene, and they lack functional SHP-1 [41 ]. Bone marrow-derived macrophages and pre-B-cell lines derived from me/me mice display enhanced IL-4 signaling when compared with their wild-type counterparts, consistent with a negative regulatory role for SHP-1 in IL-4 responses [37 ]. These mice develop profound abnormalities in their B- and T-cell function and die from hemorrhagic pneumonitis within the first few months of life [42 ]. The mice also have defects in natural killer cell and macrophage function [42 43 ]. However, me/me mast cells possess normal cell surface marker expression and degranulate normally [44 45 ]. In the present study, we examined IL-4 signaling and IL-4-dependent Fc{varepsilon}RI down-modulation in me/me and wild-type mast cells to elucidate the role of SHP-1 in IL-4 signaling and responsiveness in mast cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
BMMCs were derived from motheaten (me/me) and control CH3HeB mice as previously described [46 ]. After 4 weeks in culture, >99% of the cells were found to be mast cells by toluidine blue staining. BMMCs from me/me mice were a kind gift from Taolin Yi (Cleveland Clinic, Cleveland, OH). The mast cell lines were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 4 mM L-glutamine, 100 µg/mL of penicillin/streptomycin (BioWhittaker, Walkersville, MD), 1 mM sodium pyruvate, 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 0.1 mM nonessential amino acids (Gibco BRL Products, Gaithersburg, MD), and 15% WEHI-conditioned medium (Becton Dickenson, San Diego, CA); or 50µM ß-mercaptoethanol.

Antibodies and cytokines
Recombinant murine IL-4 was obtained from R&D Systems (Minneapolis, MN). Rabbit polyclonal anti-Stat6 antibody was the generous gift of Ulrike Schindler (Tularik Inc., San Francisco, CA). Murine IgE was obtained from PharMingen (San Diego, CA); fluorescein isothiocyanate-labeled rat anti-mouse IgE was from Southern Biotech (Birmingham, AL); and anti-murine CD16/CD23 (FC{gamma}III/II Receptor-Fc block®) was from PharMingen.

Intracellular [Ca2+] measurement
BMMCs were incubated in the presence of 5 µg/mL of IgE for 1 h at 4°C, pelleted by centrifugation, and then 2 x 106 cells/mL were loaded with 5 µM Fura-2 AM (Molecular Probes, Eugene, OR) in Hanks balanced saline solution containing 1% fetal bovine serum for 30 min at 37°C in the dark. After washing with flux buffer (145 mM NaCl, 4 mM KCl, 1 mM NaHPO4, 0.8 mM MgCl2, 1.8 mM CaCl2, 25 mM HEPES, and 22 mM glucose; pH 7.4), cells were resuspended at 2 x 106 cells/mL and maintained on ice. Next, cells were prewarmed to 35°C and stimulated with 10 µL of anti-IgE antibody EM95, kindly provided by Fred Finkelman (University of Cincinnati, Cincinnati, OH), in a RatioMaster Fluorimeter (Photon Technology, Inc., South Brunswick, NJ). Data were recorded as the relative ratio of fluorescence emitted at 510 nm after excitation at 340 and 380 nm (y-axis) over time (x-axis).

Flow-cytometric analysis
BMMCs (5 x 105) were incubated in the presence of 0.125µg of Fc block® (PharMingen) for 10 min at 4°C in a total volume of 100 µL; 0.5 µg/10 µL of murine IgE was added; after 30 min, 0.5 µg/10 µL of anti-IgE-fluorescein isothiocyanate was added, and the samples were incubated for 30 min, washed, and analyzed using FACScan® (Becton Dickenson, San Diego, CA). Control samples were stained in the absence of IgE.

Propidium iodide staining for determination of DNA content was performed as previously described [47 48 ].

