Gene expression of chemokines CX3CL1 and CXCL16 and their receptors, CX3CR1 and CXCR6, in peripheral blood mononuclear cells of patients with relapsing-remitting multiple sclerosis – A pilot study

Ljiljana Stojković; Aleksandra  Stanković; Ivan Životić; Evica  Dinčić; Dragan  Alavantić; Maja Živković

doi:10.2298/VSP180717035S

Ljiljana Stojković University of Belgrade, “Vinča” Institute of Nuclear Sciences, Laboratory for Radiobiology and Molecular Genetics, Belgrade, Serbia
Aleksandra Stanković University of Belgrade, “Vinča” Institute of Nuclear Sciences, Laboratory for Radiobiology and Molecular Genetics, Belgrade, Serbia
Ivan Životić University of Belgrade, “Vinča” Institute of Nuclear Sciences, Laboratory for Radiobiology and Molecular Genetics, Belgrade, Serbia
Evica Dinčić Military Medical Academy, Clinic for Neurology, Belgrade, Serbia
Dragan Alavantić University of Belgrade, “Vinča” Institute of Nuclear Sciences, Laboratory for Radiobiology and Molecular Genetics, Belgrade, Serbia
Maja Živković University of Belgrade, “Vinča” Institute of Nuclear Sciences, Laboratory for Radiobiology and Molecular Genetics, Belgrade, Serbia

DOI: https://doi.org/10.2298/VSP180717035S

Keywords: blood, chemokines, disease progression, gene expression, leukocytes, multiple sclerosis, rna, messenger

Abstract

Background/Aim. In vitro and in vivo studies show that CX3CL1 and CXCL16 chemokines and their specific receptors, CX3CR1 and CXCR6, respectively, mediate mechanism of neuroinflammation during the pathogenesis of multiple sclerosis (MS). The aim of this study was to investigate relative messenger ribonucleic acid (mRNA) levels of CX3CL1, CXCL16, CX3CR1 and CXCR6 in peripheral blood mononuclear cells, as potential molecular markers of relapsing-remitting (RR) MS. Methods. The study included 43 unrelated RR MS patients, 20 of them with clinically active disease (relapse) and 23 with clinically stable disease (remission), and 28 unrelated healthy subjects as controls. Real-time polymerase chain reactions (PCR) were performed using TaqMan^® gene expression assays. Relative expression (mRNA) level of each target gene in each sample of peripheral blood mononuclear cells was calculated as the mean normalized expression. Results. The levels of CX3CR1 mRNA were significantly higher in clinically active RR MS patients compared to controls [fold change = 1.38, p (Mann-Whitney U test) = 0.009], and significantly lower in clinically stable vs active RR MS patients [fold change = -1.43, p (t-test) = 0.03]. Stable RR MS patients had significantly higher CXCL16 mRNA levels than controls [fold change = 1.33, p (Mann-Whitney U test) = 0.006]. A trend of increased CXCR6 gene expression was found in active RR MS patients compared to controls [fold change = 1.23, p (Mann-Whitney U test) = 0.08]. In either active or stable RR MS patients there were no significant correlations of the clinical parameters with expression levels of the target genes. Conclusion. The current results show that increased CX3CR1 mRNA levels in peripheral blood mononuclear cells could represent a proinflammatory molecular marker of clinically active RR MS.

References

Le Thuc O, Blondeau N, Nahon JL, Rovère C. The complex con-tribution of chemokines to neuroinflammation: switching from beneficial to detrimental effects. Ann N Y Acad Sci 2015; 1351: 127−40.

Réaux-Le Goazigo A, Van Steenwinckel J, Rostène W, Mélik Par-sadaniantz S. Current status of chemokines in the adult CNS. Prog Neurobiol 2013; 104: 67−92.

Chen T, Guo ZP, Jiao XY, Jia RZ, Zhang YH, Li JY, et al. Pe-oniflorin suppresses tumor necrosis factor-α induced chemo-kine production in human dermal microvascular endothelial cells by blocking nuclear factor-κB and ERK pathway. Arch Dermatol Res 2011; 303(5): 351−60.

Shimaoka T, Kume N, Minami M, Hayashida K, Kataoka H, Kita T, et al. Molecular cloning of a novel scavenger receptor for oxidized low density lipoprotein, SR−PSOX, on macrophages. J Biol Chem 2000; 275(52): 40663−6.

Wilbanks A, Zondlo SC, Murphy K, Mak S, Soler D, Langdon P, et al. Expression cloning of the STRL33/BONZO/TYMSTR ligand reveals elements of CC, CXC, and CX3C chemokines. J Immunol 2001; 166: 5145−54.

