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Keywords:

  • cytokine;
  • interleukin 1-receptor antagonist;
  • yellow fever

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The inflammatory response in infectious and autoimmune diseases is regulated by the balance between pro- and anti-inflammatory cytokines. The IL-1 complex contains polymorphic genes coding for IL-1α, IL-1β and IL-1Ra. The IL-1Ra (variable number of tanden repeat) VNTR polymorphism has been shown to influence the capacity to produce IL-1β and IL-1Ra after in vitro stimulation. Allele 2 of this polymorphism is associated with a number of inflammatory diseases. To determine the impact of the IL-1Ra polymorphism on in vivo human cytokine synthesis, we used a yellow fever vaccination model for the induction of cytokine synthesis in healthy volunteers. Two different yellow fever vaccines were used. After administration of the RKI vaccine (34 volunteers), plasma TNF-α concentration increased from 13·4 ± 0·9 pg/ml to 23·3 ± 1·1 pg/ml (P < 0·001), and plasma IL-1Ra concentration increased from 308 ± 25 pg/ml to 1019 ± 111 pg/ml (P < 0·001), on day 2. Using Stamaril® vaccine, no increase in the plasma concentrations of either TNF-α or IL-1Ra could be detected (n = 17). Only the RKI vaccine induced TNF-α synthesis after in vitro stimulation of MNC. Carriers of allele 2 of the IL-1Ra polymorphism had increased baseline concentrations of IL-1Ra (350 ± 32 pg/ml) compared with non-carriers (222 ± 18 pg/ml, P < 0·001), and decreased concentrations of IL-1β (0·9 ± 0·2 pg/ml for carriers versus 2·8 ± 0·7 pg/ml for non-carriers, P = 0·017). After yellow fever vaccination (RKI vaccine), no significant differences in the increase of IL-1Ra plasma levels were detected between carriers and non-carriers of allele 2 of the IL-1Ra gene polymorphism. This is the first study to examine the influence of this genetic polymorphism on in vivo-induced human IL-1β and IL-1Ra synthesis. Baseline concentrations of IL-1Ra and IL-1β were significantly influenced by the IL-1Ra polymorphism. No influence of the IL-1Ra polymorphism on the in vivo-induced production of IL-1Ra and IL-1β could be detected.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The IL-1 gene complex contains genes coding for the proinflammatory cytokines, IL-1α and IL-1β, and for the physiological antagonist IL-1Ra [1]. The genes of the IL-1 complex are polymorphic. The IL-1β gene contains two biallelic base exchange polymorphisms: at position −511 in the promoter region and at position + 3953 in the fifth exon. The IL-1Ra polymorphism in intron 2 of the gene consists of a variable number of an 86-base pair repeat sequence [2]. The frequency of allele 2 of the IL-1Ra polymorphism has been demonstrated to be elevated in several diseases, such as ulcerative colitis [3,4], multiple sclerosis [5], Graves' disease [6] and diabetic nephropathy [7]. It has been suggested from in vitro and in vivo findings that the IL-1Ra polymorphism influences the capacity to produce IL-1Ra [8,9] and IL-1β[10]. Danis et al. observed increased IL-1Ra concentrations in vitro after GM-CSF stimulation of monocytes from carriers of allele 2 of the IL-1Ra polymorphism compared with non-carriers [9]. Furthermore, it has been demonstrated that alleles of the IL-1β gene are not major regulators of IL-1β production, but the IL-1Ra allele 2 affects the in vitro production of this cytokine [10]. Data from Hurme et al. suggested that the IL-1Ra plasma levels are co-ordinately regulated by both IL-1Ra and IL-1β genes in healthy humans [11].

