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IL-1 Receptor Antagonist (IL-1RA) Gene Polymorphism in Sjogren's Syndrome and Rheumatoid Arthritis
CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY
Vol. 87, No. 3, June, pp. 309–313, 1998 Article No. II984520
RAPID COMMUNICATION IL-1 Receptor Antagonist (IL-1RA) Gene Polymorphism in Sjogren’s Syndrome and Rheumatoid Arthritis S. Perrier,* C. Coussediere,† J. J. Dubost,* E. Albuisson,‡ and B. Sauvezie* *Unit of Clinical Immunology, and †Blood Transfusion Center, Hoˆpital Gabriel Montpied, Place Henri Dunant, 63003, Clermont-Ferrand, France; and ‡Department of Biomathematics and Biostatistics, Medicine University, 63003, Clermont-Ferrand, France
The gene encoding interleukin-1 receptor antagonist (IL-1ra) has a variable allelic polymorphism. The IL1RN*2 allele was recently described as a factor of severity in several autoimmune diseases and was paradoxically associated with increased production of IL1ra by monocytes in vitro. We studied this polymorphism in 36 patients with possible or definite primary Sjogren’s syndrome and found that IL1RN*2 was significantly more frequent in the definite than in the possible form. In rheumatoid arthritis, the frequency of the allele was not different from that of controls. The serum levels of IL-1ra were markedly higher in Sjogren patients than in those of healthy subjects. By contrast, the salivary IL-1ra levels were decreased. Patients with the allele generally had lower salivary levels and higher serum levels than patients without the allele. In the group of patients with the definite syndrome, CRP and TGF-b1, two in vitro stimulators of IL-1ra production, were correlated with IL-1ra serum levels. Our results suggest that IL1RN*2 is a marker of more severe forms of Sjogren’s syndrome. Its effect on salivary and serum IL-1ra may be distinct, suggesting separate regulatory mechanisms. q 1998 Academic
repeat (IL1RN*1) and 2-repeat (IL1RN*2) alleles are those most frequently found in the general population and the other three alleles are rarely observed. The number of repeats at the polymorphism site may be of functional significance because the repeated sequence contains possible binding sites for transcription factors (5). In normal monocytes in culture, the allele IL1RN∗2 was associated with increased production of IL-1ra (6). Several studies have shown a significant increase in both the frequency and carriage rate of IL1RN*2 in various diseases including systemic lupus erythematosus (SLE) (7), which shares several similarities with primary Sjogren’s syndrome (SS). The paradox of the association of the allele with inflammatory diseases and with increased production of IL-1ra has not been resolved. IL-1ra gene polymorphism and IL-1ra serum levels have not been investigated in SS and there is no report in rheumatoid arthritis (RA). We studied them along with salivary IL-1ra and serum IL-1b, TNF-a, TGF-b, and CRP which are potential inducers of IL1ra (8–11). We also studied the IL-1ra polymorphism in a group of patients with RA. PATIENTS AND METHODS
Press
Key Words: IL-1 receptor antagonist; Sjogren’s syndrome; polymorphism.
INTRODUCTION
IL-1 activity in inflammatory disorders is regulated by the natural antagonist IL-1 receptor antagonist (IL1ra) (1). IL-1ra is structurally related to IL-1a and IL1b (2). It competes with them for binding to IL-1 receptors but does not trigger intracellular signaling pathways and thus acts as an inhibitor of IL-1 activity (3). The human IL-1ra gene (IL1RN) has a variable-length polymorphism within intron 2 due to variation in the number of copies of an 86-bp sequence (4). Five distinct alleles corresponding to 2, 3, 4, 5, and 6 copies of the repeat sequence have been identified so far (5). The 4-
Patients. Blood samples were obtained from 54 patients with primary SS. According to the criteria of the European Cooperative Study (12), observations were classified as ‘‘definite’’ SS if they met 4 or more diagnostic criteria (n Å 36), and as ‘‘possible’’ SS if they met 3 diagnostic criteria (n Å 18). None of the patients had received systemic treatment with steroids, nonsteroid anti-inflammatory drugs, or cytotoxic drugs. Fortythree patients with definite RA without SS were also tested. The controls were 19 healthy blood donors (for cytokine determination) and 100 healthy bone marrow donors. They were age- and sex-matched with the patients. All patients and controls were unrelated Caucasians of northern European ancestry. DNA collection. Genomic DNA was extracted from blood EDTA by the standard proteinase K digestion
