Abstract
Idiopathic pulmonary fibrosis (IPF) is a rapidly progressive interstitial lung disease of unknown aetiology. Interleukin (IL)-1β plays an important role in inflammation and has been associated with fibrotic remodelling. We investigated the balance between IL-1β and IL-1 receptor antagonist (IL-1Ra) in bronchoalveolar lavage fluid (BALF) and serum as well as the influence of genetic variability in the IL1B and IL1RN gene on disease susceptibility and cytokine levels. In 77 IPF patients and 349 healthy controls, single nucleotide polymorphisms (SNPs) in the IL1RN and IL1B genes were determined. Serum and BALF IL-1Ra and IL-1β levels were measured using a multiplex suspension bead array system and were correlated with genotypes. Both in serum and BALF a significantly decreased IL-1Ra/IL-1β ratio was found in IPF patients compared to healthy controls. In the IL1RN gene, one SNP was associated with both the susceptibility to IPF and reduced IL-1Ra/IL-1β ratios in BALF. Our results show that genetic variability in the IL1RN gene may play a role in the pathogenesis of IPF and that this role may be more important than thought until recently. The imbalance between IL-1Ra and IL-1β might contribute to a proinflammatory and pro-fibrotic environment in their lungs.
Keywords: bronchoalveolar lavage, idiopathic pulmonary fibrosis, interleukin-1, single nucleotide polymorphism
Introduction
Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease of unknown aetiology, and is characterized by an extremely poor prognosis of 2–4 years after diagnosis [1–3]. The pathogenetic mechanisms underlying IPF are incompletely understood. The disease is characterized by abnormal repair and airway remodelling and is associated with increased proinflammatory and pro-fibrotic signals. Previous research has shown that interleukin (IL)-1 cytokines are involved in the development of fibrosis [4].
The IL-1 family consists of three structurally related proteins, of which two are agonists (IL-1α and IL-1β) and the third, IL receptor antagonist (IL-1Ra), is a competitive antagonist. IL-1Ra is the inhibitor of these IL-1 agonists and acts by competitively binding to IL-1 receptors without eliciting signal transduction [5]. IL-1β is produced by activated macrophages and epithelial cells, inducing production of other cytokines such as tumour necrosis factor (TNF)-α and IL-6.
Polymorphisms in the IL-1 receptor antagonist gene (IL1RN) and TNF have been associated with susceptibility to IPF [6,7]. Several studies suggest that IL-1β and IL-1Ra play a critical role in bleomycin-induced fibrosis in mice. Fibrosis is induced by IL-1β and neutralization of IL-1β by antibodies or specific blockage of the receptor IL-1R1 reduces the development of fibrosis [8]. In normal homeostasis, IL-1Ra production by alveolar macrophages is higher than the production of IL-1β. However, decrease in the ratio of IL-1Ra to IL-1β favours the augmentation of the pro-fibrotic function of IL-1β[9].
The aim of this study was to investigate both the predisposition and disease-modifying effects of genetic variations in the IL1B and IL1RN genes and corresponding proinflammatory cytokine levels in serum and bronchoalveolar lavage fluid (BALF) in a cohort of IPF patients.
Methods
Patients and healthy controls
Patients with IPF presenting at the Department of Pulmonology of the St Antonius Hospital in Nieuwegein between 1998 and 2007 were included in this study. From that time serum, BALF and DNA were collected from all interstitial lung disease (ILD) patients presented at our department after informed consent was given. These patients were enrolled in our database for scientific research. Retrospectively, the diagnosis of IPF was reviewed and validated using current American Thoracic Society/European Respiratory Society (ATS/ERS) guidelines. Diagnoses made before 2002 were reviewed by an experienced clinician (J.v.d.B., J.G.), and patients were included only when current ATS/ERS criteria were met. Other causes of usual interstitial pneumonia (UIP) (drugs, collagen vascular diseases) were ruled out. Seventy-seven IPF patients [mean age 60·8 years, standard deviation (s.d.) 13·6, 58 males, 19 females] were included in the present study and donated DNA. In 54 of 77 cases serum and BALF samples were also available at the time of diagnosis. At the time of serum sampling eight patients received low-dose oral corticosteroids. In 58 cases the diagnosis of UIP was confirmed on lung biopsy (75%). BALF was collected as described previously [10]. Samples were stored at −80°C until analysis. Median lung function parameters at the time of diagnosis were as follows: forced vital capacity (FVC) 75·7 % predicted [interquartile range (IQR) 61·7–87·3], DLCO 42·5 % predicted (IQR 33·1–55·6).
