A multitude of leukocyte migration events is needed to accomplish immunosurveillance in vertebrate organisms. Blood monocytes derived from central hematopoietic organs continuously seed the periphery with sentinels specialized in antigen uptake. Antigen encounter results in the mobilization of antigen-presenting cells (APC) to afferent lymphatics and their recruitment to secondary lymphoid organs, where they trigger T-cell responses. Long-distance migration of leukocytes is accomplished via blood and lymph circulation and thus requires transendothelial migration through vessel walls. The interaction of leukocytes with vascular endothelial cells during extravasation at sites of inflammation is a highly regulated process. After an initial, predominantly selectin-mediated "rolling" step, engagement of G-protein-coupled chemokine receptors leads to activation of integrins and the establishment of firm arrest, followed by diapedesis (2, 3).Recently a novel chemokine named fractalkine (FKN) (neurotactin [NTN]) was identified (1, 15) and shown to have unique properties. FKN has a CX 3 C chemokine domain and thus constitutes, according to the current chemokine nomenclature based on the spacing of N-terminal cysteines, its own CX 3 C family. Unlike any other known chemokine, the CX 3 C module was found to exist in two isoforms; one is membrane anchored and presented on an extended mucin-like stalk, and the other is a soluble form resulting from membrane-proximal proteolytic cleavage of FKN. In addition to its classical function as a chemoattractant, high-affinity interaction of FKN with its specific receptor CX 3 CR1 (8) mediates leukocyte arrest under flow conditions (4). In vitro data show that this firm adhesion is signaling independent and does not involve integrin activation, and may thus represent a novel mechanism in leukocyte trafficking (4, 7). FKN has been shown to be expressed on activated endothelial cells (1, 15), dendritic cells (DC) (9, 16), and neurons (6, 14). The FKN receptor, CX 3 CR1 (formerly V28 [18]), is a typical seven-transmembrane G-protein-coupled receptor. CX 3 CR1 is expressed on human monocytes and undefined subsets of NK and T cells (8). Expression of FKN and CX 3 CR1 in neurons and microglia, respectively, has fostered speculations that the receptor-ligand pair might be crucial for neuronal-glial cross talk (6,14).To investigate the in vivo role of FKN-CX3CR1 interactions, we generated a mouse mutant that lacks the FKN receptor. Our strategy was to replace the murine CX 3 CR1 gene with the gene encoding the enhanced green fluorescent protein (EGFP; Clontech). This approach allowed not only the generation of a mutant CX 3 CR1 locus but also the examination of the CX 3 CR1 expression pattern and migration of cells that normally express this receptor.
MATERIALS AND METHODSMolecular cloning and generation of CX 3 CR1 mutant mice. Genomic fragments of the murine CX 3 CR1 locus were isolated from a 129/Sv phage library (Stratagene, La Jolla, Calif.) by hybridization with a human CX 3 CR1 cDNA pr...
