1 Medical School, University of Tampere and Tampere University Hospital, 2 Tampere School of Public Health, University of Tampere, 3 Finnish Red Cross Blood Transfusion Service, Helsinki, and 4 Haartman Institute, Department of Virology, University of Helsinki, Finland
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Abstract |
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Methods. The genotypes of the genes of tumour necrosis factor alpha (TNF), interleukin-1
(IL-1
), IL-1ß and IL-1 receptor antagonist (IL-1RA) were analysed by polymerase chain reaction in 87 subjects, all hospital-treated for serologically confirmed acute NE. The control group comprised 400 healthy blood donors. Nineteen out of these 400 (5%) controls were PUU virus-seropositive.
Results. IL-1RA allele 2 and IL-1ß (base exchange polymorphism at position -511) allele 2 were strongly associated with each other in both groups. NE patients were more often IL-1RA-2 negative/IL-1ß-2 negative than PUU-seronegative blood donors (38 vs 27%, odds ratio 1.65, 95% confidence interval 1.02.7). However, there were no differences in the clinical severity of NE between the IL-1RA-2 negative/IL-1ß-2 negative and the other patients. The other allele frequencies studied evinced no statistically significant differences between the groups. Thirty-three out of 87 (38%) patients and 121 out of 381 (32%) seronegative controls were carriers of the high-producer genotype TNF2 allele. Several parameters showed the clinical course of NE to be more severe in TNF2 carriers than in non-carriers.
Conclusions. These data suggest that non-carriage of the IL-1RA allele 2 and IL-1ß (-511) allele 2 may contribute to susceptibility to NE. Furthermore, TNF polymorphism seems to be associated with the outcome of NE.
Keywords: cytokine; gene polymorphism; hantavirus; haemorrhagic fever with renal syndrome; nephropathia epidemica; Puumala virus
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Introduction |
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NE is clinically characterized by high fever, headache and back and abdominal pains [3]. Hypotension up to clinical shock is present in less than 10% of hospital-treated patients. Renal involvement results in transient massive proteinuria, haematuria and impairment of renal function followed by polyuria and spontaneous recovery. Transient haemodialysis is needed in a minority. Common laboratory findings are anaemia, leukocytosis, thrombocytopenia and a moderate elevation of C-reactive protein (CRP) levels [3]. There is considerable variability in the clinical severity of NE. It has previously been demonstrated that individuals with the HLA B8 DRB1*0301 haplotype suffer from a severe form of the disease [4], suggesting that host genetic factors influence the clinical picture.
The pathogenesis of NE is incompletely understood. An increase in capillary permeability is characteristic of various types of hantavirus infections, but the mechanism of vascular leakage remains to be elucidated. PUU virus causes no cytopathic effects in cultured cells but shows wide cell susceptibility in vitro. Immunological factors including cytokines may play an important role in the pathogenesis of NE [1]. Tumour necrosis factor alpha (TNF) is known to induce vascular permeability, and to increase the expression of endothelial adhesion molecules [5]. Intravenous injection of TNF
induces a number of signs and symptoms similar to those seen in NE [5], and high plasma levels of TNFa have indeed been detected during the acute phase of NE [6]. Furthermore, increased expression of intercellular and vascular cell adhesion molecules has been described in the interstitial and tubular space of NE kidneys, respectively [1].
The key cytokines participating in the regulation of inflammatory response are TNF, interleukin-1 (IL-1), IL-6, IL-10 and IL-1 receptor antagonist (IL-1RA). Functionally these cytokines can be divided into proinflammatory (IL-1, IL-6 and TNF) and anti-inflammatory (IL-1RA and IL-10) molecules. Genetic factors have a substantial influence on the production of these cytokines, and changes (polymorphisms) in the cytokine genes may determine the amount of cytokine produced in the inflammatory reaction.
Several polymorphisms have been identified inside the promoter region of the TNF gene [7]. Among these, there is a biallelic polymorphism at position -308, involving the substitution of guanine by adenosine in the uncommon allele TNF2 [8]. This latter allele has been found to correlate with enhanced TNF production [9]. A large number of studies have examined the importance of TNF genetics in susceptibility to autoimmune diseases, and some studies have also linked TNF2 polymorphism to the outcome of infections [7]. Recently, a study on TNF polymorphism in NE revealed that TNF allele 2-positive hospitalized NE patients suffered from a more severe NE than TNF allele 2-negative patients [10].
