and Immunity (2002) 3, 123–135  2002 Nature Publishing Group All rights reserved 1466-4879/02 $25.00 www.nature.com/gene RAGE and : 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 (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 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 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 joints, clinical interest, either the common RAGE 82G allele, or the vari- indices of inflammation18 were consistently lower in mice ant RAGE 82S allele. CHO cells provide a convenient treated with sRAGE vs phosphate-buffered saline (PBS) model, as they are devoid of detectable RAGE prior to or murine serum albumin (Figure 2a). In addition, histo- (not shown), or after stable transfection with pcDNA3.1 logic scoring19 revealed marked decreases in synovial vector alone (mock) (Figure 4a). Stably-transfected CHO hyperplasia/hypertrophy in the joints of sRAGE-treated cells were made with pcDNA3.1 containing either RAGE mice (Figure 2b). Further, histologic indices of cartilage 82G or RAGE 82S (Figure 4a). ELISA studies demon- and bone destruction20 were significantly decreased in strated that levels of RAGE antigen were identical in both sRAGE-treated mice compared with those animals CHO cells transfected to express RAGE 82G or RAGE 82S receiving PBS or murine serum albumin (Figure 2b). To (data not shown). Radioligand binding studies showed determine if blockade of RAGE suppressed dose-dependent binding of 125I-EN-RAGE (a prototypic immune/inflammatory responses to bovine type II col- S100/calgranulin) to CHO cells expressing RAGE 82G lagen at extra-articular sites, mice receiving vehicle or (122 ± 31 nM) and 82S receptor (Kd 77 ± 21 nM), though sRAGE were injected with bovine type II collagen (10 ␮g) the affinity of binding was greater with the 82S allele into ear tissue 6 weeks after immunization (day 42). (P = 0.008; Figure 4b). In contrast, CHO cells stably trans- Although baseline ear thickness was identical in all fected with the empty vector displayed no specific bind- groups, 18 hrs after injection of collagen, mice receiving ing of 125I-EN-RAGE (Figure 4b). That the interaction of murine serum albumin or PBS revealed significant RAGE-bearing CHO cells with 125I-EN-RAGE was spe- increases in ear thickness compared with those mice cific for interaction with RAGE was shown by sup- treated with sRAGE (Figure 2c). Levels of serum total IgG pression of specific binding in the presence of excess developed against bovine type II collagen were soluble extracellular domain of the receptor (sRAGE), or

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Figure 1 Induction of arthritis by bovine type II collagen in DBA/1 mice: increased expression of RAGE and S100/calgranulins. Six weeks after immunization (day 42), joint tissue from the hind feet (a–l) or stifle joint (m–n) was prepared for study. (a–l) Histology. In a,b,c, tissue was subjected to H&E analysis. Immunohistochemistry using anti-RAGE IgG (d,e,f); anti-S100/calgranulin IgG (g,h,i); or rabbit nonimmune IgG (j,k,l) was performed. Scale bar: a,b,c, 300 ␮m; and d,l,60␮m. (m,n) Immunoblotting: Lysates were prepared from stifle joints and subjected to immunoblotting using anti-RAGE IgG (m); or anti-S100/calgranulin IgG (n). N = 5 mice per condition were employed. Densitometric analysis of band intensity from all mice in each group was performed, and the mean ± s.d. is shown. Representative bands are shown. Statistical considerations are indicated in the figure.

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Figure 2 Blockade of RAGE suppresses inflammation in DBA/1 mice immunized/challenged with bovine type II collagen. (a) Clinical score: At the indicated times after immunization with bovine type II collagen and treatment with murine sRAGE or vehicle (murine serum albumin (MSA) or PBS), clinical scoring was performed by blinded observers. The mean ± standard deviation (s.d.) is shown; n = 10 mice per group. *indicates P Ͻ 0.05. (b) Histologic score: Histologic scoring of joints in four limbs was performed on H&E-stained sections from mice 6 weeks after immunization. The mean ± s.d. is shown; n = 10 mice per group. *indicates P Ͻ 0.05. (c) Ear swelling: Six weeks after immunization with bovine type II collagen, murine serum albumin (MSA)-, PBS- and sRAGE-treated mice were injected with bovine type II collagen into ear tissue. Ear thickness was measured by a blinded observer immediately prior to local injection, and 18 hrs later. The mean ± s.d. is shown; n = 5 mice per group. *indicates P Ͻ 0.05 vs ear thickness in PBS- and MSA-treated groups. (d) response to bovine type II collagen. At the indicated time points after immunization, plasma was retrieved from DBA/1 mice and subjected to ELISA for determination of total serum IgG levels to type II collagen. There were no statistically-significant differences in total IgG levels among the groups.

