Transcript Chemokine network in pulmonary sarcoidosis

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
 There has been little information about chemokine
network in sarcoidosis and clinical disease subtypes
Aims
 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
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
Patient characteristics
 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)
S
*
*
4.0
3.0
2.0
1.0
0.0
CCL2
CCL5
CCL7
CCL8
CCL15
CCL19
CCL22
CCL24
CXC chemokines
 Used primers/probes: Assays-on-Demand™ Gene
Expression (Applied Biosystems), LNA primers/probes
(Roche, Universal Probe Library)
C
mRNA relative expression (fold)
 Recruiment of inflammatory cells to the sarcoid lung is
mediated mainly by chemokines
C
5.0
 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)
 Relative expression was calculated using second
derivative method (RotorGene Software 6.1.71, Corbett
Research)
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
*
*
Results
S
*
*
CXCL2
CXCL3
CXCL9
CXCL10
CXCL11
CXCL12
CXCL16
Fig. 1: mRNA expression of 15 chemokines in BAL cells from
sarcoidosis patients (S) and control subjects (C). The data are
 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)
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
A
B
 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)
chemokine receptors
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.
c
7.0
mRNA relative expression (fold)
 A Th1 cell-mediated inflammatory disease characteristic
by CD4+ lymphocyte alveolitis with subsequent granuloma
formation at the site of disease
Methods
mRNA relative expression (fold)
Pulmonary sarcoidosis
CC chemokines
s
*
6.0
5.0
4.0
*
*
3.0
*
2.0
 Besides already known chemokines (CCL5/RANTES,
1.0
0.0
CCR1
CCR2variantA
CCR2variantB
CCR5
Conclusions
CXCR3
CXCR4
CXCR6
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
Support: IGA MZ CR NR/9037, MSM 6198959205
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.