The IL-1 gene family on chromosome 2q13 codes for three proteins: IL-1, IL-1ß and IL-1RA. All three genes are polymorphic. In the IL-1ß gene, there are at least two biallelic base-exchange polymorphisms: at position -511 [11] and at position +3953 [12], and in the IL-1
gene there is a base-exchange polymorphism at position -889 [13]. In intron 2 of the IL-1RA gene, there are variable numbers of an 86-bp repeat sequence; the most common allele 1 contains four repeats and allele 2 contains two repeats [14]. The frequency of IL-1RA allele 2 is increased in several diseases of inflammatory or autoimmune nature [15]. Healthy IL-1RA allele 2 carriers have been shown to have higher IL-1RA plasma levels than non-carriers [16]. However, the enhancing effect of IL-1RA allele 2 on IL-1RA plasma levels has required the presence of IL-1ß (-511) allele 2 or the absence of IL-1ß (+3953) allele 2 [16]. Furthermore, IL-1ß (-511) allele 2 has been found to be significantly associated with the presence of IL-1RA allele 2 [16]. It would, thus, seem plausible that the alleles determining a high agonist production and a high antagonist production are generally associated, which is obviously important in maintaining homeostasis.
In the present study we analysed allele frequencies and genotypes of the genes of the IL-1 complex and TNF in a group of hospitalized NE patients and in a control group of healthy blood donors to address the possibility of an association with susceptibility to disease. In addition, we sought to establish whether the clinical course of NE is influenced by the polymorphisms of these cytokine genes.
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Subjects and methods |
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The control group comprised 400 healthy blood donors. Blood samples were obtained from the Finnish Red Cross Blood Transfusion Center, Tampere, Finland. The donors were adults (1860 years of age) and had no sign of infection during a 2-week period before the blood donation. Immunoglobulin G antibodies to PUU virus were examined by immunofluorescence test as described in a recent paper by Brummer-Korvenkontio and co-workers [2]. Nineteen out of these 400 (5%) controls were PUU virus-seropositive.
Analysis of gene polymorphisms
Genomic DNA was isolated from the blood samples by the salting-out method [17]. Polymerase chain reaction (PCR)-based genotyping of TNF (base exchange polymorphism at position -308), IL-1RA (variable number of tandem repeats in exon 2), IL-1ß (base exchange polymorphisms at positions -511 and +3953) and IL-1
(base exchange polymorphism at position -889) was performed as previously described [8,1114].
Statistical analysis
Allelic frequencies (number of copies of a specific allele divided by the total number of alleles in the group) and carriage rates for infrequent 2-alleles (number of individuals with at least one copy of allele 2 divided by the total number of individuals within the group) were calculated in NE patients, and seronegative and seropositive controls. 2-test was used to analyse, if there were differences in the carriage rates between NE patients and seronegative controls, or between the groups in general. Odds ratios (OR) were calculated after the
2-test when appropriate, and 95% confidence intervals (95%CI) were determined. Tests for HardyWeinberg equilibrium were also made.
To describe the severity of NE, medians and ranges are given for skew-distributed continuous variables and percentages are used for categorical variables. The patients were grouped into carriers (including both homozygotes and heterozygotes) and non-carriers of specific alleles. Differences in clinical severity of NE between carriers and non-carriers were tested using MannWhitney U-test for numerical data and 2-test or Fisher's exact test for categorical data. OR were calculated after the
2-test where appropriate, and 95% CI were also determined. All testing was two-sided and statistically significant P-values are given. All tests were made with the SPSS (version 7.0) statistical software package.
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Results |
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The IL-1RA and IL-1ß (-511) genotypes and corresponding allele frequencies in hospitalized NE patients and PUU virus-seronegative and -seropositive healthy blood donors are shown in Table 1.
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The IL-1RA allele 2 was strongly associated with IL-1ß (-511) allele 2 both in NE patients and in PUU virus-seronegative controls (P<0.001 in both groups). However, there were differences between the groups in the allele associations. In NE patients, the IL-1RA allele 2-carriers (designated IL-1RA-2 positive) were often carriers of the IL-1ß (-511) allele 2 and vice versa, and the IL-1RA-2 negative and IL-1ß-2 negative were associated. In the PUU virus-seronegative control group the IL-1RA-2 positive/IL-1ß-2 positive association was also obvious, but the IL-1RA-2 negative population contained almost equal numbers (95 and 104, respectively) of IL-1ß (-511) allele 2-carriers and non-carriers. Thus, NE patients were more often IL-1RA-2 negative/IL-1ß-2 negative than the PUU virus-seronegative controls (38 vs 27%, OR 1.65, 95%CI 1.02.7) (Table 2). Only two out of 19 (11%) PUU virus-seropositives were IL-1RA-2 negative/IL-1ß-2 negative. When non-carriers of IL-1RA-2/IL-1ß-2 against the remaining alleles were compared between all three groups, a statistically significant difference was found (P=0.029), suggesting a difference in the allele associations between the three groups. There were no significant differences in the clinical severity of NE between the IL-1RA-2 negative/IL-1ß-2 negative and the other patients (data not shown).
