Expression of Receptor for Advanced GlycationEnd Products in Sarcoid GranulomasIlaria Campo1, Patrizia Morbini2, Michele Zorzetto1, Carmine Tinelli3, Enrico Brunetta4, Chiara Villa2,Cristina Bombieri5, Mariaclara Cuccia6, Carlo Agostini4, Valeria Bozzi6, Angelica Facoetti7, Ilaria Ferrarotti1,Paola Mazzola1, Roberta Scabini1, Gianpietro Semenzato4, Pier Franco Pignatti5, Ernesto Pozzi1, andMaurizio Luisetti1

1Laboratorio di Biochimica & Genetica, Clinica Malattie Apparto Respiratorio, 2Istituto di Anatomia ed Istologia Patologica, and3Unita di Biometria, IRCCS Policlinico San Matteo, Universita di Pavia, Pavia, Italy; 4Immunologia Clinica, Dipartimento di Medicina Clinicae Sperimentale, Universita di Padova, Padova, Italy; 5Dipartimento Materno-Infantile e di Biologia-Genetica, Universita di Verona,Verona, Italy; and 6Laboratorio di Immunogenetica, Dipartimento di Genetica e Microbiologia, and 7Dipartimento di Biologia Animale,Universita di Pavia, Pavia, Italy

Rationale: The receptor for advanced glycation end products (RAGE)engages a number of ligands implicated in inflammatory processes.The RAGE coding gene maps to the 6p21.32 region, close to thegenes DRB1 and BTNL2, which are associated with sarcoidosis.Objectives: We investigated a possible implication of RAGE in sarcoidgranulomas.Methods: RAGE and major ligands (N-ε-carboxy-methyl-lysine [CML],S100A12, and S100B) expression was investigated by immunostain-ing of 99 paraffin-embedded biopsies of sarcoid tissues, and expres-sion patterns were determined. Among the three RAGE gene single-nucleotide polymorphisms investigated, �374 T/A was selected,characterized in terms of transcriptional effect (immunocytochem-istry and real-time polymerase chain reaction), and its frequencywas determined in DNA extracted from biopsies.Measurements and Results: RAGE, CML, S100A12, and S100B immu-noreactivity was observed in all sarcoid granulomas, although atdifferent intensities. The degree of RAGE expression significantlycorrelated with the degree of S100A12 expression. The �374 TT/ATgenotypes, associated with higher RAGE transcriptional activity, weremore frequent in the sarcoidosis biopsy group than in control subjects,and the association was confirmed in a second, independent seriesof 101 patients with sarcoidosis.Conclusions: We showed the association of RAGE and its ligandswith sarcoidosis and suggest that an intrinsic genetic factor couldbe in part involved in its expression. In Italian patients, the �374T/A polymorphism seems to be significantly associated with thisdisease.

Keywords: ligands; immunohistochemistry; genetics; real-timepolymerase chain reaction

Sarcoidosis is a systemic inflammatory disorder characterizedby infiltration of CD4�-activated T cells that give rise to theformation of specific noncaseating granulomas, the hallmark ofthe disease, in a variety of affected organs (1, 2). Genes mappingwithin the HLA region are believed to play a significant role inthe pathogenesis of sarcoidosis for a number of reasons: (1)products of these genes are involved in the initial event leading

(Received in original form January 31, 2006; accepted in final form December 11, 2006 )

Supported by a Ricerca Corrente grant from IRCCS Policlinico San Matteo (M.L.and I.C.) and by the MIUR-Cofin projects 2004 (E.P.).

Correspondence and requests for reprints should be addressed to Maurizio Luisetti,M.D., Clinica Malattie Apparto Respiratorio, IRCSS Policlinico San Matteo, ViaTaramelli 5, 27100 Pavia, Italy. E-mail: m.luisetti@smatteo.pv.it

This article has an online supplement, which is accessible from this issue’s tableof contents at www.atsjounals.org

Am J Respir Crit Care Med Vol 175. pp 498–506, 2007Originally Published in Press as DOI: 10.1164/rccm.200601-136OC on December 14, 2006Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY

Scientific Knowledge on the Subject

The receptor for advanced glycation end products and itsligands represents a system for amplification of inflamma-tory processes. The RAGE gene maps close to DRB1 andBTNL2, two genes strongly associated with sarcoidosis.

