Interleukin-10 and interferon-gamma modulate surface expression of fractalkine-receptor (CX(3)CR1) via PI3K in monocytes

Abstract

The membrane-anchored form of the chemokine fractalkine (CX(3)CL1) has been identified as a novel adhesion molecule that interacts with its specific receptor (CX(3)CR1) expressed in monocytes, T cells and natural killer cells to induce adhesion. In addition, CX(3)CL1 can be cleaved from the cell membrane to induce chemotaxis of CX(3)CR1-expressing leucocytes. Recently, marked variations in CX(3)CR1 monocyte expression have been observed during several pathological conditions. Regulation of CX(3)CR1 in monocytes during basal or inflammatory/anti-inflammatory conditions is poorly understood. The aim of this study was therefore to examine CX(3)CR1 expression during monocyte maturation and the effect of soluble mediators on this process. We found that basal expression of CX(3)CR1 in fresh monocytes was reduced during culture, and that lipopolysacchairde accelerated this effect. In contrast, interleukin-10 and interferon-gamma treatment abrogated CX(3)CR1 down-modulation, through a phosphatidylinositol 3 kinase-dependent pathway. Most importantly, CX(3)CR1 membrane expression correlated with monocyte CX(3)CL1-dependent function. Taken together, our data demonstrate that CX(3)CR1 expression in monocytes can be modulated, and suggest that alterations in their environment are able to influence CX(3)CL1-dependent functions, such as chemotaxis and adhesion, leading to changes in the kinetics, composition and/or functional status of the leucocyte infiltrate.

Figure 1

Monocytes down-regulated CX₃CR1 but not CD14 during culture. Purified Mo (1 × 10⁶/ml) were cultured for 20 hr with medium or with the following protein synthesis inhibitors: cycloheximide (CHX; 1 μg/ml), actinomycin D (ActD; 0·5 μg/ml) or anisomycin (0·1 μg/ml). After washing, cells were stained with fluorescein isothiocyanate-conjugated anti-CX₃CR1, phycoerythrin-Cy5-conjugated anti-CD14 monoclonal antibodies, or isotype-matched control antibody and analysed by flow cytometry. (a) Representative histograms of surface CX₃CR1-expression in fresh Mo (broken line) or after culture 20 hr (solid line), filled histogram represents the isotype control. (b) Representative histograms of surface CX₃CR1 expression upon incubation for 20 hr with medium (black line), or with CHX (thin line) or with ActD (broken line). (c) The results are expressed as the percentage of CX₃CR1-positive (white bars) or CD14-positive (black bars) cells into the gate for Mo defined by forward (FSC) and side (SSC) scatter parameters. Each bar represents the mean ± SEM of 17 healthy donors for culture in medium, or six donors for experiments with protein synthesis inhibitors. *P < 0·05; **P < 0·01 statistically different compared with the same parameter of Mo cultured in medium for 20 hr. (d) Purified Mo (1 × 10⁶/ml) were cultured for 20 hr with medium or with the following agents: CHX (1 μg/ml), ActD (0·5 μg/ml), granulocyte–macrophage colony-stimulating factor (GM-CSF; 50 ng/ml), interleukin-10 (IL-10; 10 ng/ml). The table shows the percentage of CX₃CR1, CD14 and AnnexinV positive Mo calculated by flow cytometry, of one experiment representative of three.

Figure 2

Interleukin-10 (IL-10) avoided down-regulation of CX₃CR1 during culture. Purified monocytes (Mo; 1 × 10⁶/ml) were incubated for 20 hr with medium or with IL-10 at different doses and times, followed by flow cytometry analysis of CX₃CR1 expression. (a) Purified Mo (1 × 10⁶/ml) were incubated for 20 hr with IL-10 at the concentrations indicated below each bar. (b) Representative dot-plots for double CD14/CX₃CR1-staining showing the up-regulation of CX₃CR1 expression in Mo upon 20 hr culture with IL-10 (10 ng/ml). (c) Purified Mo (1 × 10⁶/ml) were incubated with medium or IL-10 (10 ng/ml) added during different periods, washed and then cultured in medium for up to 20 hr (or IL-10 over all 20 hr). (d) Purified Mo (1 × 10⁶/ml) were incubated with medium for different periods before adding IL-10 (time 0 represents IL-10 over all 20 hr). Each bar represents the mean ± SEM of 16 healthy donors for culture in medium or IL-10 (10 ng/ml), and three donors for each point on the time and dose curves. *P < 0·05 statistically different compared with the same parameter of Mo in medium. (e) Table showing the CX₃CR1-surface expression and the intracellular content (IC) after Mo permeabilization as detailed in the Materials and methods. Data are expressed as the mean ± SEM of three independent experiments. (f) Representative histograms showing CX₃CR1-surface and intracellular (IC) expression in Mo only upon 20 hr culture with medium or IL-10 (10 ng/ml).

