Pathway Mast Cell Function: Targeting the Stat5 1 on Β Genotype-Dependent Effects Of

Genotype-Dependent Effects of TGF-β1 on Mast Cell Function: Targeting the Stat5 Pathway

This information is current as Josephine Fernando, Travis W. Faber, Nicholas A. Pullen, of September 27, 2021. Yves T. Falanga, Elizabeth Motunrayo Kolawole, Carole A. Oskeritzian, Brian O. Barnstein, Geethani Bandara, Geqiang Li, Lawrence B. Schwartz, Sarah Spiegel, David B. Straus, Daniel H. Conrad, Kevin D. Bunting and John J. Ryan

J Immunol 2013; 191:4505-4513; Prepublished online 25 Downloaded from September 2013; doi: 10.4049/jimmunol.1202723 http://www.jimmunol.org/content/191/9/4505 http://www.jimmunol.org/ References This article cites 65 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/191/9/4505.full#ref-list-1

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Genotype-Dependent Effects of TGF-b1 on Mast Cell Function: Targeting the Stat5 Pathway

Josephine Fernando,* Travis W. Faber,* Nicholas A. Pullen,* Yves T. Falanga,* Elizabeth Motunrayo Kolawole,* Carole A. Oskeritzian,†,1 Brian O. Barnstein,* Geethani Bandara,‡ Geqiang Li,x Lawrence B. Schwartz,{ Sarah Spiegel,† David B. Straus,* Daniel H. Conrad,‖ Kevin D. Bunting,# and John J. Ryan*

We previously demonstrated that TGF-b1 suppresses IgE-mediated signaling in human and mouse mast cells in vitro, an effect that correlated with decreased expression of the high-afﬁnity IgE receptor, Fc«RI. The in vivo effects of TGF-b1 and the means by which it suppresses mast cells have been less clear. This study shows that TGF-b1 suppresses Fc«RI and c-Kit expression in vivo. « b

By examining changes in cytokine production concurrent with Fc RI expression, we found that TGF- 1 suppresses TNF pro- Downloaded from duction independent of Fc«RI levels. Rather, IgE-mediated signaling was altered. TGF-b1 signiﬁcantly reduced expression of Fyn and Stat5, proteins critical for cytokine induction. These changes may partly explain the effects of TGF-b1, because Stat5B overexpression blocked TGF-mediated suppression of IgE-induced cytokine production. We also found that Stat5B is required for mast cell migration toward stem cell factor, and that TGF-b1 reduced this migration. We found evidence that genetic background may alter TGF responses. TGF-b1 greatly reduced mast cell numbers in Th1-prone C57BL/6, but not Th2-prone 129/Sv mice. b Furthermore, TGF- 1 did not suppress IgE-induced cytokine release and did increase c-Kit–mediated migration in 129/Sv mast http://www.jimmunol.org/ cells. These data correlated with high basal Fyn and Stat5 expression in 129/Sv cells, which was not reduced by TGF-b1 treatment. Finally, primary human mast cell populations also showed variable sensitivity to TGF-b1–mediated changes in Stat5 and IgE- mediated IL-6 secretion. We propose that TGF-b1 regulates mast cell homeostasis, and that this feedback suppression may be dependent on genetic context, predisposing some individuals to atopic disease. The Journal of Immunology, 2013, 191: 4505–4513.

topic diseases, including asthma, atopic dermatitis, and kines that control cell proliferation, tissue repair, inﬂammation, allergic rhinitis, are caused by environmental and genetic migration, and angiogenesis (5, 6). The ﬁrst observation of TGF- factors (1–3). During the last three decades, the preva- b–mediated immune regulation was made two decades ago (7).

