CX3CR1, GPCR CX3C chemokine receptor 1 (CX3CR1): transmembrane receptor of therapeutic interest?

ß chemokine receptor with no ligand

 

In 1995, Combadière (1) and Raport (2) shared the paternity of a brand new membrane protein, respectively designated CMKBRL1 (chemokine ß receptor-like 1) and V28. Sequence analysis had rapidly demonstrated that this new receptor was the human ortholog of a previously described orphan receptor predicted from a cloned rat cDNA designated RBS11 (83% amino acid identity) (Harrison et al, 1994). Concordance of amino acid sequence, chromosomal location and RNA distribution have permitted to conclude that CMKBRL1/V28 was a ß chemokine receptor.

 

But its ligand was not yet known.

 

A new cytokine joins the chemokine superfamily

 

In 1997, Bazan et al were searching for novel chemokine and chemokine-like sequences from several nonhematopoietic cells and tissues using random sequencing. The screen revealed a novel sequence, but with a CX3X spacing of the characteristic cysteine motif in contrast to the traditional chemokine signatures discovered so far: CXC, CC and XC.

 

The unusual CXXXC pattern of cysteine residues led to the creation of the fourth or delta family of only one ligand to chemokine receptors. Bazan decided to baptize this unique molecule neurotactin due to its upregulation in brain inflammation, and in accordance with the nomenclature, the ligand was qualified C-X3-C motif ligand 1 or CX3CL1.

 

Nevertheless, taking a closer look to the phylogenetic tree of chemokines superfamily, the team observed that the new branch composed by CX3CL1 formed a fractal form.

 

They so decided to rename it Fractalkine.

 

Fractalkine: a very singular ligand to chemokine receptors in many respects

 

Curiously enough, Fractalkine/CX3CL1 is a type I transmembrane protein mediating cell adhesion, but from which a 85 KDa soluble form (sCX3CL1) can be generated by proteolytic cleavage by metalloproteinases. The soluble form, like CC chemokines, appeared to be a potent attractant for monocytes and lymphocytes, but not neutrophils (3).

 

In 1998, Combadière et al decided to rename CMKBRL1 CX3C chemokine receptor 1 or CX3CR1 after the name of its only ligand CX3CL1 (Nucleic acids, protein synthesis, and molecular genetics, Volume 273, Issue 37, P23799-23804, September 1998). In a rather rare way in the chemokine superfamily, the relationship between CX3CR1 and Fractalkine is exclusive: the ligand recognizes only this receptor and this receptor recognizes only this ligand.

 

Nomiyama et al (4) localized the gene coding for human CX3CL1 on chromosome 16q13. Fractalkine mRNA is expressed in most human organs, including the heart, brain, kidney, intestines and skeletal muscles.

 

At cellular level, this chemokine of 373 amino acids residues is constitutively expressed in dendritic cells, upregulated upon dendritic cell maturation (5) and is mainly produced by glomerular endothelial cells and the tubular epithelium. Intestinal epithelial cells also produce CX3CL1 and its mRNA level is enhanced in active Crohn’s disease (6). In the nervous system, this GPCR ligand is expressed in the olfactory bulb, cerebral cortex and hippocampus.

 

It is interesting to note, for the purpose of understanding immunological mechanisms and developing appropriate therapeutic strategies, that fractalkine is expressed by dendritic cells and is upregulated during dendritic cell maturation (7).

 

CX3CR1 fractalkine receptor: a 7-transmembrane receptor coupled to heterotrimeric G protein (GPCR)

 

The CX3CR1 gene, localized on chromosome 3p22.2 is one of the largest chemokine receptor genes, described as having four exons and three introns spanning over 18 kb (8). This G-protein coupled receptor is expressed by many immune cells including T CD8, mast cells, Natural Killer (NK) cells, Dendritic Cells (CDs) and monocytes/macrophages.

 

After ligand binding, the receptor-associated G protein is activated, and the α subunit dissociates from the βγ complex. This event activates several signaling pathways: PI3K kinases and MAPK kinases (JNK, ERK 1/2, p38), AKT (Ser-473 Thr-308), Src, and eNOS, which lead to different cellular responses such as migration, survival, and resistance to apoptosis.

 

Fractalkine/CX3CR1 axis in inflammation disease process

 

Since chemokines allow the migration of leukocytes to inflammatory sites, it seems quite legitimate to investigate the role of CX3CR1 in various autoimmune conditions such as Crohn's disease, asthma and rheumatoid arthritis.

