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Journal of Clinical Immunology

, Volume 32, Issue 3, pp 565–573 | Cite as

Modulation of mTOR Effector Phosphoproteins in Blood Basophils from Allergic Patients

  • Yael Gernez
  • Rabindra TirouvanziamEmail author
  • Neha Reshamwala
  • Grace Yu
  • Brittany C. Weldon
  • Stephen J. Galli
  • Leonore A. Herzenberg
  • Kari C. NadeauEmail author
Article

Abstract

The mammalian target of rapamycin (mTOR) pathway contributes to various immunoinflammatory processes. Yet, its potential involvement in basophil responses in allergy remains unclear. In this pilot study, we quantified two key mTOR effector phosphoproteins, the eukaryotic initiation factor 4E (peIF4E) and S6 ribosomal protein (pS6rp), in blood basophils from nut allergy patients (NA, N = 16) and healthy controls (HC, N = 13). Without stimulation in vitro, basophil peIF4E levels were higher in NA than HC subjects (P = 0.014). Stimulation with nut (offending) but not chicken / rice (non-offending) extract increased basophil peIF4E and pS6rp levels (+32%, P = 0.018, and +98%, P = 0.0026, respectively) in NA but not HC subjects, concomitant with increased surface levels of CD203c and CD63, both known to reflect basophil activation. Pre-treatment with the mTOR inhibitor rapamycin decreased pS6rp and CD203c responses in nut extract-stimulated basophils in NA subjects. Thus, basophil responses to offending allergens are associated with modulation of mTOR effector phosphoproteins.

Keywords

Eosinophils flow cytometry food allergy inflammation neutrophils 

Abbreviations

EDTA

ethylene diamine tetraacetic acid

eIF4E

eukaryotic Initiation Factor 4E

HC

healthy control

MFI

median fluorescence intensity

mTOR

mammalian target of rapamycin

NA

nut allergy

PBS

phosphate buffered saline

p

phosphorylated

S6rp

S6 ribosomal protein

Notes

Acknowledgments

We thank our subjects for their participation; C. Crumpton and J. Van Dyke at the Stanford FACS Facility for technical support; Drs. K. Atkuri, E. Ghosn, A. Kumar, K. Mukai, P. Sadate-Ngatchou, and M Tsai, as well as C. McDonald-Hyman for critical advice; Dr. V. Saper for recruiting two subjects; and M. Miglianico and E. Hoyte for technical assistance.

Competing interest statement

The authors declare to hold no conflict of interest with the publication of the results included in this manuscript.

Funding

Stanford School of Medicine’s Dean Fellowship (YG), Stanford Morgridge and Gallo Fellowship (YG) and the Stanford Institute of Immunity, Transplantation and Infectious Diseases Seed Grant (KCN), Orsak Family Fund (KCN), the Skippy Frank Foundation (RT and YG), the Stanford School of Medicine SPARK/SPECTRUM program (YG and KN), and United States Public Health Service grants AI23990, AI070813 and CA72074 (SJG).