A by guest on September 27, 2021 lence of atopic disease has increased in developed nations, making TGF-b1 is now linked to autoimmune diseases and cancer (5, 6, 8, them a major health concern (1–4). Although their etiology is in- 9), and has recently been shown to have a critical role in regula- completely understood, a plausible factor promoting these chronic tory T cell and Th17 differentiation (10–12). inflammatory diseases is failure of feedback mechanisms facilitat- Mast cells are important effectors of atopic disease. Once ac- ing immune homeostasis. Understanding these regulatory circuits tivated by Ag-mediated IgE cross-linkage, mast cells link the innate would provide insight for allergic and autoimmune diseases. and adaptive immune responses by altering vascular function and One means by which the immune system is regulated is through recruiting leukocytes to inflammatory sites. Mast cells can elicit cytokine signaling. Of the many factors involved, members of the TGF-b1–mediated paracrine signaling because they secrete TGF- TGF-b family are ideal candidates. TGF-bs are multipotent cyto- b1 and proteases that activate latent TGF-b1 present in blood and connective tissues (13–15). *Department of Biology, Virginia Commonwealth University, Richmond, VA 23284; We and others have found that TGF-b1 suppresses mast cell †Department of Biochemistry and Molecular Biology, Virginia Commonwealth Uni- development, function, and survival, offering homeostatic feed- ‡ versity, Richmond, VA 23298; Laboratory of Allergic Disease, National Institute of back regulation (16). TGF-b1 inhibited proliferation of mouse Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; xBoston Children’s Hospital, Harvard University, Boston, MA 02115; {Department of peritoneal mast cells and bone marrow–derived mast cells (BMMCs) Internal Medicine, Virginia Commonwealth University, Richmond, VA 23298; ‖Depart- (17, 18), and prevented stem cell factor (SCF)–mediated rescue ment of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298; and #Department of Pediatrics, Aflac Cancer and Blood Disorders Center of from apoptosis (19). TGF-b1 inhibitory effects on mast cell effector Children’s Healthcare of Atlanta and Emory University, Atlanta, GA 30322 functions have also been shown. Bissonnette et al. (20) showed that 1Current address: Department of Pathology, Microbiology and Immunology, Univer- Ag-induced histamine and TNF production from rat peritoneal mast sity of South Carolina School of Medicine, Columbia, SC. cells were inhibited by TGF-b1, whereas Meade et al. (21) showed Received for publication September 28, 2012. Accepted for publication August 3, that in vivo treatment with TGF-b1 inhibited IgE-mediated, mast 2013. cell–dependent, immediate hypersensitivity responses in mice. De- This work was supported by National Institutes of Health Grants 1R01AI59638 spite studies supporting the theory that TGF-b1 inhibits mast cell (to J.J.R.), U19AI077435 (to J.J.R., S.S., and D.H.C.), 2R01DK059380 (to K.D.B.), K01AR053186 (to C.A.O.), and 1R01AI095494 (to C.A.O.). function, contradictory evidence exists. This includes a report Address correspondence and reprint requests to Dr. John J. Ryan, Department of that reducing TGF-b1 levels diminished IgE-dependent cutaneous Biology, Virginia Commonwealth University, Box 842012, Richmond, VA 23284- anaphylaxis (22). Moreover, these studies have not revealed the 2012. E-mail address: [email protected] mechanisms by which TGF-b acts on mast cells, which could Abbreviations used in this article: AHR, airway hyperresponsiveness; BMMC, bone include decreased IgE receptor expression and/or altered signal- marrow–derived mast cell; cRPMI, complete RPMI; HSA, human serum albumin; SCF, stem cell factor; siRNA, small interfering RNA; VCU, Virginia Commonwealth ing. Furthermore, why this potentially suppressive cascade fails to University. limit mast cell responses in some individuals is unknown. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202723 4506 GENOTYPE-DEPENDENT EFFECTS OF TGF-b1 ON MAST CELL FUNCTION

We have found that Stat5 is critical for mast cell responses to TGF-b1 injection ε both SCF via c-Kit and allergens via IgE-Fc RI (23–25). We now Mice (BL/6 or 129/Sv, 8–12 wk old, n = 5/group) were injected i.p. with show that TGF-b1 suppresses Stat5 expression in mast cells derived 0.5 mg TGF-b1 or PBS twice daily for 3 d, once on day 4, and harvested 4 from Th1-prone C57BL/6 mice. Intriguingly, mast cells from Th2- h later. Peritoneal lavage cells were counted with trypan blue and stained prone 129/Sv mice had much higher Stat5 expression that was un- for flow analysis. Ear skin and small intestine sections were fixed in 4% changedbyTGF-b1 treatment. Whereas TGF-b1 suppressed IgE- paraformaldehyde and stained with chloracetate esterase before counting blinded samples with light microscopy. Mean mast cell numbers per 403 mediated cytokine production and SCF-induced migration in C57BL/ field were determined from 10 fields/mouse, after which the mean numbers 6 mast cells, it had no effect or even enhanced mast cell activation were then averaged from all 5 mice. Mast cells from entire small intestine among 129/Sv mast cells. Similarly, TGF-b1 suppressed Stat5 ex- cross sections were counted from at least three sections/mouse, and the pression and IgE-induced IL-6 secretion among approximately half mean mast cell number from each mouse was then averaged from all five mice. of the human donor–derived mast cell populations tested. These data indicate specific pathways targeted for mast cell regulation Flow cytometric analysis and suggest that genetic predisposition to atopy may include loss of homeostatic regulation by cytokines such as TGF-b1. Surface expression analysis was performed using a standard flow cytometry protocol described previously (28). To assess the percentage of peritoneal populations, we identified B cells as B220+ cells, neutrophils as Materials and Methods CD11b2/Gr1+, high side-scatter cells, and macrophages as CD11b+/Gr-12, + + Animals high forward-scatter cells. Mast cells were identified as FcεRI /Kit . For cytokine measurements, cells were first activated for 90 min with IgE + Ag,

C57BL/6 and 129S1/SvImJ (hence referred to as 129/Sv) mice were pur- then cultured for 5 h in the presence of 5 mM monensin at 37˚C, before Downloaded from chased from the Jackson Laboratory (Bar Harbor, ME). They were maintained ﬁxation in PBS/4% paraformaldehyde, and staining in the presence of in a speciﬁc pathogen-free facility at Virginia Commonwealth University 0.5% saponin with PE-conjugated anti-TNF. Stat5 was detected using (VCU). Protocols and studies involving animals were performed in accor- rabbit anti-Stat5 (clone C13341, Assay Biotech), with spectrum red- dance with the VCU Institutional Animal Care and Use Committee guidelines. conjugated rat anti-rabbit IgG (Southern Biotechnology).