 

In 2006, Brand et al (9) demonstrated that all proinflammatory stimuli (TNF-alpha, IL-1beta, LPS) significantly increased fractalkine mRNA expression in intestinal epithelial cell. The expression of the chemokine fractalkine was clearly upregulated by proinflammatory cytokines and enhanced in inflamed Crohn's disease lesions.

 

Regarding asthma, Rimaniol et al (10) observed that patients with symptomatic allergic rhinitis and asthmatic patients had increased circulating fractalkine levels, and CX3CR1 function was upregulated in circulating CD4+ T lymphocytes.

 

In the pathogenesis of rheumatoid arthritis, Blaschke et al (11) concluded that predominant expression of fractalkine in T cells with a Th1-type cytokine expression profile and MMP-2 upregulation in fractalkine-stimulated cultured synovial fibroblasts suggest a proinflammatory role.

 

Despite these various discoveries, anti-fractalkine clinical development remains in a state of semi-failure, with the example of the humanized antibody E6011 from EA Pharma/Eisai showing modest efficacy in RA patients without meeting the primary endpoints of the study.

 

CMKBRL1/CX3CR1 axis and renal diseases

 

In the excellent review by Zhuang et al (12), the authors took the time to gather all the data on renal diseases and their close links with the V28 membrane receptor. They concluded that the CX3CL1/CX3CR1 axis remains a two-sided coin in kidney disease and that therapeutic approaches should be taken with caution.

 

In fact on one side CX3CR1 reduces the inflammation-induced proliferation of TGF-B producing renal macrophages in fibrosis, and in the other side could mediate macrophage trafficking to the kidney in ischemia-reperfusion injury.

 

In some cases, CX3CR1 appears to assist in the synthesis and secretion of anti-inflammatory mediators such as IL1RA in sepsis, but at the same time promotes the survival of profibrotic macrophages in lupus or allograft rejection.

 

CX3CR1 attracts NK cells to eliminate tumor cells in renal cell carcinoma but also helps tumor cells adhesion via PI3K/Akt signaling in the same indication.

 

And the appearances of this Harvey Dent / Two-Face character seem to abound as soon as one approaches kidney disease and the CX3CL1/CX3CR1 axis.

 

Neurotactin /CX3CR1 axis and cancer

 

Cancer could be one of the promising areas for the therapeutic application of fractalkine.

 

Indeed, as early as 2003, Guo's team demonstrated that fractalkine could induce chemoattraction and adhesion of NK cells and that fractalkine expressed by genetically modified tumor cells could chemoattract more NK cells to infiltrate solid tumors. This indicates that the accumulation of NK cells in the tumor and the release of endogenous antigens from NK-lysed tumor cells could contribute to the initiation of the switch from innate to dendritic cell-mediated adaptive immunity (13).

 

Two years later, Xin et al (14) confirmed the previous results on NK cells, but noticed the appearance of a tumor-specific cytotoxic T lymphocyte (CTL) response. Indeed, they hypothesized that dendritic cells (DCs) maturation seems to be induced by fractalkine, and that mature DCs present tumor antigens and induce priming and activation of naive T cells, leading to the induction of tumor-specific adaptive immunity.

 

Thus, presence of high local concentration of fractalkine seems to be protective against tumor growth in some tumor entities and these antitumoral effects depend on NK cells, dendritic cells and T-cells.

 

But let's not put the cart before the horse.

 

Fractalkine has also shown an opposite effect and the pro-tumor observed results could be caused by fractalkine-mediated adhesion and transmigration of cancer cells.

 

Nevo et al (15) thus demonstrated that the CX3CR1–CX3CL1 axis is indeed involved in the transmigration of neuroblastoma cells through bone-marrow endothelium. It was shown that ligation of the membrane-bound CX3CL1 expressed by neuroblastoma cells by antiCX3CL1 antibodies generated a distinct pattern of tyrosine phosphorylation in these cells. These results strongly suggest that membrane form of CX3CL1 is capable of transducing outside-in signals into neuroblastoma cells.

 

In fact, it seems that the overall effect of fractalkine results from a critical balance between the activity of the secreted and membrane-anchored forms.

 

Kandera is currently developing KAND567 a small molecule, selective, non-competitive, allosteric antagonist of the fractalkine receptor CX3CR1, which is under preparation for a clinical phase IIa study in AMI patients.

 


REFERENCES

 

(1) Combadiere, Christophe, Ahuja, Sunil K., & Murphy, Philip M. (1995). Cloning, Chromosomal Localization, and RNA Expression of a Human β Chemokine Receptor-Like Gene. 

(2) Raport CJ, Schweickart VL, Eddy RL Jr, Shows TB, Gray PW. The orphan G-protein-coupled receptor-encoding gene V28 is closely related to genes for chemokine receptors and is expressed in lymphoid and neural tissues. Gene. 1995 Oct 3;163(2):295-9. doi: 10.1016/0378-1119(95)00336-5. PMID: 7590284.

