Functional Proteomics for the Characterization of Impaired Cellular Responses to Glucocorticoids in Asthma

  • Konrad PazdrakEmail author
  • Alexander Kurosky
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 795)


In chronic airway inflammatory disorders, such as asthma, glucocorticoid (GC) insensitivity is a challenging clinical problem associated with life-threatening disease progression and the potential development of serious side effects. The mechanism of steroid resistance in asthma remains unclear and may be multifactorial. Excluding noncompliance with GC treatment, abnormal steroid pharmacokinetics, and rare genetic defects in the glucocorticoid receptor (GR), the majority of GC insensitivity in asthma can be attributed to secondary defects related to GR function. Airway inflammatory cells obtained from patients with GC-resistant asthma show a number of abnormalities in cell immune responses to GC, which suggests that there is a causative defect in GR signaling in GC-resistant cells that could be further elucidated by a functional and molecular proteomics approach. Since T cells, eosinophils, and monocytes play a major role in the pathogenesis of airway inflammation, most of the work published to date has focused on these cell types as the primary therapeutic targets in GC-insensitive asthma. We herein review several distinct techniques for the assessment of (1) the cellular response to GCs including the effect of GCs on cell viability, adhesion, and mediator release; (2) the functionality of GC receptors, including phosphorylation of the GR, nuclear translocation, and binding activities; and (3) the characterization of proteins differentially expressed in steroid-resistant cells by comparative 2DE-gel electrophoresis-based techniques and mass spectrometry. These comprehensive approaches are expected to reveal novel candidates for biomarkers of steroid insensitivity, which may lead to the development of effective therapeutic interventions for patients with chronic steroid-resistant asthma.


Steroid resistance Glucocorticoids Proteomics Asthma Mass spectrometry Cell signaling pathways Biomarkers Eosinophils Airway inflammatory cells Phosphoproteomics 



