chemokine mrna expression profiling in pulmonary sarcoidosis kriegova e, fillerova r, arakelyan a,...
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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
Sarcoidosis and chemokines
• Chemokines implicated in sarcoidosis alveolitis
• Most reports on individual chemokines
• Data on chemokine expression in specific clinical phenotypes needed
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
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)
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)
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)
CC chemokines
0.0
1.0
2.0
3.0
4.0
5.0
CCL2 CCL5 CCL7 CCL8 CCL15 CCL19 CCL22 CCL24
mR
NA
rel
ativ
e ex
pre
ssio
n (
fold
)
C
S
**
mRNA expression levels of 8 candidate CC chemokines in BAL cells of sarcoidosis patients and control subjects
mRNA expression levels of 7 candidate CXC chemokines
in BAL cells of sarcoidosis patients and control subjects
CXC chemokines
0.0
1.0
2.03.0
4.0
5.0
6.0
7.08.0
9.0
10.0
CXCL2 CXCL3 CXCL9 CXCL10 CXCL12 CXCL11 CXCL16
mR
NA
rel
ativ
e ex
pre
ssio
n (
fold
)
C
S
* *
*
mRNA expression levels of other chemoattractants in
BAL cells of sarcoidosis patients and control subjects
Other molecules
0.0
0.5
1.0
1.5
2.0
2.5
MIF OPN RARRES2
mR
NA
rel
ativ
e ex
pre
ssio
n (
fold
)
C
S
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
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
Relative chemokine mRNA expression in BAL cells of S patients divided by BAL lymphocyte profile (normal:<11%, high:>11%) and in
control subjects
Results summary
Sarcoidosis as a whole: 5 upregulated chemokinesCCL 5, CCL8CXCL 9, CXCL10, CXCL12
CXR III vs I: CCL2, CCL7, CCL8
Löfgren Sy: CCL5, CXCL9, CXCL10 Lympho al.: CCL5, CXCL9, CXCL10, CCL8
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
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