María Virginia Tribulatti

Human platelets express and are activated by galectin-8

Gals (galectins) are proteins with glycan affinity that are emerging as mediators of atherosclerosis. Despite the similarities in structure and sequence, different Gals exert distinct effects on their target cells. We have shown that Gal-1 triggers platelet activation, suggesting a role for Gals in thrombus formation. Since Gal-8 is expressed upon endothelial activation and also contributes to inflammation, to understand further the role of these lectins in haemostasis, we evaluated the effect of Gal-8 on human platelets. Gal-8 bound specific glycans in the platelet membrane and triggered spreading, calcium mobilization and fibrinogen binding. It also promoted aggregation, thromboxane generation, P-selectin expression and granule secretion. GP (glycoprotein) αIIb and Ib-V were identified as putative Gal-8 counter-receptors by MS. Studies performed using platelets from Glanzmanns thromboasthenia and Bernard-Soulier syndrome patients confirmed that GPIb is essential for transducing Gal-8 signalling. Accordingly, Src, PLC2γ (phospholipase C2γ), ERK (extracellular-signal-regulated kinase) and PI3K (phosphoinositide 3-kinase)/Akt downstream molecules were involved in the Gal-8 signalling pathway. Gal-8 fragments containing either the N- or C-terminal carbohydrate-recognition domains showed that activation is exerted through the N-terminus. Western blotting and cytometry showed that platelets not only contain Gal-8, but also expose Gal-8 after thrombin activation. These findings reveal Gal-8 as a potent platelet activator, supporting a role for this lectin in thrombosis and inflammation.

Galectin-8 provides costimulatory and proliferative signals to T lymphocytes.

Galectin (Gal) constitute a family of carbohydrate‐recognizing molecules ubiquitously expressed in mammals. In the immune system, they regulate many processes such as inflammation, adhesion, and apoptosis. Here, we report the expression in the spleen of the two same Gal‐8 splice variants described previously in the thymus. Gal‐8 was found to induce two separate biological activities on T lymphocytes: a robust naive CD4+ T cell proliferation in the absence of antigen and notably, a costimulatory signal that synergized the cognate OVA peptide in DO11.10 mice transgenic for TCROVA. The antigen‐independent proliferation induced by Gal‐8 displayed increased expression of pro‐ and anti‐inflammatory cytokines, thus suggesting the polyclonal expansion of Th1 and Th2 clones. The costimulatory effect on antigen‐specific T cell activation was evidenced when the Gal and the peptide were assayed at doses suboptimal to induce T cell proliferation. By mass spectra analysis, several integrins and leukocyte surface markers, including CD45 isoforms, as well as other molecules specific to macrophages, neutrophils, and platelets, were identified as putative Gal‐8 counter‐receptors. Gal‐8 triggered pZAP70 and pERK1/2. Moreover, pretreatment with specific inhibitors of CD45 phosphatase or ERK1/2 prevented its antigen‐dependent and ‐independent T cell‐proliferative activities. This seems to be associated with the agonistic binding to CD45, which lowers the activation threshold of the TCR signaling pathway. Taken together, our findings support a distinctive role for locally produced Gal‐8 as an enhancer of otherwise borderline immune responses and also suggest that Gal‐8 might fuel the reactivity at inflammatory foci.

Redundant and Antagonistic Functions of Galectin-1, -3, and -8 in the Elicitation of T Cell Responses

Galectins, a family of mammalian lectins, have emerged as key regulators of the immune response. We previously demonstrated that galectin (Gal)-8, from the tandem-repeat subgroup, exerts two well-defined effects on mouse naive peripheral CD4 T cells: Ag-specific costimulation and Ag-independent proliferation. These stimulatory signals on naive T cells have not been described for any other Gal. Therefore, we investigated whether Gal-1 and Gal-3, two prominent members of the Gal family, share the stimulatory effects exerted by Gal-8 on naive T cells. We found that Gal-1 costimulated Ag-specific T cell responses similarly to Gal-8, as evaluated in the DO11.10 TCROVA-transgenic mouse model, by acting simultaneously on APCs and target CD4 T cells. In contrast, Gal-3 failed to costimulate Ag-specific T cell responses; moreover, it antagonized both Gal-1 and Gal-8 signals. We observed that both Gal-1 and Gal-3 were unable to induce Ag-independent proliferation; however, when two Gal-1 molecules were covalently fused, the resulting chimeric protein efficiently promoted proliferation. This finding indicates that Gal-1 might eventually induce proliferation and, moreover, stresses the requirement of a tandem-repeat structure. Remarkably, a single dose of recombinant Gal-1 or Gal-8 administered together with a suboptimal Ag dose to DO11.10 mice strengthened weak responses in vivo. Taken together, these findings argue for the participation of Gals in the initiation of the immune response and allow the postulation of these lectins as enhancers of borderline Ag responses, thus representing potential adjuvants for vaccine formulations.