Supplementary material

10875_2012_9651_MOESM1_ESM.doc (40 kb)
Supplementary Material (DOC 39 kb)
10875_2012_9651_MOESM2_ESM.jpg (88 kb)
Supplementary Figure 1 Percent increase in elF4E and S6rp upon 10-minute stimulation with nut extract in blood basophils from NA patients. Data are presented for total (T) and phosphorylated (p) forms of eIF4E and S6rp proteins. Results are shown for each of these 4 markers as % increase (mean±SE; N = 2), calculated as: 100 x ([MFI for marker upon nut extract stimulation] - [MFI for marker without stimulation]) / [MFI for marker without stimulation]. (JPEG 88 kb)
10875_2012_9651_MOESM3_ESM.jpg (165 kb)
Supplementary Figure 2 Surface CD203c and CD63 levels in blood basophils without stimulation and upon stimulation with nut (offending) or chicken / rice (non-offending) extracts. Shown are box plots (delimited by 25th and 75th percentiles, with median line inside and outside bars for 10th and 90th percentiles) for CD203c and CD63 levels in blood basophils from healthy control (HC, N = 12) and nut allergy (NA, N = 15) subjects. Each point represents an individual subject. The significance of differences between HC and NA were calculated with the Wilcoxon rank-sum test. NS for not significant. (JPEG 165 kb)
10875_2012_9651_MOESM4_ESM.jpg (43 kb)
Supplementary Figure 3 Effect of 10-minute and 30-minute stimulation with nut extract on levels of effector phosphoproteins in blood basophils from nut allergy (NA) subjects. Shown are levels of pS6rp (left) and peIF4E (right) in basophils, comparing 10-minute stimulation (black histograms) to 30-minute stimulation (grey histograms). Data are presented as % response compared to the 10-minute timepoint (mean±SE, with data at the 10-minute timepoint set at 100, by definition; N = 3 patients). (JPEG 43 kb)
10875_2012_9651_MOESM5_ESM.jpg (68 kb)
Supplementary Figure 4 Effect of rapamycin pre-treatment on blood basophil CD203c levels following nut allergen stimulation. Shown are box plots (delimited by 25th and 75th percentiles, with median line inside and outside bars marking 10th and 90th percentiles) for differential changes in CD203c in blood basophils from nut allergy (NA, N = 5) subjects, calculated as follows: differential = [MFI for CD203c upon nut allergen stimulation, without rapamycin pre-treatment] - [MFI for the same surface marker upon nut allergen stimulation, with 30-minute pre-treatment with 10 nM rapamycin]. Each point represents an individual subject. (JPEG 67 kb)
10875_2012_9651_MOESM6_ESM.jpg (463 kb)
Supplementary Figure 5 Neutrophil and eosinophil phosphoprotein profiling in blood. a: Live neutrophils and eosinophils were selected as Live/Deadlo / CD3-/ CD16+ / CD20- / CD56- / CD66b+ and CD123- and CD123+ populations, respectively. b: peIF4E and pS6rp were then quantified in gated eosinophils and neutrophils. FMO: intracellular phosphoprotein “Fluorescence Minus One” control (see Methods section above for details). Shown here is one representative NA subject, whose neutrophils and eosinophils were studied after nut extract stimulation. Blood neutrophils and eosinophils from all HC (N = 13) and NA (N = 16) subjects were successfully gated using this strategy, in all conditions studied (under no stimulation or upon stimulation with either nut or chicken / rice extracts). (JPEG 463 kb)
10875_2012_9651_MOESM7_ESM.jpg (140 kb)
Supplementary Figure 6 Phosphoprotein profiling in eosinophils without stimulation and upon stimulation with nut (offending) or chicken / rice (non-offending) extract. Shown are box plots (delimited by 25th and 75th percentiles, with median line inside and outside bars marking 10th and 90th percentiles) for levels of peIF4E (a) and pS6rp (b) in blood eosinophils from healthy control (HC, N = 13) and nut allergy (NA, N = 15) subjects. Each point represents an individual subject. The significance of differences between HC and NA were calculated with the Wilcoxon rank-sum test. NS for not significant. (JPEG 140 kb)
10875_2012_9651_MOESM8_ESM.jpg (177 kb)
Supplementary Figure 7 Phosphoprotein profiling in neutrophils without stimulation and upon stimulation with nut (offending) or chicken / rice (non-offending) extract. Shown are box plots (delimited by 25th and 75th percentiles, with median line inside and outside bars marking 10th and 90th percentiles) for levels of peIF4E (a) and pS6rp (b) in blood neutrophils from healthy control (HC, N = 12) and nut allergy (NA, N = 16) subjects. Each point represents an individual subject. The significance of differences between HC and NA were calculated with the Wilcoxon rank-sum test. NS for not significant. (JPEG 177 kb)

