In vivo structure/function and expression analysis of the CX3C chemokine fractalkine

Abstract

The CX 3 C chemokine family is composed of only one member, CX 3 CL1, also known as fractalkine, which in mice is the sole ligand of the G proteincoupled, 7-transmembrane receptor CX 3 CR1. Unlike classic small peptide chemokines, CX 3 CL1 is synthesized as a membrane-anchored protein that can promote integrin-independent adhesion. Subsequent cleavage by metalloproteases, either constitutive or induced, can generate shed CX 3 CL1 entities that potentially have chemoattractive activity. To study the CX 3 C interface in tissues of live animals, we generated transgenic mice (CX 3 CL1 cherry :CX 3 CR1 gfp ), which express red and green fluorescent reporter genes under the respective control of the CX 3 CL1 and CX 3 CR1 promoters. Furthermore, we performed a structure/function analysis to differentiate the in vivo functions of membranetethered versus shed CX 3 CL1 moieties by comparing their respective ability to correct established defects in macrophage function and leukocyte survival in IntroductionChemokine (CX 3 C motif) ligand 1 (CX 3 CL1), also known as fractalkine or neurotactin, 1,2 and its receptor CX 3 CR1 3 have been assigned their own CX 3 C chemokine family. This classification is based on the 3 amino acid gap between its N-terminal cysteines in CX 3 CL1, with no spacing in CC chemokines and only one intervening amino acid in CXC chemokines. 4 CX 3 CL1 is furthermore structurally unique in that it is synthesized as a type I transmembrane protein with the CX 3 C chemokine domain presented on an extended stalk. 1,2 Both CX 3 CL1 and CX 3 CR1 are widely expressed throughout the organism; but in given tissues, expression is often highly cell type-specific. Taking advantage of mice that harbor a targeted replacement of the CX 3 CR1 gene by a GFP reporter, 5 we could, for instance, show that CX 3 CR1 expression in the brain is restricted to microglia. CX 3 CR1 expression in the gut was found limited to lamina propria macrophages and CX 3 CR1 expression in the blood is largely restricted to monocytes, which are uniform CX 3 CR1 positive, albeit with discrete expression levels. 6 CX 3 CR1 is furthermore expressed by macrophage/ dendritic cell precursors, 7 various dendritic cell (DC) progenitors, a nonclassic CD8␣ ϩ DC subset, 8 and plasmacytoid DCs. Aside from the prominent expression in the mononuclear myeloid compartment, CX 3 CR1 receptor expression has been reported for an NK cell subset 3,9 and certain T-cell populations. 3,10,11 The in vivo expression pattern of the ligand CX 3 CL1 remains less well defined and controversial 12 but has been reported for neurons, 13 intestinal epithelium, 14 and inflamed endothelium. 2 Notably, in humans eotaxin-3/CC chemokine ligand 26 was recently reported to be a functional ligand for CX 3 CR1 15 ; in mice, the CCL26 gene, however, is a pseudogene.The analysis of CX 3 C receptor and ligand knockout mice 5,16 has revealed a number of phenotypes resulting from the lack of CX 3 CR1/ CX 3 CL1 interactions. [17][18][19][20] However, in-depth knowledge of the physiologic rol...

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“…The chemokine CX 3 CL1, which is the CX 3 CR1 ligand, is highly expressed in CNS neurons (Harrison et al, 1998), including mature neurons of the dentate gyrus (Kim et al, 2011), and is normally produced in a membrane-bound form from which the chemokine domain can be released through proteolytic cleavage (Bazan et al, 1997). The CX 3 CL1-CX 3 CR1 axis therefore represents a pathway for direct communication between neural cells and microglia.…”

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