Cytokines and reagents ELISA

Purified DNP-specific mouse IgE was purchased from BD Pharmingen (San Cytokine levels were detected by standard ELISA Kits purchased from http://www.jimmunol.org/ Diego, CA). DNP-coupled human serum albumin (HSA) was purchased PeproTech (Rocky Hill, NJ) using the manufacturer’s protocol. from Sigma Fine Chemicals (St. Louis, MO). Murine IL-3, SCF, and human TGF-b1 were purchased from PeproTech (Rocky Hill, NJ). Abs recog- Western blot analysis nizing actin were bought from Sigma-Aldrich (St. Louis, MO). Rat anti- mouse FcgRII/RIII (2.4G2), purified mouse IgE, purified anti-mouse IgE, Western blotting was performed using 50 mg total cellular protein per FITC-conjugated rat IgG isotype control, and FITC-conjugated anti-mouse sample. Protein was loaded and separated over 8–16% or 4–20% gradient CD117 (c-Kit) were purchased from BD Pharmingen. PE-conjugated rat SDS polyacrylamide gels (Bio-Rad, Hercules, CA). Proteins were trans- IgG2b isotype control and PE-conjugated anti-mouse IgE were purchased ferred to nitrocellulose (Pall Corporation, Ann Arbor, MI), and blocked for from eBioscience (San Diego, CA). Anti-Akt, -Stat5, and -Syk Abs were 60 min in 5% nonfat dry milk in TBST. Blots were incubated in 5% nonfat purchased from Cell Signaling (Danvers, MA). Anti-Fyn Ab was pur- dry milk/TBST with a 1:1000 dilution of anti-Fyn, -Syk, -STAT5, -Akt, chased from Santa Cruz Biotechnology (Santa Cruz, CA). or –b-actin overnight at 4˚C with gentle rocking. Blots were washed six by guest on September 27, 2021 times for 10 min each in TBST, followed by incubation in Blotto B con- Mouse mast cell cultures taining a 1:5000 dilution of HRP-linked anti-IgG (matched to the appro- BMMCs were derived from mice by culture in complete RPMI (cRPMI) priate primary Ab source species, from Cell Signaling, Danvers, MA). Size estimates for proteins were obtained using m.w. standards from Bio-Rad 1640 medium (Invitrogen Life Technologies, Carlsbad, CA) containing (Hercules, CA). 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 1 mM sodium pyruvate, and 10 mM HEPES (Biofluids, Rock- ville, MD), supplemented with IL-3 containing supernatant from WEHI-3 Nucleofection of small interfering RNAs cells and SCF-containing supernatant from BHK-MKL cells for 21 d. The C57BL/6 BMMCs were transfected with a Dharmacon ON-TARGET final concentration of IL-3 and SCF was adjusted to 1 and 10 ng/ml, re- SMARTpool of Stat5B or nontargeting small interfering RNAs (siR- spectively, as measured by ELISA. Peritoneal cells were extracted in PBS, NAs; Thermo Scientific). The Amaxa Nucleofector II and Cell Line then expanded in culture for 7 d as described for BMMCs. For in vitro Nucleofector Kit V (Lonza Cologne GmbH, Cologne, Germany) were analyses, BMMCs were washed and incubated at 37˚C for 4 h in cRPMI used according to the manufacturer’s protocol for human monocytes. For 1640 without cytokines to reduce background phosphorylation. Cells were 3 5 the initial nucleofection step, a concentration of 2 mM siRNA and three then plated at 5 10 cells/ml and incubated at 37˚C for 4 d in cRPMI million cells were used. The cultures were allowed to rest for 72 h b 1640 with IL-3 (5 ng/ml) with or without TGF- 1 (10 ng/ml). Cells were in growth medium added to dilute the siRNAs to a final concentration of activated by culturing overnight with 0.5 mg/ml anti-DNP IgE, followed by 57 nM. Efficiency of knockdown was determined through immunoblot cross-linking with 50 ng/ml DNP-HSA for 16 h, after which culture analyses. supernatants were harvested. Human mast cell cultures Stat5B transfection All protocols involving human tissues were approved by the human studies Stat5B, encoded by the plasmid pMSCV that bears the PGK promoter, was Internal Review Board at VCU or at the National Institute of Allergy and transfected in C57BL/6 BMMCs using the Amaxa Nucleofector, with Infectious Diseases, National Institutes of Health. Surgical skin samples program Y-001 and Amaxa reagents. Cells were cultured for 3 d before were obtained from the Cooperative Human Tissue Network of the Na- assessing Stat5 expression levels and TGF-b1 responsiveness. tional Cancer Institute or from the National Disease Research Interchange. Skin MCs were prepared and cultured as described previously (26) and Mast cell migration were used after 6–10 wk, at which time purity was essentially 100% mast cells, as determined by staining with toluidine blue. Peripheral blood hu- To measure migration, we cultured cells in IL-3 with or without TGF-b1 for man mast cells were developed from CD34+ pluripotent peripheral blood 72 h, then washed and replated them at 2 3 106 cells/ml in cRPMI progenitor cells in StemPro-34 medium with supplement (Life Technolo- lacking FCS and containing 10 mg/ml BSA. Cells (200 ml) were added to gies) containing penicillin (100 U/ml), streptomycin (100 U/ml), L-gluta- the top chamber of an 8-mm Transwell insert and assessed after a 16-h mine (2 mM), IL-3 (30 ng/ml; first week only), IL-6 (100 ng/ml), and SCF incubation in response to SCF in the bottom chamber. Live cell migration (100 ng/ml; PeproTech) for 8 wk. Human mast cells were sensitized and was assessed using timed flow cytometry–based counting of quadruplicate activated as described for BMMCs earlier, with the exception that culture samples harvested from the bottom chamber, using propidium iodide to media included soybean tryptase inhibitor (100 mg/ml) to prevent cytokine exclude dead cells. Control wells were stimulated with media alone and degradation (27). were used as the denominator to determine fold migration. The Journal of Immunology 4507

