Final Progress Report

Summary of project aims

Galectins, a growing family of animal lectins, has attracted the interest of cell biologists and immunologists as master regulators of immune cell homeostasis. Until recently the biological function of galectin-2 was unknown, however, we showed that galectins modulate the adaptive immune system in IBD patients.

Our proposal aimed to test the hypothesis that Galectin-2 is involved in the pathogenesis of IBD and may have a potential therapeutic effect in this condition. We aimed to study galectin-2 expression in the intestine of IBD patients, the effect of galectin-2 on T cell function in control and IBD patients, and the mechanisms of its regulation. Furthermore, we aimed to investigate the potential therapeutic effect of galectin-2 in models of experimental colitis, and to explore the underlying mechanisms of its action.

Accomplishments towards meeting those aims

Due to the generous support by the BRMP, we were able to accomplish nearly all of our proposed aims. As pointed out in the attached manuscripts, we were able to uncover the role of Gal-2 on T cell function in normal and IBD patients and investigated its mechanism of regulation. Furthermore, and not primarily proposed, we investigated the role galectins on epithelial cell function and epithelial wound healing. In addition, we also investigated Gal-4 and uncovered that the beneficial effect of Galectins is not restricted to Gal-2.

Regarding the original proposed aims, we have not finished yet our work regarding the Galectin expression profiles in IBD. The aim here was to investigate intrinsic Galectin protein expression in IBD patients. In longitudinal studies we want to investigate if successfully treated patients or failure of treatment is related to Galectin expression. We do have data about the original expression profile, but are still collecting data after therapy. We will finish these experiment in spring 2010.

Significant results

Sturm A, et al, J Immunol 2004:

The carbohydrate-dependent binding of galectin-2 induces apoptosis in activated T cells. Fluorogenic substrate and inhibitor assays reveal involvement of caspases-3 and -9, in accordance with cleavage of the DNA fragmentation factor. Enhanced cytochrome c release, disruption of the mitochondrial membrane potential, and an increase of the Bax/Bcl-2 ratio by opposite regulation of expression of both proteins add to the evidence that the intrinsic apoptotic pathway is triggered. Cell cycle distribution and expression of regulatory proteins remained unaffected. Notably, galectins-1 and -7 reduce cyclin B1 expression, defining functional differences between the structurally closely related galectins. Cytokine secretion of activated T cells was significantly shifted to the Th2 profile.

 

 

Galectin-2 is constitutively expressed mainly in the epithelial compartment of the mouse intestine and bind to lamina propria mononuclear cells. During colitis, galectin-2 expression was reduced, but could be restored to normal levels by immunosuppressive treatment. Galectin-2 treatment induced apoptosis of mucosal T cells and thus ameliorated acute and chronic dextran-sodium-sulfate-induced colitis and in a T-helper-cell 1-driven model of antigen-specific transfer colitis. Furthermore, the pro-inflammatory cytokine release was inhibited by galectin-2 treatment. In preliminary toxicity studies, galectin-2 was well tolerated.

 

 

Gal-2 and Gal-4 bind to epithelial cells at the E-cadherin/-catenin complex. Both galectins significantly enhanced intestinal epithelial cell restitution in vitro. This enhancement of epithelial cell restitution was TGF- β -independent. In contrast, Gal-1 decreased epithelial cell migration TGF-β dependently. By performing cell cycle analysis, we show that Gal-2 and Gal-4 increased cyclin B1 expression and consequently cell cycle progression, while Gal-1 inhibited cell cycling. Determining the influence of Gal-2 and Gal-4 on epithelial cell apoptosis, we showed no induction of apoptosis, whereas Gal-1 significantly induced apoptosis of epithelial cells caspase-independently.

 

 

Galectin-4 is selectively expressed and secreted by intestinal epithelial cells and binds potently to activated peripheral and mucosal lamina propria T-cells at the CD3 epitope. The carbohydrate-dependent binding of galectin-4 at the CD3 epitope is fully functional and inhibited T cell activation, cycling and expansion. Galectin-4 induced apoptosis of activated peripheral and mucosal lamina propria T cells via calpain-, but not caspase-dependent, pathways. Providing further evidence for its important role in regulating T cell function, galectin-4 blockade by antisense oligonucleotides reduced TNF-alpha inhibitor induced T cell death. Furthermore, in T cells, galectin-4 reduced pro-inflammatory cytokine secretion including IL-17. In a model of experimental colitis, galectin-4 ameliorated mucosal inflammation, induced apoptosis of mucosal T-cells and decreased the secretion of pro-inflammatory cytokines. Our results show that galectin-4 plays a unique role in the intestine and assign a novel role of this protein in controlling intestinal inflammation by a selective induction of T cell apoptosis and cell cycle restriction.

 

 

Galectin-2 is expressed in murine skin and is up-regulated upon cutaneous inflammation. Interestingly, treatment of mice with galectin-2 significantly reduced the contact allergy response. This effect was long-lasting since rechallenge of galectin-2-treated mice after a 14-day interval still resulted in a decreased ear swelling. We were able to demonstrate that galectin-2 induced a reduction of MHC class I-restricted immune responses in the treated animals, which was mediated by the induction of apoptosis specifically in activated CD8(+) T cells. Additionally, we report that the galectin-2-binding protein CD29 is up-regulated on the surface of activated CD8(+) T cells compared with naive CD8(+) T cells or CD4(+) T cells, suggesting that increased galectin-2/CD29 signaling might be responsible for the proapoptotic effects of galectin-2 on activated CD8(+) T cells.

Lay summary

Galectin-2 is expressed in the human gastrointestinal tract. In IBD, inflammatory cells such as T cells have a disturbed life and dead regulation. In fact, in Crohns disease and ulcerative colitis, they are resistant against signals which terminates their life (dead induction=apoptosis). This enabled dead fosters inflammation. We showed that galectin-2 induced the cell death of inflammation cells and uncovered the underlying pathways.

We also showed that Galectin-2 is present in the epithelial compartment of the intestine, the first frontier of defense in the human body. By using animal experiments, we showed that during colitis, Galectin-2 expression was reduced, but was restored to normal levels by immunosuppressive treatment. When treating the animals with Gal-2, we showed convincingly, that Galectin-2 treatment ameliorated acute and chronic experimental colitis. We also investigated the sugar-binding protein Galectin-4 which is exclusively expressed in the gastrointestinal tract and its capacity to help wound closure. When investigating Gal-2 and Gal-4, we could show that both proteins bind to epithelial cells and promote wound healing after injuries.

With regard to Gal-4, we showed that the sugar-dependent binding of Gal-4 inhibited T cell activation, cycling and expansion. Again, we could uncover underlying pathways. In a model of experimental colitis, galectin-4 ameliorated mucosal inflammation, induced apoptosis of mucosal T-cells and decreased the secretion of pro-inflammatory mediators (cytokines).

Last but not least, in cooperation with dermatologist from the University of Muenster, we showed that Galectin-2 is expressed in murine skin and is up-regulated upon cutaneous inflammation. Interestingly, treatment of mice with galectin-2 significantly reduced the contact allergy response. This effect was long-lasting since rechallenge of galectin-2-treated mice after a 14-day interval still resulted in a decreased ear swelling.