Interleukin-1 cluster and tumor necrosis factor- gene polymorphisms in polymyalgia rheumatica

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1 Interleukin-1 cluster and tumor necrosis factor- gene polymorphisms in polymyalgia rheumatica L. Boiardi, C. Salvarani, J.M Timms 1, T. Silvestri 1, P.L. Macchioni, F.S. di Giovine 1 Servizio di Reumatologia, Azienda Ospedaliera Arcispedale S. Maria Nuova, Reggio Emilia, Italy; 1 Division of Molecular and Genetic Medicine, University of Sheffield, United Kingdom. Abstract Objective To investigate whether polymorphisms in the interleukin (IL)-1 locus (human chrom. 2q13) and TNF- gene are associated with susceptibility to or severity of polymyalgia rheumatica (PMR). Methods The study included 92 consecutive PMR patients diagnosed over a 5-year period who were prospectively followed-up for at least one year and 79 healthy controls over the age of 50 residing in the same area. All the patients and controls were Caucasians of Italian origin. We tested the allelic distribution of IL-1A (+4845), IL-B (-511), IL-B (+3954), IL-1RN Intron 2 VNTR and TNFA (-308). Frequencies were compared in the patient and control groups. Results A statistically significant association between PMR patients and the IL1RN*2 allele in the homozygous state was found [OR 8.46 (95% CI )]. The polymorphisms in the other genes of the IL-1 gene cluster did not reveal any association with PMR when compared with controls. A weak association between PMR patients and the TNF2 allele was also present [OR 2.09 (95% CI )]. None of the gene variants studied was associated with the disease severity of PMR. Conclusion Our findings show that IL1RN*2 allele, particularly in the homozygous state, is associated with susceptibility to, but not with the severity of, PMR. Key words Polymyalgia rheumatica, interleukin-1 cluster gene polymorphisms, tumor necrosis factor-α, gene polymorphism, relapse/recurrence. Clinical and Experimental Rheumatology 2000; 18:

2 Cytokine gene polymorphisms in PMR / L. Boiardi et al. Luigi Boiardi, MD, PhD; Carlo Salvarani, MD; Janine M. Timms, BSc; Tania Silvestri, MD; PierLuigi Macchioni, MD; Francesco S. di Giovine, MD, PhD. Please address reprint requests to: Dr. Carlo Salvarani, Servizio di Reumatologia, Arcispedale S. Maria Nuova, V.le Umberto 1 no. 50, Reggio Emilia, Italy. salvarani.carlo@asmn.re.it Received on January 26, 2000; accepted in revised form on July 5, Copyright CLINICAL AND EXPERIMENTAL RHEUMATOLOGY Introduction Polymyalgia rheumatica (PMR) is a common disorder in the elderly characterized by aching and stiffness in the neck, shoulders and pelvic girdle, and is frequently associated with giant cell arteritis (GCA) (1, 2). The musculoskeletal symptoms appear to be linked to a nonerosive articular and extra-articular synovitis characterized by the predominance of CD68+ macrophages and CD4+ T cells (3-5). PMR is probably a multifactorial disease, with both environmental and genetic factors contributing to susceptibility and severity. To date the only genetic associations found have been with genes that lie within the HLA complex (6, 7). Additional genes, particularly those involved in the regulation of immune response, probably predispose patients to the development of PMR. Interleukin-1 (IL-1) (8, 9) and tumor necrosis factor alpha (TNF-α) (10) are potent proinflammatory agents which play a central role in various inflammatory diseases. The inflammatory response is regulated by a delicate balance between pro- and anti-inflammatory cytokines. IL-1 receptor antagonist (IL- 1 Ra) is a member of the IL-1 family that binds to the IL-1 receptor, but does not induce any intracellular response and thus acts as an inhibitor of IL-1 activity (11). Elevated serum levels of IL-1β and IL-1Ra have been found in patients with active PMR (12, 13). What is more, Weyand et al. (14) showed that patients with PMR are characterized by the in situ production of IL-1β in temporal artery specimens and by the production of this cytokine by the majority of circulating monocytes (15). Genetic polymorphisms have been characterized for the genes of these cytokines and the presence of certain alleles has been associated with the susceptibility to or severity of several diseases