Electrophoretic Mobility Shift Assay
BMMCs (2.5 x 106) were lysed in 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM ethylenediaminetetraacetate (EDTA), 1.5 mM MgCl2, 0.2% Nonidet P-40, 1.0 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride, and nuclei were reconstituted in nuclear extract buffer (20 mM HEPES [pH 7.9], 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl2, 25% glycerol, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride). Extracts (5 µg each) in equal volumes of 2x electrophoretic mobility shift assay (EMSA) reaction buffer (24 mM HEPES [pH 7.9], 8 mM Tris, 50 mM KCl, 10 mM MgCl2, 24% glycerol, 0.08 µg/mL of poly dI-dC, 2 mM EDTA, and 2 mM DTT) were incubated for 10 min on ice, and then 0.2 ng of end-labeled double-stranded Stat6 oligonucleotide (Santa Cruz Biotechnologies, Santa Cruz, CA) was added for 10 min on ice. A 100-fold excess of unlabeled nucleotide (20 ng) was used in cold competition samples, and 1.0 µL of anti-Stat6 polyclonal antibody was added to supershift samples. Extracts were resolved on a 5% polyacrylamide gel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-4-mediated down-modulation of Fc{varepsilon}RI expression in BMMCs did not occur in me/me BMMCs
Motheaten mice (me/me) are homozygous for a frameshift mutation on chromosome 6 in the SHP-1 gene and lack functional SHP-1. Although they have profound abnormalities in their B, T, and natural killer cells, their mast cells have normal morphology, cell surface marker expression, and degranulation function [44 45 ]. BMMCs derived from me/me and strain-matched control mice displayed similar morphology after toluidine blue staining (data not shown). Furthermore, stimulation of wild-type or me/me BMMCs with IgE followed by a cross-linking anti-IgE induced a rapid and equal calcium flux (data not shown). IL-4 has been shown to inhibit Fc{varepsilon}RI expression in BMMCs cultured in the presence of IL-3 [14 ]. SHP-1 has been implicated as a negative regulator of IL-4 responses in B cells [37 ]. Thus, we hypothesized that, in the absence of SHP-1-mediated down-regulation, SHP-1-deficient me/me BMMCs would display augmented IL-4-dependent inhibition of Fc{varepsilon}RI expression. IL-4 inhibited Fc{varepsilon}RI expression as predicted in the wild-type mice (Fig. 1A ), but surprisingly, IL-4 did not down-regulate Fc{varepsilon}RI expression in the me/me cells (Fig. 1B) . There was no difference in baseline Fc{varepsilon}RI expression between the wild-type and SHP-1-deficient mast cells (Fig. 1) . The decrease in Fc{varepsilon}RI expression by IL-4 has been shown to require several days of stimulation [14 ]. We examined whether the kinetics of IL-4-mediated Fc{varepsilon}RI modulation were altered in the me/me BMMCs. The wild-type cells displayed maximal IL-4-mediated inhibition at 96 h (Fig. 1A) , but the me/me BMMCs did not exhibit any change in Fc{varepsilon}RI expression even when cultured in the presence of IL-4 for 144 h (Fig. 1B) . The results of two separate experiments are shown graphically in Figure 1C . We did not observe any effect of IL-4 on Fc{varepsilon}RI expression even at doses of up to 100 ng/mL of IL-4 in SHP-1-deficient mast cells (data not shown).



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  		Figure 1. Temporal responsiveness of IL-4-mediated down-modulation of Fc{varepsilon}RI expression in me/me and wild-type murine mast cells. BMMCs derived from wild-type (A) or me/me (B) mice were treated for increasing time intervals with 20 ng/mL of IL-4 for 96 h and then surface Fc{varepsilon}RI expression was analyzed by flow cytometry (dotted line, anti-IgE alone; solid line, IgE and anti-IgE, in the absence of IL-4; bold line, IgE and anti-IgE, in the presence of IL-4). (C) Flow-cytometry data from two separate experiments using different lines (me/me, squares; wt, circles)

 
Absence of SHP-1 did not affect the sensitivity of IL-4-induced Stat6 activation in BMMCs
Since IL-4 had no effect on Fc{varepsilon}RI expression in the me/me mast cells, it was possible that IL-4 signaling was not occurring normally in these cells. Stat6 has been shown to be required for IL-4-dependent inhibition of Fc{varepsilon}RI expression [14 ]. To determine whether IL-4 signaling was intact in the SHP-1-deficient BMMCs, we examined IL-4-dependent Stat6 activation in control and me/me BMMCs by EMSA (Fig. 2 ). Stat6 activation occurred in the presence or absence of SHP-1, and the magnitude of activation was enhanced with increasing doses of IL-4. Both the wild-type and SHP-1-deficient cells exhibited identical sensitivity of IL-4-dependent Stat6 activation, with maximal Stat6 activation occurring at a dose of 10 ng/mL of IL-4. Thus, Stat6 activation occurred in the me/me BMMCs and was not sufficient for IL-4-dependent modulation of Fc{varepsilon}RI expression.