Statistics Stat5, FcεRI activates Syk kinase, which, along with Fyn, induces Data shown in each figure are the mean and SEs of the indicated number Akt activation (33). We therefore investigated TGF-b1 suppres- of samples. For comparisons of two samples, Student t test was used. For sion of these proteins. BMMCs treated with TGF-b1 for 4 d comparisons of multiple samples with a control group, one-way ANOVA showed no change in Syk or Akt expression, but had a signifi- with Dunnett’s post hoc test was used. Cluster analysis from the R sta- cant reduction in Fyn and Stat5 levels (Fig. 2A–D). There was no tistical package was used to assign human mast cell populations into two change in mRNA expression encoding any of these proteins, as groups based on TGF-mediated suppression of Stat5. judged by quantitative PCR (data not shown). As shown in Fig. 2E, these data were not restricted to in vitro–derived BMMCs, Results because peritoneal mast cells cultured for 4 d with TGF-b1 also b TGF- 1 decreases mouse mast cell numbers and c-Kit and had diminished Stat5 expression. These data indicated that TGF- « Fc RI expression in vivo b1 selectively altered FcεRI signaling, targeting the Fyn-Stat5 path- We previously found that TGF-b1 suppresses mast cell function way through a posttranscriptional mechanism. and survival in vitro (29–31). In this work, we investigated these TGF-b–mediated suppression varies with genetic background effects in vivo, first by injecting C57BL/6 mice with TGF-b1 i.p. for 4 d and assessing changes in peritoneal mast cells. When gating We have previously found that mast cells from the Th2-prone 129/ on Kit+ mast cells, we found that TGF-b1 significantly diminished Sv background are resistant to IL-10 effects (34) and express Fyn at FcεRI and c-Kit expression on peritoneal mast cells (Fig. 1A, 1B). high levels (35). To determine whether genetic background alters We also noted that mast cell IgE receptors were similarly dimin- TGF-b1 responses, we compared Th1-prone C57BL/6 and Th2- ished after only 2 d of injections, whereas c-Kit was unchanged prone 129/Sv mast cell populations. As shown in Fig. 3A and 3B, Downloaded from (data not shown). FcεRI inhibition was not related to reduced cir- TGF-b1 significantly reduced Fyn and Stat5 expression in C57BL/6 culating IgE during this short course of TGF-b1 treatment because BMMCs, as expected. In contrast, BMMCs cultured from 129/Sv we found no difference in serum IgE levels (Fig. 1C). These data mice had Fyn and Stat levels that trended down after TGF-b1 matched the changes in mast cell surface Ags we have previously culture but did not reach statistical significance (p = 0.075 for Fyn, noted in vitro (29, 30, 32). p = 0.15 for Stat5). More strikingly, 129/Sv BMMCs expressed two , We also found a 65% decrease in mast cell numbers (Fig. 1D). to three times more Fyn and Stat5 protein than C57BL/6 cells (p http://www.jimmunol.org/ Increased TGF-b1 was detectable in plasma within 4 h postin- 0.01), and post–TGF-b1 levels approached basal expression among jection and decreased to baseline levels ∼24 h later (Fig. 1E). This C57BL/6 BMMCs. These data suggest that genetic background suggested that TGF-b1 injection might alter mast cell migration, may predict TGF-b1 effects. perhaps explaining the decline in peritoneal mast cells. However, To determine whether altered Stat5 regulation is functionally mast cell numbers in the skin and intestine were unchanged (Fig. important, we overexpressed Stat5B (∼2-fold) in C57BL/6 BMMCs 1F). Therefore, it seems unlikely that the loss of peritoneal mast by transfection (Fig. 3C). TGF- b1 reduced IgE-mediated IL-6 se- cells was caused by emigration to other sites. This decline could cretion in control vector-transfected cells as expected. By contrast, be because of cell death. In support of this theory, TGF-b1 induces Stat5B-transfected cells showed a slight but significant increase in mast cell apoptosis in vitro after 4–6 d (31). Similarly, peritoneal IL-6 (Fig. 3D). Thus, increasing Stat5B can prevent suppression of by guest on September 27, 2021 mast cell numbers were unchanged after 2 d of injections (data not the IgE response. Collectively, these data suggest that both Fyn/ shown) but significantly decreased on day 4. These data demon- Stat5 expression and TGF-b1 responsiveness can vary with ge- strated that TGF-b1–mediated suppression of the mast cell com- netic background, which can contribute to loss of mast cell ho- partment can be translated to an in vivo setting. meostasis. We expanded the study of 129/Sv responses to TGF-b1 to in- b TGF- 1 suppresses the Fyn-Stat5 pathway clude in vivo responses. 129/Sv mice were injected i.p. twice daily The suppressive effects of TGF-b1 may stem from altered FcεRI for 4 d, as previously done with C57BL/6 mice. TGF-b1 sup- signaling. We recently found that Stat5, activated by Fyn kinase, is pressed mast cell FcεRI and c-Kit expression to a similar extent critical for IgE-mediated signaling (23, 24). In addition to Fyn and observed with C57BL/6 mice (Fig. 4A, 4B). We also compared the

