Abstract. Introduction. Menha Swellam 1 * Khadiga M. Abu Gabal 2 Samar Samir Youssef 3

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1 Research Communication Interleukin-1 Receptor Antagonist Gene Polymorphism and Hepcidin in Rheumatoid Arthritis: Correlations with Clinical and Laboratory Indices of Disease Activity Menha Swellam 1 * Khadiga M. Abu Gabal 2 Samar Samir Youssef 3 1 Department of Biochemistry, Genetic Engineering and Biotechnology Research Division, National Research Center, Dokki, Giza, Egypt 2 Department of Clinical and Chemical Pathology, Faculty of Medicine, Beni-Suef University, Beni-Suef, Egypt 3 Department of Microbial Biotechnology, National Research Center, Dokki, Egypt Abstract Rheumatoid arthritis (RA) is a systemic autoimmune disease, which is manifested as an inflammatory polyarthritis. Authors aimed to analyze the relationship between serum hepcidin, 25 amino acid protein, concentration and the anemia profiles of RA and to estimate whether it could reflect the disease activity of RA. Also, this study was conducted to explore the linkage between interleukin-1 (IL-1) receptor antagonist gene (IL-1RN) polymorphism, proinflammatory cytokine, and RA. One hundred and eighty five RA patients were enrolled in the study. For all, the following criteria were measured: RA disease activities, anemia profiles, serum concentration of hepcidin using enzyme-linked immunosorbent assay, and DNA samples were used to study genotypes of IL-1RN gene by polymerase chain reaction. Mean concentration of serum pro-hepcidin was ( ng/ml) in 185 RA patients. An increased frequency of the IL-1RN*1 and IL-1RN*2 alleles was relative to active RA (DAS28 > 5.1) than those with inactive to moderate RA (DAS28 5.1). Both hepcidin and IL-1RN gene showed significant correlation with each other as well with RA disease activity parameters and anemia profile. IL-1RN gene was significantly correlated with laboratory anemia profile apart from transferritin. There was a significant difference among pro-hepcidin concentration and IL-1RN frequency regarding patients with anemia of chronic disease and those without. In conclusion, both serum concentration of pro-hepcidin and IL-1RN genotypes frequency reflect the disease activity, regardless of the anemia states in RA patients, thus they may be another potential markers for disease activity of RA. VC 2013 IUBMB Life, 65(10): , 2013 Keywords: hepcidin; IL-1RN; rheumatoid arthritis; anemia; inflammation VC 2013 International Union of Biochemistry and Molecular Biology Volume 65, Number 10, October 2013, Pages *Address correspondence to: Professor Dr. Menha Swellam; Biochemistry Department, Genetic Engineering and Biotechnology Research Division, National Research Center, Tahrir Street, Dokki, Giza, 12622, Egypt. menha_m_swellam@yahoo.com Received 25 April 2013; Revised 27 June 2013; Accepted 19 July 2013 DOI /iub.1205 Published online 6 September 2013 in Wiley Online Library (wileyonlinelibrary.com) Introduction Rheumatoid arthritis (RA) is the most common chronic inflammatory joint disease of autoimmune etiology, affecting approximately 0.5% of the population. Effective treatment of RA has been impeded by a rarity of accurate diagnostic and prognostic tests, owing in part to the heterogeneity of the disease (1). Anemia is one of the common extra-articular manifestations in RA. There are possible mechanisms in the anemia of chronic inflammatory diseases in RA. First, inflammatory cytokines induce inadequate erythropoiesis in bone marrow, inhibiting erythroid progenitor differentiation and driving the apoptosis of erythroid progenitors. Second, they mediate the IUBMB Life 883

2 IUBMB LIFE inhibition of peripheral iron utilization. Third, the erythropoietin production in response to anemia is relatively impaired. Forth, red cell survival is slightly reduced in RA patients (2,3). In general, the patients with anemia are likely to have more severe joint diseases than those without anemia (4). Regulatory agents have been identified that relate to intestinal iron absorption and macrophageal iron release, the primary targets of anemia of chronic disease (ACD). These discoveries provide a molecular understanding of clinical erythrokinetics and explain many previous observations. Hepcidin was independently discovered by Krause et al., (5) and Park et al., (6) as a 25 amino acid protein with antibacterial activity, produced in the liver and excreted in the urine. Hepcidin is a type II acute-phase protein similar to ferritin (7). Production of hepcidin is regulated by interleukin-6 (IL-6) (8,9). IL-1, interferon gamma, and tumor necrosis factor alpha were considered to be responsible for the inhibition of iron release from the macrophageal system and the hypoferremia of ACD (10). IL-1 is a multifunctional proinflammatory cytokine that can be produced by nearly all cell types, including monocytes, activated macrophages, and endothelial cells (11). IL-1 plays an important role in mediating joint inflammation and destruction in RA (12), its gene family contains three related genes, IL-1A, IL-1B, and IL-1RN, which encode the proinflammatory cytokines IL-1a, IL-1b, and their naturally occurring antagonist, IL-1 receptor antagonist (IL1-RN), respectively (13). The polymorphic region within intron 2 of the IL-1RN gene contains a variable numbers of tandem repeats of 86 bp. Although allele 2 (IL-1RN*2) was found to be more frequent among patients with autoimmune diseases such as systemic lupus erythematosus, Sjogren s syndrome, or juvenile idiopathic arthritis (14,15), the data on its association with RA are still contradictory (16). The aim of this study was to analyze the relationship between serum pro-hepcidin, a prohormone of hepcidin, concentration and IL-1 receptor antagonist (IL-1RN) gene polymorphism among the anemia profiles of RA and to determine whether the pro-hepcidin and IL-1RN could reflect RA disease activity as well their correlation with each other. Materials and Methods Study Population The study was approved by the Medical Ethics Committee of the Beni-Suef University, and written informed consent was obtained from all participants. The study population included 185 patients with RA recruited from the Orthopedic and Rheumatology Departments, Beni-Suef University Hospital. All patients met the American College of Rheumatology 1987 revised criteria for the classification of RA (17). Patients were divided into two groups using DAS28 score into patients with inactive to moderate RA (DAS28 5.1, Group I were n 5 117) and those with active RA (DAS28 > 5.1, Group II were n 5 68). Laboratory Study for Anemia Laboratory analysis for anemia, such as complete blood count (CBC), hemoglobin (Hb), hematocrit (Ht), serum iron (Fe), total iron binding capacity (TIBC), ferritin, and transferrin levels, are measured using standard laboratory methods. Anemia was defined as Hb < 13 g/dl in male and <12 g/dl in female. ACD was defined as normocytic or microcytic anemia with decreased serum iron concentrations, normal or decreased transferrin levels and normal or increased serum ferritin levels (18). Patients whose anemia did not result from ACD were excluded in this study. Serum concentration of pro-hepcidin was measured using the DRG VR Hepcidin Prohormone Enzyme Immunoassay Kit (DRG Instruments, Marburg, Germany), according to the manufacturer s instructions. Pro-hepcidin is the 84 amino acid precursor of the active hepcidin peptide. Disease Activity Evaluation in RA Patients When the sera were obtained, clinical assessments, such as tender joint counts, swollen joint counts, and 100 mm-visual analogue scale (VAS) were performed by one rheumatologist. Erythrocyte sedimentation rate (ESR), CRP, the titer of IgM rheumatoid factors (RF), and the titer of antibodies to cyclic citrullinated peptide (anti-ccp Ab) were measured. From these clinical and laboratory data, Disease Activity Score 28 (DAS28) was calculated. Blood Sample and DNA Isolation Peripheral venous blood samples of 3 ml were drawn from each individual by standard venipuncture. Each blood sample was collected in sterile tubes with EDTA. Genomic DNA was isolated from buffy coats by using commercially available QIA amp DNA Blood kit (Qiagen, Hilden, Germany) (19). Concentration of the extracted DNA was measured by NanoDrop ND (NanoDrop Tech., Wilmington, DE). Genotyping of IL-1RN Gene Genomic DNA was assayed with polymerase chain reaction (PCR) for the detection of IL-1RN gene with Taq PCR core kit (Qiagen, catalogue no# 51104). To amplify the 86-bp tandem repeat region in intron 2 of IL-1RN gene, we used 2 flanking primers (Gene Bank accession number X64532) (sense 5 0 -CTCAGCCAACACTCCTAT-3 0 and antisense 5 0 -TCCTGGTCTG CAGGTAA-3 0 ) as described by Coskun et al. (20). Briefly, the PCR reaction was performed using a cocktail of 500 ng of genomic DNA. 0.5 mm of each oligonucleotide primer, 0.2 mmol/l of dntps, 1.5 mm/l MgCl 2, and 2.5 units of AmpliTaq polymerase in a final reaction volume of 50-mL, in a Mastercycler gradient thermocycler (Eppendorf, Hamburg, Germany) with an initial denaturation step of 94 C for 3 min and a final extension step 10 min at 72 C. The following thermal profile was repeated for 35 cycles: denaturation for 1 min at 94 C, annealing for 1 min at 60 C, and extension for 1 min at 72 C. PCR products were separated on 1% agarose gel and visualized by ethidium bromide with particular alleles being determined using a 100 bp size DNA ladder (Amersham Pharmacia Biotec); IL-1RN*0 (one repeat) is 150 bp, IL-1RN*1 (four 884 IL-1RA and Hepcidin in RA