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and salting out method, followed by ethanol precipitation (13). It was stored at 47C until used. The DNA was originally collected for HLA typing. IL-1ra polymorphism screening. The method was based on a PCR as previously described (5). Briefly, the oligonucleotide primers 5*-CTCAGCAACACTCCTAT3* and 5*-TCCTGGTCTGCAGGTAA-3* were used to amplify the polymorphic region of the IL1RN gene. PCR conditions were a denaturing step of 947C for 1 min and then 30 cycles at 947C for 1 min, 607C for 1 min, and 707C for 1 min. The alleles were identified by size after separation of amplified products on a 2% agarose gel stained with ethidium bromide. A control lane was performed in the absence of DNA template to assess the lack of DNA contamination. Cytokine determination. IL-1ra, IL-1b, and TGF-b1 measurements in serum samples were performed by sandwich ELISA, according to the manufacturer’s instructions. The detection limits of the assays were 6.5 pg/ml for IL-1ra (R&D Systems, Abingdon, UK), 3 pg/ ml for IL-1b (Genzyme Diagnostics, Cambridge, MA), and 7 pg/ml for TGF-b1 (R&D Systems). TNF-a levels were assessed according to a specific bioassay based on the cytotoxicity effect on L929 cells. Salivary IL-1ra was measured from unstimulated saliva collected on ‘‘salivettes’’ (Sarsted, Numbrecht, Germany) as previously described (14). CRP determination. Serum CRP levels were measured by nephelometry with a nephelometer analyzer (Behring, Germany). The detection limit of CRP in this assay was 1 mg/l (normal õ1 mg/liter). For statistical analysis, we attributed this value when the CRP level was below the detection threshold. Statistical analysis. The carriage rate of an allele is the number of individuals carrying at least one copy of the allele relative to the total number of individuals. Allele frequency is expressed as a percentage of the
total number of alleles. The x2 test and the one-tailed variance analysis were used to analyze the differences in allelic frequencies between patients and controls. The odds ratio indicates how many times more frequent the disease is in individuals carrying the allele than in individuals without the allele (equivalent to approximate relative risk) and was calculated from allele frequency and allele carriage rate with 95% confidence limits. Spearman’s rank correlation tests were used for dependent groups and the Kruskal–Wallis tests were used for independent groups. RESULTS
IL-1ra polymorphism study. The carriage rate of IL1RN*2 increased from 35% in possible SS to 68.8% in definite SS (x2 Å 4, P Å 0.04), giving an odds ratio of 2.38 with 95% confidence limits between 1 to 5.8 for definite SS compared with healthy controls (Table 1). Accordingly, the frequency of IL1RN*2 increased from 27% in controls and 20% in possible SS to 41% in the patients with the more extensive disease while the frequency of IL1RN*1 decreased from 71% in controls and 77.5% in possible SS to 53% in definite SS (x2 Å 4, P Å 0.04 and x2 Å 4.75, P Å 0.03 respectively). The frequency and the carriage rate of the allele IL1RN*2 were not significantly different among the Sjogren patients as a whole group, RA patients, and controls. Cytokine study. IL-1ra serum concentrations were significantly higher (Table 2) in SS (range 110–1767, median 483 pg/ml, P õ 0.05) than in controls (range 28–863, median 254 pg/ml) but no difference was observed between the two groups of patients (Table 2). In the group of definite Sjogren patients, there was a correlation between IL-1ra concentrations and CRP levels (Fig. 1). In possible SS, the CRP levels were frequently normal. A strong correlation was observed between IL-1ra and TGF-b1 (Fig. 2) in definite SS but
TABLE 1 Frequency and Carriage Rate of Alleles IL1RN*1 (4 Repeats) and IL1RN*2 (2 Repeats) in the Control Group, the RA Group, and the Sjogren’s Syndrome (SS) Group
IL1RN*1 Frequency (%) Carriage rate (%) IL1RN*2 Frequency (%) Carriage rate (%)
Controls (n Å 100)
RA (n Å 43)
Total SS (n Å 36)
Possible SS (n Å 20)
71 93
72 90.7
66.6 88
77.5 95
53* 81.2
27 48
25.6 44.2
29.2 50
20 35
41 68.8**
* Significant versus controls (P Å 0.04) and possible SS (P Å 0.03). ** Significant versus controls (P Å 0.04), RA (P Å 0.03) and possible SS (P Å 0.04).
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TABLE 2 Variations in Serum and Salivary Levels of IL-1ra According to the IL-1ra Gene Polymorphism
Serum IL-1ra (pg/ml) Salivary IL-1ra (ng/ml)
Controls
Total SS
SS with allele 2
SS without allele 2
254 (n Å 19) 1230 (n Å 26)
483* (n Å 40) 780** (n Å 39)
468 (n Å 11) 545 (n Å 13)
320 (n Å 5) 790 (n Å 16)
* Significant versus controls (P õ 0.05, Kruskal–Wallis test). ** Significant versus controls (P õ 0.05, Mann–Whitney test).
not in possible SS. No correlation was found between TGF-b1 and CRP. Serum IL-1b levels were below the detection limit in all except 5 patients and 4 controls, whose IL-1ra was not especially high. TNF-a measurements in 18 patients were within the range of the control levels (15 pg/ml) (data not shown). In definite SS, the mean serum level of 1L-1ra was higher in patients with at least one copy of IL1RN*2 (468 pg/ml, n Å 11, Table 2) than in those without the allele (320 pg/ml, n Å 5) (not significant). In contrast, the salivary IL-1ra concentration was decreased in patients with IL1RN*2 (545 ng/ml, n Å 13) compared to that in patients without the allele (790 ng/ml, n Å 16).