Control of angiogenesis by galectins involves the release of platelet-derived proangiogenic factors.

Platelets contribute to vessel formation through the release of angiogenesis-modulating factors stored in their α-granules. Galectins, a family of lectins that bind β-galactoside residues, are up-regulated in inflammatory and cancerous tissues, trigger platelet activation and mediate vascularization processes. Here we aimed to elucidate whether the release of platelet-derived proangiogenic molecules could represent an alternative mechanism through which galectins promote neovascularization. We show that different members of the galectin family can selectively regulate the release of angiogenic molecules by human platelets. Whereas Galectin (Gal)-1, -3, and -8 triggered vascular endothelial growth factor (VEGF) release, only Gal-8 induced endostatin secretion. Release of VEGF induced by Gal-8 was partially prevented by COX-1, PKC, p38 and Src kinases inhibitors, whereas Gal-1-induced VEGF secretion was inhibited by PKC and ERK blockade, and Gal-3 triggered VEGF release selectively through a PKC-dependent pathway. Regarding endostatin, Gal-8 failed to stimulate its release in the presence of PKC, Src and ERK inhibitors, whereas aspirin or p38 inhibitor had no effect on endostatin release. Despite VEGF or endostatin secretion, platelet releasates generated by stimulation with each galectin stimulated angiogenic responses in vitro including endothelial cell proliferation and tubulogenesis. The platelet angiogenic activity was independent of VEGF and was attributed to the concerted action of other proangiogenic molecules distinctly released by each galectin. Thus, secretion of platelet-derived angiogenic molecules may represent an alternative mechanism by which galectins promote angiogenic responses and its selective blockade may lead to the development of therapeutic strategies for angiogenesis-related diseases.

Galectin-8 tandem-repeat structure is essential for T-cell proliferation but not for co-stimulation

Gal (galectin)-8 is a tandem-repeat Gal containing N-CRDs (Nterminal carbohydrate-recognition domains) and C-CRDs (C-terminal carbohydrate-recognition domains) with differential glycan-binding specificity fused by a linker peptide. Gal-8 has two distinct effects on CD4 T-cells: at high concentrations it induces antigen-independent proliferation, whereas at low concentrations it co-stimulates antigen-specific responses. Associated Gal-8 structural requirements were dissected in the present study. Recombinant homodimers N-N (two N-terminal CRD chimaera) and C-C (two C-terminal CRD chimaera), but not single C-CRDs or N-CRDs, induced proliferation; however, single domains induced co-stimulation. These results indicate that the tandem-repeat structure was essential only for the proliferative effect, suggesting the involvement of lattice formation, whereas co-stimulation could be mediated by agonistic interactions. In both cases, C-C chimaeras displayed higher activity than Gal-8, indicating that the C-CRD was mainly involved, as was further supported by the strong inhibition of proliferation and co-stimulation in the presence of blood group B antigen, specifically recognized by this domain. Classic Gal inhibitors (lactose and thiodigalactoside) prevented proliferation but not co-stimulatory activity, which was inhibited by 3-O-β-D-galactopyranosyl-D-arabinose. Interestingly, Gal-8 induced proliferation of naïve human CD4 T-cells, varying from non- to high-responder individuals, whereas it promoted cell death of phytohaemagglutinin or CD3/CD28 pre-activated cells. The findings of the present study delineate the differential molecular requirements for Gal-8 activities on T-cells, and suggest a dual activity relying on activation state.

Binding of galectin-1 to αIIbβ3 integrin triggers “outside-in” signals, stimulates platelet activation, and controls primary hemostasis