anti-RAGE IgG, but not by bovine serum albumin (BSA) respectively; lanes 12 and 11). Thus, compared with cells or nonimmune IgG (Figure 4c). expressing the common RAGE 82G allele, CHO cells These observations led us to test the concept that the expressing the variant RAGE 82S displayed significantly 82S RAGE isoform might amplify cellular activation increased phosphorylation of MEK 1/2 and p44/p42 beyond that seen in cells bearing wild-type RAGE. Incu- MAP kinases by 1.6- and 2.2-fold (P Ͻ 0.05; Figure 4d,e bation of mock-transfected CHO cells with a physiologi- respectively; lanes 12 and 7). In both RAGE 82G and cally-relevant concentration of EN-RAGE,4 10 ␮g/ml, did RAGE 82S-transfected cells, cellular activation by EN- not significantly increase intensity of the bands corre- RAGE was due to engagement of RAGE as demonstrated sponding to phosphorylated MEK 1/2 or p44/42 MAP by suppression of phosphorylation of MEK 1/2 and kinases22–23 (Figure 4d,e, respectively; lanes 1–5). How- p44/p42 by excess sRAGE (Figure 4d,e respectively; ever, exposure of RAGE 82G CHO transfectants to EN- lanes 8 and 13), or anti-RAGE IgG (Figure 4d,e respect- RAGE increased by 2.2-fold and 1.9-fold phosphorylated ively, lanes 9 and 14). Nonimmune IgG was without MEK 1/2 and p44/42 MAP kinases, compared with cul- effect (Figure 4d,e respectively; lanes 10 and 15). In each tures incubated with BSA alone (P Ͻ 0.01; Figure 4d,e case, respective levels of total MEK 1/2 and p44/p42 respectively; lanes 7 and 6). CHO transfectants bearing MAP kinases were identical (data not shown). RAGE 82S incubated with EN-RAGE displayed 3.6-fold To further support the hypothesis that RAGE 82S and 4.1-fold increase in phosphorylated MEK 1/2 and enhanced activation of key proinflammatory signal trans- p44/42 compared with BSA (P Ͻ 0.01; Figure 4d,e duction pathways, we assessed nuclear translocation of

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Figure 3 Blockade of RAGE suppresses generation of cytokines and MMPs in DBA/1 mice immunized/challenged with bovine type II collagen. (a,b) Joint tissue: Stifle joint tissue was retrieved 6 weeks after immunization with bovine type II collagen. Lysates were subjected to ELISA for detection of murine TNF-alpha (a) and IL-6 (b). Results were normalized per mg/tissue extract. (c,d) Plasma: Plasma from mice was subjected to ELISA for levels of TNF-alpha (c) and IL-6 (d). In a,b,c,d, n = 10 mice per group. The mean ± s.d. is shown. In a– d, *indicates P Ͻ 0.05. (e–f) Zymography: Stifle joint tissue was retrieved and lysates were prepared and zymography for detection of activity of MMP-2, -9 or -13 was performed. N = 10 mice/group. Densitometric analysis of band intensity from all mice/groups was performed, and the mean ± s.d. is shown. Representative bands are shown. In f, *indicates P Ͻ 0.05 vs MSA-treated mice with collagen-induced arthritis.

NF-␬B in CHO transfectants exposed to EN-RAGE. increase in intensity in nuclear binding activity following Electrophoretic mobility shift assays (EMSA) using 32P- incubation of cultures with EN-RAGE compared to BSA labelled consensus NF-␬B probe and nuclear extracts (P Ͻ 0.05; Figure 4f, lanes 7 and 6, respectively). NF-␬B from mock-transfected CHO cells showed no increase in activation was even more striking when RAGE 82S was intensity of the gel shift band after cultures were exposed substituted for RAGE 82G. RAGE 82S CHO transfectants to EN-RAGE (Figure 4f, lane 2). In CHO transfectants displayed 5.1-fold increased intensity of the gel shift expressing RAGE 82G, there was a prominent 2.4-fold band consequent to the presence of EN-RAGE, compared

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Figure 4 CHO cells bearing RAGE 82S display increased affinity and cellular responsiveness to EN-RAGE. CHO cells, which endogenously do not express RAGE antigen, were stably-transfected with pcDNA3.1 vector containing cDNA encoding human RAGE 82G or the variant allele 82S. (a) Immunoblotting: Lysates of stably-transfected CHO cells were prepared and subjected to immunoblotting using anti-human RAGE IgG. (b,c) Radioligand binding assays: Purified EN-RAGE was radiolabelled using 125-I and radioligand binding assays were perfor- med in 96-well tissue culture dishes containing the indicated transfected CHO cells. Equilibrium binding data were analyzed according to the equation of Klotz and Hunston. Where indicated, pretreatment with either , human soluble RAGE or bovine serum albumin was performed. The mean ± standard deviation (s.d.) is shown. In (c), *indicates P Ͻ 0.01 vs respective controls. (d,e) Activation of MEK 1/2 and p44/p42 MAP kinases: The indicated stably-transfected CHO cells were incubated with EN-RAGE for 1 h. Immunoblotting of cell lysates was performed using anti-phosphorylated MEK 1/2 (d) or p44/p42 MAP kinase (e). Where indicated, pretreatment with either BSA or sRAGE, or the indicated IgG for 2 h was performed. Control immunoblotting using antibody to total MEK 1/2 and p44/p42 MAP kinase revealed that there were no differences in levels of total MEK 1/2 or p44/p42 (not shown). (f) Activation of NF-␬B. Nuclear extracts were prepared from the indicated stably-transfected CHO cells incubated with EN-RAGE for 6 h and EMSA was performed. Where indi- cated, cells were pretreated with either nonimmune/anti-RAGE IgG, soluble RAGE or BSA for 2 h prior to incubation with EN-RAGE. In d,e,f, bands were scanned into a densitometer, and band density was quantified using ImageQuant. These experiments were performed at least three times and subjected to statistical analyses as indicated in the text and the figure legend. Representative experiments are shown.

