Microglia, the resident inflammatory cells of the CNS, are the only CNS cells that express the fractalkine receptor (CX3CR1). Using three different in vivo models, we show that CX3CR1 deficiency dysregulates microglial responses, resulting in neurotoxicity. Following peripheral lipopolysaccharide injections, Cx3cr1-/- mice showed cell-autonomous microglial neurotoxicity. In a toxic model of Parkinson disease and a transgenic model of amyotrophic lateral sclerosis, Cx3cr1-/- mice showed more extensive neuronal cell loss than Cx3cr1+ littermate controls. Augmenting CX3CR1 signaling may protect against microglial neurotoxicity, whereas CNS penetration by pharmaceutical CX3CR1 antagonists could increase neuronal vulnerability.

There is increasing interest in the isolation of adult microglia to study their functions at a morphological and molecular level during normal and neuroinflammatory conditions. Microglia have important roles in brain homeostasis, and in disease states they exert neuroprotective or neurodegenerative functions. To assay expression profiles or functions of microglia, we have developed a method to isolate microglial cells and infiltrating leukocytes from adult mouse brain. This protocol uses a digestion cocktail containing collagenase and dispase, and it involves separation over discontinuous percoll gradients. Isolated cells can be used for RNA analysis, including RNase protection analysis (RPA), quantitative RT-PCR, high-density microarray, proteomic or flow cytometric characterization of cell surface markers or adoptive transfer. Cell isolation can be completed in less than 4 h.

In vitro studies have implicated chemokine receptors in consumption and clearance of specific ligands. We studied the role that various signaling chemokine receptors play during ligand homeostasis in vivo. We examined the levels of ligands in serum and CNS tissue in mice lacking chemokine receptors. Compared with receptor-sufficient controls, Cx3cr1 Ϫ/Ϫ mice exhibited augmented levels of CX3CL1 both in serum and brain, and circulating levels of CXCL1 and CXCL2 were increased in Cxcr2 Ϫ/Ϫ mice. CCR2-deficient mice showed significantly increased amounts of circulating CCL2 compared with wild-type mice. Cxcr3 Ϫ/Ϫ mice revealed increased levels of circulating and brain CXCL10 after experimental autoimmune encephalomyelitis ( IntroductionActions of chemokines through chemokine receptor signaling leads to an array of diverse functions in different tissue compartments. 1,2 Such functions go beyond the original assigned roles of chemokines in leukocyte chemoattraction to inflamed tissues, and involve physiological trafficking to localize surveillant populations in noninflamed tissues, cellular activation, proliferation, adhesion, phagocytosis, apoptosis, and angiogenesis. [3][4][5][6] Chemokine/ chemokine receptor interactions exhibit defined roles during inflammation, atherosclerosis, autoimmunity, viral pathogenesis, cancer, and neurodegeneration. [7][8][9][10][11] Even though the system exhibits apparent redundancy, modulation of chemokine function via chemokine receptor blockade is a challenging area of considerable interest for therapeutic purposes.Among the chemokine receptors, CCR2 and its ligand CCL2 (MCP-1) have been extensively studied, and their role in regulating monocyte and T-cell infiltrations is well established. In 2002, Tylaska et al showed that CCR2-knockout mice manifested extremely high levels of CCL2 at sites of alloinduced inflammation, and in vitro studies confirmed that clearance of ligand was mediated by CCR2. 12 Our group reported that CCL2 is consumed by CCR2 ϩ migrating cells in a human blood-brain barrier model using peripheral blood mononuclear cells from healthy donors. 13 These results suggest an important biologic role of chemokine receptors as scavenger molecules involved in clearance of specific ligands.Chemokine receptor-deficient mouse strains have been instrumental in understanding chemokine biology in health and disease states. 14 In the present study, we evaluated levels of chemokines in 4 receptor-deficient mice, including those lacking CC, CXC, and CX3C receptors. Both circulating and brain tissue levels were studied. Brain was selected for study as a distinct tissue compartment in which chemokines may be produced under physiological and pathological conditions. We found that the levels of circulating and-in some instances-tissue chemokines are dramatically increased in healthy chemokine receptor-deficient mice. Reconstitution with wild-type bone marrow cells restored chemokine homeostasis. Importantly, for chemokines that signal to more than one receptor, absence of one recep...

D6, a promiscuous nonsignaling chemokine binding molecule expressed on the lymphatic endothelium, internalizes and degrades CC chemokines, and D6−/− mice demonstrated increased cutaneous inflammation following topical phorbol ester or CFA injection. We report that D6−/− mice were unexpectedly resistant to the induction of experimental autoimmune encephalomyelitis due to impaired encephalitogenic responses. Following induction with myelin oligodendroglial glycoprotein (MOG) peptide 35–55 in CFA, D6−/− mice showed reduced spinal cord inflammation and demyelination with lower incidence and severity of experimental autoimmune encephalomyelitis attacks as compared with D6+/+ littermates. In adoptive transfer studies, MOG-primed D6+/− T cells equally mediated disease in D6+/+ or D6−/− mice, whereas cells from D6−/− mice transferred disease poorly to D6+/− recipients. Lymph node cells from MOG-primed D6−/− mice showed weak proliferative responses and made reduced IFN-γ but normal IL-5. CD11c+ dendritic cells accumulated abnormally in cutaneous immunization sites of D6−/− mice. Surprisingly, D6, a “silent” chemokine receptor, supports immune response generation.

Increased central nervous system (CNS) levels of monocyte chemoattractant protein 1 [CC chemokine ligand 2 (CCL2) in the systematic nomenclature] have been reported in chronic neurological diseases such as human immunodeficiency virus type 1-associated dementia, amyotrophic lateral sclerosis, and multiple sclerosis. However, a pathogenic role for CCL2 has not been confirmed, and there is no established model for the effects of chronic CCL2 expression on resident and recruited CNS cells. We report that aged (>6 months) transgenic (tg) mice expressing CCL2 under the control of the human glial fibrillary acidic protein promoter (huGFAP-CCL2hi tg+ mice) manifested encephalopathy with mild perivascular leukocyte infiltration, impaired blood brain barrier function, and increased CD45-immunoreactive microglia, which had morphologic features of activation. huGFAP-CCL2hi tg+ mice lacking CC chemokine receptor 2 (CCR2) were normal, showing that chemokine action via CCR2 was required. Studies of cortical slice preparations using video confocal microscopy showed that microglia in the CNS of huGFAP-CCL2hi tg+ mice were defective in expressing amoeboid morphology. Treatment with mutant CCL2 peptides, a receptor antagonist and an obligate monomer, also suppressed morphological transformation in this assay, indicating a critical role for CCL2 in microglial activation and suggesting that chronic CCL2 exposure desensitized CCR2 on microglia, which in the CNS of huGFAP-CCL2hi tg+ mice, did not up-regulate cell-surface expression of major histocompatibility complex class II, CD11b, CD11c, or CD40, in contrast to recruited perivascular macrophages that expressed enhanced levels of these markers. These results indicate that huGFAP-CCL2hi tg+ mice provide a useful model to study how chronic CNS expression of CCL2 alters microglial function and CNS physiology.