of an inflammatory nature (16-22). The genes coding for IL-1α and IL1β (IL-1A and IL-1B, respectively) are located on chromosome 2, in close proximity to the IL- 1RN gene (which encodes IL-1Ra) (23). Two biallelic polymorphisms in IL-1B at positions -511 and (relative to the transcriptional start codon) have been described (24, 25). A higher production of IL-1β in response to lipopolysaccharide has been reported in subjects carrying allele 2 of the polymorphism (25, 26). Two genetic polymorphisms of the human IL-1A gene at positions and have been described (27, 28). Several polymorphic sites have also been described in IL-1RN, including a variable number of tandem repeats (VNTR) within its second intron (29). Allele 2 of this polymorphism was associated with the increased production of IL-1Ra by monocytes in response to GM-CSF and with higher plasma levels (30). It has also been associated with disease severity in systemic lupus erythematosus (SLE), ulcerative colitis, and alopecia areata (16-18). The TNF-α-308 polymorphism is located in a regulatory portion of the TNF-α promoter (31), which has recently been shown to bind a novel transcription factor (A.G. Wilson, personal communication). The TNF2 allele has been found to correlate with enhanced spontaneous and stimulated TNF-α production both in vitro and in vivo (32) and to be associated with SLE, ankylosing spondylitis and, although more contradictory, with rheumatoid arthritis (RA) (33-36). In the present study we investigated allele frequencies and carriage rates of IL- 1 cluster genes [IL-1A (+4845), IL-1B (-511), IL-1B (+3954), IL-1RN Intron 2 VNTR] and the TNF-α gene (TNF -308) in a prospective cohort of 92 patients with PMR who were followed up for more than one year and in a normal healthy population in order to explore the role of these polymorphisms in determining disease susceptibility or severity. Patients and methods Study population We studied 92 consecutive new PMR patients from the Reggio Emilia area diagnosed at the Reggio Emilia Rheumatology Center over a 5-year period ( ). PMR was diagnosed when all the following were present (37): 1) persistent pain (for at least one month) involving two of the following areas: neck, shoulders, and/or pelvic girdle; 2) morning stiffness lasting more than one hour; 3) rapid response (within 72 hours) to prednisone ( 20 mg/day); and 4) absence of other diseases capable of caus- 676

3 Hypothalamus-pituitary-adrenocortical and -gonadal axis in RA / M. Cutolo Cytokine gene polymorphisms in PMR / L. EDITORIAL Boiardi et al. ing the musculoskeletal symptoms. Only patients over the age of 50 were included. All PMR patients were seronegative for rheumatoid factor. At diagnosis 82 patients had a erythrocyte sedimentation rate (ESR) < 40 mm/hr. Ten patients with typical clinical symptoms, ESR < 40 mm/hr and rapid and complete response to corticosteroids were also included. The mean age at diagnosis (± SD) was 72 ± 7 years. 75% of the PMR patients were females and 25% were males. The mean duration of therapy was 31 ± 23 months and the mean cumulative prednisone dose was 6.5 ± 4.8 gm. During the follow-up 40 patients (44%) had a relapse/recurrence. Temporal artery biopsy specimens were obtained only in patients with cranial signs or symptoms and the diagnosis of GCA was based on a positive temporal artery biopsy. Six patients (6.5%) had associated GCA. The control group consisted of 79 healthy blood donors over 50 years of age residing in the same area. No ethnic differences were present between the patients and controls. Follow-up study All 92 patients were followed up for at least one year (mean 44 ± 27 months). The patients were clinically assessed by the same physician at presentation, monthly for the first 6 months, and then every 3 months during the follow-up period. All patients received the same schedule of prednisone treatment starting with a dosage of 17.5 mg per day (occasionally the dose had to be increased to 25 mg per day to completely suppress the discomfort), and then was reduced after one month if the symptoms resolved. Small monthly decrements of 5 mg to 1 mg were successively scheduled. A standardized data collection form was used at every visit to record medical