Contents lists available at ScienceDirect Molecular Immunology journal homepage: www.elsevier.com/locate/molimm Cytokines use different intracellular mechanisms to upregulate the membrane expression of CX 3 CR1 in human monocytes Cecilia Analia Panek a , Andrea Cecilia Bruballa a,1 , Gonzalo Ezequiel Pineda a,1 , Carlos De Brasi b , Romina Jimena Fernández-Brando a , María Pilar Mejías a , María Victoria Ramos a , Marina Sandra Palermo a, ⁎ a Laboratorio de Patogénesis e Inmunología de Procesos Infecciosos, Instituto de Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas- Academia Nacional de Medicina, Buenos Aires, Argentina b Laboratorio de Genética Molecular de la Hemofilia, Instituto de Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas- Academia Nacional de Medicina, Buenos Aires, Argentina ARTICLE INFO Keywords: CX3CR1 IFN-γ IL-10 Monocytes STAT1 STAT3 mRNA ABSTRACT Membrane expression of fractalkine (CX 3 CL1)-receptor (CX 3 CR1) is relevant in monocytes (Mo) because CX 3 CR1-CX 3 CL1 interactions might participate on both, homeostatic and pathologic conditions. We have pre- viously demonstrated that CX 3 CR1 levels are decreased during culture and when Mo are differentiated into dendritic cells, but enhanced when differentiated into macrophages. Regarding soluble factors, lipopoly- saccharide (LPS) accelerated the loss of CX 3 CR1, while interleukin (IL)-10 and Interferon-gamma (IFN-γ) pre- vented it. However, the comprehensive knowledge about the intracellular pathways that underlay the level of CX 3 CR1 expression in Mo is still incomplete. In the current work, we studied the effect of anti-inflammatory cytokines (IL-4, IL-13, IL-10), alone or to- gether with IFN- γ on CX 3 CR1 expression. We found that only IL-10 and IFN-γ separately were able to prevent CX 3 CR1 down-modulation during culture of human Mo. Besides, Mo incubated with IL-10 plus IFN-γ showed the highest CX 3 CR1 expression by cell, suggesting cooperation between two different mechanism used by both cytokines. By studying intracellular mechanisms triggered by IL-10 and IFN-γ, we demonstrated that they spe- cifically induced PI3K-dependent serine-phosphorylation of signal transducer and activator of transcription (STAT)3 or STAT1, respectively. Moreover, chemical inhibitors of STAT1 or STAT3 abrogated IFN-γ or IL-10 effects on CX 3 CR1 expression. Strikingly, only IL-10 increased CX 3 CR1 mRNA level, as consequence of aug- menting mRNA stability. CX 3 CR1 mRNA increase was PI3K-dependent, supporting the causal link between the action of IL-10 at the CX 3 CR1 transcript and CX 3 CR1 protein level on Mo. Thus, both cytokines up-regulate CX 3 CR1 expression on human Mo by different intracellular mechanisms. 1. Introduction The unique member of the CX 3 C chemokine subfamily is Fractalkine (CX 3 CL1), which exists in a soluble or a membrane-anchored form. The latter functions as an adhesion molecule (Bazan et al., 1997), and is expressed on the surface of endothelial and epithelial cells (Lucas et al., 2001), dendritic cells (DC) (Papadopoulos et al., 1999) and neurons (Harrison et al., 1998) and is induced by pro-inflammatory cytokines such as interleukin (IL)-1, tumor necrosis factor (TNF)-α and Interferon- gamma (IFN-γ)(Imaizumi et al., 2004). The full-length molecule can be cleaved from the cell membrane by metalloproteases, resulting in the release of a soluble CX 3 CL1 which acts as a chemoattractant. The CX 3 CL1 receptor, CX 3 CR1, is expressed on several leukocytes, including monocytes (Mo), T cell subsets, and on the major subset of Natural https://doi.org/10.1016/j.molimm.2019.01.003 Received 8 October 2018; Received in revised form 26 December 2018; Accepted 3 January 2019 Abbreviations: CX 3 CL1, fractalkine; CX 3 CR1, fractalkine receptor; Mo, monocytes; TNF-α, tumor necrosis factor alpha; LPS, lipopolysaccharide; PI3K, phosphati- dylinositol-3-kinase; IL-, interleukin-; IFN-γ, Interferon-gamma; STAT, signal transducer and activator of transcription; JAK, Janus kinase; HRP, Horseradish per- oxidase; FITC, Fluorescein isthiocyanate; PE, Phycoerythrin; PECy5, Phycoerythrincyanin 5.1; MFI, mean fluorescence intensity; SSC/FSC, side vs. forward scattering; mAb, monoclonal antibody; ActD, actinomycin D; PFA, paraformaldehyde ⁎ Corresponding author at: Instituto de Medicina Experimental (IMEX) (CONICET), Academia Nacional de Medicina, Pacheco de Melo 3081 (C1425AUM), Buenos Aires, Argentina. E-mail address: mspalermo@hematologia.anm.edu.ar (M.S. Palermo). 1 Authors contributed equally to this work. Molecular Immunology 108 (2019) 23–33 0161-5890/ © 2019 Elsevier Ltd. All rights reserved. T