7-Aminoactinomycin D


2-Dimensional electrophoresis


A Disintegrin and metalloprotease domain family gene/protein


Apoptosis signal-regulating kinase 1


C–C motif ligand


Cyclin-dependent kinase


Chronic obstructive pulmonary disease




Glucocorticoid receptor


Glucocorticoid response element


Histone deacetylase


High-performance liquid chromatography


Heat-shock protein 90






c-JUN N-terminal kinase




MAP kinase phosphatase 1


Mass spectrometry


Peripheral blood mononuclear cells




Protein phosphatase 5


Type 2 myeloid cell


Tumor necrosis factor α


  1. Barnes PJ, Adcock IM (2003) How do corticosteroids work in asthma? Ann Intern Med 139:359–370PubMedCrossRefGoogle Scholar
  2. Bel EH, Sousa A, Fleming L, Bush A, Chung KF, Versnel J, Wagener AH, Wagers SS, Sterk PJ, Compton CH (2011) Diagnosis and definition of severe refractory asthma: an international consensus statement from the Innovative Medicine Initiative (IMI). Thorax 66:910–917PubMedCrossRefGoogle Scholar
  3. Bhavsar P, Hew M, Khorasani N, Torrego A, Barnes PJ, Adcock I, Chung KF (2008) Relative corticosteroid insensitivity of alveolar macrophages in severe asthma compared with non-severe asthma. Thorax 63:784–790PubMedCrossRefGoogle Scholar
  4. Blom M, Tool AT, Kok PT, Koenderman L, Roos D, Verhoeven AJ (1994) Granulocyte-macrophage colony-stimulating factor, interleukin-3 (IL-3), and IL-5 greatly enhance the interaction of human eosinophils with opsonized particles by changing the affinity of complement receptor type 3. Blood 83:2978–2984PubMedGoogle Scholar
  5. Bouazza B, Krytska K, Debba-Pavard M, Amrani Y, Honkanen RE, Tran J, Tliba O (2012) Cytokines alter glucocorticoid receptor phosphorylation in airway cells: role of phosphatases. Am J Respir Cell Mol Biol 47(4):464–473PubMedCrossRefGoogle Scholar
  6. Bozinovski S, Cross M, Vlahos R, Jones JE, Hsuu K, Tessier PA, Reynolds EC, Hume DA, Hamilton JA, Geczy CL, Anderson GP (2005) S100A8 chemotactic protein is abundantly increased, but only a minor contributor to LPS-induced, steroid resistant neutrophilic lung inflammation in vivo. J Proteome Res 4:136–145PubMedCrossRefGoogle Scholar
  7. Brightling CE, Green RH, Pavord ID (2005) Biomarkers predicting response to corticosteroid therapy in asthma. Treat Respir Med 4:309–316PubMedCrossRefGoogle Scholar
  8. Brode S, Farahi N, Cowburn AS, Juss JK, Condliffe AM, Chilvers ER (2010) Interleukin-5 inhibits glucocorticoid-mediated apoptosis in human eosinophils. Thorax 65:1116–1117PubMedCrossRefGoogle Scholar
  9. Butler CA, McQuaid S, Taggart CC, Weldon S, Carter R, Skibinski G, Warke TJ, Choy DF, McGarvey LP, Bradding P, Arron JR, Heaney LG (2012) Glucocorticoid receptor beta and histone deacetylase 1 and 2 expression in the airways of severe asthma. Thorax 67:392–398PubMedCrossRefGoogle Scholar
  10. Chakir J, Hamid Q, Bosse M, Boulet LP, Laviolette M (2002) Bronchial inflammation in corticosteroid-sensitive and corticosteroid-resistant asthma at baseline and on oral corticosteroid treatment. Clin Exp Allergy 32:578–582PubMedCrossRefGoogle Scholar
  11. Chen MS, Silverstein AM, Pratt WB, Chinkers M (1996) The tetratricopeptide repeat domain of protein phosphatase 5 mediates binding to glucocorticoid receptor heterocomplexes and acts as a dominant negative mutant. J Biol Chem 271:32315–32320PubMedCrossRefGoogle Scholar