The salivary concentration of IL-1ra was also lower in definite SS (550 ng/ml, n Å 11) than that in possible SS (756 ng/ml, n Å 18) (not significant). DISCUSSION
Our results show an increased frequency and carriage rate of the IL-1ra allele IL1RN*2 in primary SS. Both values were normal in RA without SS. The increase was significant in the severe form of the syndrome (definite SS). In the patients with definite or possible SS taken as a group, there was a tendency toward a correlation between the presence of IL1RN*2 and decreased salivary IL-1ra. The serum levels of IL1ra were increased in the patients with definite or possible SS taken as a group and patients with the allele tended to have a larger increase. IL1RN*2 probably has different effects in different cells. In the present work and in a previous report (15), the lower salivary levels of IL-1ra were detected in patients with severe SS, a group in which the allele is frequent. In psoriasis, the concentrations of IL-1ra were lower in severe lesions than in mild ones (16). A distinct regulation may operate in the affected tissues and in the blood. Sources of IL-1ra are epithelial cells in the oral mucosa (15) and keratinocytes in the skin (17). A similar
FIG. 1. Correlation between IL-1ra concentrations and CRP levels in patients with definite Sjogren’s syndrome (r Å 0.55, P Å 0.008, n Å 21).
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FIG. 2. Correlation between serum levels of IL-1ra and TGF-b in patients with definite Sjogren’s syndrome (r Å 0.58, P Å 0.005, n Å 21).
discrepancy between local and systemic IL-1ra may exist in other IL1RN*2-associated diseases, most of which involve epitheliums: lupus (7), psoriasis (18), lichen sclerosus (19), ulcerative colitis (20), alopecia aerata (21), Graves’ disease (22), and non-insulin-dependent diabetes mellitus (23). In these disorders, as in our patients, IL1RN*2 was related to disease severity, the first such marker outside the MHC. Circulating IL-1ra comes from mononuclear cells (24) and this source is consistent with increased serum levels in diseases associated with the allele: our patients with SS, in lupus (25), and in ulcerative colitis (26). In insulin-dependent diabetes mellitus, which has no confounding systemic inflammation, the plasma IL-1ra levels were higher in patients with the IL1RN*1/ IL1RN*2 genotype than those in patients carrying the IL1RN*1/IL1RN*1 genotype (27). TGF-b1, which stimulates IL-1ra production by monocytes (10), may add its effect to the enhancing effect of the allele on serum IL-1ra but not in keratinocytes (28). A decreased immunostaining of TGF-b especially in severe lesions of SS was described in oral mucosa (29). Serum levels of CRP were higher in the patients (especially those with definite syndrome) than those in controls and correlated with serum but not salivary IL1ra in the severe form of SS. CRP and IL-1ra were recently reported to correlate in SLE at the time of greatest disease activity (30).
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In conclusion, the allele IL1RN*2 is associated with the severity of SS. In the severe form of SS, the increase in serum IL-1ra contrasts with the decrease in salivary IL-1ra. Our results demonstrate for the first time that a condition associated with the allele can be also characterized by low concentrations of IL-1ra in at least one body fluid while IL-1ra serum levels are increased. These opposite changes are indicative of distinct regulatory and pathogenetic mechanisms. Separate regulations in separate compartments may explain the paradox of the association of the allele with increased production of IL-1ra and inflammatory diseases. ACKNOWLEDGMENTS We are greatly indebted to Annie Dosgilbert, Gael Belliot, Ousmane Traore, Marie-Claire Giraud, and Josette Meritet for their technical assistance, and to Jeffrey Watts for improving the English rendition of the manuscript. The authors thank the Direction Generale du Centre Hospitalo-Universitaire de Clermont-Ferrand for its invaluable support. REFERENCES 1. Dinarello, C. A., and Thompson, R. C., Blocking IL-1: Interleukin-1 receptor antagonist in vivo and in vitro. Immunol. Today. 12, 404–410, 1991. 2. Eisenberg, S. P., Evans, R. J., Arend, W. P., Verderber, E., Brewer, M. T., Hannum, C. H., and Thompson, R. C., Primary structure and functional expression from complementary DNA