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  		Figure 2. IL-4-dependent Stat6 activation occurs equally in me/me and wild-type murine mast cells. BMMCs derived from wild-type or me/me mice were treated with the indicated doses of IL-4 for 15 min. 10 µg of nuclear extracts were then examined for the presence of activated Stat6 by EMSA. The identity and specificity of the Stat6 band was illustrated by complete supershifting of this band using an anti-Stat6 antibody; disappearance of this band when the assay was carried out in the presence of an excess of unlabeled probe containing the Stat6 recognition sequence; and the absence of the band in unstimulated cells. The depicted experiment is representative of three separate experiments.

 
Absence of SHP-1 did not lead to enhanced or prolonged IL-4-induced Stat6 activation in BMMCs
In bone marrow-derived macrophages and pre-B-cell lines derived from wild-type mice, Stat6 activation is maximal after 30 min of IL-4 stimulation and greatly diminishes after 4 h of IL-4 treatment [37 ]. In contrast, bone marrow-derived macrophages and pre-B-cell lines from me/me mice have been reported to display enhanced and prolonged IL-4 Stat6 activation when compared with their wild-type counterparts [37 ], supporting a negative regulatory role for SHP-1 in these cell types. In the me/me-derived macrophages and pre-B-cell lines, Stat6 activation was maintained even after 4 h of IL-4 treatment and did not wane in contrast to the wild-type cells. This supported a role for SHP-1 in the deactivation of Stat6 in these cells. We examined whether this was also the case in BMMCs. In contrast to the observations made in macrophages and pre-B-cells lines, me/me BMMCs did not display enhanced Stat6 activation compared with the wild-type BMMCs (Fig. 2) . We next examined whether IL-4-dependent Stat6 activation was prolonged in SHP-1-deficient mast cells with continuous treatment of cells with 10 ng/mL of IL-4. Stat6 activation was significantly and equally decreased in both the wild-type and the me/me BMMCs after 4, 8, and 24 h (Fig. 3 ). The results were quantitated by densitometry, and there was no difference in the rate or magnitude of Stat6 deactivation between the wild-type and me/me mast cells (Fig. 3B) .



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  		Figure 3. IL-4-dependent Stat6 activation is not prolonged in the absence of SHP-1. (A) BMMCs were stimulated in the presence or absence of IL-4 for the designated time in hours. 10 µg of nuclear extracts were then examined for the presence of activated Stat6 by EMSA. The identity and specificity of the Stat6 band was illustrated by complete supershifting of this band using an anti-Stat6 antibody; disappearance of this band when the assay was carried out in the presence of an excess of unlabeled probe containing the Stat6 recognition sequence; and the absence of the band in unstimulated cells. (B) The data from panel A and a repeat experiment were quantitated by densitometry, and the relative mean amounts of activated Stat6 in densitometric units detected in lysates from the wild-type cells (solid bars) and me/me cells (hatched bars) are shown.

 
IgE-dependent modulation of Fc{varepsilon}RI expression in BMMCs was not dependent on SHP-1
SHP-1 has been reported to constitutively associate with Fc{varepsilon}RI [49 ]. To clarify whether the lack of an IL-4 effect on Fc{varepsilon}RI expression in the me/me cells was specific to IL-4 or was due to a general abnormality in Fc{varepsilon}RI modulation, we next examined IgE-dependent modulation of Fc{varepsilon}RI in wild-type and me/me BMMCs. IgE is a potent inducer of Fc{varepsilon}RI expression in murine mast cells [12 13 ]. Both control and me/me BMMCs displayed equivalent IgE-dependent up-regulation of Fc{varepsilon}RI expression (Fig. 4 ). Thus, Fc{varepsilon}RI modulation via this pathway is intact in the me/me cells and does not require SHP-1.



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  		Figure 4. Equivalent IgE-dependent up-regulation of Fc{varepsilon}RI expression in me/me and wild-type BMMCs. (A) BMMCs derived from wild-type or me/me mice were incubated in the absence (left) or presence (right) of 5 µg/mL of IgE for 5 days. (B) Surface Fc{varepsilon}RI expression was then analyzed by flow cytometry. The flow-cytometry data from three separate experiments (each bar represents the means and SE of three separate experiments) are displayed graphically in the bottom graph (wild type, filled bars; me/me, open bars).

 
Cell cycle and DNA content were not altered in SHP-1-deficient mast cells
It remained possible that there was a dysregulation of the cell cycle in the absence of SHP-1 and that this indirectly affected the response of these cells to IL-4. To directly address this possibility, we determined the relative numbers of me/me and wild-type BMMCs in different stages of the cell cycle by determining the DNA content in wild-type and me/me BMMCs. As depicted in Figure 5 , there was no significant difference in the relative proportions of cells at different stages of the cell cycle in the presence or absence of SHP-1. Notably, apoptosis was not enhanced in the SHP-1-deficient mast cells.



