Human Immunology 74 (2013) 23–27

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Preferential recognition of epitopes on AGE–IgG by the autoantibodiesin rheumatoid arthritis patients

Saman Ahmad, Safia Habib, Moinuddin, Asif Ali ⇑Department of Biochemistry, J.N. Medical College, Faculty of Medicine, Aligarh Muslim University, Aligarh 202002, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 2 May 2012Accepted 3 October 2012Available online 13 October 2012

0198-8859/$36.00 - see front matter � 2012 Americahttp://dx.doi.org/10.1016/j.humimm.2012.10.008

Abbreviations: RA, rheumatoid arthritis; AGEs, aducts; ELISA, enzyme-linked immunosorbent assay;nitroblue tetrazolium.⇑ Corresponding author. Fax: +91 571 272 0030.

E-mail address: asifali_amu@rediffmail.com (A. Al

Incubation of proteins with glucose lead to their non-enzymatic glycation ultimately resulting in the for-mation of advanced glycation end products (AGEs) in vivo. AGEs alter unique three dimensional struc-tures of various plasma proteins such as IgG. The role of oxidative stress in the pathogenesis ofrheumatoid arthritis (RA), a chronic inflammatory autoimmune disease, is well established. In view ofthis, commercially available human IgG was glycated in vitro with physiological concentration of glucose(5 mM) and the possible involvement of glycated IgG (AGE–IgG) in RA was evaluated. The RA patientswere divided into two groups on the basis of disease onset with respect to age: group I (early onset:20–32 years) and group II (late onset: 36–54 years). AGE–IgG and oxidative stress levels were detectedin RA patients and normal healthy individuals by nitroblue tetrazolium (NBT) assay and carbonyl contentestimation respectively. Binding characteristics and specificity of RA antibodies were analyzed byenzyme-linked immunosorbent assay (ELISA). We observed preferential binding of RA antibodies toAGE–IgG in comparison to native IgG. Band shift assay further substantiated the enhanced recognitionof AGE–IgG by RA antibodies. The results suggest that glycation of IgG results in the generation ofneo-epitopes, making it a potential immunogen. Our findings project AGE–IgG as one of the factors forinduction of circulating RA autoantibodies.� 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights

reserved.

1. Introduction

RA is a chronic, inflammatory, autoimmune systemic disease inwhich various joints in the body are inflamed leading to swelling,pain, stiffness and the possible loss of function. It is well knownthat oxidative stress is a typical finding in rheumatic joints [1].Detection of oxidative stress and low antioxidant concentrationsin the plasma of RA patients suggest the involvement of free radi-cals in inflammatory processes [2]. Assessment of such biochemi-cal markers as protein carbonyl content is considered as one ofthe best possible ways to monitor inflammation in RA [3].

Glycation is the non-enzymatic addition of reducing sugars(e.g., glucose, fructose, mannose, etc.) to the biological macromol-ecules (proteins, nucleic acids and lipids) [4]. In case of proteins,the reducing sugars such as glucose react with amino groups ofN-terminal amino acid or free lysine or arginine to form early-stageproducts such as Schiff base and Amadori products. Once formed,

n Society for Histocompatibility an

vanced glycation end prod-RF, rheumatoid factor; NBT,

i).

an Amadori product can undergo cyclization, dehydration, conden-sation and oxidation to generate a heterogenous class of com-pounds referred to as advanced glycation end products (AGEs).Oxidative stress in general is an important factor in the generationof AGEs [5]. Primarily studied in the diabetic population, whereAGEs appear to be implicated in tissue damage, their occurrenceand clinical significance in RA had not been much assessed previ-ously. In RA patients, AGEs represent new epitopes and containnew antigenic structures, thereby possibly contributing to the gen-eration of autoimmune responses [6]. AGEs alter unique threedimensional structures of various plasma proteins such as IgG,albumin, hemoglobin, transferrin, etc., which induce functionalabnormalities and thereby lead to several pathophysiological con-ditions [7]. IgG is a major serum protein, rich in lysine and arginine,making it a good target for glycation. The dominant factor in glyca-tion at physiological glucose concentration appears to be half-life;proteins with longer half-life showed enhanced glycation [8]. SinceIgG has longer biological half-life (24 days) so it undergoes highdegree of glycation in vivo [8]. Furthermore, IgG is continuously ex-posed to oxidative stress in RA [9]. This oxidative stress may causethe modification of IgG by AGEs in RA patients leading to the struc-tural and functional alterations in IgG making it highlyimmunogenic.

d Immunogenetics. Published by Elsevier Inc. All rights reserved.