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RAPID COMMUNICATION of a human interleukin-1 receptor antagonist. Nature 343, 341– 346, 1990. 3. Dripps, D. J., Brandhuber, B. J., Thompson, R. C., and Eisenberg, S. P., Interleukin-1 receptor antagonist binds to the 80kDa IL-1 receptor but does not initiate IL-1 signal transduction. J. Biol. Chem. 266, 10331–10336, 1991. 4. Steinkasserer, A., Koelble, K., and Sim, R. B., Length variation within intron 2 of the human IL-1 receptor antagonist protein gene (IL-1RN). Nucleic Acids Res. 19, 5095, 1991. 5. Tarlow, J. K., Blakemore, A. I. F., Lennard, A., Solari, R., Hughes, H. N., Steinkasserer, A., and Duff, G. W., Polymorphism in human IL-1 receptor antagonist gene intron 2 is caused by variable numbers of a 86-bp tandem repeat. Hum. Genet. 91, 403–404, 1993. 6. Danis, V. A., Millington, M., Hyland, V. J., and Grennan, D., Cytokine production by normal human monocytes: Inter-subject variation and relationship to an IL-1 receptor antagonist (IL-1ra) gene polymorphism. Clin. Exp. Immunol. 99, 303–310, 1995. 7. Blakemore, A. I. F., Tarlow, J. K., Cork, M. J., Gordon, C., Emery, P., and Duff, G. W., Interleukin-1 receptor antagonist gene polymorphism as a disease severity factor in systemic lupus erythematosus. Arthritis Rheum. 37, 1380–1385, 1994. 8. Jenkins, J. K., and Arend, W. P., Interleukin-1 receptor antagonist production in human monocytes is induced by IL-1 alpha, IL-3, IL-4 and GM-CSF. Cytokine 5, 407–415, 1993. 9. Van Der Poll, T., Van Deventer, J. H., Ten Cate, H., Levi, M., and Ten Cate, J. W., Tumor necrosis factor is involved in the appearance of interleukin-1 receptor antagonist in endotoxinemia. J. Infect. Dis. 169, 665–667, 1994. 10. Turner, M., Chantry, D., Katsikis, P., Berger, A., Brennan, F. M., and Feldmann, M., Induction of the interleukin-1 receptor antagonist protein by transforming growth factor-beta. Eur. J. Immunol. 21, 1635–1639, 1991. 11. Tilg, H., Vannier, E., Vachino, G., Dinarello, C. A., and Mier, J. W., Antiinflammatory properties of hepatic acute phase proteins: Preferential induction of interleukin-1 (IL-1) receptor antagonist over interleukin-1b synthesis by human peripheral blood mononuclear cells. J. Exp. Med. 178, 1629–1634, 1993. 12. Vitali, C., Bombardieri, S., Moutsopoulos, H. M., et al., Preliminary criteria for the classification of Sjo¨gren’s syndrome. Results of a prospective concerted action supported by the European Community. Arthritis Rheum. 36, 340–347, 1993. 13. Miller, S. A., Dykes, D. D., and Polesky, H. F., A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215–1218, 1988. 14. Lamey, P. J., and Nolan, A., The recovery of human saliva using the Salivette system. Eur. J. Clin. Chem. Clin. Biochem. 32, 727–728, 1994. 15. Dubost, J. J., Perrier, S., Afane, M., Viallard, J. L., Roux-Lombard, P., Baudet-Pommel, M., Begue, C., Kemeny, J. L., and Sauvezie, B., IL-1 receptor antagonist in saliva: Characterization in normal saliva and reduced concentrations in Sjogren’s syndrome (SS). Clin. Exp. Immunol. 106, 237–242, 1996. 16. Hammerberg, C., Arend, W. P., Fisher, G. J., Chan, L. S., Berger, A. E., Haskill, S., Voorhees, J. J., and Cooper, K. D., Interleukin1 receptor antagonist in normal and psoriatic epidermis. J. Clin. Invest. 90, 571–583, 1992. 17. Kristensen, M., Deleuran, B., Eedy, D. J., Feldmann, M., Breathnach, S. M., and Brennan, F. M., Distribution of interleu-
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kin-1 receptor antagonist protein (IRAP), interleukin-1 receptor, and interleukin-1 a in normal and psoriatic skin. Decreased expression of IRAP in psoriatic lesional epidermis. Br. J. Dermatol. 127, 305–311, 1992. Tarlow, J. K., Cork, M. J., Clay, F. E., Schmitt-Egenolf, M., Crane, A. M., Stierle, C., Boehncke, W.-H., Eiermann, T. H., Blakemore, A. I. F., Bleehen, S. S., Sterry, W., and Duff, G. W., Association between interleukin-1 receptor antagonist (IL-1ra) gene polymorphism and early and late-onset psoriasis. Br. J. Dermatol. 136, 147–148, 1997. Clay, F. E., Cork, M. J., Tarlow, J. K., Blakemore, A. I. F., Harrington, C. I., Lewis, F., and Duff, G. W., Interleukin-1 receptor antagonist gene polymorphism association with lichen sclerosus. Hum. Genet. 94, 407–410, 1994. Mansfield, J. C., Holden, H., Tarlow, J. K., Di Giovine, F. S., McDowell, T. L., Wilson, A. G., Holdsworth, C. D., and Duff, G. W., Novel genetic association between ulcerative colitis and the antiinflammatory cytokine interleukin-1 receptor antagonist. Gastroenterology 106, 637–642, 1994. Tarlow, J. K., Clay, F. E., Cork, M. J., Blakemore, A. I. F., McDonagh, A. J. G., Messenger, A. G., and Duff, G. W., Severity of alopecia areata is associated with a polymorphism in the interleukin-1 receptor antagonist gene. J. Invest. Dermatol. 103, 387–390, 1994. Blakemore, A. I. F., Watson, P. F., Weetman, A. P., and Duff, G. W., Association of Grave’s disease with an allele of the interleukin-1 receptor antagonist gene. J. Clin. Endocrinol. Metab. 80, 111–115, 1995. Blakemore, A. I. F., Cox, A., Gonzalez, A. M., Maskill, J. K., Hughes, M. E., Wilson, R. M., Ward, J. D., and Duff, G. W., Interleukin-1 receptor antagonist allele (IL1RN*2) associated with nephropathy in diabetes mellitus. Hum. Genet. 