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  		Figure 5. Similar DNA contents of me/me and wild-type BMMCs. Wild-type (dotted line) and me/me (solid line) mast cells were stained with propidium iodide and then analyzed by flow cytometry. Data are representative of three separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
SHP-1 is an intracellular protein tyrosine phosphatase, which is widely expressed in hematopoietic cells. It has been shown to be involved in the negative regulation of multiple cytokine receptors [31 32 33 34 35 ], to associate with IL-4R{alpha} [36 50 ], and, recently, to negatively regulate IL-4 responses in pre-B-cell lines derived from me/me mice [37 ]. Herein, we examined the role of SHP-1 in IL-4 responses in mast cells. Our data demonstrate that IL-4 was not a negative regulator of IL-4 responses in murine BMMCs and support the possibility that SHP-1 is a positive regulator of IL-4 responses in mast cells.

Pre-B-cell lines derived from me/me mice displayed enhanced and prolonged IL-4-induced Stat6 activation [37 ], supporting a role for SHP-1 in the deactivation of Stat6. In contrast, IL-4 induced Stat6 activation equally in the wild-type and me/me BMMC lines. Furthermore, the rate and magnitude of Stat6 deactivation were not altered in the absence of SHP-1. Thus, the role of SHP-1 in the regulation of IL-4 signal transduction in mast cells was quite distinct from that in B cells. In addition, mast cells must have alternative tyrosine phosphatases that are responsible for down-regulating Stat6 or can substitute for SHP-1. It is not surprising that different regulatory pathways may exist in mast cells since mast cells play a unique role in the immune response. Recently, IL-4 production in mast cells was shown to occur in the absence of c-maf, indicating that IL-4 transcription in mast cells is controlled by a more distinct mechanism than in T cells [51 ].

In BMMCs, IL-4 has been shown to inhibit Fc{varepsilon}RI expression through a Stat6-dependent mechanism [14 ]. Since SHP-1 is a negative regulator of many cytokine pathways, we hypothesized that we would observe an exaggerated response to IL-4 in me/me mast cells. Surprisingly, despite normal IL-4-dependent Stat6 activation in me/me BMMCs, IL-4-dependent negative modulation of Fc{varepsilon}RI expression did not occur in the absence of SHP-1. Thus, Stat6 activation is not sufficient for IL-4-dependent modulation of Fc{varepsilon}RI expression. Furthermore, our observations suggest that SHP-1 may also act as a positive regulator and be required for the induction of certain IL-4 responses. Recently, phosphatase activity was shown to be required for the induction of IL-4-dependent induction of IL-4R{alpha} in lymphocytes [52 ], supporting the possibility that phosphatases can be positive regulators of IL-4 responses.

The mechanism of IL-4-mediated inhibition of Fc{varepsilon}RI expression and the precise role of SHP-1 are unclear. Although Stat6 is required, it is not sufficient. One possible mechanism is that IL-4 alters Fc{varepsilon}RI expression by influencing the internalization and/or degradation pathway of Fc{varepsilon}RI through a Stat6- and SHP-1-dependent mechanism. SHP-1 has been shown to constitutively associate with Fc{varepsilon}RI and to regulate tyrosine phosphorylation of the Fc{varepsilon}RIß and Fc{varepsilon}RI{gamma} subunits [49 ]. The function of SHP-1 is unclear, but its association with Fc{varepsilon}RI supports its role in Fc{varepsilon}RI physiology. It is interesting that our data indicated that SHP-1 is not a negative regulator of IL-4 signaling in BMMCs. Rather, SHP-1 is required for IL-4-mediated Fc{varepsilon}RI down-modulation. These results demonstrate the complexity and cellular specificity of these signaling pathways and indicate a previously unrecognized role for SHP-1 in murine mast cells.


   ACKNOWLEDGEMENTS
 
This work was supported in part by NIH/NICHD grant P30HD2887 and the Glaxo-Wellcome Basic/Clinical Research Award. We are grateful to Dr. Fred D. Finkelman for many helpful discussions and to Dr. Amal Assa’ad, Dr. Fred Finkelman, Dr. Marc Rothenberg, and Dr. Jeff Whitsett for critical review of this manuscript. We thank Connie Petitt for excellent secretarial support.

Received October 19, 2000; revised December 10, 2000; accepted December 12, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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