24 S. Ahmad et al. / Human Immunology 74 (2013) 23–27

The present study has aimed to explore the role of AGE–IgG as apotential immunogen representing unique neo-epitopes againstwhich high titre autoantibodies may be present in RA patients.Keeping these points in mind, we generated neo-epitopes,in vitro, by incubating human IgG with physiological concentrationof glucose (5 mM) for 20 days at 37 �C and investigated the pres-ence of antibodies against it in both group I and group II RA pa-tients. Healthy individuals were taken as controls.

2. Materials and methods

2.1. Glycation

For the preparation of AGE–IgG, human IgG (Sigma, St. Louis,MO) at a concentration of 0.825 lM in PBS (10 mM sodium phos-phate buffer, 150 mM NaCl, pH 7.4) was incubated with 5 mM glu-cose (SRL, India) under sterile conditions at 37 �C for 20 days. Afterincubation, the solutions were extensively dialyzed against PBS toremove excess glucose.

2.2. Collection of sera

Eighty one rheumatoid factor (RF) positive samples from RA pa-tients were included in this study. These sera were obtained aftercareful clinical examination of RA patients with proven radio-graphic damage at the Department of Microbiology, J.N. MedicalCollege hospital, Aligarh Muslim University. All the patients satis-fied the criteria laid down by American Rheumatism Association[10]. Patients taking immunodepressants or any other drugs wereexcluded from the study. We divided the RA patients into twogroups on the basis of disease onset with respect to age: group I(early onset: 20–32 years) and late onset (group II: 36–54 years).Furthermore, 56 age- and sex-matched sera from normal healthyindividuals served as control. Verbal consent of patients as wellas healthy subjects was obtained before taking blood samples.The study protocol was approved by institutional ethical commit-tee. The sera were heated for 30 min at 56 �C to inactivate comple-ment proteins and stored at �20 �C with 0.1% sodium azide aspreservative.

2.3. Determination of rheumatoid factor (RF)

RF can be detected by RHELAX-RF slide test which is based onthe principle of agglutination. The test specimen is mixed withRHELAX-RF latex reagent and allowed to react. If RF is presentwithin detectable levels then a visible agglutination is observed.If RF is absent below detectable levels then no agglutination isobserved.

Around 40 ll of serum is pipette onto the glass slide and onedrop of RHELAX-RF latex reagent is added to it. The two are mixeduniformly. The slide is rocked gently and agglutination is observedmicroscopically at 2 min time.

2.4. Determination of C reactive protein (CRP) levels

CRP levels were measured using commercially available C Reac-tive Protein Human enzyme-linked immunosorbent assay (ELISA)kit (ab99995 from R&D Systems, USA), according to manufacturer’sinstructions. This assay employs an antibody specific for Human CReactive Protein coated on a 96-well plate. Standards and samplesare pipetted into the wells and C Reactive Protein present in a sam-ple binds to the wells by the immobilized antibody. The wells arewashed and biotinylated anti-Human C Reactive Protein antibodyis added. After washing away unbound biotinylated antibody,HRP-conjugated streptavidin is pipetted into the wells. The wells

are again washed, a TMB substrate solution is added to the wellsand color develops in proportion to the bound C Reactive Protein.The stop solution changes the color from blue to yellow, and theintensity of the color is measured at 450 nm.

2.5. Enzyme linked immunosorbent assay

Binding of serum autoantibodies of RA patients with native andAGE–IgG was evaluated by ELISA on polystyrene microtitre wells[11]. Antigen (native or AGE–IgG) was added to test wells and incu-bated for 2 h at 37 �C and overnight at 4 �C. The antigen coatedwells were washed thrice with TBS-T to remove unbound antigen.Unoccupied sites were blocked with 150 ll of 2.0% fat free milk for4–5 h at room temperature. The plates were washed 5–6 timeswith TBS-T. Test serum, serially diluted in TBS-T, was added to eachwell (100 ll/well) and reincubated for 2 h at 37 �C and then over-night at 4 �C. After incubation, the plates were washed thrice withTBS-T and bound antibodies were assayed with anti-human alka-line phosphatase conjugate in TBS.

