Interleukin-1 receptor antagonist polymorphism in women with peritoneal adhesions

Authors

  • Fritz Wieser,

    1. Department of Obstetrics and Gynaecology, Division of Gynaecological Endocrinology and Assisted Reproduction, University of Vienna, Vienna, Austria
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  • Clemens Tempfer,

    1. Department of Obstetrics and Gynaecology, Division of Gynaecology and Obstetrics, University of Vienna, Vienna, Austria
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  • Christian Schneeberger,

    1. Department of Obstetrics and Gynaecology, Division of Gynaecological Endocrinology and Assisted Reproduction, University of Vienna, Vienna, Austria
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  • Michael van Trotsenburg,

    1. Department of Obstetrics and Gynaecology, Division of Gynaecological Endocrinology and Assisted Reproduction, University of Vienna, Vienna, Austria
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  • Johannes Huber,

    1. Department of Obstetrics and Gynaecology, Division of Gynaecological Endocrinology and Assisted Reproduction, University of Vienna, Vienna, Austria
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  • Rene Wenzl

    Corresponding author
    1. Department of Obstetrics and Gynaecology, Division of Gynaecological Endocrinology and Assisted Reproduction, University of Vienna, Vienna, Austria
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* Dr R. Wenzl, Department of Obstetrics and Gynaecology, Division of Gynaecological Endocrinology and Assisted Reproduction, University of Vienna, Waehringer Guertel 18–20, A-1090 Vienna, Austria.

Abstract

Interleukin (IL)-1 has been shown to induce peritoneal adhesions. We determined the IL-1 receptor antagonist (IL-1RN) genotype with respect to the two most common variant alleles IL-1RN*2 and IL-1RN*3 in Caucasian women with peritoneal adhesions. One hundred seven women with surgically verified peritoneal adhesions and 79 controls without peritoneal adhesions served as controls. Univariate analysis showed an increased risk for peritoneal adhesions for Caucasian women carrying the mutant IL-1RN*2 allele (OR: 2.1; 95% CI: 1.3–3.4; P= 0.004). Multiple logistic regression analysis demonstrated an increased risk for peritoneal adhesions, which is independent of previous abdominal surgery and endometriosis. Our data suggest that IL-1RN*2 allele carriers have an increased risk for adhesion formation.

Introduction

Peritoneal adhesions cause bowel obstruction, pelvic pain and infertility and are a major cause of morbidity. In 1992, surgical adhesiolysis was performed in 400,000 patients in the United Kingdom. A variety of stimuli are known to induce adhesion formation, among them surgical trauma, infection, ischaemia and irradiation. Injury of the peritoneum leads to an inflammatory response consisting of hyperaemia, fluid exudation, recruitment of floating mesothelial cells and release of white blood cells and platelets into the peritoneal cavity. Cytokines are secreted by macrophages at the site of peritoneal injury and play an important role in regulating coagulation and fibrin formation, thereby influencing the development of adhesions1. In particular, proinflammatory cytokines (e.g. interleukin (IL)-1, IL-6, tumour growth factor (TGF)-β, and granulocyte-macrophage colony stimulating factor) regulate inflammatory and immunologic responses and are activated during peritoneal wound healing1.

IL-1 is a key mediator of the acute responses to tissue injury, inflammation and radiation2. The biologic action of IL-1 is modulated by the IL-1 receptor antagonist (IL-1RN), an anti-inflammatory protein with a molecular weight of 22 kDa. IL-1RN is secreted by macrophages and epithelial cells2. Treatment with IL-1RN and monoclonal antibodies against IL-1 reduces adhesion formation in rats3,4. The human IL-1 gene cluster on chromosome 2q contains three related genes within a 430-kilobase (kb) region, IL-1α, IL-1β and IL-1 receptor antagonist (IL-1RN), which encodes the proinflammatory cytokines IL-1α, IL-1β, and IL-1RN, respectively2. The IL-1RN gene has a VNTR polymorphism including the wild type (IL-1RN*1) and the mutant allele 2 (IL-1RN*2), which leads to a decreased production of IL-1RN2. Moreover, the presence of the IL-1RN*2 allele is associated with increased monocyte production of IL-1β2. Carriage of IL-1RN*2 is associated with a variety of chronic inflammatory diseases including ulcerative colitis, lichen sclerosus, psoriasis and diabetic nephropathy2. We determined the IL-1RN genotype with respect to the wild type allele IL-1RN*1 and the mutant alleles IL-1RN*2 and IL-1RN*3 in women with peritoneal adhesions and controls.