3 Fig 1 Interleukin-1 receptor antagonist genotypes. Agarose gel (1%) displaying IL-1RN genotypes in seven knee OA patients: heterozygotes *1/2 (lanes 1 4 and 6), homozygotes *1/1 (lanes 5), and heterozygotes *1/3 (lane 7). Lane M: molecular weight DNA ladder standard (Amersham Pharmacia Biotec). repeats) is 410 bp, IL-1RN*2 (two repeats) is 240 bp, IL-RN*3 (three repeats) is 325 bp, IL-1RN*4 (five repeats) is 500 bp, and IL-1RN*5 (six repeats) is 595 bp (21,22). As shown in (Fig. 1). Statistical Analysis Demographic and clinical data between groups were compared by Chi-square (X 2 ) test and t-test. Genotype and allele frequencies were compared by Chi-square (X 2 ) test. Odds ratios were calculated with 95 % CI. To estimate the association between genotype and RA, a logistic regression analysis was used. Statistical analysis of the data was performed with Statistical Package for Social Science software (SPSS, Chicago, IL). Two-tailed analyses were performed, and P-values less than 0.05 were considered statistically significant. Results Demographic and clinical characteristics for the study population were as follows: age yr,DAS RF was positive in 55.1% of the patients. Table 1 shows the additional clinical data of the RA patients included in the study. When the patients were divided into two groups using DAS28 score: patients with inactive to moderate RA (DAS28 5.1, Group I were n 5 117) and those with active RA (DAS28 > 5.1, Group II were n 5 68). Anemia was detected in 57 RA patients out of 117 (48.7%) and in 62 RA patients out of 68 (91.2%). Hepcidin, IL-1RN Gene, and Anemia Profiles Relative to RA Activity As reported in Table 1, hepcidin and anemia profile showed significant difference between active RA (Group II) as compared with inactive to moderate RA (Group I). Similarly, positive genotypic and allelic frequencies of the IL-RN showed significant difference among the two RA groups as shown in Table 2. Hepcidin and IL-1RN Gene Relative to Anemia Groups The laboratory profiles relative to anemia were as follows; Hb g/dl, Ht mg/dl, serum iron mg/dl, TIBC mg/dl, ferritin ng/ml, and transferrin ng/ml. When the patients were divided into anemic RA and non-anemic RA groups, the patients with ACD had lower serum iron ( vs mg/dl), lower serum TIBC ( vs mg/dl), lower transferritin ( vs ng/ml), while higher serum hepcidin ( vs ng/ml), higher ESR ( vs mm/h,), and higher ferritin ( vs ng/ml) than the patients without anemia at P < Other clinical and laboratory parameters (VAS, DAS28,), RF and anti-ccp Ab titers were significantly increased among anemic groups than non-anemic ones. Interestingly, number of patients with anemia revealed significant difference with positive IL-1RN genotypes (*1/*1, Swellam et al. 885

4 IUBMB LIFE TABLE 1 Clinical characteristics and laboratory parameters among RA patients Demographic feature Total (n 5 185) Group I (n 5 117) Group II (n 5 68) Age (yrs) Gender status (males/females) 41/144 25/92 16/52 BMI (Kg/m 2 ) Family history (negative/positive) 42/143 30/87 12/56 DAS WOMAC (0 69) (no.) a Hepcidin (ng/ml) a ESR (mm/h) a RF (IU/mL)(negative/positive) 83/102 83/34 0/68 Anti-CCP Ab (U/mL) a HB (g/dl) a Ht (mg/dl) a Iron (mg/dl) a TIBC (mg/dl) a Ferritin (ng/ml) a Transferrin (ng/ml) a a Significant at P-value < Data were expressed by mean 6 SD. BMI, body mass index; WOMAC, Western Ontario and Mcmaster Universities (WOMAC); ESR, erythrocyte sedimentation rate; RF, rheumatoid factor; Anti-CCP Ab, Anti-CCP antibody. TABLE 2 Genotypic and allelic frequencies of the IL-1RN among RA groups Group I (n 5 117) Group I (n 5 68) 2ve 1ve 2ve 1ve IL-RN N Freq.% N Freq.% N Freq.% N Freq.% X 2 P IL-1RN genotypes *1/* *1/* *2/* IL-1RN Alleles IL-RN* IL-RN* IL-1RA and Hepcidin in RA