information. Age, sex, the location of aching and morning stiffness, the presence of systemic manifestations, the dosage and duration of prednisone, and relapses and recurrences were recorded. The cumulative prednisone dose was also computed, and the presence of peripheral synovitis (swelling and tenderness of the joints) was carefully assessed. Joint radiography was performed in all patients with joint swelling at some time point during the course of the illness. Relapse and/or recurrence were considered to be present if articular signs and/ or symptoms occurred (usually with an ESR > 30 mm/hr) in a patient receiving corticosteroids or after discontinuation of treatment, respectively. The symptoms were suppressed by resumption of, or an increase in the prednisone dose. At diagnosis and during the follow-up, ESR was determined by the Westergren method. The end of the illness was considered to be the date of permanent discontinuation of therapy without subsequent relapse or recurrence. The end point of patient follow-up was either the date of the last visit or the date of death. At diagnosis and throughout the followup period, no PMR patients satisfied the modified 1987 ARA criteria for RA (38) and no clinical evidence of joint deformity or radiological evidence of erosions was observed. DNA extraction and genotyping Venous blood was drawn into EDTA tubes, and DNA was extracted by standard methods (39). DNA was aliquoted in TBE buffer and the stocks were stored at 4 C until used. We tested the IL-1A (+4845) SNP (which we have reported to be in 100% linkage disequilibrium with IL-1A -889) (19), IL-1B (+3954), IL-1B (-511), IL-1RN intron 2 VNTR and TNF (-308). Allelic frequencies at these loci were tested using previously validated PCR-based methods (39). HLA typing HLA-DRB1 alleles were determined in all the patients with PMR by polymerase chain reaction amplification and oligonucleotide hybridisation, as described elsewhere (40). Statistical analysis The overall genotype distributions at the IL-1 and TNF loci were analysed for carriage of a specific allele by chi-square analysis using the SPSS Statistical Package (SPSS Inc., Chicago, IL). Corrected P values were calculated by multiplying P by the number of alleles compared. Odds ratios and their confidence intervals were also calculated for these comparisons. The t-test for independent values was used when necessary. Results Association between cytokine alleles and susceptibility to PMR The distribution of genotypes, cytokine allele frequencies, and carriage rates in patients with PMR and healthy controls is shown in Tables I and II. All of these patients were also included in an immunogenetic study which evaluated the HLA-DRB1 alleles associated with PMR in northern Italy (40). We did not observe any association between HLA-DRB1*04, HLA-DRB1*01 alleles and rheumatoid epitope with PMR in our population. A statistically significant weak association was shown only with HLA-DR3. PMR patients had a significantly higher frequency of the IL1RN*2 allele com- Table I. Distribution of genotypes among the polymyalgia rheumatica (PMR) patients and healthy control subjects. Genotype PMR (%) Controls (%) IL-1A (-4845) * Total n CC 48 (52.2) 39 (51.3) CT 40 (43.5) 30 (39.5) TT 4 (4.3) 7 (9.2) IL-1B (+3954)** Total n /1 56 (60.9) 40 (50.6) 1/2 32 (34.8) 36 (45.6) 2/2 4 (4.3) 3 (3.8) IL-1B (-511) Total n /1 46 (50.0) 37 (48.1) 1/2 38 (41.3) 33 (42.9) 2/2 8 (8.7) 7 (9.1) IL-1RN & Total n /1 46 (50.0) 48 (60.8) 1/2 37 (40.2) 30 (38.0) 2/2 9 (9.8) 1 (1.3) TNF-α (-308) $ Total n /1 65 (70.7) 66 (83.5) 1/2 25 (27.2) 13 (16.5) 2/2 2 (2.2) 0 HLA-DR Total n SE+/SE+ 3 (3.2) 3 (2.0) SE+/SE- 25 (27.2) 33 (22.3) SE-/SE- 64 (69.6) 112 (75.7) * P = 0.43; ** P = 0.36; P = 0.97; & P = (Pcorr = 0.132); $ P = 0.09; P =