FIGURE 1. TGF-b1 decreases mouse mast cell number and c-Kit and FcεRI expression in vivo. TGF- b1 or PBS was injected i.p. twice daily for 4 d, and peritoneal lavage cells were assessed after a final injection on day 5 as described in Materials and Methods. (A and B) Peritoneal mast cell FcεRI and c-Kit expression, as determined by flow cytometry. (C)SerumIgE levels as measured by ELISA. (D) Changes in peritoneal mast cell numbers, based on FcεRI+/Kit+ gating and cell counting. Data shown in (A)–(D) are representative of two separate experiments that yielded similar results. (E) Plasma TGF-b1 levels, as determined by ELISA from five animals per group. Control received PBS. (F)Mast cell numbers in ear skin or small intestine (n = 5/group), based on histological staining, as described in Materials and Methods.DatashownaremeanandSE. 4508 GENOTYPE-DEPENDENT EFFECTS OF TGF-b1 ON MAST CELL FUNCTION Downloaded from http://www.jimmunol.org/

FIGURE 2. TGF-b1 selectively suppresses Fyn and Stat5 expression. BMMCs were cultured for 4 d in IL-3 (5 ng/ml) with or without TGF-b1 (10 ng/ ml), after which the cells were lysed. Lysates were blotted for (A) Syk, (B) Fyn, (C) Akt, or (D) Stat5. Data are representative of three populations, with mean 6 SE, after normalizing to actin loading, shown in the bottom bar charts. (E) Peritoneal cells were cultured for 4 d in IL-3 and SCF, in the presence or absence of TGF-b1. Stat5 expression on c-Kit+ mast cells was determined by ﬂow cytometry. Data shown are mean 6 SE from four mice. by guest on September 27, 2021 relative change in several immune cell lineages in the peritoneum, But the most overt difference was among mast cells, with 129/Sv which were diminished by TGF-b1 (Fig. 4C). Although the trends mice showing ,10% loss, whereas C57BL/6 mast cell numbers were similar, several differences were notable between the two declined by .70%. Thus, the in vivo response of 129/Sv mice to mouse strains. C57BL/6 mice showed signiﬁcantly diminished TGF-b1, especially within the mast cell lineage, differed from peritoneal cells, especially B cells, CD4 T cells, and neutrophils. C57BL/6 animals.

FIGURE 3. C57BL/6 and 129/Sv BMMCs show variation in TGF-b1 responsiveness. (A and B) Fyn and Stat5 expression were measured by Western blotting, using lysates from the indicated BMMC populations cultured 6 TGF-b1 for 4 d as described in Fig. 2. Fyn and Stat5 changes induced by TGF- b1 were quantiﬁed using three to ﬁve samples per point, with all points compared with control C57BL/6 samples, after normalization to actin loading. (C) Stat5B was transfected into C57BL/6 BMMCs, and Stat5 expression relative to control transfection with empty vector was determined by Western blotting. (D) Transfected BMMC populations described in (C) were cultured for 4 d 6 TGF-b1 then activated with IgE + DNP- HSA for 16 h. IL-6 concentration in supernatants was determined by ELISA. Data shown are mean 6 SE from quadruplicate samples. The Journal of Immunology 4509

expression, this curve quickly reached a plateau, indicating that a small number of receptors were needed for maximal cytokine production. This suggested that the 40–50% decrease in IgE receptor expression elicited by TGF-b1 was insufﬁcient to reduce TNF production. We additionally noted that 129/Sv mast cells produced more TNF at any given FcεRI level than C57BL/6 BMMCs (Fig. 5C), suggesting that IgE signaling in 129/Sv cells is more potent. The effects of TGF-b1 treatment were evident when we determined the fraction of cells expressing TNF. As shown in Fig. 5D and 5E, the average TNF-producing cell made only slightly less TNF when exposed to TGF-b1, whereas the fraction of TNF- producing cells decreased by 50% in C57BL/6 BMMCs. In contrast, the proportion of TNF+ cells was unchanged in the 129/Sv population. These data collectively support the hypothesis that TGF- b1 decreases mast cell responses to IgE not by downregulating its receptor, but by altering the outcome of FcεRI signaling. 129/Sv mast cells are resistant to this suppressive effect, showing no change in cytokine-producing cells. Downloaded from SCF-induced migration requires Stat5B and is blocked by TGF-b1: inﬂuence of genetic background In addition to FcεRI, TGF-b1 also suppressed c-Kit expression. SCF, the ligand for c-Kit, is a potent mast cell survival and migration factor (36–39). We previously found that SCF activates