After incubation for 2 h, the plates were washed 4 times withTBS-T and the color development was initiated by the addition of100 ll of 2.2 mmol/L p-nitrophenyl phosphate substrate in carbon-ate–bicarbonate buffer, pH 9.6. The absorbance in each well wasmonitored at 410 nm on an automatic microplate reader. Eachsample was run in duplicate. The control wells were devoid of anti-gen. Results were expressed as a mean of Atest–Acontrol.

2.6. Determination of protein carbonyl content in serum

Carbonyl content in the sera of RA patients was quantitated bythe protein carbonyl assay [12]. Briefly, 100 ll of sera were incu-bated with 500 ll of 10 mM 2,4-dinitrophenylhydrazone (DNPH)(dissolved in 2N HCl). After one hour incubation at room tempera-ture, 0.5 ml of 20% v/v trichloroacetic acid (TCA) was added, andcentrifuged for 3 min at 11,000g. The pellet was washed with1 ml of ethanol–ethyl acetic acid mixture (1:1). The protein pelletwas finally suspended in 250 ll of 6 M guanidinium chloride in20 mM phosphate buffer pH 2.3 (adjusted with trifluoroacetic acid)and samples were incubated at 37 �C for 15–30 min and then cen-trifuged. The absorbance of the supernatants was read at 360 nmon a Shimadzu spectrophotometer (Japan). Protein carbonyls werecalculated using a molar extinction coefficient of 22,000 M�1 cm�1.

2.7. Isolation of serum IgG on protein A-agarose column

Serum IgG of RA patients was isolated by affinity chromatogra-phy on protein A-agarose affinity column [13]. Serum (0.3 ml) di-luted with equal volume of PBS (pH 7.4) was applied on top ofthe column pre-equilibrated with the same buffer. The washthrough was recycled 2–3 times and unbound material was re-moved by extensive washing with PBS, pH 7.4. The bound IgGwas eluted with 0.58% acetic acid in 0.85% sodium chloride andcollected in a tube containing 1.0 ml of 1.0 M Tris–HCl (pH 8.5).Three ml fractions were collected and read at 278 nm. The IgG con-centration was determined considering 1.4 OD278 = 1.0 mg IgG/ml.The isolated IgG was dialyzed against PBS, pH 7.4 and stored at�20 �C with 0.1% sodium azide.

2.8. Nitroblue tetrazolium (NBT) colorimetric method

This method has been used to estimate the glycation in thepurified IgG samples from RA patients with slight modifications[14]. Fifty micro liters of the purified IgG was added to the wellsof 96-well microtitre plates, in duplicate. Standard curves wereestablished with glycated IgG (IgG modified in vitro with glucoseas mentioned in ‘Glycation’) in the range of 0–4 mg/ml. Hundred

Fig. 1. Direct binding ELISA of serum antibodies from group I and II rheumatoidarthritis (RA) patients to native IgG (h) and AGE–IgG (j). Serum samples fromnormal healthy subjects (NHS) served as control. The microtitre plates were coatedwith the respective antigens (10 lg/ml). Each bar represents the mean ± SD ofanalysed samples.

S. Ahmad et al. / Human Immunology 74 (2013) 23–27 25

micro liters of the NBT reagent [250 lM NBT in 0.1 M carbonatebuffer (pH 10.35)] was added to each well, and then incubated at37 �C for 2 h. The plate was read on an ELISA plate reader at550 nm. The amount of AGE-modified IgG (AGE–IgG) in the sam-ples was calculated from the standard curves prepared from gly-cated IgG.