References

  1. 1.
    Gough NR. Focus Issue: demystifying mTOR signaling. Sci Signal. 2009;2:eg5.PubMedCrossRefGoogle Scholar
  2. 2.
    Ma XM, Blenis J. Molecular mechanisms of mTOR-mediated translational control. Nat Rev Mol Cell Biol. 2009;10:307–18.PubMedCrossRefGoogle Scholar
  3. 3.
    Ruvinsky I, Meyuhas O. Ribosomal protein S6 phosphorylation: from protein synthesis to cell size. Trends Biochem Sci. 2006;31:342–8.PubMedCrossRefGoogle Scholar
  4. 4.
    Alessi DR, Pearce LR, Garcia-Martinez JM. New insights into mTOR signaling: mTORC2 and beyond. Sci Signal. 2009;2:pe27.PubMedCrossRefGoogle Scholar
  5. 5.
    Kim MS, Kuehn HS, Metcalfe DD, Gilfillan AM. Activation and function of the mTORC1 pathway in mast cells. J Immunol. 2008;180:4586–95.PubMedGoogle Scholar
  6. 6.
    Li Q, Rao RR, Araki K, Pollizzi K, Odunsi K, Powell JD, Shrikant PA. A central role for mTOR kinase in homeostatic proliferation induced CD8+ T cell memory and tumor immunity. Immunity. 2011;34:541–53.PubMedCrossRefGoogle Scholar
  7. 7.
    Araki K, Turner AP, Shaffer VO, Gangappa S, Keller SA, Bachmann MF, Larsen CP, Ahmed R. mTOR regulates memory CD8 T-cell differentiation. Nature. 2009;460:108–12.PubMedCrossRefGoogle Scholar
  8. 8.
    Yang CS, Song CH, Lee JS, Jung SB, Oh JH, Park J, Kim HJ, Park JK, Paik TH, Jo EK. Intracellular network of phosphatidylinositol 3-kinase, mammalian target of the rapamycin/70 kDa ribosomal S6 kinase 1, and mitogen-activated protein kinases pathways for regulating mycobacteria-induced IL-23 expression in human macrophages. Cell Microbiol. 2006;8:1158–71.PubMedCrossRefGoogle Scholar
  9. 9.
    Makam M, Diaz D, Laval J, Gernez Y, Conrad CK, Dunn CE, Davies ZA, Moss RB, Herzenberg LA, Tirouvanziam R. Activation of critical, host-induced, metabolic and stress pathways marks neutrophil entry into cystic fibrosis lungs. Proc Natl Acad Sci U S A. 2009;106:5779–83.PubMedCrossRefGoogle Scholar
  10. 10.
    Kuehn HS, Jung MY, Beaven MA, Metcalfe DD, Gilfillan AM. Prostaglandin E2 activates and utilizes mTORC2 as a central signaling locus for the regulation of mast cell chemotaxis and mediator release. J Biol Chem. 2011;286:391–402.PubMedCrossRefGoogle Scholar
  11. 11.
    Meng Q, Ying S, Corrigan CJ, Wakelin M, Assoufi B, Moqbel R, Kay AB. Effects of rapamycin, cyclosporin A, and dexamethasone on interleukin 5-induced eosinophil degranulation and prolonged survival. Allergy. 1997;52:1095–101.PubMedCrossRefGoogle Scholar
  12. 12.
    Miura K, MacGlashan DW. Phosphatidylinositol-3 kinase regulates p21ras activation during IgE-mediated stimulation of human basophils. Blood. 2000;96:2199–205.PubMedGoogle Scholar
  13. 13.
    Sha Q, Poulsen LK, Gerwien J, Dum N, Skov PS. Signaling transduction pathways involved in basophil adhesion and histamine release. Chin Med J (Engl). 2006;119:122–30.Google Scholar
  14. 14.
    Rudders SA, Banerji A, Vassallo MF, Clark S, Camargo Jr CA. Trends in pediatric emergency department visits for food-induced anaphylaxis. J Allergy Clin Immunol. 2010;126:385–8.PubMedCrossRefGoogle Scholar
  15. 15.
    Burks AW. Peanut allergy. Lancet. 2008;371:1538–46.PubMedCrossRefGoogle Scholar
  16. 16.
