Specific inhibition of T-cell adhesion to extracellular matrix and proinflammatory cytokine secretion by human recombinant galectin-1

Abstract

The migration of immune cells through the extracellular matrix (ECM) towards inflammatory sites is co-ordinated by receptors recognizing ECM glycoproteins, chemokines and proinflammatory cytokines. In this context, galectins are secreted to the extracellular milieu, where they recognize poly-N-acetyllactosamine chains on major ECM glycoproteins, such as fibronectin and laminin. We investigated the possibility that galectin-1 could modulate the adhesion of human T cells to ECM and ECM components. T cells were purified from human blood, activated with interleukin-2 (IL-2), labelled, and incubated further with intact immobilized ECM and ECM glycoproteins in the presence of increasing concentrations of human recombinant galectin-1, or its more stable, related, C2-S molecule obtained by site-directed mutagenesis. The presence of galectin-1 was shown to inhibit T-cell adhesion to intact ECM, laminin and fibronectin, and to a lesser extent to collagen type IV, in a dose-dependent manner. This effect was specifically blocked by anti-galectin-1 antibody and was dependent on the lectin's carbohydrate-binding properties. The inhibition of T-cell adhesion by galectin-1 correlates with the ability of this molecule to block the re-organization of the activated cell's actin cytoskeleton. Furthermore, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production was markedly reduced when IL-2-activated T cells were incubated with galectin-1 or its mutant. This effect was prevented by beta-galactoside-related sugars. The present study reveals an alternative inhibitory mechanism for explaining the suppressive properties of the galectin-1 subfamily on inflammatory and autoimmune processes.

Figure 1

IL-2-induced human T-cell adhesion to ECM components is specifically inhibited by galectin-1. ⁵¹[Cr]-labelled human T cells were incubated for 30 min in microtitre plates pre-coated with intact ECM (a), FN (b), LN (c) and CO-IV (d) in the presence of IL-2 (10 U/ml) and the indicated concentrations of recombinant galectin-1. In some wells, T cells were also incubated in the presence of IL-2 (10 U/ml), galectin-1 (4 μg/ml) and an antibody raised against galectin-1 (diluted 1:50). Non-stimulated T cells were used as negative controls. T cell-adhesion was then analysed. Results are expressed as mean percentage±SD of bound T cells from quadruplicate wells per experimental group. One experiment representative of four is shown.

Figure 2

The morphology and shape of FN-adherent T cells exposed to IL-2 (a), PMA (b), IL-2 and galectin-1 (c), PMA and galectin-1 (d) or medium alone (e). Photographs were taken 30–60 min after seeding of cells on FN-coated microtitre wells and exposure to the activating or inhibiting compounds.

Figure 3

IL-2-induced proinflammatory cytokine secretion by T cells is inhibited by galectin-1. Human T cells were purified and cultured in the absence or presence of IL-2 (100 U/ml) and recombinant galectin-1 at concentrations ranging from 0·04 to 4 μg/ml for 18 hr at 37°. In some treatments, galectin-1 (4 μg/ml) was added together with a specific antibody (diluted 1:50) or a carbohydrate competitor (100mm TDG). Unstimulated T cells were used as negative controls Supernatants were collected and assayed for TNF-α (a) and IFN-γ (b) secretion by standard ELISA. Results are expressed as mean±SD from quadruplicate wells per experimental group. One experiment representative of four is shown.