to incubation with BSA (P Ͻ 0.001; Figure 4f, lanes 12 and rare number of subjects homozygous for 82S in our study 11, respectively). Thus, RAGE-mediated NF-␬B activation populations, we limited our studies to those individuals due to EN-RAGE was significantly enhanced by 2.1-fold homozygous for the common RAGE allele 82G, or those comparing RAGE 82S to 82G; P Ͻ 0.01. That activation of subjects heterozygous for 82G and 82S alleles. ELISA NF-␬B in transfected CHO cells by EN-RAGE resulted revealed that basal levels of RAGE antigen did not differ from ligation of common or variant RAGE was confirmed between MPs bearing 82G or 82G/82S (data not shown). by its suppression in the presence of sRAGE (Figure 4f, Signalling was compared in MPs from subjects bearing lanes 8 and 13, respectively), or anti-RAGE IgG RAGE 82G and RAGE 82G/82S by assessing activation (Figure 4f, lanes 9 and 14, respectively), but not by non- of MEK 1/2 and p44/p42 MAP kinases. In the presence immune IgG (Figure 4f, lanes 10 and 15, respectively). of EN-RAGE, MPs isolated from individuals bearing A critical test of the concept that S100/calgranulin- RAGE 82G/82S displayed an 4.5- and 4.3-fold increase in RAGE 82S interaction might enhance proinflammatory phosphorylated MEK 1/2 and p44 and p42 MAP kinases, effects was whether mononuclear phagocytes (MPs) respectively, compared with unstimulated cells (P Ͻ 0.01; retrieved from human subjects bearing the variant RAGE Figure 5a–d). However, MPs bearing the common RAGE 82S allele displayed heightened activation of signal trans- 82G allele exposed to EN-RAGE revealed a significant, duction molecules in the presence of ligand. Due to the although smaller (2.6- and 2.3-fold) increase in activation

