Chemokine network in pulmonary sarcoidosis Petrek 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

Methods

Aims

Pulmonary sarcoidosis

Results

Conclusions

Support: IGA MZ CR NR/9037, MSM 6198959205

Patient characteristics

Molecules of interest

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

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 Mol Biol. 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 investigate mRNA 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 specific clinical phenotypes

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)

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.

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

Sarcoidosis patients (S, n=61) (dg. - Statement on Sarcoidosis, 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)

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

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 receptors

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

CCR1 CCR2-variantA

CCR2-variantB

CCR5 CXCR3 CXCR4 CXCR6

mR

NA

re

lati

ve

ex

pre

ss

ion

(fo

ld)

c

s

*

**

*

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.

A B

CC chemokines

0.0

1.0

2.0

3.0

4.0

5.0

CCL2 CCL5 CCL7 CCL8 CCL15 CCL19 CCL22 CCL24

mR

NA

re

lati

ve

ex

pre

ss

ion

(fo

ld) C

S

* *

CXC chemokines

0.0

1.0

2.0

3.0

4.0

5.06.0

7.0

8.0

9.0

10.0

CXCL2 CXCL3 CXCL9 CXCL10 CXCL11 CXCL12 CXCL16

mR

NA

re

lati

ve

ex

pre

ss

ion

(fo

ld)

C

S

*

**

*