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