2.9. Competition ELISA

Fine specificity of the autoantibodies in RA sera was determinedby competition ELISA [15]. Varying amounts of inhibitors (0–20 lg/ml) were mixed with a constant amount of antibody (IgG)and the mixture was incubated at room temperature for 2 h andovernight at 4 �C. The immune complex thus formed was addedto wells instead of the serum/IgG. The remaining steps were thesame as in direct binding ELISA. Percent inhibition was calculatedusing the following formula;

Percent inhibition ¼ 1� Ainhibited

Auniniited� 100

2.10. Band shift assay

Antigen–antibody interaction was further confirmed by bandshift assay in polyacrylamide gel. A constant amount of antigen(native IgG or AGE–IgG) was mixed with varying amounts of IgGfrom RA patients and the interaction was allowed for 2 h at 37 �Cand then overnight at 4 �C [16]. At the end of incubation, one-tenthvolume of sample buffer (10% glycerol, 2% SDS, 0.5 M Tris pH 6.8and 0.002% bromophenol blue) was added to antigen–antibodycomplex and the non-reducing SDS–PAGE was carried out on 10%SDS–polyacrylamide for 3–5 h at 80 volts. The protein bands werevisualized by silver nitrate staining.

2.11. Statistical analysis

Data are presented as mean ± SD. Statistical significance of datawas determined by Student’s t test as well as by 1-way ANOVA. A pvalue of <0.05 was considered as significant.

3. Results

In the present study, RA patients were divided into two groupsbased on the disease onset with respect to age-group I (early onset:20–32 years) and group II (late onset: 36–54 years). All the patientsselected for the study had normal blood glucose level. Patients hav-ing ESR of a minimum 28 mm/h and a serum C-reactive proteinconcentration of at least 20 mg/L were selected [10]. Sex- andage-matched control serum samples were obtained from 56healthy individuals. 21 serum samples of normal healthy subjects(NHS1) were used as control for group I patients and 35 serumsamples (NHS2) were used as control for group II patients.

3.1. Direct binding ELISA

All the sera were tested for binding to native and AGE-IgG by di-rect binding ELISA. Majority of RA serum antibodies showed strongbinding to AGE–IgG over native IgG at 1:100 dilution (p < 0.05). Noappreciable binding was observed with the sera from normalhealthy subjects. The average absorbance at 410 nm (±SD) in groupI (n = 29) and group II (n = 52) patients sera binding to AGE-IgG was0.55 ± 0.2 and 0.75 ± 0.2, respectively (p < 0.05). Whereas, the aver-age absorbance of the same group I and II patients’ sera binding tonative IgG was 0.26 ± 0.04 and 0.37 ± 0.07, respectively (p < 0.05)(Fig. 1). The results point towards better recognition of AGE–IgG

by autoantibodies in RA patients. This was more pronounced forgroup II patients.

3.2. Carbonyl content estimation

Carbonyl content in group I and II patients was determined toassess the effect of in vivo oxidative stress in normal subjects aswell as in RA patients. The average carbonyl content of group Iand II patients was found to be 2.93 ± 0.21 nmol/mg protein and3.45 ± 0.24 nmol/mg protein respectively (p < 0.05) compared with1.67 ± 0.17 nmol/mg protein in NHS1 and 1.97 ± 0.19 nmol/mgprotein in NHS2 (p < 0.05) (Table 1). Biochemical and immunolog-ical details of RA patients have been summarized in Table 1.

3.3. IgG isolation

To substantiate the above results, IgG was isolated from the ser-um of group I and II patients and normal healthy subjects on Pro-tein A-agarose affinity column. The purified IgG eluted as asymmetrical single peak on the affinity column (Fig. 2). Purity ofIgG was confirmed by the presence of a single homogenous bandin SDS-PAGE under non-reducing conditions (Fig. 2 inset).

3.4. NBT assay

The average concentration of AGE–IgG in the purified IgG sam-ples of group I patients, estimated by NBT assay, was found to be0.11 ± 0.04 mg/ml while for group II patients it was0.13 ± 0.06 mg/ml (p < 0.05). However, the AGE-IgG concentrationwas 0.048 ± 0.02 and 0.074 ± 0.02 mg/ml in NHS1 and NHS2respectively (p < 0.05) (Table 1).