97, 369–374, 1996. Nerad, J. L., Griffiths, J. K., Van der Meer, J. W. M., Endres, S., Poutsiaka, D. D., Keusch, G. T., Bennish, M., Salam, M. A., Dinarello, C. A., and Cannon, J. G., Interleukin-1b (IL-1b), IL1 receptor antagonist, ant TNF-a production in whole blood. J. Leukocyte Biol. 52, 687–692, 1992. Suzuki, H., Takemura, H., and Kashiwagi, H., Interleukin-1 receptor antagonist in patients with active systemic lupus erythematosus. Arthritis Rheum. 38, 1055–1059, 1995. Andus, T., Gross, V., and Scholmerich, J., Elevated circulatory interleukin-1 receptor antagonist levels in serum of patients with active Crohn’s disease and ulcerative colitis. Gastroenterology 104, A660, 1993. [Abstract] Mandrup-Poulsen, T., Pociot, F., Molvig, J., Shapiro, L., Nillson, P., Emdal, T., Roder, M., Kjems, L. L., Dinarello, C. A., and Nerup, J., Monokine antagonism is reduced in patients with IDDM. Diabetes 43, 1242–1247, 1994. Phillips, W. G., Feldmann, M., Breathnach, S. M., and Brennan, F. M., Modulation of the IL-1 cytokine network in keratinocytes by intracellular IL-1a and IL-1 receptor antagonist. Clin. Exp. Immunol. 101, 177–182, 1995. Ogawa, N., Dang, H., Lazaridis, K., McGuff, H. S., Aufdemorte, T. B., and Talal, N., Analysis of transforming growth factor beta and other cytokines in autoimmune exocrinopathy (Sjogren’s syndrome). J. Interferon Cytokine Res. 15, 759–767, 1995. Sturfelt, G. K., Roux-Lombard, P., Dayer, J.-M., and Wolheim, F. A., Interleukin-1 receptor antagonist in the course of SLE: Low levels coincide with kidney involvement. Arthritis Rheum. 39, S296, 1996. [Abstract]
Received December 23, 1997; accepted January 9, 1998
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Vol. 87, No. 3, June, pp. 309–313, 1998 Article No. II984520
RAPID COMMUNICATION IL-1 Receptor Antagonist (IL-1RA) Gene Polymorphism in Sjogren’s Syndrome and Rheumatoid Arthritis S. Perrier,* C. Coussediere,† J. J. Dubost,* E. Albuisson,‡ and B. Sauvezie* *Unit of Clinical Immunology, and †Blood Transfusion Center, Hoˆpital Gabriel Montpied, Place Henri Dunant, 63003, Clermont-Ferrand, France; and ‡Department of Biomathematics and Biostatistics, Medicine University, 63003, Clermont-Ferrand, France
The gene encoding interleukin-1 receptor antagonist (IL-1ra) has a variable allelic polymorphism. The IL1RN*2 allele was recently described as a factor of severity in several autoimmune diseases and was paradoxically associated with increased production of IL1ra by monocytes in vitro. We studied this polymorphism in 36 patients with possible or definite primary Sjogren’s syndrome and found that IL1RN*2 was significantly more frequent in the definite than in the possible form. In rheumatoid arthritis, the frequency of the allele was not different from that of controls. The serum levels of IL-1ra were markedly higher in Sjogren patients than in those of healthy subjects. By contrast, the salivary IL-1ra levels were decreased. Patients with the allele generally had lower salivary levels and higher serum levels than patients without the allele. In the group of patients with the definite syndrome, CRP and TGF-b1, two in vitro stimulators of IL-1ra production, were correlated with IL-1ra serum levels. Our results suggest that IL1RN*2 is a marker of more severe forms of Sjogren’s syndrome. Its effect on salivary and serum IL-1ra may be distinct, suggesting separate regulatory mechanisms. q 1998 Academic
repeat (IL1RN*1) and 2-repeat (IL1RN*2) alleles are those most frequently found in the general population and the other three alleles are rarely observed. The number of repeats at the polymorphism site may be of functional significance because the repeated sequence contains possible binding sites for transcription factors (5). In normal monocytes in culture, the allele IL1RN∗2 was associated with increased production of IL-1ra (6). Several studies have shown a significant increase in both the frequency and carriage rate of IL1RN*2 in various diseases including systemic lupus erythematosus (SLE) (7), which shares several similarities with primary Sjogren’s syndrome (SS). The paradox of the association of the allele with inflammatory diseases and with increased production of IL-1ra has not been resolved. IL-1ra gene polymorphism and IL-1ra serum levels have not been investigated in SS and there is no report in rheumatoid arthritis (RA). We studied them along with salivary IL-1ra and serum IL-1b, TNF-a, TGF-b, and CRP which are potential inducers of IL1ra (8–11). We also studied the IL-1ra polymorphism in a group of patients with RA. PATIENTS AND METHODS
Press
Key Words: IL-1 receptor antagonist; Sjogren’s syndrome; polymorphism.