4 Cytokine gene polymorphisms in PMR / L. Boiardi et al. pared with controls (P = 0.04), but this relationship turned into a trend when the P value was corrected for the number of alleles tested (P corr = 0.08) (Table II). The carriage rate of IL1RN*2 also increased, from 39.3% in healthy controls to 50% in PMR patients (OR = 1.55). However, this difference was not significant. The carriage rate of IL1RN*1 was significantly lower in patients than in controls (P = 0.02, P corr = 0.04). IL1- RN*2 homozygosity was significantly higher in patients than in controls (9.8% versus 1.3%, P = 0.02, P corr = 0.04), and the OR was 8.46 (95% CI ). IL1RN*2 homozygosity increased mainly in shared epitope-negative compared to shared epitope-positive patients (12.5% versus 3.6%). However, this difference was not significant. The polymorphisms in the other genes of the IL-1 gene cluster did not reveal any association with PMR when compared with the healthy controls. The frequency and the carriage rate of the allele TNF2 were significantly higher in cases than in controls, but this relationship turned into a trend when the P value was corrected for the number of alleles tested (P corr = 0.06 and P corr = 0.08). The TNF2 allele was significantly correlated with HLA-DR3 (p < ). No other associations between HLA alleles and TNF-α genotypes could be demonstrated. The carriage rates of the TNF2 allele were higher in shared epitope-negative compared to shared epitope-positive patients (34.4% versus 17.9%). However, the difference was not significant. We did not find any significant difference in the allelic frequencies or carriage rates of the IL-1 cluster genes and the TNF-α gene between shared epitopepositive and -negative, DR4-positive and -negative, or DR1-positive and -negative patients, or between patients with high ( 30 mm/hr) or low (< 30 mm/hr) ESR levels at PMR diagnosis (data not shown). We also investigated the possible associations between alleles of the IL-1RN gene and the IL-1B gene (positions -511 and +3954) in both PMR patients and healthy controls. IL-1B-511 allele 2 was significantly associated with the presence of IL-1RN allele 2 both in PMR patients (73.9% vs. 28.3%, p = ) and controls (73.3% versus 38.3%, p = 0.003). However, IL-1B allele 2 was significantly less frequent in the carriers of the IL-1RN allele 2 among the PMR patients (28.3% versus 50.0%, p = 0.03), but not among the controls (41.9% versus 54.2%, p = NS). Furthermore, given the functional role attributed to IL-1RN allele 2 and IL-1B (-511 and +3954) alleles 2, we investigated all the possible associations between these alleles, and compared the different frequencies between PMR patients and controls (Table III). The IL-1RN allele 2+/ IL-1B+3954 allele 2- association had a significantly higher frequency in PMR patients compared with controls (p = 0.05). The frequency of the IL-1RN allele 2+/ IL-1B- 511 allele 2+ association was higher in PMR patients, but the difference was not significant. Association between cytokine alleles and PMR severity No significant associations were observed between any of the 5 polymorph- Table II. Comparison of allelic frequencies and carriage rates observed in polymyalgia rheumatica (PMR). Allelic frequency Carriage rate Gene, allele PMR Controls P OR (95% CI) PMR Controls P OR (95% CI) no. (%) no. (%) no. (%) no. (%) IL-1A (-4845) C 136 (73.9) 108 (71.1) NS 1.15 ( ) 88 (95.7) 69 (90.8) NS 2.23 ( ) T 48 (26.1) 44 (28.9) 0.87 ( ) 44 (47.8) 37 (48.7) NS 0.97 ( ) IL-1B (+3954) (78.3) 116 (73.4) NS 1.30 ( ) 88 (95.7) 76 (96.2) NS 0.87 ( ) 2 40 (21.7) 42 (26.6) 0.77 ( ) 36 (39.1) 39 (49.4) NS 0.66 ( ) IL-1B (-511) (70.7) 107 (69.5) NS 1.06 ( ) 84 (91.3) 70 (90.9) NS 1.05 ( ) 2 54 (29.3) 47 (30.5) 0.95 ( ) 46 (50.0) 40 (52.0) NS 0.92 ( ) IL-1RN (70.1) 126 (79.7) 0.04* 0.60 ( ) 83 (90.2) 78 (98.7) 0.02** 0.12 ( ) 2 55 (29.9) 32 (20.3) 1.68 ( ) 46 (50.0) 31 (39.3) NS 1.55 ( ) TNF-α (-308) (84.2) 145 (91.8) 0.03 & 0.48 ( ) 90 (97.9) 79 (100) NS (15.8) 13 (8.2) 2.09 ( ) 27 (29.4) 13 (16.5) ( ) SE on DR Positive 31 (16.7) 39 (13.2) NS 1.33 ( ) 28 (30.4) 36 (24.3) NS 1.4 ( ) Negative 153 (82.3) 257 (86.8) 0.75 ( ) 64 (69.6) 112 (75.7) NS 0.73 ( ) *P corr = 0.08; **P corr = 0.04; & P corr = 0.06; P corr =