What This Study Adds to the Field

RAGE and its ligands are expressed in sarcoid granulomas.The RAGE �374 T/A polymorphism is significantly associ-ated with sarcoidosis.

to granuloma formation (i.e., presentation of an unknown anti-gen or antigens by antigen-presenting cells to T lymphocytes)(1–3); (2) various genetic studies have shown that HLA class IIalleles are associated with sarcoidosis (4–8); and (3) from ananalysis of 55 German families with siblings suffering from sar-coidosis, Schurmann and coworkers demonstrated a strong asso-ciation maximum multipoint nonparametric linkage score 3.2with the marker D6S1666 mapping in HLA region at the junctionbetween class II and class III (6p21) (9).

Subsequently, a subregion corresponding to the butyrophilin-like 2 (BTNL2) gene was found to be associated with sarcoidosis(10). The gene coding for the receptor for advanced glycation endproducts (RAGE) maps to the same 6p21.32 position. RAGErecognizes tertiary structures rather than amino acid sequencesand therefore acts as a multiligand receptor with the abilityto engage classes of molecules (11, 12). Major ligands, termed“advanced glycation end products” (AGEs), are a heteroge-neous group of compounds deriving from nonenzymatic glyca-tion and oxidation of proteins and lipids. Generically, AGEs aretriggered by oxidant stress and can accumulate in the vessel wall(13). They have been reported to form in several pathophysio-logic situations associated with inflammation. Moreover, RAGEbinds proinflammatory cytokine–like mediators of the S100/calgranulin family (14), which have been repeatedly associatedwith granulomatous disorders, including sarcoidosis (15). RAGEis, therefore, heavily implicated in inflammatory processes, par-ticularly contributing to their amplification (11).

Considering the previously mentioned points, we hypothe-sized that RAGE could be involved in the inflammatory processrelated to granuloma formation in sarcoidosis. In this article,we describe a series of experiments showing that RAGE isexpressed in sarcoid granulomas, that its naturally occurring

Campo, Morbini, Zorzetto, et al.: RAGE in Sarcoidosis 499

ligands are present in the sarcoidosis inflammatory process, andthat the intensity of RAGE expression in sarcoid granulomasvaries, and we speculate that a functional polymorphism in thepromoter region of the RAGE gene could be implicated, at leastpartly, in the different degrees of RAGE expression at sitesof sarcoidosis activity. Some of the results of this study werepreviously reported in abstract form (16).

METHODS

Immunohistochemistry

Paraffin-embedded tissue specimens, obtained from patients with sar-coidosis for biopsy confirmation between 1992 and 2003 at the PathologyDepartment of Pavia, were selected. All subjects were white and fromNorthern Italy. Anti-RAGE goat antibody and antibodies againstRAGE ligands N-ε-carboxy-methyl-lysine (CML), S100B, and S-100A12were immunoreacted on paraffin tissue sections. RAGE immunoreac-tivity was determined by means of quantitative optical density analysis;RAGE ligand expression was assessed semiquantitatively. Additionalinformation is available in the online supplement.

RAGE and BTNL Gene Single-Nucleotide Polymorphisms

Genomic DNA from four populations was used for this investigation:

1. DNA extracted from the paraffin-embedded sarcoid tissues.

2. DNA extracted from peripheral blood mononuclear cells(PBMCs) of a second, replication series of patients with sarcoido-sis, followed up at centers in Pavia and Padua. The diagnosis ofsarcoidosis was made according to international standards (17).None of the subjects in the replication series was included in thetissue specimen series.

3. DNA from PBMCs of healthy laboratory and clinical staff andof blood and bone marrow donors. The subjects in this last group,who acted as control subjects, were ethnically and geographicallymatched with the subjects in the first two populations of patientswith sarcoidosis and were enrolled in two consecutive series toobtain a second control population.

4. Three series of “positive” control subjects (patients with lungdisease different from sarcoidosis): asthma, idiopathic pulmonaryfibrosis, and systemic sclerosis. Characteristics of some of thesepatients have been reported (18, 19).