3.5. Competitive inhibition ELISA

The binding specificity of isolated autoantibodies from highbinding sera of both the groups was evaluated by competition ELI-SA using native and AGE–IgG as inhibitors. The microtitre plateswere coated with AGE–IgG. IgG samples from RA patients exhib-ited higher binding towards AGE–IgG over the native form. Thecompetitive inhibition ELISA showed higher inhibition (and hencemore recognition) of antibodies from group II patients by the AGE–IgG as compared to group I patients. The average percent inhibition(±SD) in the binding of antibodies from group I (n = 29) and group II(n = 52) RA patients to AGE–IgG was 54.8 ± 6.2 and 71.8 ± 8.5%,respectively (p < 0.05). Native IgG caused 33.2 ± 4.1 and40.9 ± 5.1% inhibition in group I and group II antibodies respec-tively (p < 0.05). The results have been summarized in Tables 2and 3.

Table 1Biochemical and immunological details of study subjects.

Parameters NHS1 Group I patients NHS2 Group II patients

RA patients, n 21 29 35 52Average age (years) 23 ± 2.9 25 ± 2.6 45 ± 3.8 48 ± 3.5Sex 8M/13F 11M/18F 13M/22F 18M/34FRF test � + � +Glucose (mmol/L)* 5.15 ± 0.3 5.2 ± 0.4 5.32 ± 0.3 5.4 ± 0.6ESR (mm/h)* 14.5 ± 2.8 37.9 ± 4.2# 22.2 ± 3.6 44.5 ± 4.8#

CRP (mg/L)* 0.00 24.8 ± 2.8# 0.00 29.7 ± 3.3#

AGE–IgG (mg/ml)* 0.048 ± 0.02 0.11 ± 0.04# 0.074 ± 0.02 0.13 ± 0.06#

Carbonyl content (nmol/mg)* 1.67 ± 0.17 2.93 ± 0.21# 1.97 ± 0.19 3.45 ± 0.24#

NHS1: Sex- and age-matched normal human subjects used as control for group I patients; NHS2: Sex- and age-matched normal human subjects used as control for group IIpatients; RA: rheumatoid arthritis; n: number of samples tested; M: male; F: female; RF: rheumatoid factor;* Data expressed as average of all the values reported in the patients included in this study.

# Statistically significant at p < 0.05 versus respective normal human sera.

Fig. 2. Elution profile of antibodies (IgG) from the serum of group I patient onProtein A-agarose affinity column. Inset: Non-reducing SDS–PAGE of purified IgG on10% polyacrylamide gel. A similar pattern was obtained when antibodies werepurified from other sera of group I and group II RA patients.

Table 2Competitive inhibition of IgG from group I RA patients.

Sera no Maximum percent inhibition at 20 lg/ml

Native IgG AGE–IgG

5 28.4 52.88 32.3 60.514 35.9 46.617 39.1 62.323 34.8 57.526 29.2 49.6Mean±SD 33.2 ± 4.1 54.8 ± 6.2

The microtitre plates were coated with AGE–IgG (10 lg/ml). The inhibitors werenative IgG and AGE–IgG.

Table 3Competitive inhibition of IgG from group II RA patients.

Sera No Maximum percent inhibition at 20 lg/ml

Native IgG AGE–IgG

2 45.5 59.45 32.5 63.88 38.8 68.911 46.2 66.614 41.5 74.620 44.2 88.621 49.2 69.524 44.3 62.427 39.2 80.731 33.5 72.633 42.5 78.642 37.3 65.843 40.7 85.749 33.3 74.250 45.2 66.8Mean ± SD 40.9 ± 5.1 71.8 ± 8.5

The microtitre plates were coated with AGE–IgG (10 lg/ml). The inhibitors werenative IgG and AGE–IgG.

Fig. 3. Band shift of IgG from group II RA patients using (A) AGE–IgG and (B) nativeIgG as the antigenic candidates. Varying concentrations of RA IgG were incubatedwith a constant amount (10 lg) of native or AGE–IgG for 2 h at 37 �C and overnightat 4 �C. Electrophoresis was carried out on 10% polyacrylamide gel for 3 h. Lane 1contains native or AGE–IgG while lanes 2–5 contain native or AGE–IgG with 10, 20,30 and 40 lg of RA IgG. Lane 6 contains RA IgG.