5 Hypothalamus-pituitary-adrenocortical and -gonadal axis in RA / M. Cutolo Cytokine gene polymorphisms in PMR / L. EDITORIAL Boiardi et al. isms studied or the rheumatoid epitope and the occurrence of at least one relapse/ recurrence, the duration of corticosteroid treatment, or the cumulative prednisone dose (data not shown). The frequency of the IL1RN*2 carriage rate was higher in the 23 patients with a treatment duration of more than 4 years compared to the 20 patients with a treatment duration of less than 2 years and at least one year of follow up without treatment after corticosteroid suspension (Table IV). However, the difference was not significant. Similarly, the frequency of the rheumatoid epitope was higher in the patients with more than 4 years of corticosteroid treatment, but the difference was not significant. The combination of an HLA-DR allele expressing the rheumatoid epitope and the presence of the IL1RN*2 allele was not found to be significantly higher in patients with more than 4 years of corticosteroid treatment. None of the other polymorphic systems studied was associated with more than 4 years of corticosteroid treatment. To investigate possible associations between IL-1RN allele 2 and IL-1B (-511 and +3954) alleles 2 and disease severity (the presence of at least one relapse/ recurrence, the duration of corticosteroid treatment and the cumulative prednisone dose), patients were stratified into 4 groups, depending on whether they were carriers or non-carriers of allele 2 of either gene. No significant associations were found (data not shown). Discussion To date, the only genetic associations found for PMR susceptibility or severity have been with genes that lie within the HLA complex (6, 7). HLA-DRB1*04 and 01 alleles have been found to be associated with disease susceptibility and severity (6, 7). In the northern Italian population we did not find any association between HLA-DRB1 alleles and PMR (40). We analyzed 5 polymorphisms involving IL-1 cluster genes [IL- Table III. Frequencies of the IL-1B allele 2 carriage in control subjects and patients with polymyalgia rheumatica in relation to carriage of IL-1RN allele 2 (*p = 0.05). Carriers of IL-1RN Carriers of IL-1B Controls PMR patients Allele 2 Allele 2 (-511) n (%) n (%) (+) (-) (+) (+) (-) (-) (-) (+) Carriers of IL-1RN Carriers of IL-1B Controls PMR patients Allele 2 Allele 2 (+3954) n (%) n (%) (+) (-) * * (+) (+) (-) (-) (-) (+) Table IV. Frequency of the rheumatoid epitope, of allele 2 of the interleukin-1 receptor antagonist gene polymorphism, and of the combination of both in patients with > 4 years of corticosteroid treatment and in patients with < 2 years of corticosteroid treatment (and at least one year of follow up without treatment after the suspension of corticosteroid). Patients with Patients with > 4 years of < 2 years of CS therapy CS therapy P OR (95% CI) Epitope positive NS 1.6 ( ) IL-1RN NS 1.90 ( ) Epitope positive and IL-1RN2 positive NS 1.57 ( ) 1A(+4845), IL-1B(-511), IL-1B(+3954), IL-1RN Intron 2 VNTR] and TNF-α gene [TNFA(-308)] in order to identify possible associations between particular alleles and susceptibility to or severity of PMR. We observed a significant association between PMR patients and the IL1RN*2 allele; in particular IL1RN*2 homozygosity confers an OR for PMR of We also considered the possibility that the frequency of the ILRN*1 and IL- RN*2 alleles could be different in our controls compared to those observed in other studies. However, the frequency of these alleles in our healthy controls was similar to that observed in the controls of other European Caucasoid populations (41, 42). The type 2 IL-1RN allele has been previously found in association with a variety of autoimmune diseases, including ulcerative colitis, SLE, and alopecia areata (16-18). It has recently been demonstrated that carriers of the ILRN*2 allele had higher plasma levels of IL-1Ra than non-carriers, but this required the presence of the IL-1B-511 allele 2 or absence of the IL- 1B+3954 allele 2 (43). This indicates the participation of IL-1B genes in the regulation of IL-1Ra production. In addition, IL-1RN allele 2 strongly increased the in vitro production of IL-1β, regardless of the presence or absence of IL-1B genes (44). Higher production of IL-1β in response to LPS has been reported in subjects carrying allele 2 of the polymorphism (26). This allele was not higher in PMR patients compared to healthy controls. However, IL-1B+3954 allele 2 was significantly less frequent in the carriers of the IL-1RN allele 2 in PMR patients and the IL-1RN allele 2+/IL-1B+3954 allele 2- association was significantly higher in PMR patients compared with healthy controls. Taken together, the functional consequences of these polymorphisms of the genes of the IL-1 complex may explain the elevated serum levels of IL-1β and IL-1Ra, the increased production of IL- 1β by circulating monocytes, and the in situ production of IL-1β in temporal artery specimens observed in patients with PMR (12-15). 679