  12. Cho YJ, Lee KE (2003) Decreased glucocorticoid binding affinity to glucocorticoid receptor is important in the poor response to steroid therapy of older-aged patients with severe bronchial asthma. Allergy Asthma Proc 24:353–358PubMedGoogle Scholar
  13. Cidlowski JA, Bortner CD, Gomez-Angelats M, Mann C, Scoltock A, Huang J, Evans-Storms R (2000) The cell and molecular biology of apoptosis in T-lymphocytes. Ann Endocrinol (Paris) 61:138Google Scholar
  14. Clarke M, Dodson PM (2007) PKC inhibition and diabetic microvascular complications. Best Pract Res Clin Endocrinol Metab 21:573–586PubMedCrossRefGoogle Scholar
  15. Cox G (1995) Glucocorticoid treatment inhibits apoptosis in human neutrophils. Separation of survival and activation outcomes. J Immunol 154:4719–4725PubMedGoogle Scholar
  16. Deykin A, Lazarus SC, Fahy JV, Wechsler ME, Boushey HA, Chinchilli VM, Craig TJ, DiMango E, Kraft M, Leone F, Lemanske RF, Martin RJ, Pesola GR, Peters SP, Sorkness CA, Szefler SJ, Israel E (2005) Sputum eosinophil counts predict asthma control after discontinuation of inhaled corticosteroids. J Allergy Clin Immunol 115:720–727PubMedCrossRefGoogle Scholar
  17. Durham A, Adcock IM, Tliba O (2011) Steroid resistance in severe asthma: current mechanisms and future treatment. Curr Pharm Des 17:674–684PubMedCrossRefGoogle Scholar
  18. Galliher-Beckley AJ, Williams JG, Collins JB, Cidlowski JA (2008) Glycogen synthase kinase 3beta-mediated serine phosphorylation of the human glucocorticoid receptor redirects gene expression profiles. Mol Cell Biol 28:7309–7322PubMedCrossRefGoogle Scholar
  19. Godard P, Chanez P, Siraudin L, Nicoloyannis N, Duru G (2002) Costs of asthma are correlated with severity: a 1-yr prospective study. Eur Respir J 19:61–67PubMedCrossRefGoogle Scholar
  20. Goleva E, Li LB, Eves PT, Strand MJ, Martin RJ, Leung DY (2006) Increased glucocorticoid receptor beta alters steroid response in glucocorticoid-insensitive asthma. Am J Respir Crit Care Med 173:607–616PubMedCrossRefGoogle Scholar
  21. Goleva E, Li LB, Leung DY (2009) IFN-gamma reverses IL-2- and IL-4-mediated T-cell steroid resistance. Am J Respir Cell Mol Biol 40:223–230PubMedCrossRefGoogle Scholar
  22. Goleva E, Jackson LP, Gleason M, Leung DY (2012) Usefulness of PBMCs to predict clinical response to corticosteroids in asthmatic patients. J Allergy Clin Immunol 129:687–693PubMedCrossRefGoogle Scholar
  23. Green RH, Brightling CE, McKenna S, Hargadon B, Parker D, Bradding P, Wardlaw AJ, Pavord ID (2002) Asthma exacerbations and sputum eosinophil counts: a randomised controlled trial. Lancet 360:1715–1721PubMedCrossRefGoogle Scholar
  24. Gross KL, Lu NZ, Cidlowski JA (2009) Molecular mechanisms regulating glucocorticoid sensitivity and resistance. Mol Cell Endocrinol 300:7–16PubMedCrossRefGoogle Scholar
  25. Hallsworth MP, Soh CP, Lane SJ, Arm JP, Lee TH (1994) Selective enhancement of GM-CSF, TNF-alpha, IL-1 beta and IL-8 production by monocytes and macrophages of asthmatic subjects. Eur Respir J 7:1096–1102PubMedGoogle Scholar
  26. Haselkorn T, Fish JE, Zeiger RS, Szefler SJ, Miller DP, Chipps BE, Simons FE, Weiss ST, Wenzel SE, Borish L, Bleecker ER (2009) Consistently very poorly controlled asthma, as defined by the impairment domain of the Expert Panel Report 3 guidelines, increases risk for future severe asthma exacerbations in The Epidemiology and Natural History of Asthma: Outcomes and Treatment Regimens (TENOR) study. J Allergy Clin Immunol 124:895–902PubMedCrossRefGoogle Scholar