All individuals gave informed consent before entering the study, whichwas approved by the ethical committees of the institutions involved.The ethical committees approved the use of human tissue for thisinvestigation. RAGE �374 T/A (rs1800624), �429 C/T, 1704 G/T genepolymorphisms, and BTNL2 rs2076530 single-nucleotide polymorphisms(SNPs) were analyzed (details available in the online supplement).

Determination of the Functional Expression of the RAGE –374T/A Polymorphism

Immunocytochemistry. Goat polyclonal anti-RAGE antibody was used.Blood samples, collected in ethylenediaminetetraacetic acid, were takenfrom 58 healthy donors previously genotyped for the –374 T/A polymor-phism. PBMCs were isolated by centrifugation on a 1,077 density gradient(Istopaque; Gibco BRL, Corning, NY) and immunostained accordingto the standard streptavidin-biotin peroxidase technique using a cell andtissue staining kit (R&D Systems, Minneapolis, MN). Two independentobservers semiquantitatively evaluated the relative degree of staining.Different values of positivity were scored as weak, moderate, and intense.

Statistical Analysis

Quantitative data are presented as means and SDs. The Hardy-Weinbergequilibrium was assessed by the goodness-of-fit test for biallelic mark-ers. Frequencies were compared with the �2 test or Fisher’s test (whenappropriate), and by the odds ratio (ORs), calculated by Woolf’smethod, with their 95% confidence limits. Genotype–phenotype corre-lations were performed by Mann-Whitney U test. A p value of lessthan 0.05 was considered statistically significant. All tests were twosided. Analyses were performed with Statistica for Windows software(StatSoft, Inc., Tulsa, OK). Linkage disequilibrium and haplotype fre-

quencies were estimated with the EHWIN software program (Rocke-feller University, New York, NY) (20).

RESULTS

Immunohistochemistry

Ninety-nine consecutive sarcoid biopsy samples fulfilling histo-logic and clinical criteria for sarcoidosis were collected for theimmunohistochemical characterization of RAGE and its majorligands in granulomas. Specimens were obtained from 47 maleand 52 female patients, with a mean age of 40 � 3 years (range,18–71 yr), and included tissues from peripheral lymph nodes(n � 58), lung (n � 20), skin (n � 16), and liver (n � 5). Insarcoid granulomas, RAGE was expressed in the cytoplasm ofepithelioid histiocytes and multinucleated giant cells. RAGEimmunoreactivity was observed in all sarcoid granulomas; itsmean optical density (OD) differed in different cases (range,0.035612–0.380846), whereas it was fairly homogeneous withineach single case (SD range, 0.004–0.05) (Figure 1). Skin and lunggranulomas showed the strongest degree of immunoreactivity.RAGE was also expressed in tissue outside the granulomas(details are available in the online supplement).

Immunohistochemical investigations were extended to themajor RAGE ligands CML, S100A12, and S100B (Figure 2).CML immunoreactivity was present in epithelioid histiocytesand giant cells, with the intensity of reactivity ranging fromlow to high. The expression of S100A12 was highly variable inhistiocytes and giant cells, ranging from negative to stronglypositive. Epithelioid and giant cells of sarcoid granulomas didnot express S100B, although this RAGE ligand was presentin dendritic cells infiltrating granulomas. At semiquantitativeevaluation, we observed an overall higher degree of RAGEligand expression in granulomas with higher mean RAGE opti-cal density. However, statistical analysis only confirmed a sig-nificant correlation between the degree of expression ofRAGE and of S100A12 (p � 0.05) (Figure 3) but not of S100B(p � 0.05) or CML (p � 0.05). No correlation was found betweenthe stage of granuloma evolution and RAGE expression.

Functional Variant of the RAGE Gene

We examined the mechanisms underlying the varying degree ofRAGE expression within sarcoid granulomas. We hypothesizedthat a possible intrinsic factor within the RAGE gene might playa role. We assessed the frequency of three RAGE SNPs (–374T/A, –429 T/C, and 1704 G/T) in a control population (Table1). The first two polymorphisms were selected based on the factthat analysis of the RAGE promoter sequence revealed thepresence of potential transcription factor binding sites, whichcould interfere with the modulation of gene expression (21).The 1704 G/T polymorphism was investigated because of thepreviously reported involvement in the oxidative stress (13). Wethen selected the RAGE –374 T/A polymorphism for furtherinvestigation because it was the only SNP investigated with asignificant minor allele frequency (47%) in healthy individuals(Table 1).