6 Cytokine gene polymorphisms in PMR / L. Boiardi et al. Arend et al. speculated that IL-1Ra may be an acute phase protein (APP) because injection of humans with IL-1 or interleukin-6 (IL-6), which are known to regulate the production of APP by the liver, led to a rapid rise in blood IL-1Ra levels (11). The elevated serum levels of IL-1Ra in PMR patients at diagnosis (12) may be indicative of the strong inflammatory process present in PMR patients. Polymorphism at position of the IL-1A gene was not found to be associated with PMR, suggesting that this polymorphism plays no role in the predisposition to PMR. Several polymorphisms have been identified inside the TNF-α promoter (31, 45-47). Among these, the polymorphism at nucleotide position -308 directly affects TNF-α expression and production in vitro and in vivo (32, 48, 49). The uncommon allele TNF2 of this polymorphism has been reported to be strongly linked to HLA-DR3 in Caucasians (31). This association was also confirmed in PMR patients. Our study found a weak association between the TNF2 allele and PMR. It seems probable that the observed association between the DR3 genotype and PMR was due to a linkage disequilibrium between TNF2 allele and HLA-DR3. This linkage disequilibrium may explain the high TNF-α production in patients carrying the HLA-DR3 genotype. Circulating TNF-α concentrations in PMR patients were similar to those in normal donors (12, 50). However, detectable plasma TNF-α does not represent the concentration of cytokine locally produced at the site of inflammation. There is no data on the expression of this cytokine on PMR shoulder synovial membrane. In temporal arteritis, a disease closely linked to PMR, circulating TNFα concentrations were not different from those in normal subjects (50). However, using immunohistochemical techniques, TNF-α was demonstrated in up to 60% of the cells in all areas of inflamed arteries (51). There seem to be two subsets of PMR patients. One subset presents with mild, limited disease, while the other has persistent disease requiring long-term treatment (> 2 years). Patients with more severe disease constituted about 30% of the subjects in our series and experienced one or more episodes of relapse/recurrence during the follow-up. Increased frequency of the IL-1RN allele 2 has been associated with disease severity in ulcerative colitis, SLE, alopecia areata, psoriasis, and adult periodontitis (16-18, 52). The frequency of the IL1RN*2 carriage rate was higher in the patients with more than 4 years of corticosteroid treatment compared to those treated with corticosteroids for less than 2 years and followed up for at least one year without treatment. However, the difference was not significant. The combination of rheumatoid epitope with the presence of the IL1RN*2 allele was not found to be significantly higher in patients with more than 4 years of corticosteroid treatment. None of the other polymorphic systems studied was associated with more than 4 years of corticosteroid treatment. In particular, polymorphism, whose allele 2 is associated with higher production of IL-1ß in response to LPS, was not found to have any prognostic value. In contrast, it may be noted that a rise in the levels of this cytokine was reported by Pountain et al. to be associated with relapses of PMR/GCA (13). We also investigated the possible associations between IL-1RN allele 2 and IL- 1B (-511 and +3954) allele 2 and disease severity. The patients were stratified into 4 groups, depending upon whether they were carriers or non-carriers of allele 2 of either gene. No significant associations were found with the presence of at least one relapse/recurrence, the duration of corticosteroid treatment or the cumulative prednisone dose. In conclusion, in this study we observed an association between PMR and the IL1RN*2 allele, particularly in the homozygous state. 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