The control group consisted of 349 healthy Caucasian volunteers (mean age 39·2 years, s.d. 12·4, 139 males, 210 females). In 36 cases in the control group, BAL was performed and in those controls cytokine levels in serum and BALF were measured. The study protocol was approved by the Ethical Committee of the St Antonius Hospital and all subjects gave written informed consent.
Genotyping
Three haplotype tagging single nucleotide polymorphisms (SNPs) for each gene were selected using the Tagger program for the gene region of IL1B and IL1RN ± 2500 base pairs (bp) on genome build 35. Preferential picking of SNPs was conducted under the pairwise tagging option, with a minimum allele frequency of 25% and a high Illumina design score. The algorithm was set to select tags that would cover the Caucasian HapMap panel with an r2 of 0·8 or greater [11]. Furthermore, for both genes one additional custom SNP was selected on the basis of previously published association studies or presumed functionality. The following SNPs were genotyped in the IL1B gene; rs1143627 (tag), rs1143634 (tag), rs1143643 (tag) and rs1799916 (custom); IL1RN: rs11677397 (custom), rs2637988 (tag), rs408392 (tag), rs397211 (tag). DNA was extracted from whole blood samples and SNP typing was conducted using a custom Illumina goldengate bead SNP assay in accordance with the manufacturer's recommendations (Illumina Inc., San Diego, CA, USA).
Cytokine levels
Serum and BALF levels of IL-1β and IL-1Ra were determined using a multiplex suspension bead array system according to the manufacturer's protocol (Bio-Rad Laboratories, Hercules, CA, USA). Data analysis was performed using the Bioplex 100 system and Bioplex Manager software version 4·1 (Bio-Rad Laboratories). The lower limit of detection was 0·3 pg/ml for IL-1β and 2·2 pg/ml for IL-1Ra. Because the variation in BALF retrieval in healthy controls was not significantly different from retrieval in IPF patients, we did not correct for that.
Statistical analysis
Genotype frequencies were tested for Hardy–Weinberg equilibrium (http://ihg2.helmholtz-muenchen.de/ihg/snps.html). Genotype and allele frequencies in the IPF group were compared with the control population using the χ2 test. Haplotypes and linkage disequilibrium (LD) were calculated (Haploview 4·1; Broad Institute of MIT and Harvard, Cambridge, MA, USA). Serum and BALF data were expressed as median and IQR. Differences in serum or BALF concentrations between patients and controls were analysed using a Mann–Whitney U-test. For analysis of correlation, log-transformation was used to reach near-normal distribution. The correlation between cytokines in BALF and clinical data was assessed using Pearson's correlation coefficients. The differences between cytokine levels in different genotypes were assessed with the Kruskal–Wallis test. Statistical analysis was performed using spss version 15·0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5·0 (GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was considered at a value of P < 0·05.
Results
IL-1 levels in serum and BALF
Serum levels of IL-1β in IPF patients were increased significantly compared to healthy controls, while serum levels of IL-1Ra were decreased (Table 1). Furthermore, BALF levels of both IL-1β and IL-1Ra were increased significantly in IPF patients compared to healthy controls. In the IPF group there were eight patients receiving low-dose corticosteroids, the median serum and BALF IL-1Ra levels were significantly higher in the patients who were on corticosteroids; serum IL-1Ra 284·3 (IQR 202·3–515·3) versus 214·3 (IQR 175·6–255·2), P = 0·006; BALF IL-1Ra 152·9 (IQR 67·2–622·3) versus 74·0 (IQR 37·0–121·4), P = 0·026. IL-1β levels were not affected by corticosteroids.