    Krutzik PO, Irish JM, Nolan GP, Perez OD. Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clin Immunol. 2004;110:206–21.PubMedCrossRefGoogle Scholar
  17. 17.
    Tirouvanziam R, Diaz D, Gernez Y, Laval J, Crubezy M, Makam M, Herzenberg LA: An integrative approach for immune monitoring of human health and disease by advanced flow cytometry methods. In: Tuchin V, editor. Advanced optical flow cytometry: methods and disease diagnoses. Wiley-VCH Verlag GmbH & Co.; 2011. p. 333–362Google Scholar
  18. 18.
    Gernez Y, Tirouvanziam R, Nguyen KD, Herzenberg LA, Krensky AM, Nadeau KC. Altered phosphorylated signal transducer and activator of transcription profile of CD4+CD161+ T cells in asthma: modulation by allergic status and oral corticosteroids. J Allergy Clin Immunol. 2007;120:1441–8.PubMedCrossRefGoogle Scholar
  19. 19.
    Tirouvanziam R, Gernez Y, Conrad CK, Moss RB, Schrijver I, Dunn CE, Davies ZA, Herzenberg LA. Profound functional and signaling changes in viable inflammatory neutrophils homing to cystic fibrosis airways. Proc Natl Acad Sci U S A. 2008;105:4335–9.PubMedCrossRefGoogle Scholar
  20. 20.
    Nowak-Wegrzyn A, Assa’ad AH, Bahna SL, Bock SA, Sicherer SH, Teuber SS. Work Group report: oral food challenge testing. J Allergy Clin Immunol. 2009;123:S365–83.PubMedCrossRefGoogle Scholar
  21. 21.
    Gernez Y, Tirouvanziam R, Yu G, Ghosn EE, Reshamwala N, Nguyen T, Tsai M, Galli SJ, Herzenberg LA, Nadeau KC. Basophil CD203c levels are increased at baseline and can be used to monitor omalizumab treatment in subjects with nut allergy. Int Arch Allergy Immunol. 2010;154:318–27.PubMedCrossRefGoogle Scholar
  22. 22.
    Reiling JH, Sabatini DM. Stress and mTORture signaling. Oncogene. 2006;25:6373–83.PubMedCrossRefGoogle Scholar
  23. 23.
    Tung JW, Heydari K, Tirouvanziam R, Sahaf B, Parks DR, Herzenberg LA. Modern flow cytometry: a practical approach. Clin Lab Med. 2007;27:453–68.PubMedCrossRefGoogle Scholar
  24. 24.
    Kawakami T, Galli SJ. Regulation of mast-cell and basophil function and survival by IgE. Nat Rev Immunol. 2002;2:773–86.PubMedCrossRefGoogle Scholar
  25. 25.
    Botana LM, MacGlashan DW. Differential effects of cAMP-elevating drugs on stimulus-induced cytosolic calcium changes in human basophils. J Leukoc Biol. 1994;55:798–804.PubMedGoogle Scholar
  26. 26.
    Pramod SN, Venkatesh YP, Mahesh PA. Potato lectin activates basophils and mast cells of atopic subjects by its interaction with core chitobiose of cell-bound non-specific immunoglobulin E. Clin Exp Immunol. 2007;148:391–401.PubMedCrossRefGoogle Scholar
  27. 27.
    Shichijo M, Shimizu Y, Hiramatsu K, Inagaki N, Tagaki K, Nagai H. Cyclic AMP-elevating agents inhibit mite-antigen-induced IL-4 and IL-13 release from basophil-enriched leukocyte preparation. Int Arch Allergy Immunol. 1997;114:348–53.PubMedCrossRefGoogle Scholar
  28. 28.
    Tsujimura Y, Obata K, Mukai K, Shindou H, Yoshida M, Nishikado H, Kawano Y, Minegishi Y, Shimizu T, Karasuyama H. Basophils play a pivotal role in immunoglobulin-G-mediated but not immunoglobulin-E-mediated systemic anaphylaxis. Immunity. 2008;28:581–9.PubMedCrossRefGoogle Scholar
  29. 29.
