1. HCR : A FUNCTIONAL ORPHAN CC CHEMOKINE RECEPTOR IN HUMAN ASTROCYTES 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. 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. 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. - 100ng/ml 0.01 mM 0.1 mM 1 mM HCR GAPDH B C A 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. 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 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. Results Aim of the study The goal of the current project is the functional characterization and localization of HCR in cultured adult human astrocytes. Mike W. Zuurman*, Knut Biber, Hendrikus W.G.M. Boddeke Department of Medical Physiology, University of Groningen, The Netherlands * m.w.zuurman@med.rug.nl 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 4. Chemokine induced reorganization of F-actin in human astrocytes