Shashkin P, Simpson D, Mishin V, Chesnutt B, Ley K. Expression of CXCL16 in human T cells. Arterioscler Thromb Vasc Biol 2003; 23(1): 148−9.

Imai T, Hieshima K, Haskell C, Baba M, Nagira M, Nishimura M, et al. Identification and molecular characterization of frac-talkine receptor CX3CR1, which mediates both leukocyte mi-gration and adhesion. Cell 1997; 91(4): 521−30.

Wehr A, Baeck C, Heymann F, Niemietz PM, Hammerich L, Mar-tin C, et al. Chemokine receptor CXCR6-dependent hepatic NK T Cell accumulation promotes inflammation and liver fi-brosis. J Immunol 2013; 190(10): 5226−36.

Rennert K, Heisig K, Groeger M, Wallert M, Funke H, Lorkowski S, et al. Recruitment of CD16(+) monocytes to endothelial cells in response to LPS-treatment and concomitant TNF re-lease is regulated by CX3CR1 and interfered by soluble frac-talkine. Cytokine 2016; 83: 41−52.

Wang JH, Su F, Wang S, Lu XC, Zhang SH, Chen D, et al. CXCR6 deficiency attenuates pressure overload-induced monocytes migration and cardiac fibrosis through downregu-lating TNF-α-dependent MMP9 pathway. Int J Clin Exp Pathol 2014; 7(10): 6514−23.

Bazan JF, Bacon KB, Hardiman G, Wang W, Soo K, Rossi D, et al. A new class of membrane−bound chemokine with a CX3C motif. Nature 1997; 385(6617): 640‒4.

Ludwig A, Weber C. Transmembrane chemokines: versatile ‘special agents’ in vascular inflammation. Thromb Hae-most 2007; 97(5): 694−703.

Sunnemark D, Eltayeb S, Nilsson M, Wallstrom E, Lassmann H, Olsson T, et al. CX3CL1 (fractalkine) and CX3CR1 expression in myelin oligodendrocyte glycoprotein−induced experimental autoimmune encephalomyelitis: kinetics and cellular origin. J Neuroinflammation 2005; 2: 17.

Blauth K, Zhang X, Chopra M, Rogan S, Markovic-Plese S. The role of fractalkine (CX3CL1) in regulation of CD4(+) cell migration to the central nervous system in patients with re-lapsing−remitting multiple sclerosis. Clin Immunol 2015; 157(2): 121−32.

Kastenbauer S, Koedel U, Wick M, Kieseier BC, Hartung HP, Pfist-er HW. CSF and serum levels of soluble fractalkine (CX3CL1) in inflammatory diseases of the nervous system. J Neuroimmunol 2003; 137(1‒2): 210−7.

Huang D, Shi FD, Jung S, Pien GC, Wang J, Salazar-Mather TP, et al. The neuronal chemokine CX3CL1/fractalkine selective-ly recruits NK cells that modify experimental autoimmune en-cephalomyelitis within the central nervous system. FASEB J 2006; 20(7): 896−905.

Broux B, Pannemans K, Zhang X, Markovic−Plese S, Broekmans T, Eijnde BO, et al. CX(3)CR1 drives cytotoxic CD4(+)CD28(−) T cells into the brain of multiple sclerosis patients. J Autoimmun 2012; 38(1): 10−9.

Ludwig A, Schulte A, Schnack C, Hundhausen C, Reiss K, Brodway N, et al. Enhanced expression and shedding of the transmem-brane chemokine CXCL16 by reactive astrocytes and glioma cells. J Neurochem 2005; 93(5): 1293−303.

Wojkowska DW, Szpakowski P, Ksiazek-Winiarek D, Leszczynski M, Glabinski A. Interactions between neutrophils, Th17 cells, and chemokines during the initiation of experimental model of multiple sclerosis. Mediators Inflamm 2014; 2014: 590409.

Fukumoto N, Shimaoka T, Fujimura H, Sakoda S, Tanaka M, Kita T, et al. Critical roles of CXC chemokine ligand 16/scavenger receptor that binds phosphatidylserine and oxidized lipopro-tein in the pathogenesis of both acute and adoptive transfer experimental autoimmune encephalomyelitis. J Immu-nol 2004; 173(3): 1620−7.

Le Blanc LM, van Lieshout AW, Adema GJ, van Riel PL, Verbeek MM, Radstake TR. CXCL16 is elevated in the cerebrospinal fluid versus serum and in inflammatory condi-tions with suspected and proved central nervous system in-volvement. Neurosci Lett 2006; 397(1‒2): 145−8.

Stojković L, Stanković A, Djurić T, Dinčić E, Alavantić D, Zivković M. The gender-specific association of CXCL16 A181V gene polymorphism with susceptibility to multiple sclerosis, and its effects on PBMC mRNA and plasma soluble CXCL16 levels: preliminary findings. J Neurol 2014; 261(8): 1544−51.

