2nd Annual BMRP Investigator Meeting - Abstract

An Anti-glycan Antibody Blocks Colitis, Cytokine Production and Induces Apoptosis of Activated Macrophage

Geetha Srikrishna¹, Olga Turovskaya², Raziya Shaikh², Simon Murch³, Mitchell Kronenberg² and Hudson H. Freeze¹,a

¹Glycobiology and Carbohydrate Chemistry Program, The Burnham Institute (La Jolla, California, U.S.A.); ²Developmental Immunology Laboratory, La Jolla Institute of Allergy and Immunology (California, U.S.A.); ³Centre for Paediatric Gastroenterology, Royal Free and University College Medical School (London, United Kingdom)

The Receptor for Advanced Glycation End Products (RAGE) uses unusual carboxylated N-glycans for ligand binding.  Since blocking RAGE-ligand binding quenches colitis in IL-10 knockout mice, we hypothesized that the glycans contribute to inflammatory bowel disease.  Anti-glycan mAbGB3.1. binds macrophage micro aggregates and activated dendritic cells from Crohn's disease patients’ inflammatory lesions.  mAbGB3.1 also blocks the onset of Th-1 mediated colitis in CD4+ CD45RB^high T cell transfer to Rag-1^-/- immune deficient mice.

Animals given a control antibody lost ~26% body weight and had severe diarrhea.  Mice treated with mAbGB3.1 were healthy, had minimal weight loss, and no diarrhea.  All mice treated with the control antibody had extensive inflammatory cell infiltration, hyperplasia, mucin depletion, and crypt abscesses, but >70% of the mAbGB3.1 treated mice had normal colonic architecture and minimal or no inflammation. mAbGB3.1 specifically reduced accumulation of CD4⁺ T-cells in the colonic lamina propria, but not in lung, spleen, or small intestine mesenteric lymph nodes.  Treated mice expressed gut-homing a4b7, but T-cell recruiter, MAdCAM, was reduced in inflamed tissue.  TNFa, IFNg, and NFkBp65 expression were greatly reduced.  These results show that mAbGB3.1-reactive glycans on antigen presenting cells are involved in the development of colitis in this model.  Giving mAbGB3.1 after onset of disease also impaired its progression.

To understand the mechanism of mAbGB3.1 action, we studied mAbGB3.1 epitope expressing RAW264.7 murine macrophage, which produce pro- and anti-inflammatory cytokines, and reactive oxygen species when activated with lipopolysaccharide.  mAbGB3.1 blocked expression and secretion of TNFa, IL-12, IL-23 and nitric oxide, but had no effect on the secretion of macrophage-migration inhibitory factor or IL-10.  mAbGB3.1 also blocked activation of NF-kB p65, and rapidly induced apoptosis of activated macrophages.

NF-kB regulates many immune response genes including TNFa.  Constitutive and inducible NF-kB activation preserves macrophage survival.  These results suggest that mAbGB3.1 acts by blocking receptor-mediated signaling and induction of NF-kB, which subsequently blocks pro-inflammatory cytokine production and induces apoptosis.  Whether GB3.1 blocks sustained activation of NF-kB by blocking RAGE ligation is unproven.

^aPrincipal Investigator