We performed two experiments to assess the transcriptionaleffect of the –374 T/A RAGE polymorphism. In the first experi-ment, immunocytochemical analysis of PBMCs isolated from58 healthy donors showed only weak immunostaining for RAGEin subjects with the AA genotype (Figure 4B), suggesting a lowlevel of RAGE expression on the cell surface, whereas PBMCsfrom subjects with the AT and TT genotypes (Figures 4C and 4D,respectively) showed stronger immunoreactivity. In the secondexperiment, we used real-time polymerase chain reaction tomake a semiquantitative analysis of PBMCs isolated from 58healthy blood donors. In our conditions, we identified a trend

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Figure 1. Immunohistochemical assess-ment of receptor for advanced glycationend products (RAGE) expression in sar-coid granulomas in two different cases ofnodal sarcoidosis (case 1: A, C, E; case 2:B, D, F ), one showing a high degree ofRAGE immunoreactivity (brown stain) (E )and the other showing a lower intensityof expression (F ). Representative areas ofhematoxylin-eosin–stained sections andof negative control samples of RAGE im-munoreactions of the two cases are pre-sented in A and B, respectively, and in Cand D, where tissue details are visible dueto light hematoxylin counterstain. (Origi-nal magnification: 20.)

in which the AA genotype seemed to correlate with a lowertranscriptional activity, whereas the TT/AT genotypes seemedto be associated with higher RAGE expression, although thisdifference did not reach statistical significance (as shown in Fig-ure E1 in the online supplement).

The Frequency of RAGE and BTNL2 Polymorphisms

The frequency of the RAGE gene –374 T/A polymorphism in theseries of sarcoid biopsies is reported in Table 2. The frequency ofthe �374 TT/AT genotypes in the group of sarcoidosis biopsieswas significantly higher than that in the control subjects (90 vs.76%; �2 � 8.2781; p � 0.004; OR, 2.7590; 95% confidence interval[CI], 1.3523–5.6291). The –374 AA genotype was significantlyless frequent in biopsies from patients with sarcoidosis than incontrol subjects (OR, 0.3625; 95% CI, 0.1776–0.7395). Afterstratifying for sex, the –374 AA genotype frequencies remainedsignificantly different between sarcoidosis biopsies from femalesubjects and control subjects (�2 � 4.4817; p � 0.034). This kindof female sex–restricted association is not unusual in our Italianseries of patients with sarcoidosis (5). The investigation wasextended to a second, confirmatory series of patients with sar-coidosis (n � 101), whose clinical characteristics have been re-ported (22), and it was compared with a second group of healthycontrol subjects (n � 65). The findings were confirmed in this

second group of subjects, among whom the frequency of theTT/AT genotypes was 94% (�2 � 7.7127; p � 0.005 vs. controlsubjects) (Table 3). Genotypes of the patients with sarcoidosisand of control subjects agreed with the Hardy-Weinberg equilib-rium, whereas the genotypes of sarcoidosis biopsies were slightlyout of equilibrium (p � 0.0231). When the frequency of the–374 T/A RAGE polymorphism was analyzed in function of theclinical characteristics of the series of patients with sarcoidosis(radiologic stage, relapses, and prognosis), we were unable todetect any significant correlation (data not shown). We alsoincluded three series of “positive” controls (i.e., patients with alung disease whose pathogenesis is believed to be different fromthat of sarcoidosis). The frequencies in 74 patients with idio-pathic pulmonary fibrosis (TT/AT, 80%; AA, 20%) and in 117patients with systemic sclerosis (TT/AT, 75%; AA, 25%) didnot significantly differ from the 327 healthy control subjects,whereas in 101 patients with asthma, the frequencies (TT/AT,86%; AA, 14%) were slightly but significantly different fromhealthy control subjects (�2 � 3.858; p � 0.0459).