INTRODUCTION
IL-1 activity in inflammatory disorders is regulated by the natural antagonist IL-1 receptor antagonist (IL1ra) (1). IL-1ra is structurally related to IL-1a and IL1b (2). It competes with them for binding to IL-1 receptors but does not trigger intracellular signaling pathways and thus acts as an inhibitor of IL-1 activity (3). The human IL-1ra gene (IL1RN) has a variable-length polymorphism within intron 2 due to variation in the number of copies of an 86-bp sequence (4). Five distinct alleles corresponding to 2, 3, 4, 5, and 6 copies of the repeat sequence have been identified so far (5). The 4-
Patients. Blood samples were obtained from 54 patients with primary SS. According to the criteria of the European Cooperative Study (12), observations were classified as ‘‘definite’’ SS if they met 4 or more diagnostic criteria (n Å 36), and as ‘‘possible’’ SS if they met 3 diagnostic criteria (n Å 18). None of the patients had received systemic treatment with steroids, nonsteroid anti-inflammatory drugs, or cytotoxic drugs. Fortythree patients with definite RA without SS were also tested. The controls were 19 healthy blood donors (for cytokine determination) and 100 healthy bone marrow donors. They were age- and sex-matched with the patients. All patients and controls were unrelated Caucasians of northern European ancestry. DNA collection. Genomic DNA was extracted from blood EDTA by the standard proteinase K digestion
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and salting out method, followed by ethanol precipitation (13). It was stored at 47C until used. The DNA was originally collected for HLA typing. IL-1ra polymorphism screening. The method was based on a PCR as previously described (5). Briefly, the oligonucleotide primers 5*-CTCAGCAACACTCCTAT3* and 5*-TCCTGGTCTGCAGGTAA-3* were used to amplify the polymorphic region of the IL1RN gene. PCR conditions were a denaturing step of 947C for 1 min and then 30 cycles at 947C for 1 min, 607C for 1 min, and 707C for 1 min. The alleles were identified by size after separation of amplified products on a 2% agarose gel stained with ethidium bromide. A control lane was performed in the absence of DNA template to assess the lack of DNA contamination. Cytokine determination. IL-1ra, IL-1b, and TGF-b1 measurements in serum samples were performed by sandwich ELISA, according to the manufacturer’s instructions. The detection limits of the assays were 6.5 pg/ml for IL-1ra (R&D Systems, Abingdon, UK), 3 pg/ ml for IL-1b (Genzyme Diagnostics, Cambridge, MA), and 7 pg/ml for TGF-b1 (R&D Systems). TNF-a levels were assessed according to a specific bioassay based on the cytotoxicity effect on L929 cells. Salivary IL-1ra was measured from unstimulated saliva collected on ‘‘salivettes’’ (Sarsted, Numbrecht, Germany) as previously described (14). CRP determination. Serum CRP levels were measured by nephelometry with a nephelometer analyzer (Behring, Germany). The detection limit of CRP in this assay was 1 mg/l (normal õ1 mg/liter). For statistical analysis, we attributed this value when the CRP level was below the detection threshold. Statistical analysis. The carriage rate of an allele is the number of individuals carrying at least one copy of the allele relative to the total number of individuals. Allele frequency is expressed as a percentage of the
total number of alleles. The x2 test and the one-tailed variance analysis were used to analyze the differences in allelic frequencies between patients and controls. The odds ratio indicates how many times more frequent the disease is in individuals carrying the allele than in individuals without the allele (equivalent to approximate relative risk) and was calculated from allele frequency and allele carriage rate with 95% confidence limits. Spearman’s rank correlation tests were used for dependent groups and the Kruskal–Wallis tests were used for independent groups. RESULTS
IL-1ra polymorphism study. The carriage rate of IL1RN*2 increased from 35% in possible SS to 68.8% in definite SS (x2 Å 4, P Å 0.04), giving an odds ratio of 2.38 with 95% confidence limits between 1 to 5.8 for definite SS compared with healthy controls (Table 1). Accordingly, the frequency of IL1RN*2 increased from 27% in controls and 20% in possible SS to 41% in the patients with the more extensive disease while the frequency of IL1RN*1 decreased from 71% in controls and 77.5% in possible SS to 53% in definite SS (x2 Å 4, P Å 0.04 and x2 Å 4.75, P Å 0.03 respectively). The frequency and the carriage rate of the allele IL1RN*2 were not significantly different among the Sjogren patients as a whole group, RA patients, and controls. Cytokine study. IL-1ra serum concentrations were significantly higher (Table 2) in SS (range 110–1767, median 483 pg/ml, P õ 0.05) than in controls (range 28–863, median 254 pg/ml) but no difference was observed between the two groups of patients (Table 2). In the group of definite Sjogren patients, there was a correlation between IL-1ra concentrations and CRP levels (Fig. 1). In possible SS, the CRP levels were frequently normal. A strong correlation was observed between IL-1ra and TGF-b1 (Fig. 2) in definite SS but
TABLE 1 Frequency and Carriage Rate of Alleles IL1RN*1 (4 Repeats) and IL1RN*2 (2 Repeats) in the Control Group, the RA Group, and the Sjogren’s Syndrome (SS) Group
IL1RN*1 Frequency (%) Carriage rate (%) IL1RN*2 Frequency (%) Carriage rate (%)
Controls (n Å 100)
RA (n Å 43)
Total SS (n Å 36)
Possible SS (n Å 20)
71 93
72 90.7
66.6 88
77.5 95
53* 81.2
27 48
25.6 44.2
29.2 50
20 35
41 68.8**
* Significant versus controls (P Å 0.04) and possible SS (P Å 0.03). ** Significant versus controls (P Å 0.04), RA (P Å 0.03) and possible SS (P Å 0.04).