To study the regulation of the synthesis of proinflammatory cytokines, investigators have predominantly used in vitro systems such as stimulated peripheral blood mononuclear cells (MNC). However, it is not known to what extent kinetics of cytokine synthesis in blood leucocytes stimulated in vitro reflect in vivo conditions. To circumvent this problem, we have previously described a model for the easy and safe in vivo stimulation of synthesis of cytokines using live yellow fever vaccination [12]. We have demonstrated a reproducible induction of tumour necrosis factor (TNF)-α and IL-1Ra synthesis following vaccination with the yellow fever vaccine from the Robert Koch Institute. In the present study, we aimed (i) to compare our previous findings using two different yellow fever vaccines in a larger group of vaccinees, and (ii) to use this model to define the influence of the IL-1Ra allele 2 on in vivo production of IL-1Ra.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Healthy subjects 18–60 years of age consulting the outpatient clinic for vaccination against yellow fewer were included. The study was approved by the local ethics committee. Thirty-four vaccinees received live attenuated yellow fever vaccine from the Robert Koch Institute (RKI), Berlin, and 17 vaccinees received Stamaril® (Pasteur Mérieux, Germany) subcutaneously in the left upper arm. The RKI vaccine is derived from the 17 D strain and contains 1·5 × 104 plaque-forming units (pfu)/0·5 ml. Stamaril is derived from the 17 D strain and contains 6·3 × 103 pfu/0·5 ml. Plaques on PS cells tend to be smaller than those generated by the RKI vaccine (Prof. L‘age-Stehr, RKI, Berlin, personal communication). Those persons with acute or chronic diseases were excluded. The vaccinees had not taken any medicine or received other vaccinations for a minimum of 6 weeks prior to vaccination. They had not received yellow fever vaccination earlier.

Cytokine ELISA

Blood samples (EDTA) were collected in the morning, immediately centrifuged and the plasma stored at −80°C. IL-1Ra and IL-1β plasma concentrations were measured using commercially-available ELISA kits as described [12]. The IL-1Ra ELISA has a lower limit of detection of 14 pg/ml, and a percentage coefficient of variation (CV%) of 6·2% at a mean concentration of 153 pg/ml and 3·1% at a mean concentration of 1916 pg/ml. The IL-1β ELISA has a lower limit of detection of 0·1 pg/ml, and a CV% of 10·2% at a mean concentration of 0·48 pg/ml and 6·4% at a mean concentration of 4·56 pg/ml. In the in vitro experiments, TNF-α was measured by an ELISA developed in the laboratory showing good correlation with a commercially-available TNF-α ELISA (human-TNF-alpha EASIA, Medgenix Diagnostics, Belgium), which was used for the vaccinees’ samples. The lower limit of detection of the Medgenix TNF-α ELISA is 3 pg/ml. The CV% is 5·2% at a mean concentration of 88 pg/ml according to the manufacturer. The lower limit of detection for our own TNF-α ELISA is 25 pg/ml, and the CV% is 6·7% at a mean concentration of 912 pg/ml and 10·9% at a mean concentration of 106 pg/ml. Limulus amoebocyte lysate assay (Coatest®, Chromogenix, Essen, Germany) was performed to exclude endotoxin contamination of vaccines. It had a lower detection limit of 5 pg/ml (= 0·06 endotoxin units/ml).

PCR analysis of IL-1Ra gene polymorphism

IL-1Ra polymorphism was analysed in n = 46 volunteers as described [2]. In brief, genomic DNA was isolated and PCR was performed using primers (5′CTCAGCAACACTCCTAT3′) and (5′TCCTGGTCTGCAGGTAA3′) under the following conditions: denaturation at 94°C for 3 min, followed by 94°C (1 min), 65°C (1 min) and 72°C (1 min) for two cycles, 94°C (1 min), 63°C (1 min) and 72°C (1 min) for two cycles, followed by 94°C (1 min), 60°C (1 min) and 72°C (1 min) for 30 cycles. Final extension was carried out at 72°C for 5 min. The PCR product was analysed on a 2% agarose gel stained with ethidium bromide.

MNC isolation and culture

MNC were isolated from healthy donors by centrifugation through a Ficoll density gradient (Biochrom, Berlin, Germany) in Leukosep® tubes (Greiner, Frickenhausen, Germany). Cells (5 × 105) were seeded in 24-well plates at a final volume of 2 ml, and lipopolysaccharide (LPS; Sigma, Deisenhofen, Germany) was added at a concentration of 10 ng/ml. Stamaril® and the RKI vaccine were added at a 1:10 dilution from stock. For heat inactivation, vaccines were heated to 56°C for 30 min.