We evaluated the linkage disequilibrium between the twoRAGE and BTNL2 SNPs (rs1800624 and rs2076530) and ob-tained a D value of 0.2127 (p � 0.028) for control subjects anda D value of 0.325 (p � 0.028) for patients with sarcoidosis.Table 4 reports the deduced haplotype frequencies in patients

Campo, Morbini, Zorzetto, et al.: RAGE in Sarcoidosis 501

Figure 2. Expression of RAGE and RAGEligands in pulmonary sarcoidosis. Repre-sentative areas of a hematoxylin-eosin–stained section and of negative controlof immunoreactions are presented, re-spectively, in A and B, where tissue detailsare visible due to light hematoxylincounterstain. Epithelioid histiocytes andgiant cells of sarcoid granulomas arestrongly immunoreactive for RAGE (C,brown stain), for N-ε-carboxy-methyl-lysine (D, brown stain), and for s100A12(E, brown stain). s100B is expressed indendritic cells (arrows, F, brown stain).(Originalmagnification:A–E, 20; F, 40.)

with sarcoidosis and in healthy subjects. The frequency of thededuced haplotype BTNL2 G and RAGE –374 T was signifi-cantly different between the two groups (�2 � 4.0015; p � 0.045vs. healthy control subjects).

Genotype–Phenotype Correlates

We looked for correlations between the –374 T/A polymorphismand the degree of RAGE expression in sarcoidosis granulomas.

Figure 3. The relationship between RAGE and the level of expres-sion of its ligand S100A12, scored as follows: 0 � absent; 1 � low;2 � intermediate; 3 � high; 4 � very high.

We found a wide dispersion of data (Figure 5). Despite a trendfor the group of subjects carrying the T allele to include moresamples with OD � 0.2, no statistically significant correlationwas found.

DISCUSSION

We provide evidence that RAGE and its ligands are expressedin sarcoid granulomas. RAGE expression has been extensively

TABLE 1. GENOTYPE AND ALLELE FREQUENCIES FOR THREERECEPTOR FOR ADVANCED GLYCATION END PRODUCTSSINGLE-NUCLEOTIDE POLYMORPHISMS (–374 T/A, –429 T/C,AND 1704 G/T) IN HEALTHY SUBJECTS

–374 T/A –429 T/C 1704 G/T(n � 262) n (% ) (n � 227) n (% ) (n � 148) n (% )

GenotypeT T 79 (30) TT 167 (73.6) GG 122 (82.4)AT 121 (46) TC 59 (26) GT 25 (16.9)AA 62 (24) CC 1 (0.4) TT 1 (0.7)

Allele, n (%)T 279 (53) T 393 (86.6) G 269 (91)A 245 (47) C 61 (13.4) T 27 (9)

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Figure 4. Representative immunocyto-chemistry patterns for the expression ofRAGE in peripheral blood mononuclearcells from healthy donors. (A ) Controlstaining. (B ) The AA genotype showed aweak immunostaining on cell surface, ascompared with the AT (C ) and TT (D )genotypes. (Original magnification: 40.)

documented in normal human tissues (23) and in several in-flammatory conditions associated with diabetes (24), amyloidosis(25), and rheumatologic disorders. The concomitant expressionof RAGE and its ligands has never been investigated in chronicgranulomatous diseases such as sarcoidosis, with the exceptionof one early report dealing with the presence of S100 immunore-activity in mononuclear phagocytes and in epithelioid cells ofmature sarcoid granulomas (15). We observed that RAGE isconsistently expressed, with variable intensity, in epithelioid his-tiocytes and giant cells in sarcoidosis and that its expression isassociated with that of its major proinflammatory ligands. Thepresence of RAGE in giant cells and epithelioid histiocytes ofsarcoid granulomas of mononuclear lineage is in accordance withthe documented RAGE expression in mononuclear inflamma-tory cells and phagocytes. Macrophages have also been reportedto express high levels of AGEs, produced by intracellular oxida-tive degradation of glycated proteins or by endocytosis of recep-tor-bound proteins in normal and pathologic conditions (26),