Stojković L, Djurić T, Stanković A, Dinčić E, Stančić O, Veljković N, et al. The association of V249I and T280M fractalkine re-ceptor haplotypes with disease course of multiple sclerosis. J Neuroimmunol 2012; 245(1-2): 87−92.

Polman CH, Reingold SC, Banwell B, Clanet M, Cohen JA, Filippi M, et al. Diagnostic criteria for multiple sclerosis: 2010 revi-sions to the McDonald criteria. Ann Neurol 2011; 69(2): 292−302.

Lublin FD, Reingold SC. Defining the clinical course of multi-ple sclerosis: results of an international survey. National Mul-tiple Sclerosis Society (USA) Advisory Committee on clinical trials of New Agents in Multiple Sclerosis. Neurol 1996; 46(4): 907−11.

Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurol 1983; 33(11): 1444−52.

Roxburgh RH, Seaman SR, Masterman T, Hensiek AE, Sawcer SJ, Vukusic S, et al. Multiple Sclerosis Severity Score: using disa-bility and disease duration to rate disease severity. Neurol 2005; 64(7): 1144−51.

Andersen CL, Jensen JL, Ørntoft TF. Normalization of re-al−time quantitative reverse transcription−PCR data: a mod-el−based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004; 64(15): 5245−50.

Zimmermann AK, Simon P, Seeburger J, Hoffmann J, Ziemer G, Aebert H, et al. Cytokine gene expression in monocytes of pa-tients undergoing cardiopulmonary bypass surgery evaluated by real−time PCR. J Cell Mol Med 2003; 7(2): 146−56.

Pfaffl MW, Horgan GW, Dempfle L. Relative expression soft-ware tool (REST) for group−wise comparison and statistical analysis of relative expression results in real−time PCR. Nucl Ac Res 2002; 30(9): e36.

Blaschke S, Koziolek M, Schwarz A, Benöhr P, Middel P, Schwarz G, et al. Proinflammatory role of fractalkine (CX3CL1) in rheumatoid arthritis. J Rheumatol 2003; 30(9): 1918−27.

Hurst LA, Bunning RA, Couraud PO, Romero IA, Weksler BB, Sharrack B, et al. Expression of ADAM−17, TIMP−3 and fractalkine in the human adult brain endothelial cell line, hCMEC/D3, following pro−inflammatory cytokine treat-ment. J Neuroimmunol 2009; 210(1‒2): 108−12.

Infante-Duarte C, Weber A, Kratzschmar J, Prozorovski T, Pikol S, Hamann I, et al. Frequency of blood CX3CR1−positive natu-ral killer cells correlates with disease activity in multiple scle-rosis patients. FASEB J 2005; 19(13): 1902−4.

Hendrickx DA, Koning N, Schuurman KG, van Strien ME, van Eden CG, Hamann J, et al. Selective upregulation of scavenger receptors in and around demyelinating areas in multiple sclero-sis. J Neuropathol Exp Neurol 2013; 72(2): 106−18.

Calabresi PA, Yun SH, Allie R, Whartenby KA. Chemokine re-ceptor expression on MBP-reactive T cells: CXCR6 is a mark-er of IFNgamma-producing effector cells. J Neuroimmunol 2002; 127(1‒2): 96−105.

Haji Abdolvahab M, Mofrad MR, Schellekens H. Interferon beta: from molecular level to therapeutic effects. Int Rev Cell Mol Biol 2016; 326: 343−72.

Derbigny WA, Shobe LR, Kamran JC, Toomey KS, Ofner S. Iden-tifying a role for Toll−like receptor 3 in the innate immune re-sponse to Chlamydia muridarum infection in murine oviduct epithelial cells. Infect Immun 2012; 80: 254−65.

Tamtaji OR, Kouchaki E, Salami M, Aghadavod E, Akbari E, Tajabadi-Ebrahimi M, et al. The effects of probiotic supple-mentation on gene expression related to inflammation, insulin, and lipids in patients with multiple sclerosis: a randomized, double-blind, placebo-controlled trial. J Am Coll Nutr 2017; 36(8): 660−5.

Mindur JE, Valenzuela RM, Yadav SK, Boppana S, Dhib-Jalbut S, Ito K. IL-27: a potential biomarker for responders to glati-ramer acetate therapy. J Neuroimmunol 2017; 304: 21−8.

Ciriello J, Tatomir A, Hewes D, Boodhoo D, Anselmo F, Rus V, et al. Phosphorylated SIRT1 as a biomarker of relapse and re-sponse to treatment with glatiramer acetate in multiple sclero-sis. Exp Mol Pathol 2018; 105(2): 175−80.