Table 1.
Serum levels in idiopathic pulmonary fibrosis (IPF) patients and healthy controls
| IPF patients | Healthy controls | |
|---|---|---|
| n = 54 | n = 36 | |
| Serum levels (median, IQR) | ||
| IL-1β (pg/ml) | 3·2 (2·3–4·1)* | 1·4 (0·6–2·1) |
| IL-1Ra (pg/ml) | 224·6 (179·3–312·0)* | 406·7 (309·5–690·7) |
| BALF levels (median, IQR) | ||
| IL-1β (pg/ml) | 0·6 (0·3–2·7)* | < 0·3 |
| IL-1Ra (pg/ml) | 87·2 (43·1–138·1)* | 36·4 (26·3–48·3) |
P < 0·05 compared to healthy controls. BALF: bronchoalveolar lavage fluid; IL: interleukin; IQR: interquartile range.
As IL-1Ra inhibits the physiological activities of IL-1β by occupying the IL-1 receptor, we evaluated IL-1Ra in relation to IL-1β through calculation of the IL-1Ra/IL-1β ratio. IPF patients showed a 3·5-fold decrease in the IL-1Ra/IL-1β ratio in BALF (215·7; IQR 58·6–437·9) compared to healthy controls (771·4; IQR 337·4–5210·0), P < 0·0001. A similar decrease in the IL-1Ra/IL-1β ratio was found in serum from patients (77·9; IQR 51·5–110·9) compared to healthy controls (293·5; IQR 201·1–1054·0), P < 0·0001 (Fig. 1).
Fig. 1.
Bronchoalveolar lavage fluid (BALF) (a) and serum (b) interleukin (IL)-1Ra/IL-1β ratios in patients with idiopathic pulmonary fibrosis (IPF) and healthy controls, both P < 0·0001. Data are shown as median with interquartile ranges.
The IL-1Ra/IL-1β ratio in serum was affected significantly by the use of corticosteroids; the eight patients who were on corticosteroids had a significantly higher IL-1Ra/IL-1β ratio: 101·7 (IQR 77·2–143·4) versus 71·5 (IQR 51·0–102·2), P = 0·01. In BALF there was no significant difference.
Polymorphisms in cytokine genes
Table 2 summarizes allelic and genotype frequencies in IPF patients and controls. Both populations were in Hardy–Weinberg equilibrium for all genotypes. One SNP in the IL1RN gene was associated with IPF. The frequency of the rs2637988 allele 2 (G) in the IL1RN gene was increased in the IPF group (47%) compared to the controls (38%), P = 0·04. The best-fitting genetic model was a risk conferred by the carriage of allele 2 compared to non-carriers; odds ratio (OR) 1·95 [95% confidence interval (CI): 1·11–3·42; P = 0·02]. Frequency of the rs408392 allele 2 (T) was increased in IPF patients and showed a trend towards significance; allele 2 occurred in 32% of the IPF patients compared to 26% in controls, P = 0·09. For carriage of allele 2 versus non-carriers, the OR was 1·58 (95% CI: 0·96–2·60, P = 0·07). There was significant linkage disequilibrium between the two SNPs; D′ = 0·94, r2 = 0·46. Additionally, haplotype frequencies were calculated. Haplotype analysis was of no superior value compared to single SNP analysis.
Table 2.