TABLE 2. FREQUENCIES OF THE RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS GENE–374 T/A POLYMORPHISM IN BIOPSIES FROM PATIENTS WITH SARCOIDOSIS AND INHEALTHY CONTROL SUBJECTS

Healthy Control Subjects Sarcoidosis Biopsies

Total Sample Male Subjects Female Subjects Total Sample Male Subjects Female Subjects(n � 262) (n � 114) (n � 148) (n � 99) (n � 47) (n � 52)

GenotypeT T/AT, n (%) 200 (76) 90 (79) 110 (74) 89 (90)* 43 (91.5) 46 (88.5)†

AA, n (%) 62 (24) 24 (21) 38 (26) 10 (10) 4 (8.5) 6 (11.5)Allele

T, n (%) 279 (53) 133 (58) 146 (49) 120 (60.6) 56 (59.6) 64 (61.5)‡

A, n (%) 245 (47) 95 (42) 150 (51) 78 (39.4) 38 (40.4) 40 (38.5)

* �2 � 8.2781; p � 0.004; odds ratio (OR), 2.7590; 95% confidence interval (CI), 1.3523–5.6291.† �2 � 4.4817; p � 0.034; OR, 2.6485; 95% CI, 1.0478–6.6942.‡ �2 �4.6040; p � 0.032; OR, 1.6438; CI, 1.0418–2.5937.

although AGE expression in epithelioid histiocytes has not beendocumented. The S100/calgranulins are a family of closely re-lated calcium-binding polypeptides that have been implicated inthe regulation of protein phosphorylation, calcium ion homeo-stasis, enzyme and transcription factor activities, cell growth anddifferentiation, and in the inflammatory response (27). S100A12and S100-B have been shown to activate endothelial cells, vascu-lar smooth muscle cells, monocytes, and T cells via RAGE,resulting in the generation of cytokines and proinflammatoryadhesion molecules (14, 12). Moreover, the serum level ofS100A12 is increased in conditions associated with chronic im-mune/inflammatory responses, in which immunohistochemicalstudies have shown its selective expression in polymorphonu-clear granulocytes (28–30), and in mononuclear phagocytes inexperimental settings (31). We observed S100A12 not only ingranulocytes but also in granulomas in over the half of the cases,indicating that the protein can be produced in vivo by stimulatedmonocytes. Regarding the receptor/ligand interaction, it has

Campo, Morbini, Zorzetto, et al.: RAGE in Sarcoidosis 503

TABLE 3. FREQUENCIES OF THE RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS GENE–374 T/A POLYMORPHISM IN CONFIRMATORY GROUPS OF PATIENTS WITH SARCOIDOSISAND HEALTHY CONTROL SUBJECTS

Healthy Control Subjects Patients with Sarcoidosis

Total Sample Male Subjects Female Subjects Total Sample Male Subjects Female Subjects(n � 65) (n � 24) (n � 41) (n � 101) (n � 45) (n � 56)

GenotypeTT/AT, n (%) 52 (80) 18 (75) 34 (83) 95 (94)* 42 (94)† 53 (94)AA, n (%) 13 (20) 6 (25) 7 (17) 6 (6) 3 (6) 3 (6)

AlleleT, n (%) 73 (56) 24 (50) 49 (60) 141 (70)‡ 59 (67) 82 (72)§

A, n (%) 57 (44) 24 (50) 33 (40) 61 (30) 31 (33) 30 (28)

* �2 � 7.7127; p � 0.005; odds ratio (OR), 3.9583; 95% confidence interval (CI), 1.4206–11.0294.† �2 � 4.6388; p � 0.031; OR, 4.6667; 95% CI, 1.0498–20.7453.‡ �2 � 6.4311; p � 0.011; OR, 1.8049; 95% CI, 1.1411–2.8548.§ �2 �3.9102; p � 0.048; OR, 1.8408; 95% CI, 1.0022–3.3813.

been shown that RAGE–ligand binding initiates a sustainedperiod of cellular activation mediated by receptor-dependentsignaling (32), leading to increased expression of the receptoritself. The net result is a positive feedback loop in which theligand-receptor binding increases expression of the receptor.