  27. Heaney RK, Robinson DS (1976) The isolation and characterisation of hyaluronic acid in egg shell. Biochim Biophys Acta 451:133–142PubMedCrossRefGoogle Scholar
  28. Hirsch G, Lavoie-Lamoureux A, Beauchamp G, Lavoie JP (2012) Neutrophils are not less sensitive than other blood leukocytes to the genomic effects of glucocorticoids. PLoS One 7:e44606PubMedCrossRefGoogle Scholar
  29. Horgan GW (2007) Sample size and replication in 2D gel electrophoresis studies. J Proteome Res 6:2884–2887PubMedCrossRefGoogle Scholar
  30. Huang ST, Cidlowski JA (2002) Phosphorylation status modulates Bcl-2 function during glucocorticoid-induced apoptosis in T lymphocytes. FASEB J 16:825–832PubMedCrossRefGoogle Scholar
  31. Ismaili N, Garabedian MJ (2004) Modulation of glucocorticoid receptor function via phosphorylation. Ann N Y Acad Sci 1024:86–101PubMedCrossRefGoogle Scholar
  32. Kam JC, Szefler SJ, Surs W, Sher ER, Leung DY (1993) Combination IL-2 and IL-4 reduces glucocorticoid receptor-binding affinity and T cell response to glucocorticoids. J Immunol 151:3460–3466PubMedGoogle Scholar
  33. Keatings VM, Collins PD, Scott DM, Barnes PJ (1996) Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 153:530–534PubMedCrossRefGoogle Scholar
  34. Kelly EA, Busse WW, Jarjour NN (2000) Inhaled budesonide decreases airway inflammatory response to allergen. Am J Respir Crit Care Med 162:883–890PubMedCrossRefGoogle Scholar
  35. Krouwels FH, Hol BE, Bruinier B, Lutter R, Jansen HM, Out TA (1996) Cytokine production by T-cell clones from bronchoalveolar lavage fluid of patients with asthma and healthy subjects. Eur Respir J Suppl 22:95s–103sPubMedGoogle Scholar
  36. Kupczyk M, Wenzel S (2012) U.S. and European severe asthma cohorts: what can they teach us about severe asthma? J Intern Med 272:121–132PubMedCrossRefGoogle Scholar
  37. Kurowska-Stolarska M, Stolarski B, Kewin P, Murphy G, Corrigan CJ, Ying S, Pitman N, Mirchandani A, Rana B, van Rooijen N, Shepherd M, McSharry C, McInnes IB, Xu D, Liew FY (2009) IL-33 amplifies the polarization of alternatively activated macrophages that contribute to airway inflammation. J Immunol 183:6469–6477PubMedCrossRefGoogle Scholar
  38. Lane SJ, Wilkinson JR, Cochrane GM, Lee TH, Arm JP (1993) Differential in vitro regulation by glucocorticoids of monocyte-derived cytokine generation in glucocorticoid-resistant bronchial asthma. Am Rev Respir Dis 147:690–696PubMedCrossRefGoogle Scholar
  39. Leung DY, Martin RJ, Szefler SJ, Sher ER, Ying S, Kay AB, Hamid Q (1995) Dysregulation of interleukin 4, interleukin 5, and interferon gamma gene expression in steroid-resistant asthma. J Exp Med 181:33–40PubMedCrossRefGoogle Scholar
  40. Leung DY, Hamid Q, Vottero A, Szefler SJ, Surs W, Minshall E, Chrousos GP, Klemm DJ (1997) Association of glucocorticoid insensitivity with increased expression of glucocorticoid receptor beta. J Exp Med 186:1567–1574PubMedCrossRefGoogle Scholar
  41. Louis R, Sele J, Henket M, Cataldo D, Bettiol J, Seiden L, Bartsch P (2002) Sputum eosinophil count in a large population of patients with mild to moderate steroid-naive asthma: distribution and relationship with methacholine bronchial hyperresponsiveness. Allergy 57:907–912PubMedCrossRefGoogle Scholar
  42. Lu NZ, Cidlowski JA (2004) The origin and functions of multiple human glucocorticoid receptor isoforms. Ann N Y Acad Sci 1024:102–123PubMedCrossRefGoogle Scholar
  43. Lu NZ, Cidlowski JA (2005) Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes. Mol Cell 18:331–342PubMedCrossRefGoogle Scholar
