Irisin supports integrin-mediated cell adhesion of lymphocytes

Abstract

Irisin, a myokine released from skeletal muscle, has recently been found to act as a ligand for the integrins αVβ5, αVβ1, and α5β1 expressed on mesenchymal cells, thereby playing an important role in the metabolic remodeling of the bone, skeletal muscle and adipose tissues. Although the immune-modulatory effects of irisin in chronic inflammation have been documented, its interactions with lymphocytic integrins have yet to be elucidated. Here, we show that irisin supports the cell adhesion of human and mouse lymphocytes. Cell adhesion assays using a panel of inhibitory antibodies to integrins have shown that irisin-mediated lymphocyte adhesion involves multiple integrins including not only α4β1 and α5β1, but also leukocyte-specific αLβ2 and α4β7. Importantly, mouse lymphocytic TK-1 cells that lack the expression of β1 integrins have exhibited αLβ2- and α4β7-mediated cell adhesion to irisin. Irisin has also been demonstrated to bind to purified recombinant integrin αLβ2 and α4β7 proteins. Thus, irisin represents a novel ligand for integrin αLβ2 and α4β7, capable of supporting lymphocyte cell adhesion independently of β1 integrins. These results suggest that irisin may play an important role in regulating lymphocyte adhesion and migration in the inflamed vasculature.

Keywords: Cell adhesion; ECM, extracellular matrix; FNDC-5, fibronectin type III domain-containing protein 5; Fc, fragment crystallizable; Fibronectin type III domain-containing protein 5; ICAM-1, intercellular adhesion molecule-1; Integrins; Irisin; LFA-1, lymphocyte function-associated antigen 1; LPAM-1, lymphocyte Peyer's patch adhesion molecules; MAdCAM-1, mucosal addressin cell adhesion molecule-1; PBMCs, peripheral blood mononuclear cells; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Fig. 1

Cell adhesion of lymphocytic Jurkat cells to irisin, in comparison to ICAM-1, and fibronectin (A) A representative SDS-PAGE image of recombinant irisin-Fc fusion protein; lane 1 protein molecular weight maker; lane 2, control-Fc protein; lane 3, irisin-Fc protein. (B) Jurkat cells showed concentration-dependent binding to fibronectin (FN, blue), inter-cellular adhesion molecule-1 (ICAM-1, red), and irisin (gray). Data are expressed as the mean ± SEM of three independent experiments carried out in triplicate. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Cell adhesion of human and mouse lymphocytes to irisin (A) Jurkat cells (B) PBMCs, and (C) TK1 cells. Data are expressed as the mean ± SEM of three independent experiments carried out in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 compared with Fc.

Fig. 3

Immunofluorescent cytometry analysis of integrin expression. Representative FACS histograms of flow cytometry analysis illustrating the expression of different integrin subunits on (A) Jurkat cells, (B) PBMCs, and (C) TK1 cells. The background staining with isotype control antibodies is shown as black lines, whereas specific monoclonal antibody staining is shown as red lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Specific integrins involved in lymphocyte adhesion to irisin studied using a panel of inhibitory antibodies. Binding of (A) Jurkat cells and (B) PBMCs to Fc, BSA, fibronectin, ICAM-1-Fc and irisin-Fc in the presence or absence of blocking anibodies (C) Binding of TK1 cells to Fc, BSA, ICAM-1-Fc, MAdCAM-1-Fc and irisin-Fc in the presence or absence of blocking antibodies. (A–C) Data are expressed as the mean ± SEM of three independent experiments carried out in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 compared with an isotype control in each group.

Fig. 5

Purified protein based-ELISA type experiments to study the interaction of irisin with integrin αLβ2 and α4β7. Binding of irisin- or control-Fc to immobilized proteins such as; mouse αLβ2 (A), human αLβ2 (B), human α4β7 (C), and BSA as a control (A–C); was examined. (A–C) Data are expressed as the mean ± SEM of three independent experiments carried out in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control Fc in each group.