Because the antibody used for the RAGE investigations isspecific for the extracellular V-type domain of the protein, whichis present on the full-length transmembrane receptor and onthe C-terminally truncated secretory forms (sRAGE), we wereunable to discriminate the active form of the transmembranereceptor from the secretory forms stored in the cytoplasm. How-ever, it has been shown there is more full-length, membrane-bound receptor than soluble variants in tissue with a low mitoticactivity, such as lung (33). Exogenous administration of the solu-ble splice variant has been demonstrated in experimental settingsto have a negative role on RAGE-mediated proinflammatoryaction because it prevents RAGE ligands from binding to theactive, membrane-bound receptor (34). A recent report hasshown that sRAGE is the prevalent form expressed intracellu-larly in normal human tissues (23), suggesting that it may haveroles other than binding AGEs; however, the relative distribu-tion of different RAGE isoforms and the antiinflammatory roleof endogenous sRAGE in vivo have not been clarified.

Analogous to what is observed at sites of diabetic microvas-cular injury (35), we reported the coexpression of the severalRAGE ligands in RAGE-reactive granulomas, suggesting thattheir interaction may contribute to inflammatory processes re-lated to granuloma formation or progression, at least in someof the sarcoidosis lesions. The significant correlation betweenthe degree of expression of RAGE and of S100A12 (see Figure

TABLE 4. DEDUCED HAPLOTYPE FREQUENCIES OF THEBUTYROPHILIN-LIKE 2 (BTNL2) RS2076530 AND RECEPTORFOR ADVANCED GLYCATION END PRODUCTS (RAGE) –374T/A POLYMORPHISMS

Haplotype (BTNL2-RAGE) Sarcoidosis* Healthy Control Subjects

GA 0.10 0.12GT 0.27† 0.20AA 0.30 0.36AT 0.33 0.32

* The sarcoidosis group for this analysis includes DNA from biopsy and patientsgroups.

† �2 � 4.0015; p � 0.045; odds ratio, 1.4393; 95% confidence interval, 1.0065–2.0583.

3) suggests that the latter has a predominant role in RAGEbinding in sarcoid giant and epithelioid cells. However, the acti-vation of the RAGE inflammatory pathway is not specific tosarcoid granuloma because overexpression of RAGE and itsligands was recently described in a series of nonsarcoidoiticgranulomatous and of nongranulomatous lung inflammatory dis-orders (36). These findings are consistent with the hypothesisthat RAGE is not a disease-specific causative or initiating factor,but rather a mediator in the amplification of chronic cellularactivation and dysfunction (11). The resulting so-called two-hitpathway (11) has been also proposed as a model for sustainedinflammation in Crohn’s disease and ulcerative colitis (37). Thisgroup of inflammatory bowel diseases shares many immunopa-thophysiologic aspects with sarcoidosis (38).

We have shown that the intensity of RAGE immunoreactivityin sarcoid tissue varies widely from strong to absent (see Figures1 and 2). Although the previously mentioned role of ligand–receptor interaction in the induction of receptor expressionmight be one plausible explanation, we also explored the possi-bility of an intrinsic RAGE regulatory factor. According to ourRAGE SNP investigation (see Table 1) and according to theconcept that the promoter region of a given gene is characterizedby a high density of variants potentially affecting the gene’sexpression (39), we focused on the –374 T/A (rs1800624) poly-morphism. Our cytochemistry experiments (see Figure 4) andreal-time polymerase chain reaction analysis (see Figure E1)on PBMCs from healthy control subjects seemed to confirm adifference in the degree of RAGE expression according to the�374 T/A genotype, with the ranking being AA � AT/TT. Theex vivo correlates (Figure 5) were less convincing, suggestingthat, in the complex inflammatory network within the sarcoidgranuloma, the –374 T/A genotype is not the major factor modu-lating RAGE expression.

We expanded our genetic investigations to a possible role ofthe RAGE �374 T/A polymorphism as a risk factor in sarcoido-sis. When we looked at the allele frequencies of the polymor-phism, we found that the T allele was significantly more frequentin sarcoid tissue DNA than in a large series of control subjects,whereas the AA genotype was less frequently represented inthe sarcoid samples (see Table 2). Similar findings were obtainedwhen a second confirmatory, independent series of patients withsarcoidosis were included in the analysis (see Table 3). The excessof the T allele, in turn related to overexpression of RAGE,would be therefore an intrinsic mechanism for further sustainedinflammation in sarcoid granulomas.