Stat5 and requires this protein to elicit survival (25, 40). The role of http://www.jimmunol.org/ Stat5 in SCF-induced migration is unknown. Because TGF-b1 suppressed Stat5 as well as c-Kit, we examined the importance of Stat5 in SCF-induced migration and how TGF-b1altersthisre- sponse. Using siRNA, we demonstrated that SCF-mediated migra- FIGURE 4. Genetic background affects TGF-b1 effects in vivo. (A and tion was reduced to background levels when Stat5B was suppressed B) 129/Sv mice (n = 5) were injected with TGF-b1 for 4 d as described in (Fig. 6A). Loss of Stat5B in this short-term assay had no impact on Fig. 1. Anti-IgE and anti–c-Kit staining intensity among FcεRI+/c-Kit+ survival, which did not differ between control- and Stat5B- peritoneal mast cells was measured by flow cytometry. (C) The fold change suppressed cells under the migration conditions (data not shown). in number of each peritoneal cell lineage was measured by flow cytometry, This pathway, like SCF-mediated survival or IgE-induced cytokine by guest on September 27, 2021 using mice of the indicated genotype injected with PBS as the reference secretion, appears to be dependent on Stat5B. Materials and sample. Cell populations were identified as described in We next examined the ability of TGF-b1 to suppress SCF- Methods. n = 5. The indicated p values were obtained by Student t test. induced migration in C57BL/6 or 129/Sv BMMCs. As shown in Fig. 6B, there was a striking contrast between these populations. These in vivo data partly confirmed the TGF-b1–resistant Prior culture in TGF-b1 for 3 d reduced SCF-mediated migration phenotype of 129/Sv mast cells. However, they also demonstrated by .50% in C57BL/6 BMMCs. However, 129/Sv mast cells ex- that TGF-b1 reduced FcεRI and c-Kit expression, indicating that posed to TGF-b1 for 3 d showed at least 5-fold greater migration some TGF-mediated suppressive effects persist on the 129/Sv back- in response to SCF. These data showed that TGF-b1 effects can ground. Why responses vary and how they affect mast cell func- diverge greatly among genetically different mast cell populations, tion remained unclear. We found that BMMCs derived from the impacting the functional effects of both IgE and SCF. two mouse strains had similar expression of TGF-bRI and TGF- b bRII (Fig. 5A), indicating that TGF-b1 resistance was not related Variable responsiveness to TGF- 1 is consistent in primary to loss of TGF-bR expression. We next addressed the functional human mast cells relevance of TGF-b1 effects by examining IgE-mediated cytokine We extended our study of the Fyn-Stat5 cascade, assessing TGF- production. Each BMMC population was cultured for 4 d in the b1–mediated suppression in three preparations of primary human presence or absence of TGF-b1, then activated by IgE XL. As we skin mast cells. Surprisingly, none of these isolates showed a and others have previously noted (20, 29, 32), TGF-b1 reduced change in Fyn expression when cultured with TGF-b1 for 4 d IgE-induced cytokine secretion by at least 50% among C57BL/6 (data not shown). In contrast, Stat5 expression was diminished 50– BMMCs. In contrast, 129/Sv cells cultured with TGF-b1 showed 60% in two mast cell isolates, whereas a third showed little change no loss of TNF production, and actually secreted more IL-6 (Fig. 5B). (Fig. 7A). By expanding this study to a total of 9 donors, we found Hence 129/Sv mast cells were functionally resistant to TGF-b1– variable effects on Stat5 expression that fell into two groups upon mediated suppression. cluster analysis. As shown in Fig. 7B, mast cells from three donors The ability of TGF-b1toreduceFcεRI expression without sup- (group A) showed no decrease in Stat5 after culture in TGF-b1. pressing cytokine secretion led us to investigate the importance of By contrast, cells from six donors (group B) decreased Stat5, with FcεRI suppression under these conditions. We examined the re- a significant difference between the two groups. These data de- lationship between FcεRI and TNF levels, using intracellular monstrated that the variable Stat5 suppression in C57BL/6 and staining to examine groups of cells with similar FcεRI expression 129/Sv mast cells may have a human correlate. levels. Among TNF+ cells, those receiving TGF-b1 produced as Because of low cell numbers, we were not able to test all human much TNF as untreated cells at each FcεRI level measured (Fig. mast cell populations for both Stat5 expression and IgE responses 5C). Whereas TNF levels initially increased in parallel with FcεRI after TGF-b1 stimulation. We did find that among five prepara- 4510 GENOTYPE-DEPENDENT EFFECTS OF TGF-b1 ON MAST CELL FUNCTION Downloaded from http://www.jimmunol.org/ FIGURE 5. TGF-b1 effects on IgE-mediated cytokine production. (A) TGF-bRI and TGF-bRII surface expression on C57BL/6 and 129/Sv BMMCs were measured by flow cytometry. Data shown are means 6 SEM of 6–15 samples. (B) IgE-induced IL-6 and TNF production was assessed by ELISA after 4 d of culture in IL-3 6 TGF-b1. Data are means 6 SEM of three populations. (C) BMMCs were cultured 6 TGF-b1 for 4 d, then activated with IgE XL and assessed for TNF production by intracellular staining as described in Materials and Methods.FcεRI expression levels were determined using anti-IgE staining, after gating on TNF+ cells. Data shown are geometric mean fluorescence intensity (gMFI) of seven to nine samples per point. *p , 0.05 when using t test to compare TNF staining of C57BL/6 cells with 129/Sv cells cultured in the absence of TGF-b1. (D and E) The TNF+ populations from (C) were used to determine the gMFI of TNF staining among all TNF+ cells and the fraction of cells producing TNF. Data shown are means 6 SEM from eight to nine samples per point. by guest on September 27, 2021 tions from different donors, three showed resistance to TGF-b1, basis, TNF-producing cells make similar amounts regardless of as demonstrated by continued IL-6 secretion in response to IgE TGF-b1 exposure. In addition, the relationship between FcεRI lev- cross-linkage. In contrast, two populations showed a significant els and TNF secretion was not altered by TGF-b1 (Fig. 5). There- decrease in IgE-mediated IL-6 production (Fig. 7C). Two mem- fore, although TGF-b1 suppresses FcεRI expression, this is not bers of group A, which showed no change in Stat5, were included causally linked to reduced cytokine production. These data are in these experiments. One population was TGF-b1 resistant, logical: because TGF-b1 reduces FcεRI surface expression 50– whereas the other was sensitive; hence TGF-b1 may inhibit mast 60% (29), and human mast cells express ∼200,000 IgE receptors cell function even in the absence of Stat5 suppression. (47), an excess of receptors needed for activation would still be available. By comparison, the productive use of anti-IgE therapy Discussion (e.g., omalizumab) reduces FcεRI levels by $95% (48, 49). In- Mast cells are known as key initiators of the atopic response. We stead, the fraction of TNF-producing mast cells is halved by TGF- have sought to understand how factors such as TGF-b1 control b1 exposure, suggesting that a subset of the population has de- mast cell homeostasis. Human and mouse mast cells secrete TGF- fective IgE signaling. b1 along with proteases such as chymase that activate latent TGF- Supporting this theory, we demonstrate that TGF-b1 reduces b1 (16). This has direct consequences for allergic disease, because Fyn and Stat5 expression in mouse mast cells. Among peritoneal mast cell–derived TGF-b1 has been associated with the induction cells treated ex vivo (Fig. 2A), we noted a decrease in both per- of regulatory T cells (41), smooth muscle contractility (42), and cell Stat5 levels and the percentage of the population expressing fibroblast proliferation and collagen deposition (43–45). More- Stat5. As we have recently shown, Fyn associates with Stat5 and is over, TGF-b1 has many direct effects on mast cells, including required for Stat5 tyrosine phosphorylation (24). Because both increased mouse mast cell protease-1 and -7 expression, en- Fyn and Stat5 are essential for IgE-induced cytokine secretion (23, hanced adhesion, and increased migration. However, we and 50), a blockade in this pathway could explain our observations. others have also noted that TGF-b1 has suppressive activities on Unlike IL-10, which suppresses Syk and Akt in addition to Fyn mast cells, including decreased proliferation, FcεRI expression, and Stat5 (28), the effects of TGF-b1 were more selective. We cytokine production, and reduced c-Kit expression, which some also noted no change in Fyn or Stat5 mRNA expression (data not correlated with the induction of apoptosis (reviewed in Refs. 46 shown), suggesting alterations in protein production. We have and 16). How these suppressive activities are carried out has not previously found that TGF-b1 reduces the translational efficiency been determined. of FcεRI b (29) and hypothesize that this mechanism may also be Our data allow us to postulate a mechanism by which TGF-b1 in place here. acts on mast cells. We show that C57BL/6 BMMCs exposed to TGF-b1 is one of the more enigmatic factors that contribute to TGF-b1 produce less TNF as a population, but that on a per-cell atopic disease. Several studies have noted polymorphisms in the The Journal of Immunology 4511