Understanding noncanonical mechanisms of platelet activation represents an important challenge for the identification of novel therapeutic targets in bleeding disorders, thrombosis, and cancer. We previously reported that galectin‐1 (Gal‐1), a β‐galactoside‐binding protein, triggers platelet activation in vitro. Here we investigated the molecular mechanisms underlying this function and the physiological relevance of endogenous Gal‐1 in hemostasis. Mass spectrometry analysis, as well as studies using blocking antibodies against the anti‐αIIb subunit of αIIbβ3 integrin or platelets from patients with Glanzmanns thrombasthenia syndrome (αIIbβ3 deficiency), identified this integrin as a functional Gal‐1 receptor in platelets. Binding of Gal‐1 to platelets triggered the phosphorylation of β3‐integrin, Syk, MAPKs, PI3K, PLCγ2, thromboxane (TXA2) release, and Ca2+ mobilization. Not only soluble but also immobilized Gal‐1 promoted platelet activation. Gal‐1‐deficient (Lgals1–/–) mice showed increased bleeding time (P< 0.0002, knockout vs. wild type), which was not associated with an abnormal platelet count. Lgals1–/– platelets exhibited normal aggregation to PAR4, ADP, arachidonic acid, or collagen but abnormal ATP release at low collagen concentrations. Impaired spreading on fibrinogen and clot retraction with normal levels of αIIbβ3 was also observed in Lgals1–/– platelets, indicating a failure in the “outside‐in” signaling through this integrin. This study identifies a noncanonical mechanism, based on galectin‐integrin interactions, for regulating platelet activation.—Romaniuk, M. A., Croci, D. O., Lapponi, M. J., Tribulatti, M. V., Negrotto, S., Poirier, F., Campetella, O., Rabinovich, G. A., Schattner, M. Binding of galectin‐1 to αIIbβ3 integrin triggers “outside‐in” signals, stimulates platelet activation, and controls primary hemostasis. FASEB J. 26, 2788–2798 (2012). www.fasebj.org

Galectin-8 elicits pro-inflammatory activities in the endothelium

Galectins (Gals), a family of mammalian lectins, play diverse roles under physiological and pathological conditions. Here, we analyzed the tandem-repeat Gal-8 synthesis, secretion and effects on the endothelium physiology. Gal-8M and Gal-8L isoforms were secreted under basal conditions by human microvascular endothelial cells (HMEC-1). However, expression and secretion of the Gal-8M isoform, but not Gal-8L, were increased in response to bacterial lipopolysaccharide (LPS) stimulus and returned to control values after LPS removal. Similarly, cell surface Gal-8 exposure was increased after stimulation with LPS. To evaluate Gal-8 effects on the endothelium physiology, HMEC-1 cells were incubated in the presence of recombinant Gal-8M. Pretreated HMEC-1 cells became proadhesive to human normal platelets, indicating that Gal-8 actually activates endothelial cells. This effect was specific for lectin activity as it was prevented by the simultaneous addition of lactose, but not by sucrose. Endothelial cells also increased their exposition of von Willebrand factor after Gal-8 treatment, which constitutes another feature of cell activation that could be, in turn, responsible for the observed platelet adhesion. Several pro-inflammatory molecules were abundantly produced by Gal-8 stimulated endothelial cells: CXCL1 (GRO-α), GM-CSF, IL-6 and CCL5 (RANTES), and in a lower degree CCL2 (MCP-1), CXCL3 (GRO-γ) and CXCL8 (IL-8). In agreement, Gal-8M induced nuclear factor kappa B phosphorylation. Altogether, these results not only confirm the pro-inflammatory role we have already proposed for Gal-8 in other cellular systems but also suggest that this lectin is orchestrating the interaction between leukocytes, platelets and endothelial cells.

Galectin‐8 activates dendritic cells and stimulates antigen‐specific immune response elicitation

Galectin‐8 (Gal‐8) is a mammalian β‐galactoside‐binding lectin, endowed with proinflammatory properties. Given its capacity to enhance antigen‐specific immune responses in vivo, we investigated whether Gal‐8 was also able to promote APC activation to sustain T cell activation after priming. Both endogenous [dendritic cells (DCs)] and bone marrow‐derived DCs (BMDCs) treated with exogenous Gal‐8 exhibited a mature phenotype characterized by increased MHC class II (MHCII), CD80, and CD86 surface expression. Moreover, Gal‐8‐treated BMDCs (Gal‐8–BMDCs) stimulated antigen‐specific T cells more efficiently than immature BMDCs (iBMDCs). Proinflammatory cytokines IL‐3, IL‐2, IL‐6, TNF, MCP‐1, and MCP‐5, as well as growth factor G‐CSF, were augmented in Gal‐8–BMDC conditioned media, with IL‐6 as the most prominent. Remarkably, BMDCs from Gal‐8‐deficient mice (Lgals8−/− BMDC) displayed reduced CD86 and IL‐6 expression and an impaired ability to promote antigen‐specific CD4 T cell activation. To test if Gal‐8‐induced activation correlates with the elicitation of an effective immune response, soluble Gal‐8 was coadministrated with antigen during immunization of BALB/cJ mice in the experimental foot‐and‐mouth disease virus (FMDV) model. When a single dose of Gal‐8 was added to the antigen formulation, an increased specific and neutralizing humoral response was developed, sufficient to enhance animal protection upon viral challenge. IL‐6 and IFN‐γ, as well as lymphoproliferative responses, were also incremented in Gal‐8/antigen‐immunized animals only at 48 h after immunization, suggesting that Gal‐8 induces the elicitation of an inflammatory response at an early stage. Taking together, these findings argue in favor of the use of Gal‐8 as an immune‐stimulator molecule to enhance the adaptive immune response.