26 S. Ahmad et al. / Human Immunology 74 (2013) 23–27

3.6. Band shift assay

Binding of IgG from group II patients to AGE–IgG and native IgGwas further ascertained through band shift assay (Fig. 3). Onincreasing the concentration of RA IgG from 10 to 40 lg, we ob-served a shift or retarded movement in the bands as well as a pro-portional decrease of the unbound antigen. The effect was morepronounced when AGE–IgG was used as antigen as compared tonative IgG. This reflects better formation of immune complexes be-tween RA IgG and AGE–IgG; thereby exhibiting better recognitionof AGE–IgG by the IgG from group II patients (Fig. 3A and B). How-ever, due to lesser binding in case of group I patients, we did notget any significant band shift.

4. Discussion

Generation of AGEs is an evitable process in vivo and can beaccelerated under pathological conditions such as hyperglycemia

and/or oxidative stress [17]. At physiological glucose concentra-tion, increased AGE formation has been correlated with oxidativestress [18]. In our previous study, human IgG incubated with phys-

S. Ahmad et al. / Human Immunology 74 (2013) 23–27 27

iological concentration of glucose for 20 days exhibited extensivedamage analyzed by various physicochemical techniques [19].AGE–IgG was found to be a potent antigenic stimulus inducinghigh titre antibodies in rabbits as compared to native IgG [20].

RA is a chronic, systemic, inflammatory disorder of unknownetiology in which antioxidant defense system is shown to be com-promised resulting in constant oxidative stress in RA patients [21–23]. AGEs formation is accelerated by oxidative stress and they arethought to contribute directly to the joint pathology [24]. Duringinflammation in RA, proteins can be damaged by non-enzymaticglycation [25]. Studies have shown that AGE-damaged IgG can bedetected in patients suffering from RA [26–29].

In the light of available literature, we hypothesized that IgGmodified by AGEs in RA patients under oxidative stress at physio-logical glucose concentration results in the conformational altera-tions of the protein generating neo-epitopes on IgG which maycause production of autoantibodies in RA. To test this hypothesis,we screened the RA serum samples for the antibodies reactive tonative and AGE–IgG. The samples were divided into two groups,i.e. early onset (group I; n = 29) and late onset (group II; n = 52).The data of direct binding ELISA showed that antibodies presentin the sera of group II patients showed preferentially high bindingto AGE–IgG as compared to group I patients. No appreciable bind-ing was observed with the antibodies from normal subjects. Thelikely participation of oxidative stress in modifying the proteinstructure by AGEs was supported by high carbonyl content in thesera of RA patients as compared to normal healthy individuals.However, carbonyl content estimated in group II patients was sig-nificantly higher than group I suggesting increased oxidative stressin group II patients as compared to group I. High carbonyl contentmay be involved in the initiation/progression/secondary complica-tions of the disease leading to the generation or exposure of neo-antigenic determinants upon glycation and antigen driven autoim-mune responses leading to the production of autoantibodies.

Furthermore, competition ELISA and band shift assay with affin-ity purified RA IgG reiterated that AGE–IgG possess preferred epi-topes more for the group II RA IgG as compared to group I IgG.Quantitation of AGE–IgG in purified IgG samples of RA patientsand healthy subjects was carried out by NBT colorimetric method.Group II sera showed a high level of AGE–IgG than group I sera andit was much less in normal subjects. We observed that level ofAGE–IgG had a good correlation with the disease activity, whichagrees with earlier studies [29]. Hence, the increased oxidativestress in group II patients could be the reason for high AGE–IgGconcentration in group II as compared to group I. The autoantibod-ies against AGE–IgG might be helpful in monitoring the progress ofdisease from mild to chronic stage and preventive measures couldbe exercised in time. Furthermore, level of AGE–IgG in combinationwith other clinical features of the RA might be a powerful diagnos-tic tool.

In conclusion, our results clearly demonstrate the presence ofAGEs-damaged IgG in RA patients. Furthermore, we report the pos-sible involvement of AGE–IgG in the generation of anti-IgG autoan-tibodies in RA patients. Thus, AGE–IgG is a potent immunogen thatappears to be a more suitable trigger for the antigen-driven induc-tion of autoantibodies in RA. The AGE–IgG levels may be used as amarker for disease severity and may also form a tool for the eval-uation of the efficacy of a therapeutic approach.

Acknowledgments

This study was supported by a research grant (62/3/2008-BMS)from the Indian Council of Medical Research, New Delhi. DST-FIST

infrastructure facilities in the department are also dulyacknowledged.

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