    Tsai M, Chen RH, Tam SY, Blenis J, Galli SJ. Activation of MAP kinases, pp 90rsk and pp70–S6 kinases in mouse mast cells by signaling through the c-kit receptor tyrosine kinase or Fc epsilon RI: rapamycin inhibits activation of pp70–S6 kinase and proliferation in mouse mast cells. Eur J Immunol. 1993;23:3286–91.PubMedCrossRefGoogle Scholar
  30. 30.
    Kim MS, Radinger M, Gilfillan AM. The multiple roles of phosphoinositide 3-kinase in mast cell biology. Trends Immunol. 2008;29:493–501.PubMedCrossRefGoogle Scholar
  31. 31.
    Gibbs BF, Grabbe J. PI 3-kinase and MEK kinase inhibitors differentially affect IgE-dependent and IgE-independent basophil mediator release. Inflamm Res. 2001;50:S53–4.PubMedGoogle Scholar
  32. 32.
    Gibbs BF, Plath KE, Wolff HH, Grabbe J. Regulation of mediator secretion in human basophils by p38 mitogen-activated protein kinase: phosphorylation is sensitive to the effects of phosphatidylinositol 3-kinase inhibitors and calcium mobilization. J Leukoc Biol. 2002;72:391–400.PubMedGoogle Scholar
  33. 33.
    MacGlashan D Jr., Undem BJ: Inducing an anergic state in mast cells and basophils without secretion. J Allergy Clin Immunol. 2008;121:1500–1506, 1506Google Scholar
  34. 34.
    Zaidi AK, Saini SS, Macglashan Jr DW. Regulation of Syk kinase and FcεRI expression in human basophils during treatment with omalizumab. J Allergy Clin Immunol. 2010;125:902–8.PubMedCrossRefGoogle Scholar
  35. 35.
    Aerts NE, Dombrecht EJ, Bridts CH, Hagendorens MM, de Clerck LS, Stevens WJ, Ebo DG. Simultaneous flow cytometric detection of basophil activation marker CD63 and intracellular phosphorylated p38 mitogen-activated protein kinase in birch pollen allergy. Cytometry B Clin Cytom. 2008;76B:8–17.PubMedGoogle Scholar
  36. 36.
    Perez OD, Krutzik PO, Nolan GP. Flow cytometric analysis of kinase signaling cascades. Methods Mol Biol. 2004;263:67–94.PubMedGoogle Scholar
  37. 37.
    Mourani PM, Garl PJ, Wenzlau JM, Carpenter TC, Stenmark KR, Weiser-Evans MC. Unique, highly proliferative growth phenotype expressed by embryonic and neointimal smooth muscle cells is driven by constitutive Akt, mTOR, and p70S6K signaling and is actively repressed by PTEN. Circulation. 2004;109:1299–306.PubMedCrossRefGoogle Scholar
  38. 38.
    Furic L, Rong L, Larsson O, Koumakpayi IH, Yoshida K, Brueschke A, Petroulakis E, Robichaud N, Pollak M, Gaboury LA, Pandolfi PP, Saad F, Sonenberg N. eIF4E phosphorylation promotes tumorigenesis and is associated with prostate cancer progression. Proc Natl Acad Sci USA. 2010;107:14134–9.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Yael Gernez
    • 1
  • Rabindra Tirouvanziam
    • 2
    • 3
    Email author
  • Neha Reshamwala
    • 2
  • Grace Yu
    • 2
  • Brittany C. Weldon
    • 2
  • Stephen J. Galli
    • 4
  • Leonore A. Herzenberg
    • 1
  • Kari C. Nadeau
    • 2
    Email author
  1. 1.Department of GeneticsStanford University School of MedicineStanfordUSA
  2. 2.Department of PediatricsStanford University School of MedicineStanfordUSA
  3. 3.Emory+Children’s Center for Cystic Fibrosis Research, Department of PediatricsEmory University School of Medicine and Children’s Healthcare of AtlantaAtlantaUSA
  4. 4.Department of PathologyStanford University School of MedicineStanfordUSA

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