BACKGROUND AND PURPOSE:Induction of cellular migration is the primary effect of chemokine receptor activation. However, several chemokine receptor-like proteins bind chemokines without subsequent induction of intracellular signalling and chemotaxis. It has been suggested that they act as chemokine scavengers, which may control local chemokine levels and contribute to the function of chemokines during inflammation. This has been verified for the chemokine-like receptor proteins D6 and DARC as well as CCX-CKR. Here, we provide evidence for an additional biological function of human (h)CCX-CKR. EXPERIMENTAL APPROACH:We used transfection strategies in HEK293 and human T cells. KEY RESULTS:Co-expression of hCCX-CKR completely inhibits hCXCR3-induced chemotaxis. We found that hCCX-CKR forms complexes with hCXCR3, suggesting a relationship between CCX-CKR heteromerization and inhibition of chemotaxis. Moreover, negative binding cooperativity induced by ligands both for hCXCR3 and hCCX-CKR was observed in cells expressing both receptors. This negative cooperativity may also explain the hCCX-CKR-induced inhibition of chemotaxis. CONCLUSIONS AND IMPLICATIONS:These findings suggest that hCCX-CKR prevents hCXCR3-induced chemotaxis by heteromerization thus representing a novel mechanism of regulation of immune cell migration.

1 Leiden/Amsterdam Center for Drug Research (LACDR), Division of Medicinal Chemistry, Faculty of Sciences, VU University Amsterdam, Amsterdam, The Netherlands 2 Laboratory for Molecular Endocrinology-GPCRs, Western Australian Institute for Medical Research (WAIMR) and Centre for Medical Research, The University of Western Australia, Nedlands, Australia 3 Department of Medical Physiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 4 Molecular Pharmacology & DMPK, MSD, Merck Research Laboratories, Oss, The Netherlands 5 Dimerix Bioscience Pty Ltd, Nedlands, Australia

Background and PurposeThe C‐X‐C chemokine receptors 3 (CXCR3) and C‐X‐C chemokine receptors 4 (CXCR4) are involved in various autoimmune diseases and cancers. Small antagonists have previously been shown to cross‐inhibit chemokine binding to CXCR4, CC chemokine receptors 2 (CCR2) and 5 (CCR5) heteromers. We investigated whether CXCR3 and CXCR4 can form heteromeric complexes and the binding characteristics of chemokines and small ligand compounds to these chemokine receptor heteromers.Experimental ApproachCXCR3–CXCR4 heteromers were identified in HEK293T cells using co‐immunoprecipitation, time‐resolved fluorescence resonance energy transfer, saturation BRET and the GPCR‐heteromer identification technology (HIT) approach. Equilibrium competition binding and dissociation experiments were performed to detect negative binding cooperativity.Key ResultsWe provide evidence that chemokine receptors CXCR3 and CXCR4 form heteromeric complexes in HEK293T cells. Chemokine binding was mutually exclusive on membranes co‐expressing CXCR3 and CXCR4 as revealed by equilibrium competition binding and dissociation experiments. The small CXCR3 agonist VUF10661 impaired binding of CXCL12 to CXCR4, whereas small antagonists were unable to cross‐inhibit chemokine binding to the other chemokine receptor. In contrast, negative binding cooperativity between CXCR3 and CXCR4 chemokines was not observed in intact cells. However, using the GPCR‐HIT approach, we have evidence for specific β‐arrestin2 recruitment to CXCR3‐CXCR4 heteromers in response to agonist stimulation.Conclusions and ImplicationsThis study indicates that heteromeric CXCR3–CXCR4 complexes may act as functional units in living cells, which potentially open up novel therapeutic opportunities.

1 Department of Neuroscience, Section Medical Physiology, University Medical Center Groningen, University of Groningen, The Netherlands. 2 Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit, Amsterdam, The Netherlands. 3 Department of Psychiatry and Psychotherapy, Section of Molecular Psychiatry, University of Freiburg, Freiburg, Germany 4 Department of Neurosciences, metabolic and biomedical sciences, University of Modena and Reggio Emilia, Modena, Italy. 5 Department of Laboratory Medicine, Laboratory of Clinical Chemistry, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. * Both authors contributed equally to the present study

Multiple sclerosis lesion activity concurs with the extent of inflammation, demyelination and axonal suffering. Pro-inflammatory myeloid cells contribute to lesion development, but the self-limiting nature of lesions implies as yet unidentified anti-inflammatory mechanisms. We addressed the hypothesis that myelin ingestion by myeloid cells induces a foamy appearance and confers anti-inflammatory function. First, we show that myelin-containing foam cells in multiple sclerosis lesions consistently express a series of anti-inflammatory molecules while lacking pro-inflammatory cytokines. Second, unique location-dependent cytokine and membrane receptor expression profiles imply functional specialization allowing for differential responses to micro-environmental cues. A novel human in vitro model of foamy macrophages functionally confirmed that myelin ingestion induces an anti-inflammatory programme. Foamy macrophages are unable to respond to prototypical inflammatory stimuli but do express molecules involved in suppression of inflammation. These findings provide novel insights into the mechanisms of lesion control and may open new roads to intervention.