Methods

The study group comprised 107 Caucasian women (median age: 36 years; range: 19–80) with surgically confirmed peritoneal adhesions. A consecutive series of 79 Caucasian women without peritoneal adhesions were included in the control group (median age: 34; range: 18–76 years). Study patients underwent laparoscopy or laparotomy because of benign ovarian masses (n= 44), infertility (n= 23), chronic pelvic pain (n= 17), fibroids (n= 10), endometriosis (n= 8), ectopic pregnancy (n= 2), polycystic ovarian disease (n= 2) and tubal ligation (n= 1). We included a surgical control group of Caucasian women with surgically confirmed absence of adhesions, who were admitted for laparoscopy or laparotomy because of infertility (n= 27), benign ovarian masses (n= 23), fibroids (n= 12), chronic pelvic pain (n= 9), ectopic pregnancy (n= 5), menorrhagia (n= 2) and polycystic ovarian disease (n= 1). Subjects were excluded from the study if they had history of radiation or chronic inflammatory diseases. Genomic DNA was extracted from whole peripheral blood samples using the QIAGEN System (QIAmp DNA Blood Midi Kit, Hilden, Germany). A genomic DNA fragment was amplified by the polymerase chain reaction (PCR) to determine IL-1RN genotypes as described previously (Fig. 1)2. Genotype and allele frequencies were compared between the study and the control groups using the Pearson χ2 test of independence with Yates' continuity correction or the Fisher's exact test. The confounding influence of previous abdominal surgery was tested by multiple logistic regression analysis. Logistic regression models were generated by three covariates, the genotype frequency, allele frequency and the confounder previous abdominal surgery or endometriosis. The corresponding P values were calculated by the Wald statistic. Odds ratios and corresponding 95% confidence intervals are results of the proportional hazard method. All P values are two tailed. The significance level was set at 0.05.

Figure 1.

Photograph of 3% agarose gel used to resolve the IL-1RN*1, IL-1RN*2 and IL-1RN*3 alleles of IL-1RN. Lane 1: the homozygous IL-1RN*2 allele pattern (IL-1RN2/2). Lane 2: the heterozygous pattern for the IL-1RN*1 and IL-1RN*3 allele (IL-1RN1/3). Lane 3: the homozygous IL-1RN*1 allele pattern (IL-1RN1/1).

Results

In the study and the control group, 65 and 27 women had previous abdominal surgery. Univariate analysis showed that carriage of the mutant IL-1RN*2 allele is associated with the presence of peritoneal adhesions (OR: 2.1; 95% CI: 1.3–3.4; P= 0.004) (Table 1). Logistic regression analysis (Table 2) demonstrated an increased risk for peritoneal adhesions in women carrying the mutant IL-1RN*2 allele (OR: 2.2; 95% CI 1.3–3.8; P= 0.002), which is independent of previous abdominal surgery (OR: 3.2; 95% CI 2.0–5.0; P < 0.001) and endometriosis (OR: 1.7; 95% CI 1.1–2.6; P= 0.020)

Table 1.  Univariate analysis of the IL-1RN genotype and alleles, previous abdominal surgery and endometriosis among women with peritoneal adhesions (n= 107) and controls (n= 79). Frequency values are given as n (%).
Univariate analysesFrequencyOdds ratio (95% CI)P value
Women with PA*Controls
  1. Fisher's exact test.

  2. *PA = peritoneal adhesions.

  3. **Reference group.

  4. OR was calculated vs 1/1 genotype.

  5. §Case number is too low for statistical calculations.