Characterization of a double-CRD-mutated Gal-8 recombinant protein that retains co-stimulatory activity on antigen-specific T-cell response

Galectins (Gals) constitute a family of mammalian lectins with affinity for β-galactosides, characterized by the presence of conserved CRDs (carbohydrate-recognition domains). We have found previously that Gal-8, from the tandem-repeat group with two linked CRDs, exerts two separate actions on CD4(+)T-cells: antigen-independent proliferation and, at lower concentration, antigen-specific co-stimulation. Whereas proliferation can be ascribed to the pro-inflammatory role of Gal-8, the co-stimulatory activity of borderline T-cell-specific responses allows the proposal of Gal-8 as an adjuvant in vaccination. To study the relevance of glycan-lectin interaction to these T-cell activities, we generated a double-mutated protein (Gal-8mut) by replacing canonical arginine residues on each CRD, so as to abolish sugar-binding capacity. As expected, Gal-8mut was unable to bind to lactosyl-Sepharose, confirming that lactose recognition was precluded; however, preservation of lectin activity was still evident since Gal-8mut displayed haemoagglutinatory effects and binding capacity to the T-cell surface. To search for glycan affinity, a glycan microarray analysis was conducted which revealed that Gal-8mut lost most low- and intermediate-, but retained high-, affinity interactions, mainly to polylactosamines and blood group antigens. These findings were supported further by molecular modelling. Regarding biological activity, Gal-8mut was unable to induce T-cell proliferation, but efficiently co-stimulated antigen-specific responses, bothin vitroandin vivo.Therefore Gal-8mut represents a useful tool to dissect the specificities of lectin-glycan interactions underlying distinctive Gal-8 activities on T-cell biology. Moreover, given its distinguishing properties, Gal-8mut could be used to enhance borderline immune responses without the non-specific pro-inflammatory activity or other potential adverse effects.

Interleukin-6 signalling mediates Galectin-8 co-stimulatory activity of antigen-specific CD4 T-cell response

Galectin‐8 (Gal‐8) is a mammalian lectin endowed with the ability to co‐stimulate antigen‐specific immune responses. We have previously demonstrated that bone‐marrow‐derived dendritic cells produce high levels of interleukin‐6 (IL‐6) in response to Gal‐8 stimulation. As IL‐6 is a pleiotropic cytokine that has a broad effect on cells of the immune system, we aimed to elucidate whether IL‐6 was involved in Gal‐8‐dependent co‐stimulatory signals during antigen recognition by specific CD4 T cells. With this aim, splenocytes from DO11.10 mice were incubated with a low dose of the cognate ovalbumin peptide in combination with Gal‐8. Interleukin‐6 was found significantly increased in cultures stimulated with Gal‐8 alone or Gal‐8 plus cognate peptide. Moreover, IL‐6 signalling was triggered during Gal‐8‐induced co‐stimulation, as determined by phosphorylation of signal transducer and activator of transcription 3. Interleukin‐6 blockade by neutralizing monoclonal antibody precluded Gal‐8 co‐stimulatory activity but did not affect the antigen‐specific T‐cell receptor activation. Different subsets of dendritic cells, as well as macrophages and B cells, were identified as the cellular source of IL‐6 during Gal‐8‐induced co‐stimulation. To confirm that IL‐6 mediated the Gal‐8 co‐stimulatory effect, antigen‐presenting cells from IL‐6‐deficient or wild‐type mice were co‐cultured with purified CD4 T cells from OTII mice in the presence of cognate peptide and Gal‐8. Notably, Gal‐8‐induced co‐stimulation, but not the antigen‐specific response, was significantly impaired in the presence of IL‐6‐deficient antigen‐presenting cells. In addition, exogenous IL‐6 fully restored Gal‐8‐induced co‐stimulation. Taken together, our results demonstrate that IL‐6 signalling mediates the Gal‐8 immune‐stimulatory effect.