a review of TGF-b1 effects on atopic disease, see Refs. 51–53). Work in mouse asthma models has implicated TGF-b1asanex- acerbating agent. For example, blocking TGF-b1 signaling through overexpression of the Smad7 antagonist reduced inﬂammation (54), as did treatment with anti–TGF-b1 Ig (55) in murine airway hyperresponsiveness (AHR) models. A recent study found that AHR treatment with the antimalarial drug mepacrine suppressed airway remodeling and correlated with reduced TGF-b1 production (56). It has recently been suggested that targeting TGF-b1maybeather- apeutic intervention, based on data such as these (51–53). In contrast, other studies ﬁnd that transient increases in TGF-b1 levels may suppress AHR in mouse models. Fu and coworkers (57) used a TGF-b1–expressing plasmid to suppress AHR. A recent study by Nemeth and colleagues (58) suggests that TGF-b1 secretion by bone marrow stromal cells correlates with reduced eosinophilia, mucus production, and cytokines in mouse AHR. Fur- thermore, genotypes associated with low TGF-b1 production have been linked with asthma (59). Separate from asthma, food allergy

studies have demonstrated that oral TGF can suppress atopic in- Downloaded from ﬂammation (60). Finally, regulatory T cell function, including allergy suppression, has been postulated to occur partly via TGF production (61). A unifying hypothesis is that TGF-b1, although immunosuppressive and hence beneﬁcial to asthma amelioration, also exacerbates the remodeling phase of chronic asthma, hence

short-term exposure to TGF-b1 is beneficial, whereas long-term http://www.jimmunol.org/ exposure is detrimental. Perhaps the most striking observation in this study was the im- FIGURE 6. The role of Stat5 in SCF-mediated migration and its portance of genetic background in TGF-b1 responsiveness. When regulation by TGF-b1. (A) C57BL/6 BMMCs were transfected with human mast cell populations were exposed to TGF-b1, we noted nontargeting (NT) or Stat5B-targeting (siB) siRNAs before measuring two groups: one with significantly reduced Stat5 levels after incu- SCF-induced migration as described in Materials and Methods. Data bation with TGF-b1, compared with another group that resisted shown are mean 6 SEM of n = 4 from one of two representative suppression. Similarly, we noted responsive and resistant groups experiments. Inset is Western blotting to show efficacy and specificity when assessing TGF-b1–mediated effects on IL-6 secretion. Fur-