Genes and Immunity RAGE and inflammatory arthritis MA Hofmann et al 129 of MEK 1/2 and p44/p42 MAP kinases, respectively RAGE 82S allele and association with rheumatoid (P Ͻ 0.01; Figure 5a–d). Importantly, EN-RAGE-triggered arthritis activation of MEK 1/2 and p44/p42 MAP kinases was Subjects from a previously-reported Caucasian popu- significantly increased in heterozygous RAGE 82G/82S- lation of RA patients (n = 205) and matched controls bearing MPs vs those MPs expressing RAGE 82G (n = 169)31 were evaluated. The RAGE 82S allele exhibited (P Ͻ 0.05; Figure 5b,d). an association with RA with relative risk (RR) = 2.6, To assess the functional consequences of enhanced P Ͻ 0.001 (Table 1a). Further analyses were performed to binding and activation of signal transduction molecules account for the linkage disequilibrium described pre- stimulated upon ligation of RAGE by EN-RAGE, we viously between RAGE 82S and an RA-associated allele, examined production of key inflammatory and tissue- HLA-DRB*0401.9,32–34 To assess whether the RAGE 82S degradative mediators linked to RA24–26 by MPs bearing allele confers risk independent of DRB1*0401, we com- common or variant RAGE alleles. Exposure of RAGE pared the frequency of the RAGE 82S allele in 0401+ 82G-bearing MPs to EN-RAGE caused a significant patients and controls. increase in generation of TNF-alpha detected in culture This analysis did not reveal a significant independent supernatant compared with quiescent cultures (470 ± 53 effect of RAGE 82S on RA disease susceptibility; P = 0.13 vs 15 ± 2 pg/ml; P Ͻ 0.001) (Figure 5e). However, upon by Fisher’s exact test (Table 1b). However, DRB1*0401 incubation of human MPs bearing RAGE 82G/82S, an continued to exhibit a significant association with RA in even greater increase in elaborated TNF-alpha was the absence of the RAGE 82S allele (Table 1c). observed in culture supernatants compared with basal levels (850 ± 64 vs 17 ± 2 pg/ml; P Ͻ 0.01) (Figure 5e). Discussion Importantly, although basal levels of TNF-alpha did not differ between RAGE 82G and 82G/82S-bearing MPs, S100/calgranulin molecules have been extensively linked levels of TNF-alpha in EN-RAGE-stimulated MPs were to immune/inflammatory diseases. Although their intra- increased 1.8-fold in the presence of RAGE 82G/82S com- cellular functions are relatively well-defined, such as cal- pared with RAGE 82G (P Ͻ 0.01; Figure 5e). Similarly, cium-dependent chemotaxis, degranulation and phago- 5,6,35 RAGE 82G-bearing MPs exposed to EN-RAGE displayed cytosis by activated granulocytes, an increasingly a small, but significant increase in generation of IL-6 com- recognized view is that these molecules may exhibit dis- 35 pared with basal expression (55 ± 7 vs 15 ± 2 pg/ml; tinct functions in the extracellular milieu. These con- P Ͻ 0.01) (Figure 5f). However, MPs bearing RAGE cepts are consistent with the observations that the degree 82G/82S revealed augmented generation of IL-6 upon of elevation of S100/calgranulins in inflamed foci may mirror the extent of disease activity. For example, in incubation with EN-RAGE compared with unstimulated 4 controls (160 ± 14 vs 12 ± 2 pg/ml; P Ͻ 0.01) (Figure 5f). addition to their association with disease severity in RA, S100/calgranulins have been linked to disease pro- EN-RAGE-stimulated MPs bearing RAGE 82G/82S gen- 36,37 erated increased amounts of IL-6 compared with cells gression in inflammatory bowel disease. Furthermore, strongly increased expression of a member of this family from subjects bearing RAGE 82G (2.9-fold) (P Ͻ 0.01; of molecules, psoriasin, has been demonstrated in psori- Figure 5f). atic lesions compared with adjacent unaffected skin in A central means by which structural elements of joints human subjects,38 thus further supporting the premise are degraded in unchecked RA is by generation of MMPs, that S100/calgranulins may not simply be ‘innocent such as MMP-9.27–30 We hypothesized that RAGE- bystanders’, but, rather, by virtue of their engagement of mediated MP activation would augment generation of RAGE,1 active participants in the host inflammatory MMP activity on cells bearing RAGE 82G/82S vs RAGE response. We speculate that triggered by interaction with 82G. We tested activity of MMP-9 in MPs retrieved from accumulating S100/calgranulins, activation of RAGE subjects bearing the common or variant RAGE alleles. engages key signalling pathways linked to proinflamma- MPs from subjects bearing RAGE 82G displayed a 2.3- tory responses, such as MAP kinases and NF-␬B, thereby fold increase in MMP-9 activity in the presence of EN- Ͻ amplifying expression of molecules linked to chronic RAGE compared with basal expression (P 0.01; cellular perturbation and tissue destruction. This hypoth- Figure 5g,h). However, MPs isolated from individuals esis appears to be supported by our studies in DBA/1 bearing RAGE 82G/82S demonstrated a 4.3-fold increase mice immunized/challenged with bovine type II col- in EN-RAGE-mediated MMP-9 activity compared with lagen, as activation of RAGE mediated, at least in part, Ͻ baseline (P 0.01; Figure 5g,h). Although basal levels of increased generation of inflammatory cytokines and acti- MMP-9 activity did not differ among 82G- or 82G/82S- vation of MMPs. Indeed, blockade of the receptor was bearing MPs, the extent of EN-RAGE-mediated enhanced associated with decreased clinical and histologic indices MMP-9 activity was significantly enhanced, 1.9-fold, in of inflammation and bone/cartilage destruction in this MPs bearing RAGE 82G/82S vs RAGE 82G (P Ͻ 0.05; model. Figure 5g,h). Polymorphic differences in the RAGE gene may lead Taken together, our studies in DBA/1 mice suggest to varied effects of RAGE in inflammatory settings. This that activation of RAGE amplifies cytokine generation led us to investigate the effects of the G82S polymor- and activation of MMPs in joint tissue primed by phism in the binding domain of RAGE on receptor func- immunization with bovine type II collagen. These con- tion and on the inflammatory response. In transfected siderations, together with our findings in vitro suggesting CHO cells, increased ligand affinity was found to be con- that S100/calgranulin-RAGE interaction modifies cellular ferred by the 82S isoform, which, upon further analysis, responses, especially in the presence of the 82S allele, led demonstrated upregulation of intracellular signalling us to evaluate the association of the RAGE 82S allele pathways linked to modulation of proinflammatory with RA. genes upon interaction with S100/calgranulin. Using

Genes and Immunity RAGE and inflammatory arthritis MA Hofmann et al 130

Figure 5 Human peripheral blood mononuclear phagocytes (MPs) expressing RAGE 82S display increased responsiveness to EN-RAGE. MPs were purified from the blood of human subjects bearing RAGE (G82G) (n = 5) or heterozygotes (G82S) (n = 5). (a–d) Activation of MEK 1/2 and p44/p42 MAP kinases: The indicated MPs were incubated with EN-RAGE or no mediator for 1 h. Cell lysates were prepared and immunoblotting performed using anti-phosphorylated MEK 1/2 (a,b) or p44/p42 MAP kinase (c,d). Densitometric analysis was perfor- med and mean ± s.d. is shown in b and d. Immunoblotting using antibody to total MEK 1/2 and p44/p42 MAP kinase revealed that there were no differences in levels of total MEK 1/2 or p44/p42 (not shown). (e,f) Generation of TNF-alpha and IL-6: Human MPs bearing the indicated RAGE alleles were cultured in the presence of either no mediator or EN-RAGE for 14 h. Supernatants were retrieved and levels of TNF-alpha (e) and IL-6 (f) determined by ELISA. The mean ± s.d. is shown. (g,h) MMP-9 activity: Human MPs bearing the indicated RAGE alleles were cultured in the presence of either no mediator or EN-RAGE for 14 h. Supernatants were retrieved and subjected to zymography to assess levels of activated MMP-9. In b, d, and h, bands from n = 5 G82G and n = 5 G82S subjects were scanned into a densitometer and band density was quantified. Representative experiments are shown. The mean ± s.d. is shown. In b, d, e, f and h, *indicates P Ͻ 0.01 vs baseline.