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TABLE 2 Variations in Serum and Salivary Levels of IL-1ra According to the IL-1ra Gene Polymorphism
Serum IL-1ra (pg/ml) Salivary IL-1ra (ng/ml)
Controls
Total SS
SS with allele 2
SS without allele 2
254 (n Å 19) 1230 (n Å 26)
483* (n Å 40) 780** (n Å 39)
468 (n Å 11) 545 (n Å 13)
320 (n Å 5) 790 (n Å 16)
* Significant versus controls (P õ 0.05, Kruskal–Wallis test). ** Significant versus controls (P õ 0.05, Mann–Whitney test).
not in possible SS. No correlation was found between TGF-b1 and CRP. Serum IL-1b levels were below the detection limit in all except 5 patients and 4 controls, whose IL-1ra was not especially high. TNF-a measurements in 18 patients were within the range of the control levels (15 pg/ml) (data not shown). In definite SS, the mean serum level of 1L-1ra was higher in patients with at least one copy of IL1RN*2 (468 pg/ml, n Å 11, Table 2) than in those without the allele (320 pg/ml, n Å 5) (not significant). In contrast, the salivary IL-1ra concentration was decreased in patients with IL1RN*2 (545 ng/ml, n Å 13) compared to that in patients without the allele (790 ng/ml, n Å 16).
The salivary concentration of IL-1ra was also lower in definite SS (550 ng/ml, n Å 11) than that in possible SS (756 ng/ml, n Å 18) (not significant). DISCUSSION
Our results show an increased frequency and carriage rate of the IL-1ra allele IL1RN*2 in primary SS. Both values were normal in RA without SS. The increase was significant in the severe form of the syndrome (definite SS). In the patients with definite or possible SS taken as a group, there was a tendency toward a correlation between the presence of IL1RN*2 and decreased salivary IL-1ra. The serum levels of IL1ra were increased in the patients with definite or possible SS taken as a group and patients with the allele tended to have a larger increase. IL1RN*2 probably has different effects in different cells. In the present work and in a previous report (15), the lower salivary levels of IL-1ra were detected in patients with severe SS, a group in which the allele is frequent. In psoriasis, the concentrations of IL-1ra were lower in severe lesions than in mild ones (16). A distinct regulation may operate in the affected tissues and in the blood. Sources of IL-1ra are epithelial cells in the oral mucosa (15) and keratinocytes in the skin (17). A similar
FIG. 1. Correlation between IL-1ra concentrations and CRP levels in patients with definite Sjogren’s syndrome (r Å 0.55, P Å 0.008, n Å 21).
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FIG. 2. Correlation between serum levels of IL-1ra and TGF-b in patients with definite Sjogren’s syndrome (r Å 0.58, P Å 0.005, n Å 21).
discrepancy between local and systemic IL-1ra may exist in other IL1RN*2-associated diseases, most of which involve epitheliums: lupus (7), psoriasis (18), lichen sclerosus (19), ulcerative colitis (20), alopecia aerata (21), Graves’ disease (22), and non-insulin-dependent diabetes mellitus (23). In these disorders, as in our patients, IL1RN*2 was related to disease severity, the first such marker outside the MHC. Circulating IL-1ra comes from mononuclear cells (24) and this source is consistent with increased serum levels in diseases associated with the allele: our patients with SS, in lupus (25), and in ulcerative colitis (26). In insulin-dependent diabetes mellitus, which has no confounding systemic inflammation, the plasma IL-1ra levels were higher in patients with the IL1RN*1/ IL1RN*2 genotype than those in patients carrying the IL1RN*1/IL1RN*1 genotype (27). TGF-b1, which stimulates IL-1ra production by monocytes (10), may add its effect to the enhancing effect of the allele on serum IL-1ra but not in keratinocytes (28). A decreased immunostaining of TGF-b especially in severe lesions of SS was described in oral mucosa (29). Serum levels of CRP were higher in the patients (especially those with definite syndrome) than those in controls and correlated with serum but not salivary IL1ra in the severe form of SS. CRP and IL-1ra were recently reported to correlate in SLE at the time of greatest disease activity (30).