Genotype and allele frequencies of the IL1B and IL1RN polymorphisms in idiopathic pulmonary fibrosis (IPF) patients and healthy controls
| IPF (n = 77) | Controls (n = 349) | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Major/minor allele | Genotype frequency | Minor allele carriership | Genotype frequency | Minor allele carriership | |||||
| 1·1 | 1·2 | 2·2 | 2 | 1·1 | 1·2 | 2·2 | 2 | ||
| IL1B | |||||||||
| rs1143627 | T/C | 36 (28) | 48 (37) | 16 (12) | 64 (49) | 44 (153) | 45 (158) | 11 (38) | 56 (196) |
| rs1143634 | C/T | 53 (41) | 39 (30) | 8 (6) | 47 (36) | 56 (197) | 36 (125) | 8 (27) | 44 (152) |
| rs1143643 | G/A | 55 (42) | 35 (27) | 10 (8) | 45 (35) | 44 (152) | 46 (161) | 10 (36) | 56 (197) |
| rs1799916 | T/G | 100 (77) | 0 (0) | 0 (0) | 0 (0) | 100 (349) | 0 (0) | 0 (0) | 0 (0) |
| IL1RN | |||||||||
| rs11677397 | C/T | 49 (38) | 43 (33) | 8 (6) | 51 (39) | 56 (196) | 37 (129) | 7 (24) | 44 (153) |
| rs2637988* | A/G | 25 (19) | 56 (43) | 19 (15) | 75 (58) | 39 (136) | 45 (158) | 16 (55) | 61 (213) |
| rs397211 | T/C | 45 (35) | 45 (35) | 9 (7) | 55 (42) | 51 (178) | 40 (140) | 9 (31) | 49 (171) |
| rs408392† | G/T | 44 (34) | 47 (36) | 9 (7) | 56 (43) | 56 (194) | 37 (130) | 7 (25) | 44 (155) |
Minor allele carriership IPF versus healthy controls, P = 0·02.
Minor allele carriership IPF versus healthy controls, P = 0·07.
Genotype frequencies and minor allele carriership are shown in percentages, absolute numbers are shown in parentheses. 1 = major allele, 2 = minor allele.
The polymorphisms in the IL1RN and IL1B genes did not significantly influence BALF or serum IL-1Ra or IL-1β levels in IPF patients and healthy controls. However, differences were seen between genotypes of the rs2637988 polymorphism and the BALF IL-1Ra/IL-1β ratio; AA 1856 (IQR 1421–3730), AG 223·7 (IQR 84·6–384·9), GG 29·3 (IQR 6·95–130), P = 0·005 (Fig. 2). A less significant effect was found when genotypes of the rs408392 polymorphism were compared (P = 0·09). Other SNPs were not associated with the IL-1Ra/IL-1β ratio in serum or BALF.
Fig. 2.
Bronchoalveolar lavage fluid (BALF) interleukin (IL)-1Ra/IL-1β ratio in idiopathic pulmonary fibrosis (IPF) patients according to genotype of the rs2637988 polymorphism, AA (n = 19), AG (n = 43), GG (n = 15). Data are shown as median with interquartile ranges. BALF IL-1Ra/IL-1β ratios are dependent on the rs2637988 polymorphism, P = 0·005 (Kruskal–Wallis test).
Cellular profiles in BALF
The total cell count and absolute numbers of macrophages, lymphocytes, neutrophils and eosinophils in BALF were increased significantly in IPF patients compared to healthy controls (all P < 0·001; Table 3). The relationship between BALF cellular profiles and IL-1β and IL-1Ra is shown to illustrate the relevance in clinical perspective. In healthy controls, there was no correlation between BALF IL-1β levels or IL-1Ra and absolute neutrophil counts. However, in IPF patients absolute neutrophil counts were correlated with both BALF levels of IL-1β (r = 0·32, P = 0·05) and IL-1Ra (r = 0·65, P < 0·001) (Fig. 3).
Table 3.
Cellular profiles in bronchoalveolar lavage fluid (BALF) in idiopathic pulmonary fibrosis (IPF) patients and healthy controls
| IPF patients | Healthy controls | |
|---|---|---|
| n = 54 | n = 36 | |
| Cellular profiles in BALF (median, IQR) | ||
| Total cell count (×106) | 20·9 (13·1–30·1)* | 11·8 (7·2–20·3) |
| Macrophages (×106) | 17·1 (12·7–25·2)* | 6·4 (6·1–18·8) |
| Lymphocytes (×106) | 1·6 (0·6–3·6)* | 0·9 (0·4–1·3) |
| Neutrophils (×106) | 1·3 (0·5–3·8)* | 0·2 (0·1–0·3) |
| Eosinophils (×106) | 0·8 (0·4–2·7)* | 0·03 (0·01–0·08) |
P < 0·05 compared to healthy controls. IQR: interquartile range.
