1. Chemokine mRNA expression profiling in pulmonary sarcoidosis Kriegova E, Fillerova R, Arakelyan A, Mrazek F, Hutyrova B, Kolek V, du Bois R*, Petrek M Immunology and Respiratory Medicine, Palacky University and Faculty Hospital Olomouc, Czech Republic; *National Jewish, Denver, Co, USA

2. Sarcoidosis and chemokines Chemokines implicated in sarcoidosis alveolitis Most reports on individual chemokines Data on chemokine expression in specific clinical phenotypes needed

3. Aims Measure expression of genes for a spectrum of chemokines and new chemotactic molecules in sarcoidosis - already reported chemokines - new chemokine candidates - other chemotactic molecules Subanalyse expression data in clinical phenotypes

4. Methods I - study subjects Patients: n=61 Diagnosis: statement on sarcoidosis. Control subjects: n=17 (11 males, 6 females; 9 non-smokers; age 44.0 ± 16.0 yrs)

7. Methods II – molecules of interest Already reported chemokines (6): CCL2, CCL5, CCL19, CXCL9, CXCL10 and CXCL11 Candidate chemokines (9): CCL7, CCL8, CCL15, CCL22, CCL24, CXCL2, CXCL3, CXCL12, CXCL16 Other chemoattractants (3): osteopontin, RARRES, Macrophage Migration Inhibitory Factor (MIF)

10. Methods III - analytical quantitative RT-PCR on mRNA from unseparated BAL cells Normalisation to a stable reference gene PSMB2 (Kriegova et al. BMC Mol.Biol. 2008) Relative expression levels, fold change±SD division of average relative gene expression in patient group/subgroups to its average expression in control group (=1)

11. * * mRNA expression levels of 8 candidate CC chemokines in BAL cells of sarcoidosis patients and control subjects

12. mRNA expression levels of 7 candidate CXC chemokines in BAL cells of sarcoidosis patients and control subjects * * *

13. mRNA expression levels of other chemoattractants in BAL cells of sarcoidosis patients and control subjects

14. Comparison between the relative mRNA expression (target gene/reference PSMB2 gene) of chemokines CCL2, CCL7, CCL8 in unseparated BAL cells from sarcoidosis patients subdivided according the CXR stages I, II, III

15. Comparison between the relative mRNA expression of chemokines CCL5, CXCL9 and CXCL10 in BAL cells from sarcoidosis patients presenting with/without Löfgren's syndrome and in control subjects

16. Relative chemokine mRNA expression in BAL cells of S patients divided by BAL lymphocyte profile (normal: 11%) and in control subjects

17. Results summary Sarcoidosis as a whole : 5 upregulated chemokines CCL 5, CCL8 CXCL 9, CXCL10, CXCL12 CXR III vs I: CCL2, CCL7, CCL8 L ö fgren Sy: CCL5, CXCL9, CXCL10 Lympho al.: CCL5, CXCL9, CXCL10, CCL8

18. Conclusions Besides already known mediators, two novel chemokines are implicated in sarcoidosis: CCL8 and CXCL12 Different chemokines are upregulated in distinct sarcoidosis clinical phenotypes Protein work now needed to confirm and extend the present data

19. Novel chemokines CCL8 belongs to MCP family in lung only in alveolar proteinosis cell source and function in sarcoidosis? CXCL12 „homeostatic“ chemokine fibrotic milieu (fibrocyte migration) CCL7 (part of advanced dis. profile) – associated with experimental fibrosis, regulated by CCL5