Figure 3

Influence of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K) chemical inhibitors on cytokine-mediated CX₃CR1 regulation. (a) Purified Mo (1 × 10⁶/ml) were pre-incubated for 1 hr with the corresponding kinase-inhibitors (PD98059, 20 μm; SB203580, 30 μm; Wortmannin, 100 nm; LY294002, 25 μm) before interleukin-10 (IL-10; 10 ng/ml) addition, and cultured for 20 hr. The results are expressed as the percentage of CX₃CR1-positive cells. Each bar represents the mean ± SEM of five to seven healthy donors. #P < 0·05 statistically different compared with IL-10 alone. (b) Purified Mo (1 × 10⁶/ml) were cultured for 20 hr with medium or interferon-γ (IFN-γ; 240 U/ml) alone or with LY294002 (25 μm); and/or IL-10 (10 ng/ml). Then, CD14 and CX₃CR1 expression was analysed by flow cytometry. The results are expressed as the percentage of CX₃CR1-positive (white bars) or CD14-positive (black bars) cells into the gate of Mo. Each bar represents the mean ± SEM of five to seven healthy donors, except for medium and IFN-γ bars which each correspond to 11 blood samples. *P < 0·05; statistically different compared with the same parameter of Mo in medium; **P < 0.05 statistically different compared with IFN-γ alone. (c) Representative histograms of CX₃CR1 expression after culture with medium, IL-10, IFN-γ or both cytokines for 20 hr. Filled histogram corresponds to isotype-control. Data in the table are the mean ± SEM of CX₃CR1 mean fluorescence intensity (MFI) of n experiments. *P < 0·05 statistically different compared with medium; **P < 0·05 statistically different compared with IFN-γ alone.

Figure 4

Phorbol 12-myristate 13-acetate (PMA) -maturated THP-1 cells down-modulated CX₃CR1 expression. (a) THP-1 cells (1 × 10⁶/ml) were differentiated with PMA (5 ng/ml) for 48 hr, then they were incubated with medium, interleukin-10 (IL-10; 10 ng/ml) or interferon-γ (IFN-γ; 240 U/ml) during 20 hr. Some cell samples were pre-incubated for 1 hr with LY294002 (25 μm). All samples were analysed for CX₃CR1 and CD14 expression by flow cytometry. The results are expressed as the percentage of CX₃CR1-positive (white bars) or CD14-positive (black bars) THP-1 cells. Each bar represents the mean ± SEM of five independent experiments. *P < 0·05 statistically different compared with the same parameter of THP-1 cells in medium + PMA; **P < 0·05 statistically different compared with IFN-γ alone; #P < 0·02 statistically different compared with IL-10 alone. (b) LY294002 impaired IL-10- or IFN-γ-dependent Akt activation. PMA-maturated THP-1 cells (1 × 10⁶/ml) were pre-incubated during 60 min with medium or LY294002 (25 μM), and 60 min with IL-10 (10 ng/ml) or IFN-γ (240 U/ml). Then protein cell extracts were assayed for Akt phosphorylation by immunoblotting as described in the Materials and methods. Cells incubated with lipopolysaccharide (1 μg/ml) during 60 min were assayed as a positive control.

Figure 5

CX₃CL1-dependent functionality. (a) Peripheral blood mononuclear cells (PBMC) were loaded with Fluo3-AM, as indicated in the Materials and methods. Then cells were treated with sCX₃CL1 (100 ng/ml) or fMLP (10⁻⁷m). Changes in [Ca²⁺]_i were followed by monitoring the change in the percentage of positive cells inside the monocyte (Mo) gate. The figure shows one representative experiment out of three. (b) Purified Mo (1 × 10⁶ cells/ml) cultured with medium, interleukin-10 (IL-10; 10 ng/ml), interferon-γ (IFN-γ; 240 U/ml) or IL-10 + IFN-γ for 20 hr, were incubated with CX₃CL1 (0·2; 2 ng/ml) for 15 min at 37°. Mo were gated out based on forward and side scatter parameters. CD11b expression, measured as mean fluorescence intensity (MFI), was expressed as percentage above baseline (i.e. incubation with each cytokine without sCX₃CL1). The figure shows one representative experiment out of three.