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Figure 5. Correlation between the degree of RAGE ex-pression and the –374 T/A polymorphism in 99 biopsyspecimens containing sarcoidosis tissue. OD � opticaldensity.

The RAGE gene is located in a hot spot region of the shortarm of chromosome 6, where there is a high density of sarcoido-sis-related genes (Figure 6). It lies approximately 400 kb down-stream of DRB1, a major genetic factor for sarcoidosis (40–42),and only 211 kb from BTNL2 (10, 43); a gene–gene interactionin this region is, therefore, plausible. Valentonyte and colleagues(10) has suggested that DRB1 is a significant risk factor forsarcoidosis only in the presence of the truncating BTNL2 allele,whereas Rybicki and colleagues (43) suggested that, in whiteethnic groups, BTNL2 is a risk factor independently of an associ-ation of DRB1 but that a negative interaction exists betweenBTNL2 and HLA class II alleles in African Americans. Althoughwe did not screen for DRB1 in our study, the combined RAGEand BTNL genotyping revealed a weak linkage disequilibriumbetween the BTNL2 G and RAGE –374 T variants. This is notsurprising because the BTNL2 locus is close to the RAGE geneand because it has been extensively documented that the HLAregion is characterized by strong linkage disequilibrium. Theproximity of these genes mapping within the chromosome 6hot spot region makes the interpretation of association studiescomplicated.

We have no data on the possible role of RAGE, its ligands,and the RAGE polymorphism on the clinical course of the sar-coidosis. This represents a major limitation of this study; how-ever, sarcoidosis in the Italian population always shows a mild

Figure 6. Position map of the rs1800624 and rs2076530SNPs and their relative distance from the microsatellitemarker D6S1666. K � kilobases.

clinical course, which also explains our negative results in previ-ous studies aimed at correlating its course with biallelic polymor-phisms in other genes, such as ACE (44) or CR1 (22). By con-trast, significant correlations were found when we investigatedhighly polymorphic genes, such as HLA class I and II genes(40). Nevertheless, it would be interesting to explore the distribu-tion of RAGE polymorphism in different ethnic groups in whichsarcoidosis is characterized by a less favorable outcome (5).Regarding the therapeutic implications of our findings, in vivoand in vitro studies have suggested that the proinflammatoryeffects of RAGE activation in an inflamed environment couldbe markedly attenuated by blocking the RAGE–ligand interac-tion. It has been demonstrated that blockade of RAGE, byusing anti-RAGE F(ab)2 or sRAGE, suppresses inflammationin acute and chronic models (14, 45). In particular, administrationof sRAGE to mice decreased infiltration of the tissue by immune/inflammatory cells. Moreover, it was shown that sRAGE treat-ment in diabetic mice suppressed levels of IL-6, tumor necrosisfactor-�, and matrix metalloproteinases-2, -3, and -9 (46). Con-firming a role of RAGE or its natural ligands in the progressionof sarcoidosis or its resistance to therapy could open new thera-peutic perspectives targeted against RAGE.

In conclusion, we show here for the first time that RAGE isexpressed by sarcoid granulomas. This finding is corroboratedby the simultaneous expression of its natural ligands. Although

Campo, Morbini, Zorzetto, et al.: RAGE in Sarcoidosis 505

RAGE overexpression might be mediated by a possible feed-back loop induced by ligand binding, we have observed indica-tions that an intrinsic genetic factor could be in part involvedin regulating RAGE expression. Finally, we show that carriageof the –374 T allele, which is likely to contribute to increasedRAGE expression, is significantly associated with sarcoidosis,at least in our series of Italian patients.

Conflict of Interest Statement : None of the authors has a financial relationshipwith a commercial entity that has an interest in the subject of this manuscript.

Acknowledgment : The authors thank Marina Gorrini for the development of themanuscript and Dr. Rachel Stenner for editing.

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