Genes and Immunity RAGE and inflammatory arthritis MA Hofmann et al 131 Table 1 (a)Allele frequencies of the RAGE 82S polymorphism in recent reports indicate that additional genes in the TNF = Ͻ RA patients and control subjects. Relative Risk (RR) 2.6, P 0.001 region may influence disease susceptibility, independent of DRB1, and unrelated to DR4 haplotypes.51–52 In our RAGE 82S (+) RAGE 82S (−) = view, a full understanding of the role of the MHC in RA RA patients (n 205) 52 153 and other autoimmune diseases will require a more Controls (n = 169) 19 150 detailed analysis of this genetic region, since there are many genes within the MHC, in addition to RAGE, (b) RAGE 82S is not associated with RA in the absence of DRB1* 0401. RR = 3.2, P = NS which may influence the immune/inflammatory response.53 RAGE 82S (+) RAGE 82S (−) Lastly, in addition to the 82S variant of the RAGE gene, RA patients (DRB1 0401 (−)) 5 90 it will be important to examine the potential role of other − Controls (DRB1 0401 ( )) 2 132 variants of RAGE in disease susceptibility and/or sever- ity in immune/inflammatory diseases. In this context, (c) DRB1*0401 is associated with RA, independent of RAGE 82S. = Ͻ recently identified polymorphisms within the promoter RR 5.0, P 0.001 of the gene encoding RAGE may hold the key to unique DRB1*0401 (+) DRB1*0401 (−) elements controlling enhanced transcription of the RAGE 54 RA patients (RAGE 82S (−)) 63 90 gene and increased expression of cell surface RAGE. As Controls (RAGE 82S (−)) 18 132 a number of studies have suggested that expression of RAGE is increased at sites of ligand accumulation such as diabetic atherosclerotic lesions, infected periodontium or Alzheimer disease brain,13,18,55 polymorphisms within isolated MPs from human subjects, we demonstrated sig- regulation elements and/or ligand-binding regions may, nificant differences in inflammatory responses between alone or in combination, orchestrate RAGE’s availability cells isolated from homozygous wild-type G82G and het- and/or functional potential in a given milieu.56 erozygous G82S subjects. These effects were quite In conclusion, we propose that these observations in marked between wild-type and heterozygote subjects, an animal model of inflammatory arthritis and in thereby suggesting that the 82S allele alone may impart S100/calgranulin-stimulated activation of 82S RAGE- an even greater effect, a premise which requires further bearing cells highlight ligand-RAGE engagement as an study. amplifier of proinflammatory mechanisms in immune/ Finally, we investigated the prevalence of the 82S allele inflammatory diseases. These findings provide a compel- in a population of subjects with RA. Our results demon- ling rationale to expand the analysis of RAGE polymor- strated a statistically-significant increase in prevalence of phisms and investigate their potential contribution to the 82S allele in subjects with RA. Sub-analysis for the susceptibility and/or progression of the inflammatory presence/absence of DRB1*0401 led to the loss of an stat- response in diverse diseases with an immune/ istically-significant association of 82S with RA (P = 0.13 inflammatory component, such as RA, despite odds ratio 3.2). Conversely, DRB1*0401 continued and diabetes. to exhibit a significant association with RA in the absence of the RAGE 82S allele. Clearly, the large genetic suscep- tibility to RA related to the MHC39,40 cannot be ascribed Materials and methods solely to the RAGE 82S allele. Due to the relatively low frequency of the 82S allele and low subject numbers in Murine model of bovine collagen type II-induced these Caucasian cases and controls after sub-analysis, it arthritis remains possible that the RAGE G82S polymorphism is related independently to RA. This issue requires further Induction of arthritis: Male DBA/1 mice were purchased study. from the Jackson Laboratories (Bar Harbor, ME, USA). The literature is conflicting with respect to a role for On day 0, mice weighing 20–30 gms were injected intra- DR4 haplotypes in disease severity.41–50 In view of the dermally at the base of the tail with bovine type II col- linkage disequilibrium between DRB1*0401 and the lagen, 200 ␮g (Sigma, St Louis, MO, USA), dissolved in RAGE 82S allele, it will be interesting to specifically test acetic acid (0.01 M) and emulsified in complete Freund’s whether DR4 haplotypes containing the RAGE 82S allele adjuvant (ICN Biochemcials, Costa Mesa, CA, USA). have an influence on disease progression and outcome. Three weeks later (day 22), mice were challenged by By analogy with our observations in cultured CHO and injection of bovine collagen type II (200 ␮g) in incomplete human mononuclear cells, we speculate it may be Freund’s adjuvant.15–17 Beginning 3 weeks after immuni- possible to identify a subset of subjects with RA parti- zation (day 22), mice were treated with murine soluble cularly vulnerable to the sequelae of heightened RAGE1 (100 ␮g per day by intraperitoneal route). Vehicle production/activation of proinflammatory mediators. treatment consisted of murine serum albumin (Sigma), Specifically, in parallel with outcome measures of clinical 100 ␮g per day by intraperitoneal injection, or equal vol- severity, such as numbers of bony erosions, the extent of umes of PBS. Treatment was continued daily until sacri- elevation of TNF-alpha, IL-6 and MMP activities in rheu- fice. All murine studies were performed in accordance matoid joint fluid and/or RA plasma must be correlated with the policies of the university’s Institutional Animal with subject genotype. Care and Use Committee. A general implication of these studies is that the classi- cal DRB1 associations with RA may not completely Determination of anti-collagen IgG: ELISA was performed explain the underlying reasons for the strong linkage and to determine levels of IgG to bovine type II collagen in association of the MHC with RA. Indeed, a number of dba/1 mice. Plastic Maxisorp 96-well plates (Nunc,