  44. Lu NZ, Cidlowski JA (2006) Glucocorticoid receptor isoforms generate transcription specificity. Trends Cell Biol 16:301–307PubMedCrossRefGoogle Scholar
  45. Macek B, Gnad F, Soufi B, Kumar C, Olsen JV, Mijakovic I, Mann M (2008) Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation. Mol Cell Proteomics 7:299–307PubMedGoogle Scholar
  46. Matthews JG, Ito K, Barnes PJ, Adcock IM (2004) Defective glucocorticoid receptor nuclear translocation and altered histone acetylation patterns in glucocorticoid-resistant patients. J Allergy Clin Immunol 113:1100–1108PubMedCrossRefGoogle Scholar
  47. Meagher LC, Cousin JM, Seckl JR, Haslett C (1996) Opposing effects of glucocorticoids on the rate of apoptosis in neutrophilic and eosinophilic granulocytes. J Immunol 156:4422–4428PubMedGoogle Scholar
  48. Naseer T, Minshall EM, Leung DY, Laberge S, Ernst P, Martin RJ, Hamid Q (1997) Expression of IL-12 and IL-13 mRNA in asthma and their modulation in response to steroid therapy. Am J Respir Crit Care Med 155:845–851PubMedCrossRefGoogle Scholar
  49. Pazdrak K, Olszewska-Pazdrak B, Stafford S, Garofalo RP, Alam R (1998) Lyn, Jak2, and Raf-1 kinases are critical for the antiapoptotic effect of interleukin 5, whereas only Raf-1 kinase is essential for eosinophil activation and degranulation. J Exp Med 188:421–429PubMedCrossRefGoogle Scholar
  50. Pazdrak K, Young TW, Stafford S, Olszewska-Pazdrak B, Straub C, Starosta V, Brasier A, Kurosky A (2008) Cross-talk between ICAM-1 and granulocyte-macrophage colony-stimulating factor receptor signaling modulates eosinophil survival and activation. J Immunol 180:4182–4190PubMedGoogle Scholar
  51. Petersen BC, Budelsky AL, Baptist AP, Schaller MA, Lukacs NW (2012) Interleukin-25 induces type 2 cytokine production in a steroid-resistant interleukin-17RB + myeloid population that exacerbates asthmatic pathology. Nat Med 18:751–758PubMedCrossRefGoogle Scholar
  52. Poon AH, Eidelman DH, Martin JG, Laprise C, Hamid Q (2012) Pathogenesis of severe asthma. Clin Exp Allergy 42:625–637PubMedCrossRefGoogle Scholar
  53. Prasse A, Germann M, Pechkovsky DV, Markert A, Verres T, Stahl M, Melchers I, Luttmann W, Muller-Quernheim J, Zissel G (2007) IL-10-producing monocytes differentiate to alternatively activated macrophages and are increased in atopic patients. J Allergy Clin Immunol 119:464–471PubMedCrossRefGoogle Scholar
  54. Quaedvlieg V, Sele J, Henket M, Louis R (2009) Association between asthma control and bronchial hyperresponsiveness and airways inflammation: a cross-sectional study in daily practice. Clin Exp Allergy 39:1822–1829PubMedCrossRefGoogle Scholar
  55. Rhen T, Cidlowski JA (2005) Antiinflammatory action of glucocorticoids–new mechanisms for old drugs. N Engl J Med 353:1711–1723PubMedCrossRefGoogle Scholar
  56. Rider CF, King EM, Holden NS, Giembycz MA, Newton R (2011) Inflammatory stimuli inhibit glucocorticoid-dependent transactivation in human pulmonary epithelial cells: rescue by long-acting beta2-adrenoceptor agonists. J Pharmacol Exp Ther 338:860–869PubMedCrossRefGoogle Scholar
  57. Sale R, Sabatini F, Silvestri M, Serpero L, Petecchia L, Rossi GA (2004) Concentration-dependent activity of mometasone furoate and dexamethasone on blood eosinophils isolated from atopic children: modulation of Mac-1 expression and chemotaxis. Int Immunopharmacol 4:1687–1696PubMedCrossRefGoogle Scholar
  58. Schleimer RP (1993) An overview of glucocorticoid anti-inflammatory actions. Eur J Clin Pharmacol 45(Suppl 1):S3–S7PubMedCrossRefGoogle Scholar
