title: In search of secreted protein biomarkers for the anti-inflammatory effect of beta2-adrenergic receptor agonists: application of DIGE technology in combination with multivariate and univariate data analysis tools.
abstract: Two-dimensional difference gel electrophoresis (DIGE) in combination with univariate (Student's t-test) and multivariate data analysis, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to study the anti-inflammatory effects of the beta(2)-adrenergic receptor (beta(2)-AR) agonist zilpaterol. U937 macrophages were exposed to the endotoxin lipopolysaccharide (LPS) to induce an inflammatory reaction, which was inhibited by the addition of zilpaterol (LZ). This inhibition was counteracted by addition of the beta(2)-AR antagonist propranolol (LZP). The extracellular proteome of the U937 cells induced by the three treatments were examined by DIGE. PCA was used as an explorative tool to investigate the clustering of the proteome dataset. Using this tool, the dataset obtained from cells treated with LPS and LZP were separated from those obtained from LZ treated cells. PLS-DA, a multivariate data analysis tool that also takes correlations between protein spots and class assignment into account, correctly classified the different extracellular proteomes and showed that many proteins were differentially expressed between the proteome of inflamed cells (LPS and LZP) and cells in which the inflammatory response was inhibited (LZ). The Student's t-test revealed 8 potential protein biomarkers, each of which was expressed at a similar level in the LPS and LZP treated cells, but differently expressed in the LZ treated cells. Two of the identified proteins, macrophage inflammatory protein-1beta (MIP-1beta) and macrophage inflammatory protein-1alpha (MIP-1alpha) are known secreted proteins. The inhibition of MIP-1beta by zilpaterol and the involvement of the beta(2)-AR and cAMP were confirmed using a specific immunoassay.
authors.affiliation: TNO Pharma, Utrechtseweg 48, P.O. Box 360, 3700AJ Zeist, The Netherlands. Verhoeckx@pharma.tno.nl
authors.forename: Kitty C M
authors.initials: KC
authors.lastname: Verhoeckx
authors.forename: Marco
authors.initials: M
authors.lastname: Gaspari
authors.forename: Sabina
authors.initials: S
authors.lastname: Bijlsma
authors.forename: Jan
authors.initials: J
authors.lastname: van der Greef
authors.forename: Renger F
authors.initials: RF
authors.lastname: Witkamp
authors.forename: Robert P
authors.initials: RP
authors.lastname: Doornbos
authors.forename: Richard J T
authors.initials: RJ
authors.lastname: Rodenburg
mesh_terms: D000322:Adrenergic Agonists / Q000494:pharmacology*; D058666:Adrenergic beta-2 Receptor Agonists; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D015415:Biomarkers; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D016000:Cluster Analysis; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007118:Immunoassay; D007249:Inflammation; D008070:Lipopolysaccharides / Q000378:metabolism; D019402:Macrophage Inflammatory Proteins / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D013058:Mass Spectrometry; D009000:Monocytes / Q000378:metabolism; D015999:Multivariate Analysis; D025341:Principal Component Analysis; D011433:Propranolol / Q000494:pharmacology; D020543:Proteome; D040901:Proteomics / Q000379:methods; D013223:Statistics as Topic; D014297:Trimethylsilyl Compounds / Q000494:pharmacology; D020298:U937 Cells; D015854:Up-Regulation
chemical_list: D000322:Adrenergic Agonists; D058666:Adrenergic beta-2 Receptor Agonists; D000319:Adrenergic beta-Antagonists; D000893:Anti-Inflammatory Agents; D015415:Biomarkers; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D008070:Lipopolysaccharides; D019402:Macrophage Inflammatory Proteins; D020543:Proteome; D014297:Trimethylsilyl Compounds; C475840:Zilpaterol; D011433:Propranolol