Fig. 3.
Scatter-plot illustrating the correlation between absolute neutrophil count in idiopathic pulmonary fibrosis (IPF) patients with interleukin (IL)-1Ra in bronchoalveolar lavage fluid (BALF). Values on the x- and y-axes represent log-transformed values; r = 0·65, P < 0·001.
Discussion
Disease development in IPF is thought to result from repetitive injury to epithelial cells and an abnormal fibrotic response. Proinflammatory mediators, such as IL-1β, are known to promote fibrosis, but can be regulated by the receptor antagonist IL-1Ra. In the present study, we found that the ratio between IL-1Ra and IL-1β was decreased in both serum and BALF of IPF patients compared to healthy controls. Furthermore, we showed that one SNP in IL1RN, rs2637988, associated with susceptibility to IPF and with the IL-1Ra/IL-1β ratio in BALF.
A predisposing effect of genetic variation in IL1RN was described previously by Whyte et al., who found an increased risk of fibrosing alveolitis in an Italian and a British population [6]. They investigated the IL1RN + 2018 SNP, which in the Caucasian Hapmap panel is in complete linkage disequilibrium with our tag rs408392 (r2 = 1). In our study, rs408392 was not the most significantly associated SNP, although carriership of allele 2 of rs408392 was more common in patients with IPF (P = 0·07). In other studies the variable number of tandem repeats (VNTR) in intron 2 of IL1RN was investigated and found to be in linkage disequilibrium with the IL1RN + 2018 SNP. However, both a small Australian [7] and an independent Czech cohort [12] did not reveal any association between the VNTR and IPF susceptibility [13]. Functional effects of IL1RN + 2018 alleles have been demonstrated by Carter et al. They showed that IL1RN + 2018 allele 2 not only correlated with the susceptibility to ulcerative colitis, but also to a significantly decreased ratio between the protein and mRNA content of IL-1Ra and total IL-1 in the colonic mucosa [14].
Although we found the same trend as reported in the Italian and British cohorts, our data suggest that carriership of the G allele of IL1RN rs2637988 is associated more strongly with IPF. Carriership of the G-allele is higher in IPF patients (75%) compared to controls (61%), P = 0·02. In addition, we showed that IPF patients carrying the rs2637988 G-allele had a significantly lower IL-1Ra/IL-1β ratio in BALF, suggesting a relative shortage of IL-1Ra compared to IL-1β. This implies that presence of the G allele has a pathogenic role in IPF.
The balance between IL-1 and IL-1Ra seems crucial in inflammatory diseases [15–18]. Although IPF is not primarily an inflammatory disease, IPF is characterized by high levels of inflammatory parameters. The balance between IL-1 and IL-1Ra has rarely been studied in IPF, but extensively in inflammatory diseases. In inflammatory bowel disease, changes in the IL-1Ra/IL-1β ratio have also been studied. Protein levels in the colonic mucosa of IL-1Ra, IL-1α and IL-1β were higher than in controls, but the ratio between IL-1Ra and total IL-1 was decreased significantly [14,19]. Similarly, it was found that the protein and gene transcript ratio between IL-1Ra and IL-1β in cultured alveolar macrophages of patients with interstitial lung diseases were significantly lower in comparison with healthy controls [9]. We found that IL-1Ra levels in BALF of IPF patients were increased, but this was not enough to equal the vast increase in local IL-1β. Altogether, this resulted in a 3·5-fold decrease in the IL-1Ra/IL-1β ratio in IPF patients compared to healthy controls.
