RAGE and Arthritis: the G82S Polymorphism Amplifies the Inflammatory Response
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Genes and Immunity (2002) 3, 123–135 2002 Nature Publishing Group All rights reserved 1466-4879/02 $25.00 www.nature.com/gene RAGE and arthritis: the G82S polymorphism amplifies the inflammatory response MA Hofmann1, S Drury1, BI Hudson2, MR Gleason1,WQu1,YLu1, E Lalla1, S Chitnis3, J Monteiro3, MH Stickland2, LG Bucciarelli1, B Moser1, G Moxley4, S Itescu1, PJ Grant2, PK Gregersen3, DM Stern1 and AM Schmidt1 1College of Physicians and Surgeons, Columbia University, New York, NY, USA; 2Academic Unit of Molecular Vascular Medicine, University of Leeds, Leeds, UK; 3North Shore Long Island Jewish Research Institute, Manhasset, New York, NY, USA; 4McQuire VA Medical Center and Medical College of Virginia, Richmond, VA, USA The receptor for advanced glycation end products (RAGE) and its proinflammatory S100/calgranulin ligands are enriched in joints of subjects with rheumatoid arthritis (RA) and amplify the immune/inflammatory response. In a model of inflammatory arthritis, blockade of RAGE in mice immunized and challenged with bovine type II collagen suppressed clinical and histologic evidence of arthritis, in parallel with diminished levels of TNF-alpha, IL-6, and matrix metalloproteinases (MMP) 3, 9 and 13 in affected tissues. Allelic variation within key domains of RAGE may influence these proinflammatory mechanisms, thereby predisposing individuals to heightened inflammatory responses. A polymorphism of the RAGE gene within the ligand-binding domain of the receptor has been identified, consisting of a glycine to serine change at position 82. Cells bearing the RAGE 82S allele displayed enhanced binding and cytokine/MMP generation following ligation by a prototypic S100/calgranulin compared with cells expressing the RAGE 82G allele. In human subjects, a case-control study demonstrated an increased prevalence of the 82S allele in patients with RA compared with control subjects. These data suggest that RAGE 82S upregulates the inflammatory response upon engagement of S100/calgranulins, and, thereby, may contribute to enhanced proinflammatory mechanisms in immune/inflammatory diseases. Genes and Immunity (2002) 3, 123–135. DOI: 10.1038/sj/gene/6363861 Keywords: receptor; inflammation; polymorphism; S100/calgranulin; joint Introduction inflammatory disorders,5,6 may be released by activated inflammatory effector cells such as monocytes,7 thereby The receptor for advanced glycation end products freeing them to engage cell surface RAGE and amplify (RAGE), a multi-ligand member of the immunoglobulin host inflammatory responses. Indeed, accumulation of superfamily of cell surface molecules, has been impli- S100/calgranulins in synovial fluid and plasma of sub- 1 cated in amplification of proinflammatory responses. jects with RA has been correlated with indices of disease S100/calgranulins, proinflammatory signal transduction severity, such as bony erosions.4 ligands of the receptor, are enriched in joints of subjects Recent studies have highlighted the possibility that 2–4 with rheumatoid arthritis (RA). Members of this family polymorphisms within key domains of RAGE may of molecules, long-associated with classic immune/ influence its function, so that under conditions of increased ligand accumulation, individuals may be pre- disposed to heightened inflammatory responses.8,9 Correspondence: Dr AM Schmidt, College of Physicians & Surgeons, Columbia University, 630 W. 168th Street, P&S 17–401, New York, New Importantly, a polymorphism of the RAGE gene has been York 10032, USA. E-mail: ams11Ȱcolumbia.edu identified within the V-type immunoglobulin domain in This work is supported, in part, by the Surgical Research Fund of the extracellular region of the receptor, consisting of a the College of Physicians & Surgeons, Columbia University, and glycine to serine change at position 82.8 In previous stud- by grants from the United States Public Health Service to DMS and ies, we found that the ligands of RAGE, including AMS. Support for PKG and the North American Rheumatoid S100/calgranulins, engage the V-domain of the receptor Arthritis Consortium (NARAC) is provided by the National Arthritis Foundation and by the United States Public Health Ser- and activate signal transduction pathways, thereby mod- 1,10–14 vice. BIH, MHS and PJG gratefully acknowledge the assistance of ulating gene expression. These considerations led us Dr Mark S Shearman at Merck, Sharpe and Dohme. BIH is a junior to hypothesize that activation of RAGE contributed to research fellow of the British Heart Foundation (Junior Research proinflammatory responses in immune/inflammatory Fellowship FS/2000007). MAH and LGB are postdoctoral research disorders such as RA, and that RAGE 82S might further fellows of