Statistics

Results are expressed as means ± standard error of the mean. Analysis of variance (anova) was performed for the in vivo data. If a given anova was significant at a P < 0·05, means were compared using the SchefféF-test on Stat-View software (Abacus Concepts, Calabas, CA). Differences were considered significant at P < 0·05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Two different yellow fever vaccines were used, one from the Robert Koch Institute (RKI) and Stamaril® from Pasteur Mérieux. A significant increase in the plasma TNF-α concentration was observed in a group of 34 healthy volunteers receiving the RKI vaccine (from 13·4 ± 0·9 pg/ml to 23·3 ± 1·1 pg/ml, P < 0·001, Fig. 1a), confirming the results of our previous study [12]. While IL-1β concentrations were not increased, IL-1Ra concentrations showed a significant increase on day 2 after vaccination (from 308 ± 25 pg/ml to 1019 ± 111 pg/ml, P < 0·001, Fig. 1b). Baseline levels of IL-1Ra and IL-1β did not correlate significantly. Using Stamaril® (n = 17), no increase in the plasma concentrations of either TNF-α or IL-1Ra could be detected (Fig. 1a, b). In order to study the capacity of both vaccines to induce TNF-α production in vitro, 5 × 105 MNC were co-incubated with either the RKI vaccine or Stamaril®, with or without prior heat inactivation of the vaccine preparation. Lipopolysaccharide was used as a positive control. Again, only the RKI vaccine induced TNF-α synthesis, while Stamaril® and both heat-inactivated vaccines did not (Fig. 2).

image

Figure 1.  (a) Plasma concentrations (means ± s.e.m.) of TNF-α in healthy volunteers before and 2 days after yellow fever vaccination with either the RKI vaccine (▪, n = 34) or Stamaril® vaccine (□, n = 17). (b) Plasma concentrations (means ± s.e.m.) of IL-1RA in healthy volunteers before and 2 days after yellow fever vaccination with either the RKI vaccine (▪, n = 34) or Stamaril® (□, n = 17).

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image

Figure 2.  Concentration of TNF-α in the supernatant fluid of MNC after incubation with the indicated vaccines and controls for up to 48 h (one representative out of three experiments, each performed in duplicates). (□) RKI; (▪) RKI inactivated; (○) Stamaril; (●) Stamaril inactivated; (▴) negative control.

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For all further experiments, we used the RKI vaccine. We next characterized the IL-1Ra polymorphism genotypes and baseline plasma levels of IL-1Ra and IL-1β in 46 healthy volunteers. We confirmed the results of Hurme et al. [11] who demonstrated increased plasma concentrations of IL-1Ra in carriers of allele 2 of the IL-1Ra polymorphism. In our study, plasma IL-1Ra concentration was 350 ± 32 pg/ml for carriers and 222 ± 18 pg/ml for non-carriers of allele 2, P < 0·001 (Fig. 3a). Furthermore, in our group, IL-1β plasma concentration was significantly lower in carriers of allele 2 of the IL-1Ra polymorphism (0·9 ± 0·2 pg/ml) compared with non-carriers (2·8 ± 0·7 pg/ml; P = 0·017; Fig. 3b).

image

Figure 3.  (a) Baseline IL-1RA concentrations in the plasma (healthy volunteers) of allele 2 carriers (n = 20) and of non-carriers (n = 26). (b) Baseline IL-1β concentrations in the plasma (healthy volunteers) of allele 2 carriers (n = 20) and of non-carriers (n = 26).

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After yellow fever vaccination using the RKI vaccine (n = 33), IL-1Ra plasma levels increased from 357 ± 34 pg/ml to 1060 ± 126 pg/ml in carriers, and from 261 ± 32 pg/ml to 1004 ± 163 pg/ml in non-carriers of allele 2. Thus, there was no difference in in vivo induction of this cytokine between the allele groups (Fig. 4). In homozygote allele 2 carriers, the IL-1Ra increased from 279 to 1066 pg/ml, but there were only two individuals in this subgroup, precluding a statistically-valid comparison.