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Discussion |
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The prevalence of PUU virus antibodies in the control group of blood donors was the same as previously reported in the Finnish population [2]. Unfortunately, the voluntary nature of blood donating made it impossible to establish retrospectively whether these seropositive controls had previously been hospitalized because of an acute PUU virus infection. It is likely, however, that most of them had had a mild or asymptomatic disease, as comparison of diagnosis incidences and seroprevalences in fertile-aged women shows that in Finland on average only 13% of all PUU hantavirus infections are serodiagnosed [2]. In the present study, cytokine genotype distribution in this group of seropositive controls (with a probable mild disease) differed markedly from the genotype distribution of hospital-treated NE patients (obviously with a more severe form of disease). There were more IL-1ß (-511) 2-carriers and less non-carriers of IL-1RA allele 2/IL-1ß (-511) allele 2 among the seropositive controls than among patients. They also showed a tendency towards a higher carriage rate of IL-1RA allele 2 and a lower carriage rate of TNF allele 2 compared with patients (Tables 1
3
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Recently, Cox and co-workers have identified a common, eight-locus haplotype of the IL-1 gene cluster [18]. Their data indicate a linkage-disequilibrium across the region of chromosome 2q13. In the present study, a strong association emerged between IL-1RA allele 2 and IL-1ß (-511) allele 2. Functionally, the significance of this association is yet not entirely clear, but there are data suggesting that both of these alleles are required to maintain high IL-1RA plasma levels [16]. We found that the frequency of non-carriers of IL-1RA allele 2/IL-1ß (-511) allele 2 was increased in the hospitalized NE patients compared with the group of healthy, PUU virus-seronegative blood donors (Table 2). Hence, the present data suggest that IL-1RA allele 2/IL-1ß (-511) allele 2-polymorphism may contribute to susceptibility to NE. Associations between IL-1 complex genes have recently been analysed also in another viral disease, namely in EpsteinBarr virus infection. A significantly higher number of non-carriers of IL-1RA allele 2/IL-1ß (-511) allele 2 have been found among EpsteinBarr virus-seronegative than seropositive blood donors [19].
Our data suggest that TNF polymorphism is unlikely to be of significance in susceptibility to NE, although there was a slight tendency towards an increased frequency of allele TNF2 in NE patients compared with controls. Previously, high-producer TNF alleles have been associated with susceptibility to autoimmune diseases, in which TNF is of pathophysiological importance [7]. Recently, Mira and colleagues demonstrated that the TNF2 allele occurs with an increased frequency in patients with septic shock compared with a group of blood donors [20]. In addition, mortality due to septic shock, was shown to be increased in patients with this allele, and every patient who was TNF2 homozygous had a fatal outcome [20].
Previously, the TNF gene polymorphism of 59 NE patients has been analysed by Kanerva and co-workers [10]. They showed that the clinical course of NE was more severe in TNF2 carriers than in non-carriers [10]. The same result was also predictably found in the present material that included the 59 patients of the earlier study [10]. Especially, both TNF2 homozygous patients evinced marked renal failure requiring transient dialysis therapy. Cytokines, including TNF, may play an important role in the pathogenesis of NE [1]. It is, therefore, possible that high-producer allele TNF2 contributes to the outcome of NE. Individuals with the HLA B8 DRB1*0301 haplotype have previously been shown to suffer from a severe form of NE [4], and the TNF2 allele is known to be in strong linkage disequilibrium with this HLA haplotype in a northern European population [7]. It is, thus, possible that TNF2 allele is not an independent risk factor for severe NE but a passive component in the extended HLA haplotype. This issue cannot be evaluated from the present data, since the HLA haplotypes of the patients were not studied.
In conclusion, the results here show that non-carriage of IL-1RA allele 2 and IL-1ß (-511) allele 2 may contribute to susceptibility to NE. The data also support the hypothesis that the alleles determining high agonist and high antagonist production are generally associated. Furthermore, TNF polymorphism seems to regulate the clinical course of NE. It would be interesting to carry out genetic studies on the more severe forms of hantavirus diseases and also on PUU virus infections with a subclinical or asymptomatic course.
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Acknowledgments |
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Notes |
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References |
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