HCR : A FUNCTIONAL

1
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
2. Chemokine-induced calcium gradients in HCR
expressing HEK cells
3. Chemotactic responses to different chemokines
of HCR expressing HEK cells
A
- 100ng/ml 0.01 mM
0.1 mM 1 mM
MM C LPS detanonoate(NO)
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
HCR-immunohistochemistry in cultured human
astrocytes. B. unspecific fluorescent background
staining. C. HCR immunofluorescence, arrow
indicates a strong signal along an astrocytic
process. TRITC-immunofluorescent staining. Space
bar represents 30 ?m.
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 students t-test with n6.
4. Chemokine induced reorganization of F-actin in
human astrocytes
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
m.w.zuurman_at_med.rug.nl