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Figure 4.  IL-1RA concentration in the plasma of healthy volunteers before and 2 days after yellow fever vaccination (RKI vaccine), grouped for carriers (n = 18) and non-carriers (n = 15) of allele 2 of the IL-1RA VNTR polymorphism. Significant differences (P < 0·05) are indicated by an asterisk.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Confirming findings from an earlier study [12], we have shown a significant increase of TNF-α and IL-1Ra plasma concentrations in 34 healthy volunteers two days after vaccination with the RKI yellow fever vaccine. Using Stamaril®, no such increase could be detected. In vitro experiments using MNC stimulated with the two vaccines confirmed the in vivo results.

Endotoxin contamination is unlikely to explain the differences between the two vaccines because (i) no endotoxin was detected by the limulus amoebocyte assay and (ii) short heat inactivation, which inactivated the virus but not the endotoxin, abolished the effect. According to the manufacturer, it is known that the RKI vaccine generates higher numbers of pfu than Stamaril®, and that the plaques generated by the RKI vaccine are larger than those generated by Stamaril®. From these data, we would argue that a threshold number of pfu is necessary to induce the in vivo cytokine response. Another possible explanation for the differences in the induction of TNF-α and IL-1Ra production between the two vaccines could be the higher number of passages of the Stamaril® vaccine.

Next, we addressed the question of whether there are correlations between the amounts of IL-1Ra and IL-1β synthesis after vaccination with the RKI vaccine and the carrier status of allele 2 of the VNTR polymorphism in the IL-1Ra gene [2]. An increased risk of developing severe sepsis has been reported for carriers of this allele [13]. Moreover, El-Omar et al. described an increased risk of gastric cancer for homozygote carriers of allele 2 of the VNTR polymorphism [14]. These findings strongly suggest that there is an influence of these polymorphisms on the production of IL-1β and IL-1Ra which can then affect the susceptibility to, course and outcome of different diseases.

For allele 2 of the IL-1Ra polymorphism, an association with enhanced IL-1β production in vitro[10] has been reported. However, the data concerning IL-1Ra production are conflicting. Danis et al. [9] have described an increased production of IL-1Ra after GM-CSF stimulation of MNC derived from carriers of allele 2, and Mandrup-Poulsen et al. found higher IL-1Ra production in diabetic patients carrying the allele 2 than in non-carriers [15]. However, Andus et al. detected decreased IL-1Ra concentrations in the mucosa of patients with IBD carrying allele 2 [8]. In another study, allele 2 of the IL-1Ra polymorphism was associated with decreased production of IL-1Ra in cultured peripheral MNC from both inflammatory bowel disease patients and controls [4].

In the present study, we confirmed the data of Hurme et al. [11] which demonstrated higher baseline levels of IL-1Ra in carriers of allele 2 of the IL-1Ra polymorphism. For the first time, we have demonstrated significantly lower baseline IL-1β levels in carriers of allele 2 compared with non-carriers.

Using our in vivo model, carriers and non-carriers of allele 2 showed similar increases in IL-1RA plasma concentrations after live yellow fever vaccination. Therefore, we could not confirm in vitro results showing increased IL-1RA production in carriers of allele 2 [9]. The incongruous results between the in vitro and in vivo studies suggest that the reported association with allele 2 of the IL-1RA polymorphism is dependent on stimulus and cell compartment. In our in vivo model, IL-1Ra is probably induced via a sequence of mediators, with only some of them functionally linked with alleles of the IL-1Ra VNTR polymorphism. In conclusion, using this unique human in vivo model, which may be closer to the physiological state than in vitro systems, we could not identify an association of the induction of IL-1Ra plasma concentrations with the carrier status of allele 2 of the VNTR polymorphism in the IL-1Ra gene.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Thomas Löscher for making this study possible; A. Eigler, B. Siegmund and C. Bidlingmaier for most helpful discussions. This work was supported in part by Pasteur Merieux. The experimental data of this study are part of the dissertation of S. Erhardt (Medizinische Fakultät der LMU München, unpublished).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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