Genes and Immunity RAGE and inflammatory arthritis MA Hofmann et al 132 Naperville, IL, USA) were coated with bovine type II col- automated DNA sequencer (Perkin Elmer Biosystems, lagen (5 ␮g/ml) in bicarbonate/carbonate buffer; pH 9.3 Foster City, CA, USA) to confirm the inserted sequence for 16 h at 4°C. Unbound sites were blocked with PBS changes and to ensure that no other mutations were cre- containing BSA (1%) for 8 h at 4°C. Wells were washed ated. RAGE 82G and 82S cDNAs were excised from five times in PBS and then incubated for 16 h in samples pCR2.1TOPO using EcoR I and subcloned into the of mouse plasma (dilution, 1:50) in PBS at 4°C. Wells pcDNA3.1 expression vector (Invitrogen). Cells were were washed five times with PBS and incubated with PBS transfected with plasmid DNA using lipofectamine (Life containing 1% BSA and horse radish peroxidase-labelled Technologies) encoding the following: pcDNA3.1 con- anti-mouse IgG (Sigma; 1:500 dilution) for 2 h at room taining full-length RAGE cDNA (−82G−), pcDNA3.1 con- temperature. Wells were washed with PBS five times and taining RAGE cDNA (−82S−), or pcDNA3.1 containing no developed using o-phenylenediamine dihydrochloride insert (mock). Twenty-four hours after transfection, selec- (Sigma). A standard curve was prepared using serial tion was begun using G418 (1 mg/ml) (Life dilutions from a mouse antibody cocktail against bovine Technologies). RAGE expression was assessed by immu- type II collagen (Chemicon, Temecula, CA, USA). noblotting in stably-transfected cells after 6 weeks. Cells were incubated with EN-RAGE (Extracellular Newly- Assessment of arthritis: Arthritis was evaluated by clinical Identified RAGE binding protein), a prototypic and histologic scoring by observers blinded to the experi- S100/calgranulin molecule previously-identified to be a mental conditions. Clinical severity of arthritis in each of signal transduction ligand for RAGE,1 or the indicated four limbs was assessed by the following scale:19 mediators. Assessment of activation of phosphorylated 0 = normal; 1 = slight erythema and swelling; 2 = pro- MEK 1/2 and p44/p42 MAP kinase by immunoblotting nounced edematous swelling; and 3 = joint deformity or nuclear translocation of NF-kB was performed. All with ankylosis, resulting in a maximum score of 12 per reagents were tested for endotoxin content using the animal. Histologic scoring of arthritis and cartilage and limulus amebocyte assay (Sigma). Any endotoxin ident- bone destruction was performed on hematoxylin and ified in these assays was removed by chromatography eosin-stained sections of extremity joints by the following of the protein solutions onto Detox-igel columns (Pierce, scale:20 (i) synovial lesions: 0, no lesions; 1: mild effect; 2: Arlington Hts, IL, USA). The material was re-tested in moderate effect/proliferation; 3: severe lesions with the limulus amebocyte assay for the absence of detect- destruction; (ii) cartilage destruction: 0, none; 1, mild; 2, able endotoxin. moderate; 3, severe destruction with loss or complete fragmentation of cartilage; and (iii) bone destruction: 0, Radioligand binding assays: Purified EN-RAGE was radio- none; 1, mild destruction of subchondral bone; 2, moder- labelled using 125-I and Iodobeads (Pierce, Arlington ate destruction; and 3, severe destruction with loss of Heights, IL, USA) to a specific activity of approximately large areas of bone. In other studies, 6 weeks after initial 5000 cpm/ng. Radioligand binding assays were perfor- sensitization, bovine type II collagen (10 ␮g) was injected med in 96-well tissue culture dishes containing the indi- into the ear of each mouse. Eighteen hours later, thick- cated transfected CHO cells in the presence of the indi- ness of the ear was assessed using calipers by an observer cated concentration of radiolabelled EN-RAGE ± a 50- blinded to the experimental conditions. fold molar excess of unlabelled EN-RAGE in PBS contain- ing calcium/magnesium and BSA, 0.2%, for 3 h at 37°C. Immunoblotting and ELISA: Mice were sacrificed 3 or 6 Wells were washed rapidly with washing buffer (PBS weeks after challenge. The stifle joint was removed and containing Tween 20 (0.05%)). Elution of bound material homogenized in tris-buffered saline containing protease was performed in a solution containing heparin, inhibitors (Complete Protease Inhibitor, Boehringer- 1 mg/ml. The aspirate was counted in a gamma counter Mannheim, Indianapolis, IN, USA). SDS-PAGE and (LKB, Gaithersburg, MD, USA). Equilibrium binding data immunoblotting were performed on extracts of stifle joint were analyzed according to the equation of Klotz and tissue using the following antibodies: anti-RAGE IgG and Hunston:57 B = nKA/1 + KA, where B = specifically anti-S100/calgranulin IgG as previously described1 (4.7 bound ligand (total binding, wells incubated with tracer and 2.0 ␮g/ml, respectively). ELISA for TNF-alpha and alone, minus nonspecific binding, wells incubated with IL-6 was performed using kits from R&D Systems tracer in the presence of excess unlabelled material), (Minneapolis, MN, USA). n = sites/cell, K = the dissociation constant, and A = free ligand concentration) using nonlinear least-squares Zymography: Zymography for detection of MMP-3, 9 and analysis (Prism, San Diego, CA, USA). Where indicated, 13 activity was performed using gelatin or casein-laden pretreatment with antibodies (70 ␮g/ml), or human sol- gels from Novex/Invitrogen (Carlsbad, CA, USA). uble RAGE (50-fold molar excess), was performed.