B by guest on September 27, 2021 of siRNA treatment. ( ) BMMCs of the indicated background were ther work is needed to discern a link between reduced Stat5 and 6 cultured in IL-3 TGF-b1 for 3 d before measuring SCF-induced mi- attenuated cytokine production. However, treatment of one prep- gration. Data shown are means 6 SEM from 14 samples analyzed in 2 aration of human skin mast cells with TGF-b1 did not reduce Stat5 experiments. but still suppressed IL-6 secretion. Thus, we conclude that Stat5 suppression may not be an absolute requirement for blocking mast TGF-b1 promoter that associate with asthma. TGF-b1 levels are cell function in human mast cells. Importantly, our interpretations are increased in atopic dermatitis and in asthma, whereas TGF-b1is limited by access to a small number of human mast cell populations. associated with lung remodeling and collagen deposition (for This study demonstrates the plausibility of TGF-b1–sensitive and

FIGURE 7. Variation in human mast cell responsiveness to TGF-b1. (A) Mast cells cultured from human skin donors were cultured 6 TGF-b1 for 4 d as described in Materials and Methods, and Stat5 expression was assessed by Western blotting. The change in Stat5 expression was determined by comparison with the control lane, after normalizing to actin loading. Data are from three independent experiments. (B)TGF-b1–mediated change in Stat5 expression, as determined in (A), from a total of nine subjects. (C) Human mastcellswereculturedfor4dinthe presence or absence of TGF-b1, before activation with IgE and Ag for 16 h. Culture supernatants were analyzed for the production of IL-6. Data shown are mean 6 SE from three resistant and two sensitive populations, each analyzed in triplicate. 4512 GENOTYPE-DEPENDENT EFFECTS OF TGF-b1 ON MAST CELL FUNCTION

–resistant individuals; further work with a larger number of donors Acknowledgments is needed to fully characterize these groups. We thank Dr. Michael Gurish, Harvard Medical School, for kind assistance These data suggested that Stat5 is a target of TGF-b1, and that with histology samples. genetic variation might alter TGF-b1 effects. We reasoned that the question of genetic influence could be addressed using in-bred mouse Disclosures strains with known Th1- or Th2-prone responses. Because Th2- The authors have no financial conflicts of interest. prone 129/Sv mice were previously shown to have increased mast cell numbers and histamine release in vivo (35), we compared their TGF-b1 response with Th1-prone C57BL/6 mice, the source References of BMMCs for the majority of our studies. We found that 129/Sv 1. Oettgen, H. C., and R. S. Geha. 1999. IgE in asthma and atopy: cellular and molecular connections. J. Clin. Invest. 104: 829–835. and C57BL/6 BMMCs expressed TGF-b1 receptors at comparable 2. Umetsu, D. T., J. J. McIntire, O. Akbari, C. Macaubas, and R. H. DeKruyff. levels, but that 129/Sv mast cells were completely resistant to sup- 2002. Asthma: an epidemic of dysregulated immunity. Nat. Immunol. 3: 715– 720. pression of IgE-induced cytokines. In fact, IL-6 production increased 3. von Mutius, E. 2009. Gene-environment interactions in asthma. J. Allergy Clin. among 129/Sv BMMCs treated with TGF-b1. The explanation for Immunol. 123: 3–11, quiz 12–13. this enhanced response is not known, but we postulate that TGF-b1 4. Wilson, D. H., R. J. Adams, G. Tucker, S. Appleton, A. W. Taylor, and R. E. Ruffin. 2006. Trends in asthma prevalence and population changes in South could enhance activity of the Fyn-Stat5 cascade. TGF-b1 resistance Australia, 1990-2003. Med. J. Aust. 184: 226–229. correlated with 2- to 3-fold greater Fyn and Stat5 expression in 129/ 5. Yoshimura, A., Y. Wakabayashi, and T. Mori. 2010. Cellular and molecular basis Sv BMMCs, levels that were not statistically decreased by TGF-b1 for the regulation of inflammation by TGF-beta. J. Biochem. 147: 781–792. 6. Yang, L., Y. Pang, and H. L. Moses. 2010. TGF-beta and immune cells: an treatment. Fyn was previously reported to be highly expressed in important regulatory axis in the tumor microenvironment and progression. Downloaded from 129/Sv mast cells (35). Our corroboration of these data, along with Trends Immunol. 31: 220–227. 7. Kehrl, J. H., L. M. Wakefield, A. B. Roberts, S. Jakowlew, M. Alvarez-Mon, enhanced Stat5 expression, fits their TGF-b1 resistance. These data R. Derynck, M. B. Sporn, and A. S. Fauci. 1986. Production of transforming suggest that genetic background contributes to the ultimate effects of growth factor beta by human T lymphocytes and its potential role in the regu- TGF-b1 on mast cell homeostasis and implicate the Fyn-Stat5 lation of T cell growth. J. Exp. Med. 163: 1037–1050. 8. Blobe, G. C., W. P. Schiemann, and H. F. Lodish. 2000. Role of transforming pathway as a contributing factor. growth factor beta in human disease. N. Engl. J. Med. 342: 1350–1358.

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