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HCR : A FUNCTIONAL
ORPHAN CC CHEMOKINE RECEPTOR
IN HUMAN ASTROCYTES
Mike W. Zuurman*, Knut Biber, Hendrikus W.G.M. Boddeke
Department of Medical Physiology, University of Groningen, The Netherlands
Introduction
Chemokines are a family of chemotactic cytokines that orchestrate the immune response, ranging from homing of
immune cells to areas of inflammation to activation of immune cells. The family is divided into four subfamilies based on
their molecular structure and chemokines activate G-protein coupled chemokine receptors. To date 20 different
chemokine receptors have been identified. In addition, some orphan chemokine receptors exist that have not yet been
functionally classified. Recently, we have characterized the orphan chemokine receptor L-CCR in murine astrocytes and
have suggested a possible role for L-CCR in neuroinflammation. As the human orphan chemokine receptor HCR shows
high sequence homology to L-CCR, we have have examined the functional characteristics of HCR in human astrocytes.
Aim of the study
The goal of the current project is the functional characterization and localization of HCR in cultured adult human
astrocytes.
Results
1. HCR expression in human
astrocytes
-
C
100ng/ml 0.01 mM 0.1 mM
LPS
A
1 mM
detanonoate(NO)
B
100nM CCL5
0.8
0.7
HCR
100nM CCL2
GAPDH
0.5
0.60
0
10
20
30
40
50
0
10
Time(s)
C
20
30
Time(s)
D
1 M CCL4
0.70
100 M Carbachol
Ratio
Ratio
0.7
0.60
0.6
0.55
Figure 1. mRNA and protein expression of HCR in
cultured human astrocytes. RT-PCR (A) of mRNA
isolated from human astrocytes, stimulated with
lipopolysaccharide(100 ng/ml) or detanonoate (0.01,
0.1 and 1 mM). Confocal images of HCRimmunohistochemistry in cultured human astrocytes.
B. unspecific fluorescent background staining. C. HCR
immunofluorescence, arrow indicates a strong signal
along
an
astrocytic
process.
TRITCimmunofluorescent staining. Space bar represents 30
m.
Migration (% of control±SEM)
CCL2
177±11*
CCL5
180±10*
CCL7
147±18*
CCL8
161±15*
CCL4
112±13
XC3CL1
98±6
XCL1
110±5
CXCL8
78±15
CXCL9
102±10
CXCL12
132±8
0.8
0.65
C
Chemokine
0.65
0.6
B
3. Chemotactic responses to
different chemokines of
HCR expressing HEK cells
0.70
Ratio
MM
Ratio
A
2. Chemokine-induced calcium
gradients in HCR expressing
HEK cells
0.50
0.5
0
10
20
30
Time(s)
40
50
0
10
20
30
40
50
Time(s)
Figure 2. Detection of intracellular calcium
gradients in HEK cells transiently transfected
with full length HCR. 100 nM solutions of CCL5
(A) and CCL2 (B) produced a strong increase in
intracellulair calcium (in 50% and 30% of the
cells respectively), while up to 1 M of CCL4
produced no effect at all (C). 100 M carbachol
(D) was used as a positive control.
Table 1. Chemotaxis assays (Boyden chamber) with
HEK cells transiently transfected with HCR. Values
represent the mean number of migrating cells in
response to 10 different chemokines tested, expressed
as percentage of control (background migration) and
the standard errors of the mean. Cells responded with
chemotaxis to CCL2, CCL5, CCL7 and CCL8. Asterixes
indicate significant migration when compared to
control after a student’s t-test with n=6.
4. Chemokine induced reorganization of F-actin in human astrocytes
A
B
C
D
Figure 3. Fluorescence microscopy images of
cultured human astrocytes stained for F-actin after
stimulation with 10-8 M CCL2 for different time
periods: 0 s (A), 30 s (B), 1 min (C) and 5 min (D).
Note the redistribution of F-actin from parallel fiber
organization to rim-like polarization (arrows).
Phalloidine-TRITC staining. Space bar represents 50
m.
Conclusions
- HCR is constitutively expressed in cultured adult human astrocytes on a mRNA and protein level
- HCR mRNA expression is upregulated by nitric oxide
- HEK cells transiently transfected with HCR show functional responses to CCL2, CCL5, CCL7 and CCL8.
- CCL2 induces rapid F-actin redistribution in cultured adult human astrocytes
Overal conclusion : HCR is a functional chemokine receptor that may play a role in neuroinflammation
* [email protected]