Chinese hamster ovary (CHO) cell studies Activation of pMEK 1/2 and p44/p42 MAP kinases: CHO CHO cells were obtained from ATCC (Rockville, MD, cells were incubated with EN-RAGE, 10 ␮g/ml, for 1 h. USA) and cultured in F12 medium containing fetal bov- Cells were lysed in lysis buffer (New England Biolabs, ine serum (10%) (Life Technologies, Grand Island, NY, Beverly, MA, USA) and lysate was subjected to centrifug- USA). To generate the 82S allele, the cDNA encoding ation. Protein concentration of the supernatant was human RAGE10 was cloned using the TOPO TA cloning determined by Bio-Rad assay (Bio-Rad, Hercules, CA, system into pCR2.1TOPO vector for mutagenesis USA). Equal amounts of protein were subjected to SDS- (Invitrogen). Site-directed mutagenesis to insert the PAGE (Novex/Invitrogen). Contents of the gels were (Ϫ82GϪ)to(Ϫ82SϪ) change was performed using the transferred to nitrocellulose and immunoblotting perfor- GeneEditor In Vitro SDM System (Promega, Madison, med using anti-phosphorylated/total p44/p42 MAP kin- WI, USA). Sequencing was performed using an ABI310 ase and MEK 1/2 (New England Biolabs) (1 ␮g/ml).

Genes and Immunity RAGE and inflammatory arthritis MA Hofmann et al 133 Electrophoretic mobility shift assay: Nuclear extracts were USA)) were then standardized using untreated cells as prepared using the NE kit (Pierce) and EMSA performed control, and assigned a value of ‘1’. Similarly, in animal employing consensus 32P-labelled probe for NF-kB as studies, an arbitrary value of ‘1’ was assigned to bands described.1 Where indicated, cells were treated with (immunoblotting or zymography) in assays from animals either nonimmune/anti-RAGE IgG, or soluble RAGE.1,21 without collagen-induced arthritis). Statistical analyses to identify the incidence of G82S in human subjects were Peripheral blood-derived mononuclear phagocyte performed as previously described.59–60 P Ͻ 0.05 was con- (MPs) studies sidered statistically significant.