Genotypes
IL-1RN1/1**44 (41.1)49 (62.0)
IL-1RN1/243 (49.4)22 (31.0)2.2 (1.1–4.2)0.023
IL-1RN2/211 (20.0)4 (7.5)3.1 (0.9–10.3)0.094
IL-1RN2/3§3 (6.4)0 (0.0)
IL-1RN3/3§0 (0.0)0 (0.0)
IL-1RN1/2/IL-1RN2/254 (55.1)26 (34.7)2.3 (1.2–4.3)0.009
IL-1RN1/2/IL-1RN2/2/IL-1RN2/357 (56.4)26 (34.7)2.4 (1.3–4.5)0.006
IL-1RN1/2/IL-1RN1/3/IL-1RN2/2/IL-1RN2/363 (58.9)30 (38.0)2.3 (1.3–4.2)0.007
IL-1RN1/3§6 (12.0)4 (7.5)
Additional influence factors
Previous abdominal surgery65 (60.7)27 (33.8)3.0 (1.7–5.6)<0.001
Endometriosis59 (55.1)35 (43.8)1.6 (0.9–2.8)0.141
Alleles
IL-1RN*1**137 (66.8)124 (80.5)
IL-1RN*268 (33.2)30 (19.5)2.1 (1.3–3.4)0.004
IL-1RN*39 (6.2)4 (3.1)2.0 (0.6–6.8)0.269
IL-1RN*2/IL-1RN*377 (36.0)34 (21.5)2.1 (1.3–3.3)0.003
Table 2.  Multiple logistic regression analyses for peritoneal adhesions as response variable predicted by IL-1 genotypes and alleles adjusted by previous abdominal surgery and endometriosis as additional categorical predictors. The backward selection procedure did not eliminate any predictor at a removal value of 0.1. The adjustment in the multiple analyses revealed a suppressive effect of the confounders previous abdominal surgery and endometriosis compared with the univariate analyses, which means the odds ratio of IL-1 genotypes containing allele*2 was raised, after neutralisation of the influence of previous abdominal surgery and endometriosis.
Model predictor variables for PA*P value**Odds ratio (95% CI)
  1. *PA = peritoneal adhesions.

  2. **P values were calculated using the Wald statistic.

  3. Not significant but confounder with trend to significance (P < 0.1). Although the predicting effect on the response variable was not significant, the confounding influence on other predictors is of interest.

  4. Eliminated from the regression model during the backward selection procedure using the Wald statistic for the removal P value of 0.1.

Multiple analyses for genotypes
1IL-1RN1/2/IL-1RN2/2/IL-1RN2/30.0042.6 (1.4–5.0)
Previous abdominal surgery<0.0013.2 (1.7–6.1)
Endometriosis0.0891.7 (0.9–3.3)
2IL-1RN1/2/IL-1RN2/20.0072.4 (1.3–4.7)
Previous abdominal surgery<0.0013.2 (1.7–6.2)
Endometriosis0.0821.8 (0.9–3.4)
3IL-1RN1/20.0232.2 (1.1–4.5)
Previous abdominal surgery<0.0013.4 (1.7–6.7)
Endometriosis0.0701.9 (1.0–3.7)
Multiple analyses for alleles
4IL-1RN*20.0022.2 (1.3–3.8)
Previous abdominal surgery<0.0013.2 (2.0–5.0)
Endometriosis0.0201.7 (1.1–2.6)
5IL-1RN*3n.s. 
Previous abdominal surgery<0.0013.2 (1.9–5.2)
Endometriosis0.0681.6 (1.0–2.6)
6IL-1RN*2/IL-1RN*30.0022.2 (1.3–3.6)
Previous abdominal surgery<0.0013.1 (2.0–4.8)
Endometriosis0.0321.6 (1.0–2.5)

Discussion

The search for a specific genetic marker for peritoneal adhesions is rudimentary. Apart from the association of intestinal adhesions with HLA subtypes A24 and DR11, no data are available5. We describe a novel genetic association between the IL-1RN*2 allele and peritoneal adhesions.

The balance of fibrin deposition and fibrin breakdown seems to be crucial in the early phase of peritoneal repair. If the fibrin matrix is not degraded within the first days of their occurrence, fibrous adhesions are formed by collagen deposition within a week1. IL-1 and other IL-1-induced cytokines, including TGF-β and IL-6, contribute to adhesion formation by increasing coagulation and decreasing fibrinolysis during peritoneal tissue repair1. Among other biochemical pathways, IL-1 was shown to decrease fibrinolytic activity by increasing the release as well as the synthesis of the coagulatory factor plasminogen activator inhibitor type 1 in human mesothelial cells1.

IL-1 has been shown to induce peritoneal adhesions in animal models and to aggravate radiation-induced adhesions3. In accordance, rats treated with IL-1RN or antibodies against IL-1 prior to surgery developed significantly less surgically induced adhesions compared with controls3,4. In this context, our data suggest that carriage of the IL-1RN*2 allele and subsequently elevated IL-1β levels and low anti-inflammatory IL-1RN levels in IL-1RN*2 carriers predisposes to peritoneal adhesion formation. The IL-1RN*3 allele frequency was shown to be low in our study (0.06) and control (0.03) groups and in North American controls (0.02), therefore it seems not to play an important clinical role.

In conclusion, our results suggest that IL-1RN*2 allele carriers have an increased risk for adhesion formation. Our findings may have clinical implications for testing recombinant IL-1RN as a novel therapeutic intervention to prevent the formation of adhesions subsequent to abdominal surgery in genetically predisposed women.

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