(3) Bazan, J. F., Bacon, K. B., Hardiman, G., Wang, W., Soo, K., Rossi, D., … Schall, T. J. (1997). A new class of membrane-bound chemokine with a CX3C motif. Nature, 385(6617), 640–644. doi:10.1038/385640a0 

(4) Nomiyama H, Imai T, Kusuda J, Miura R, Callen DF, Yoshie O. Human chemokines fractalkine (SCYD1), MDC (SCYA22) and TARC (SCYA17) are clustered on chromosome 16q13. Cytogenet Cell Genet. 1998;81(1):10-1. doi: 10.1159/000015000. PMID: 9691168.

(5) Kanazawa N, Nakamura T, Tashiro K, Muramatsu M, Morita K, Yoneda K, Inaba K, Imamura S, Honjo T. Fractalkine and macrophage-derived chemokine: T cell-attracting chemokines expressed in T cell area dendritic cells. Eur J Immunol. 1999 Jun;29(6):1925-32. doi: 10.1002/(SICI)1521-4141(199906)29:06<1925::AID-IMMU1925>3.0.CO;2-U. PMID: 10382755.

(6) Muehlhoefer A, Saubermann LJ, Gu X, Luedtke-Heckenkamp K, Xavier R, Blumberg RS, Podolsky DK, MacDermott RP, Reinecker HC. Fractalkine is an epithelial and endothelial cell-derived chemoattractant for intraepithelial lymphocytes in the small intestinal mucosa. J Immunol. 2000 Mar 15;164(6):3368-76. doi: 10.4049/jimmunol.164.6.3368. PMID: 10706732.

(7) Papadopoulos, E. J., Sassetti, C., Saeki, H., Yamada, N., Kawamura, T., Fitzhugh, D. J., Saraf, M. A., Schall, T., Blauvelt, A., Rosen, S. D. et al., Fractalkine, a CX3C chemokine, is expressed by dendritic cells and is up-regulated upon dendritic cell maturation. Eur. J. Immunol. 1999. 29: 2551–2559.

(8) Garin A, Pellet P, Deterre P, Debré P, Combadière C. Cloning and functional characterization of the human fractalkine receptor promoter regions. Biochem J. 2002 Dec 15;368(Pt 3):753-60. doi: 10.1042/BJ20020951. PMID: 12234253; PMCID: PMC1223041.

(9) Brand S, Hofbauer K, Dambacher J, Schnitzler F, Staudinger T, Pfennig S, Seiderer J, Tillack C, Konrad A, Göke B, Ochsenkühn T, Lohse P. Increased expression of the chemokine fractalkine in Crohn's disease and association of the fractalkine receptor T280M polymorphism with a fibrostenosing disease Phenotype. Am J Gastroenterol. 2006 Jan;101(1):99-106. doi: 10.1111/j.1572-0241.2005.00361.x. PMID: 16405540.

(10) Rimaniol AC, Till SJ, Garcia G, et al. The CX3C chemokine fractalkine in allergic asthma and rhinitis. J Allergy Clin Immunol 2003;112(6):1139-46

(11) Blaschke S, Muller GA. Fractalkine–a proinflammatory chemokine in rheumatoid arthritis. Zeitschrift fur Rheumatologie 2008;67(5):424-8

(12) Zhuang Q, Cheng K, Ming Y. CX3CL1/CX3CR1 Axis, as the Therapeutic Potential in Renal Diseases: Friend or Foe?. Curr Gene Ther. 2017;17(6):442-452. doi:10.2174/1566523218666180214092536

(13) Guo, J., Chen, T., Wang, B., Zhang, M., An, H., Guo, Z., … Cao, X. (2003). Chemoattraction, adhesion and activation of natural killer cells are involved in the antitumor immune response induced by fractalkine/CX3CL1. Immunology Letters, 89(1), 1–7. doi:10.1016/s0165-2478(03)00101-9 

(14) Xin H, Kikuchi T, Andarini S, et al. Antitumor immune response by CX3CL1 fractalkine gene transfer depends on both NK and T cells. Eur J Immunol 2005;35(5):1371-80

(15) Nevo, I., Sagi-Assif, O., Meshel, T., Ben-Baruch, A., Jöhrer, K., Greil, R., … Witz, I. P. (2009). The involvement of the fractalkine receptor in the transmigration of neuroblastoma cells through bone-marrow endothelial cells. Cancer Letters, 273(1), 127–139. doi:10.1016/j.canlet.2008.07.029