Cellular isolation: Venous blood was obtained from heal- thy volunteers (30 ml) bearing RAGE 82G or 82G/82S in References accordance with the policies of the university’s Insti- tutional Review Board. Due to the rare numbers of sub- 1 Hofmann MA, Drury S, Fu C et al. RAGE mediates a novel proinflammatory axis: a central for jects homozygous for the 82S allele, our studies were con- S100/calgranulin polypeptides. Cell 1999; 97: 889–901. fined to those bearing solely 82G or heterozygous 2 Chilosi M, Mombello A, Montagna L et al. Multimarker immu- 82G/82S. Mononuclear cells were isolated using Histo- nohistochemical staining of calgranulin, chloroacetate esterase, paque 1077 (Sigma) and cultured on plastic dishes for 3 h and S100 for simultaneous demonstration of inflammatory cells at 37°C. Nonadherent cells were removed by washing in on paraffin sections. J Histochem Cytochem 1990; 38: 1669–1675. PBS. Adherent cells were removed by incubation with 3 Youssef P, Roth J, Frosch MP et al. Expression of myeloid related EDTA (2 mM) for 15 mins at 37°C. Cells (Ͼ95% MPs as proteins (MRP) 8 and 14 and the MRP8/14 heterodimer in rheu- assessed by immunostaining with anti-CD68 IgG) were matoid arthritis synovial membrane. J Rheumatol 1990; 26: seeded in tissue culture-coated wells for study. 2523–2528. 4 Frosch M, Strey A, Vogl T et al. Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and Activation of phosphorylated MEK 1/2 and p44/p42 MAP kin- activated endothelium and are useful markers for monitoring ases and generation of IL-6 and TNF-alpha: MPs were seeded disease activity in pauciarticular-onset juvenile rheumatoid onto the wells of 24-well tissue culture plates at a density arthritis. Arthritis Rheum 2000; 43: 628–637. of 5 × 105 cells per well. Cells were stimulated with either 5 Zimmer DB, Cornwall EH, Landar A, Song W. The S100 protein BSA or EN-RAGE (10 ␮g/ml), and immunoblotting for family: history, function, and expression. Brain Res Bul 1995; 37: detection of phosphorylated/total MEK 1/2 and p44/p42 417–429. MAP kinases performed as above. Supernatant was 6 Schafer BW, Heinzmann CW. The S100 family of EF-hand cal- assayed for IL-6 and TNF-alpha using ELISA kits from cium-binding proteins: functions and pathology. TIBS 1996; 21: 134–140. R&D systems (Minneapolis, MN, USA). 7 Rammes A, Roth J, Goebeler M, Klempt M, Hartmann M, Sorg C. Myeloid-related protein (MRP) 8 and MRP14, calcium-bind- Patient population and detection of 82S ing proteins of the S100 family, are secreted by activated mono- polymorphism cytes via a novel, tubulin-dependent pathway. J Biol Chem 1997; RA patients used for association studies meet the criteria 272: 9496–9502. of the American College of Rheumatology58 and were 8 Hudson BI, Stickland MH, Grant PJ. Identification of polymor- taken from patient populations collected previously at phisms in the Receptor for Advanced Glycation End Products the Medical College of Virginia, Virginia Commonwealth (RAGE) gene. Diabetes 1998; 47: 1155–1157. University.31 The following primers were synthesized for 9 Prevost G, Fajardy I, Fontaine P, Danze PM, Besmond C. detection of the glycine82serine (G82S) polymorphism Human RAGE Gly82Ser dimorphism and HLA class II DRB1- 8 ′ DQA1-DQB1 haplotypes in type 1 diabetes. Eur J Immunogenet of the RAGE gene: sense primer: 5 GTAAGCG 1999; 26: 343–348. GGGCTCCTGTTGCA-3′ and the antisense primer: ′ ′ 10 Neeper M, Schmidt AM, Brett J et al. Cloning and expression of 5 GGCCAAGGCTGGGGTTGAAGG-3 . Whole blood RAGE: a cell surface receptor for advanced glycosylation end (20 ␮l) was obtained from human volunteers in accord- products of proteins. J Biol Chem 1992; 267: 14998–15004. ance with the standards and policies of the Institutional 11 Schmidt AM, Vianna M, Gerlach M et al. Isolation and charac- Review Boards of the institutions. Genomic DNA was terization of binding proteins for advanced glycosylation end- prepared using a kit from Qiagen (Valencia, CA, USA); products from tissue which are present on the endothelial cell surface. J Biol Chem 1992; 267: 14987–14997. 10 ng was amplified using Taq DNA polymerase (Life ⑀ Technologies) in a final volume of 25 ␮l and PCR perfor- 12 Kislinger T, Fu C, Huber B et al. N carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced gly- med as follows: 94°C for 30 secs, 62°C for 45 s, and 72°C ␮ cation endproducts that activate cell signalling pathways and for 60 s for a total of 35 cycles. PCR product (25 l) was modulate gene expression. J Biol Chem 1999; 274: 31740–31749. digested with Alu I (Life Technologies), 3 U for 16 h at 13 Yan SD, Chen X, Fu J et al. RAGE and peptide 37°C, followed by electrophoresis on 2% agarose gels. neurotoxicity in Alzheimer’s disease. Nature 1996; 382: 685–691. 14 Hori O, Brett J, Slattery T et al. The receptor for advanced gly- Statistical analysis cation endproducts (RAGE) is a cellular binding site for ampho- Statistical comparisons were determined using one-way terin: mediation of neurite outgrowth and coexpression of analysis of variance (ANOVA); where indicated, individ- RAGE and amphoterin in the developing nervous system. J Biol ual comparisons were performed using Student’s t-test. Chem 1995; 270: 25752–25761. For cell culture studies, immunoblotting, zymography 15 Courtenay JS, Dallman MJ, Dayan AD, Martin A, Mosedale B. Immunization against heterologous type II collagen induces and electrophoretic mobility shift assays were performed arthritis in mice. Nature 1980; 283: 666–668. at least three times. Statistical analyses were performed 16 Trentham DE, Townes AS, Kang AH. Autoimmunity to type II across each of the experiments by scanning gels/ auto- collagen an experimental model of arthritis. J Exp Med 1997; 146: radiograms into a densitometer. Arbitrary units of inten- 857–868. sity (Image Quant/Molecular Dynamics (Foster City, CA, 17 Cathcart ES, Hayes KC, Gonnerman WA, Lazzari AA, Franzblau

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