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In conclusion, the allele IL1RN*2 is associated with the severity of SS. In the severe form of SS, the increase in serum IL-1ra contrasts with the decrease in salivary IL-1ra. Our results demonstrate for the first time that a condition associated with the allele can be also characterized by low concentrations of IL-1ra in at least one body fluid while IL-1ra serum levels are increased. These opposite changes are indicative of distinct regulatory and pathogenetic mechanisms. Separate regulations in separate compartments may explain the paradox of the association of the allele with increased production of IL-1ra and inflammatory diseases. ACKNOWLEDGMENTS We are greatly indebted to Annie Dosgilbert, Gael Belliot, Ousmane Traore, Marie-Claire Giraud, and Josette Meritet for their technical assistance, and to Jeffrey Watts for improving the English rendition of the manuscript. The authors thank the Direction Generale du Centre Hospitalo-Universitaire de Clermont-Ferrand for its invaluable support. REFERENCES 1. Dinarello, C. A., and Thompson, R. C., Blocking IL-1: Interleukin-1 receptor antagonist in vivo and in vitro. Immunol. Today. 12, 404–410, 1991. 2. Eisenberg, S. P., Evans, R. J., Arend, W. P., Verderber, E., Brewer, M. T., Hannum, C. H., and Thompson, R. C., Primary structure and functional expression from complementary DNA
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RAPID COMMUNICATION of a human interleukin-1 receptor antagonist. Nature 343, 341– 346, 1990. 3. Dripps, D. J., Brandhuber, B. J., Thompson, R. C., and Eisenberg, S. P., Interleukin-1 receptor antagonist binds to the 80kDa IL-1 receptor but does not initiate IL-1 signal transduction. J. Biol. Chem. 266, 10331–10336, 1991. 4. Steinkasserer, A., Koelble, K., and Sim, R. B., Length variation within intron 2 of the human IL-1 receptor antagonist protein gene (IL-1RN). Nucleic Acids Res. 19, 5095, 1991. 5. Tarlow, J. K., Blakemore, A. I. F., Lennard, A., Solari, R., Hughes, H. N., Steinkasserer, A., and Duff, G. W., Polymorphism in human IL-1 receptor antagonist gene intron 2 is caused by variable numbers of a 86-bp tandem repeat. Hum. Genet. 91, 403–404, 1993. 6. Danis, V. A., Millington, M., Hyland, V. J., and Grennan, D., Cytokine production by normal human monocytes: Inter-subject variation and relationship to an IL-1 receptor antagonist (IL-1ra) gene polymorphism. Clin. Exp. Immunol. 99, 303–310, 1995. 7. Blakemore, A. I. F., Tarlow, J. K., Cork, M. J., Gordon, C., Emery, P., and Duff, G. W., Interleukin-1 receptor antagonist gene polymorphism as a disease severity factor in systemic lupus erythematosus. Arthritis Rheum. 37, 1380–1385, 1994. 8. Jenkins, J. K., and Arend, W. P., Interleukin-1 receptor antagonist production in human monocytes is induced by IL-1 alpha, IL-3, IL-4 and GM-CSF. Cytokine 5, 407–415, 1993. 9. Van Der Poll, T., Van Deventer, J. H., Ten Cate, H., Levi, M., and Ten Cate, J. W., Tumor necrosis factor is involved in the appearance of interleukin-1 receptor antagonist in endotoxinemia. J. Infect. Dis. 169, 665–667, 1994. 10. Turner, M., Chantry, D., Katsikis, P., Berger, A., Brennan, F. M., and Feldmann, M., Induction of the interleukin-1 receptor antagonist protein by transforming growth factor-beta. Eur. J. Immunol. 21, 1635–1639, 1991. 11. Tilg, H., Vannier, E., Vachino, G., Dinarello, C. A., and Mier, J. W., Antiinflammatory properties of hepatic acute phase proteins: Preferential induction of interleukin-1 (IL-1) receptor antagonist over interleukin-1b synthesis by human peripheral blood mononuclear cells. J. Exp. Med. 178, 1629–1634, 1993. 12. Vitali, C., Bombardieri, S., Moutsopoulos, H. M., et al., Preliminary criteria for the classification of Sjo¨gren’s syndrome. Results of a prospective concerted action supported by the European Community. Arthritis Rheum. 36, 340–347, 1993. 13. Miller, S. A., Dykes, D. D., and Polesky, H. F., A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215–1218, 1988. 14. Lamey, P. J., and Nolan, A., The recovery of human saliva using the Salivette system. Eur. J. Clin. Chem. Clin. Biochem. 32, 727–728, 1994. 15. Dubost, J. J., Perrier, S., Afane, M., Viallard, J. L., Roux-Lombard, P., Baudet-Pommel, M., Begue, C., Kemeny, J. L., and Sauvezie, B., IL-1 receptor antagonist in saliva: Characterization in normal saliva and reduced concentrations in Sjogren’s syndrome (SS). Clin. Exp. Immunol. 106, 237–242, 1996. 16. Hammerberg, C., Arend, W. P., Fisher, G. J., Chan, L. S., Berger, A. E., Haskill, S., Voorhees, J. J., and Cooper, K. D., Interleukin1 receptor antagonist in normal and psoriatic epidermis. J. Clin. Invest. 90, 571–583, 1992. 17. Kristensen, M., Deleuran, B., Eedy, D. J., Feldmann, M., Breathnach, S. M., and Brennan, F. M., Distribution of interleu-
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Received December 23, 1997; accepted January 9, 1998
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