  59. Schleimer RP, Bochner BS (1994) The effects of glucocorticoids on human eosinophils. J Allergy Clin Immunol 94:1202–1213PubMedCrossRefGoogle Scholar
  60. Schleimer RP, Derse CP, Friedman B, Gillis S, Plaut M, Lichtenstein LM, MacGlashan DW Jr (1989) Regulation of human basophil mediator release by cytokines. I. Interaction with antiinflammatory steroids. J Immunol 143:1310–1317PubMedGoogle Scholar
  61. Silverstein AM, Galigniana MD, Chen MS, Owens-Grillo JK, Chinkers M, Pratt WB (1997) Protein phosphatase 5 is a major component of glucocorticoid receptor.hsp90 complexes with properties of an FK506-binding immunophilin. J Biol Chem 272:16224–16230PubMedCrossRefGoogle Scholar
  62. Spahn JD, Szefler SJ, Surs W, Doherty DE, Nimmagadda SR, Leung DY (1996) A novel action of IL-13: induction of diminished monocyte glucocorticoid receptor-binding affinity. J Immunol 157:2654–2659PubMedGoogle Scholar
  63. Staples KJ, Hinks TS, Ward JA, Gunn V, Smith C, Djukanovic R (2012) Phenotypic characterization of lung macrophages in asthmatic patients: overexpression of CCL17. J Allergy Clin Immunol 130:1404–1412PubMedCrossRefGoogle Scholar
  64. Straub C, Pazdrak K, Young TW, Stafford SJ, Wu Z, Wiktorowicz JE, Haag AM, English RD, Soman KV, Kurosky A (2009) Toward the proteome of the human peripheral blood eosinophil. Proteomics Clin Appl 3:1151–1173PubMedCrossRefGoogle Scholar
  65. Straub C, Burnham JP, White AC Jr, Pazdrak K, Sanchez C, Watanabe LC, Kurosky A, Montes M (2011) Altered eosinophil proteome in a patient with hypereosinophilia from acute fascioliasis. Clin Vaccine Immunol 18:1999–2002PubMedCrossRefGoogle Scholar
  66. Syed F, Bingham B, Johnson M, Markham AF, Morrison JF (1998) The CD4+ T lymphocyte is a site of steroid resistance in asthma. QJM 91:567–572PubMedCrossRefGoogle Scholar
  67. Tliba O, Damera G, Banerjee A, Gu S, Baidouri H, Keslacy S, Amrani Y (2008) Cytokines induce an early steroid resistance in airway smooth muscle cells: novel role of interferon regulatory factor-1. Am J Respir Cell Mol Biol 38:463–472PubMedCrossRefGoogle Scholar
  68. Wang Z, Frederick J, Garabedian MJ (2002) Deciphering the phosphorylation “code” of the glucocorticoid receptor in vivo. J Biol Chem 277:26573–26580PubMedCrossRefGoogle Scholar
  69. Wang Z, Chen W, Kono E, Dang T, Garabedian MJ (2007) Modulation of glucocorticoid receptor phosphorylation and transcriptional activity by a C-terminal-associated protein phosphatase. Mol Endocrinol 21:625–634PubMedCrossRefGoogle Scholar
  70. Wenzel S (2012) Severe asthma: from characteristics to phenotypes to endotypes. Clin Exp Allergy 42:650–658PubMedCrossRefGoogle Scholar
  71. Yoshikawa H, Nakajima Y, Tasaka K (1999) Glucocorticoid suppresses autocrine survival of mast cells by inhibiting IL-4 production and ICAM-1 expression. J Immunol 162:6162–6170PubMedGoogle Scholar
  72. Zubiaga AM, Munoz E, Huber BT (1992) IL-4 and IL-2 selectively rescue Th cell subsets from glucocorticoid-induced apoptosis. J Immunol 149:107–112PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular Biology, Institute for Translational SciencesNHLBI Proteomics Center for Airway Inflammation, University of Texas Medical BranchGalvestonUSA
  2. 2.Department of Biochemistry and Molecular Biology, Institute for Translational SciencesNHLBI Proteomics Center for Airway Inflammation, University of Texas Medical BranchGalvestonUSA

Personalised recommendations