5 TABLE 3 Positive genotypic and allelic frequencies of the IL-1RN among anemic groups Non-anemic RA (n 5 66) Anemic RA (n 5 119) 2ve 1ve 2ve 1ve IL-RN N Freq.% N Freq.% N Freq.% N Freq.% X 2 P IL-1RN genotypes *1/* *1/* *2/* IL-1RN Alleles IL-RN* IL-RN* and 1*/2*) and positive allele IL-RN*1 as compared to nonanemic RA patients, Table 3. Correlation Between Hepcidin and IL-1RN Gene Among RA Group Among RA group, both hepcidin and IL-1RN gene showed significant correlation with each other as well with RA disease activity parameters (WOMAC, DAS 28, and VAS) [data not shown] and anemia profile (negatively correlated with Hb, Ht, iron, TIBC, and transferritin, at P < , positively correlated with ESR, RF, and ferritin, at P < ). Regarding active RA patient, hepcidin was correlated only with ferritin and transferritin (R , P , and R , P , respectively). IL-1RN gene was significantly correlated with laboratory anemia profile apart from transferritin. Discussion RA is the most common inflammatory arthropathy (23), biological therapeutics are expensive and can have life-threatening side effects; therefore, biomarkers predictive of progression to severe RA would be useful for alerting clinicians for early initiation of aggressive treatment regimens. Although it is a reliable predictor of further joint destruction in established RA, the presence of cartilage and bone erosion is not a useful prognostic biomarker in early RA, when erosions have yet to appear (1). In this study, the enrolled population (n 5 185) were suffering from RA ranged from patients with inactive to moderate RA (group I; n 5 117) and those with active (severe) RA (group II; n 5 68). According to our results, hepcidin level was significantly increased in group II as compared to group I, moreover, its level were significantly correlated with RA disease activity score parameters. These findings agreed with previously reported studies (4,24). Indicating that hepcidin is a useful biomarker for detection of active inflammation and it reflects disease activity of RA patients. Hepcidin regulates intestinal iron absorption, recycling, and tissue storage (25). Among the enrolled RA patients, anemia was detected in 57 RA patients out of 117 (48.7%) and in 62 RA patients out of 68 (91.2%). Hepcidin levels were significantly higher in anemic RA patients as compared to non-anemic RA ones. In addition, hepcidin was significantly correlated with laboratory anemia profile indicating that hepcidin binds to and induces the degradation of the unique iron transporter ferroportin, reduces intestinal absorption of iron (26) and blocks mobilization of iron from hepatocytes and macrophages, thereby causing low serum iron and hypo-proliferative anemia through decreased iron reutilization (27). These findings indicate that measurement of hepcidin in biological fluids is, therefore, a promising tool in the diagnosis and management of medical conditions in which iron metabolism is affected (28). It has been reported that in RA patients, synovial fibroblastic cells produced large amounts of IL-6 when stimulated by inflammatory cytokines such as IL-1 (29), to the best of our knowledge, this study is the first to investigate IL-1RN gene among RA patients. The results of this study revealed a significant increase in IL-1RN genotypes 1*/2 and 2*/2* in group II (active RA) as compared to group I (inactive to moderate RA), the same was observed for IL-1RN allele 2*. Alternatively, IL- 1R genotype 1*/1* and IL-1RN allele 1* have showed significant increase in group I as compared to group II. These findings indicate that IL-1RN may has a diagnostic role in RA, however, further study on large population is in progress to elucidate the exact responsibility of the IL-1R among RA patients. According to the present results, positive IL-1RN genotypes (1*/2* and 2*/2*) and alleles frequencies were significantly correlated with severe RA especially those with anemia. These results indicate that in anemia of inflammation, Swellam et al. 887

6 IUBMB LIFE proinflammatory cytokines affect iron metabolism, particularly iron turnover and ferritin synthesis causing low serum iron without evidence of iron deficiency (28,30). Interestingly, both hepcidin and IL-1RN gene were significantly correlated in RA patients. This finding emphasizes that IL-1RA play a significant role in the pathogenesis of RA and has a significant role in the anemia of inflammation by up-regulating hepcidin (31). In conclusion, hepcidin concentration was closely associated with markers of iron status and was elevated among RA participants with the anemia compared with non-anemic RA. Moreover, IL-1 receptor antagonist gene up-regulates hepcidin and both of them can reflect RA disease activity. Acknowledgement The authors acknowledge Dr. Maher Hamed, Consultant Professor in Health insurance Hospital for his help in the supplying and diagnosing the clinical samples. References [1] Lindstrom, T. M. and Robinson, W. H. 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