Methods

1. * * * Methods * * * * * * * Pulmonary sarcoidosis Results A B Molecules of interest Conclusions Aims Patient characteristics Fig. 3: Chemokine expression profiles in patient subgroup according to A) CXR stages, B) presence of LS.The data are presented as a mean fold change of relative expression compared to controls (normalized to 1); the whiskers on each box represent the SD values. Fig. 2:mRNA expression of studied chemokine receptors in BAL cells from sarcoidosis patients (S) and control subjects(C). The data are presented as a mean fold change of relative expression compared to controls (normalized to 1); the whiskers on each box represent the SD values. * p< 0.05 Chemokine network in pulmonary sarcoidosisPetrek M,Kriegova E, Fillerova R, Arakelyan A, Mrazek F, Hutyrova B, Kolek V, du Bois RM*Laboratory of Immunogenomics and proteomics & Dept. of Respiratory Medicine, Palacky University, Olomouc, Czech Republic;*National Jewish Health, Denver, CO, USA • A Th1 cell-mediated inflammatory disease characteristic by CD4+ lymphocyte alveolitis with subsequent granuloma formation at the site of disease • Recruiment of inflammatory cells to the sarcoid lung is mediated mainly by chemokines • There has been little information about chemokine network in sarcoidosis and clinical disease subtypes • Quantitative RT-PCR (RotorGene 3000 system, Corbett Research)was used to investigate mRNA expression of studied molecules in unseparated BAL cells, PSMB2 was used as a reference gene (Kriegova et al. BMC MolBiol. 2008) • Used primers/probes: Assays-on-Demand™ Gene Expression (Applied Biosystems), LNA primers/probes (Roche, Universal Probe Library) • Relative expression was calculated using second derivative method (RotorGene Software 6.1.71, Corbett Research) • To investigatemRNA expression of 15 candidate chemokines and 6 chemokine receptors in unseparated bronchoalveolar lavage (BAL) cells from sarcoidosis patients and control subjects by quantitative RT-PCR • To analyse chemokine expression profiles in patient subgroups based on specificclinical phenotypes Fig. 1:mRNA expression of 15 chemokines in BAL cells from sarcoidosis patients (S) and control subjects(C). The data are presented as a mean fold change of relative expression compared to controls (normalized to 1); the whiskers on each box represent the SD values. * p< 0.05 • Of 15 studied chemokines, mRNA expression of CCL5 (p=0.0001), CCL8 (p=0.002), CXCL9 (p=0.001), CXCL10 (p=0.0001), CXCL11 (p=0.013) and CXCL12 (p=0.045) was up-regulated in BAL cells from sarcoidosis patients vs. controls (Fig. 1) • Of 6 studied chemokine receptors,mRNA expression of CCR2-variantA (p=0.003),CCR5 (p=0.002), CXCR3 (p=0.001) and CXCR6(p=0.00004) was up-regulated in sarcoidosis vs. controls (Fig. 2) • Patient groups subdivided according to their CXR-stages and presence of LS differ in chemokine/receptor expression profiles (Fig. 3A, 3B) • Candidate chemokines (15): CCL2, CCL5, CCL7, CCL8, CCL15, CCL19, CCL22,CCL24, CXCL2, CXCL3, CXCL9, CXCL10, CXCL11, CXCL12, CXCL16 • Candidate chemokine receptors (6): CCR1, CCR2, CCR5, CXCR3, CXCR4, CXCR6 • Sarcoidosispatients(S, n=61) (dg. - Statement onSarcoidosis, 1999), clinical features+granuloma on biopsy+CD4+ lymphocytic alveolitis) • Control subjects (C, n=17) (patients without inflammation, normal BAL profile) • Subgroups based on disease phenotypes: • as assessed by chest X-ray (CXR) stage: CXR stage I (S-I, n=17), CXR stage II (S-II, n=34), CXR stage III (S-III, n=10) • patients presenting with/without Löfgren´s syndrome (LS, n=11/nonLS, n=50) • Besides already known chemokines (CCL5/RANTES, CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC), two novel chemokines are implicated in sarcoidosis: CCL8/MCP-2 and CXCL12/SDF-1. • Our data provide further evidence that chemokine/receptor profile changes during the disease course in sarcoidosis. Support: IGA MZ CR NR/9037, MSM 6198959205