the Juvenile Diabetes Research Foundation. AMS is a enhance generation of inflammatory mediators linked to recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research. chronic tissue injury. Here, we tested these concepts in a Received 9 January 2002; revised 27 January 2002; accepted 28 Janu- murine model of polyarticular arthritis, and in cultured ary 2002 Chinese hamster ovary (CHO) cells or human peripheral RAGE and inflammatory arthritis MA Hofmann et al 124 blood-derived monocytes bearing RAGE 82S. Finally, we unchanged in the presence of sRAGE vs PBS or murine performed a preliminary study of the distribution of the serum albumin, thereby indicating that blockade of G82S polymorphism in subjects with RA. RAGE did not suppress primary immunization (Figure 2d). To dissect the molecular mechanisms underlying the Results protection afforded by RAGE blockade, we assessed levels of cytokines and matrix metalloproteinases RAGE and murine collagen-induced arthritis (MMPs) in the tissues. ELISA on extracts prepared from As a first test of the hypothesis that RAGE contributed stifle joints revealed a significant reduction in levels of to inflammatory and tissue-destructive mechanisms in TNF-alpha (4.3-fold) and IL-6 (3.1-fold) from mice immu- arthritis, we induced arthritis in DBA/1 mice by sensitiz- nized with bovine type II collagen treated with sRAGE ation and challenge with bovine type II collagen, the pre- vs murine serum albumin (Figure 3a,b, respectively). dominant protein of articular cartilage.15–17 Bovine type Similarly, levels of plasma TNF-alpha and IL-6 antigens II collagen was emulsified in complete Freund’s adjuvant were significantly reduced, 3.5- and 3.6-fold, in sRAGE- and injected intradermally at the base of the tail (day 0). treated mice compared with those animals receiving Three weeks later, mice were challenged with bovine murine serum albumin (Figure 3c,d, respectively). type II collagen (day 22). The contribution of RAGE to As induction of TNF-alpha and other inflammatory the pathogenesis of arthritis was studied by treating ani- cytokines sets in motion events leading to activation of mals with soluble (s) RAGE,1,18 the extracellular ligand- latent/proenzyme MMPs,21 we assessed activity of binding domain of the receptor, 100 g per day, begin- MMPs 3, 9 and 13 in joint tissue. Compared with control ning 3 weeks after initial immunization (day 22). In pre- joints, tissue retrieved from murine serum albumin- vious studies, blockade of RAGE at this dose effected the treated mice undergoing the collagen-induced arthritis greatest decrease in the proinflammatory phenotype in a protocol revealed an 2.5-fold, 4.0-fold and 5.3-fold murine model of delayed-type hypersensitivity.1 increase in activity of MMPs 3, 9 and 13, respectively, by The relevance of RAGE-ligand interaction to murine zymography (Figure 3e,f). That activation of RAGE was collagen-induced arthritis was underscored by the critical in this process was demonstrated by the signifi- increased expression of RAGE and S100/calgranulins in cant reduction in activation of these MMPs in joint tissue joint tissue. Six weeks after immunization, RAGE and of sRAGE-treated mice (Figure 3e,f, respectively). S100/calgranulin expression was increased in joints from Taken together, these observations suggested that mice with arthritis compared with controls, especially interaction of S100/calgranulins and RAGE, both within the proliferating synovium (RAGE, Figure 1e,d; enriched in joint tissues in arthritis, activates proinflam- S100/calgranulin, Figure 1h,g, respectively). By immuno- matory and tissue-destructive mediators, thereby sup- blotting, levels of RAGE antigen were enhanced 2.2-fold porting roles for these molecules in the evolution of in arthritis vs control joints (Figure 1m). Although levels joint destruction. of S100/calgranulins were quite low in joint tissue of con- trol DBA/1 mice without collagen-induced arthritis, RAGE 82S allele enhances binding/signalling/gene expression of these proinflammatory mediators was expression by S100/calgranulins induced in murine serum albumin-treated mice Polymorphisms within the RAGE gene may influence immunized/challenged with bovine type II collagen these effects by altering proinflammatory pathways. To (Figure 1n). In mice subjected to blockade of RAGE with study this, we tested whether the glycine to serine poly- sRAGE, levels of RAGE and S100/calgranulin antigens morphism (G82S) within the ligand-binding domain of by immunoblotting were significantly reduced compared RAGE displayed enhanced ligand affinity and activation to mice treated with vehicle (Figure 1m,n respectively). of signal transduction pathways. We prepared stably- In parallel with reduced expression of RAGE and transfected CHO cells bearing the two RAGE alleles of S100/calgranulins in sRAGE-treated