In animal studies it has been shown that alterations in the balance between IL-1β and IL-1Ra cause the development of lung fibrosis. Mice with bleomycin-induced fibrosis have an up-regulated expression of IL-1β mRNA after instillation of bleomycin [20], and addition of recombinant IL-1β induces fibrotic remodelling [8]. Overexpression of IL-1β in rat lungs after intratracheal administration of bleomycin was associated with severe progressive tissue fibrosis in the lung, characterized by the presence of myofibroblasts, fibroblast foci and significant extracellular accumulations of collagen and fibronectin [4]. Other studies showed that administration of exogenous IL-1Ra prevented or even reversed the generation of pulmonary and synovial fibrosis [21–23]. The pathogenetic processes in bleomycin-induced fibrosis are simply a model for IPF and results cannot be extrapolated to human IPF. However, in patients with acute myocardial infarction, there is evidence that IL-1 blockade with IL-1Ra suppresses the inflammatory response and positively affects tissue remodelling [24].
IL-1 ligands such as IL-1α, IL-1β and IL-1Ra all bind to the IL-1 receptor (IL-1R1). Mice lacking the IL-1R1 receptor showed significantly reduced cellular infiltrates, alveolar wall destruction and collagen deposition. Moreover, blockade of the IL-1R1 receptor by exogenous IL-Ra (anakinra) dramatically reduced neutrophil influx and the formation of bleomycin-induced fibrosis in mice [8]. Altogether, IL-1 seems to be a critical cytokine and may possibly be a therapeutic target in IPF.
There are different hypotheses about the role of inflammation and thus proinflammatory cytokines such as IL-1β in the role of pulmonary fibrosis. Historically, the hypothesis was that inflammation in response to an unknown agent was the key process in IPF, ultimately resulting in fibrosis. The current concept is that IPF is a result of repeated episodes of lung injury, with a minor role for inflammation. This concept states that inflammation in IPF could be a consequence of the architectural remodelling, rather than a cause. The increased parameters of inflammation such as neutrophilia in BALF may be a reflection of remodelling and traction bronchiectasis due to fibrosis [25]. However, this does not exclude a role for inflammation in an earlier stage of the disease. An interesting paper in this context is the study by Flaherty et al. [26], in which the co-existence of UIP and non-specific interstitial pneumonia (NSIP) has been described in a considerable amount of patients who had multiple lung biopsies, demonstrating the presence of chronic inflammation and fibrosis next to each other. This pleads for a hypothesis in which UIP and NSIP are two different entities in one continuum. Before discarding the role of inflammation in the pathogenesis of IPF, we first need to understand the natural history of UIP [27]. Our hypothesis states the association of a SNP in the IL1RN gene with IPF predisposition; this suggests a role for IL-1 in the beginning of the pathogenetic process.
The present study is one of the more expanded studies evaluating IL-1Ra and IL-1β cytokine polymorphisms and corresponding protein levels in IPF. However, a limitation of this study is that the number of IPF patients is relatively small for genetic associations. Conversely, the results are in line with previously published literature [6,28]. Although our data suggest no effect of age or gender on the IL-1Ra/IL-1β ratio (results not shown), more studies are needed to confirm the role of a decreased ratio in IPF. Another point that needs attention is that the rs2637988 polymorphism influenced the IL-1Ra/IL-1β ratio of but not the individual cytokine levels. The cytokine values of IL-1Ra and IL-1β were not influenced significantly, but a mild trend is present. Carriers of the G allele had a slightly lower BALF IL-1Ra level (P = 0·21) and a higher BALF IL-1β level (P = 0·16). Although both not significant, when the ratio is calculated this effect is enhanced. A hypothetical explanation is that the balance between pro- and anti-inflammatory cytokines is of more biological importance than the absolute concentrations of IL-1Ra and IL-1β. Carter et al. [14] showed that carriage of the IL1RN + 2018 allele 2 was associated with a reduced colonic IL-1Ra protein level and a reduced IL-1Ra/total IL-1 ratio. It is likely that in our population a similar effect is present; however, our population might not be big enough to illustrate this with significant results, and this should be replicated in a larger cohort.
In conclusion, this study showed that variation in the IL1RN associates with susceptibility to IPF. The subsequent imbalance between IL-1β and IL-1Ra might have a significant pathogenetic effect in IPF patients. Better understanding of the role of these mediators in the context of disease susceptibility and progression is important, as it may help us to find rational for newly available therapies.
Acknowledgments
The authors thank Annette van der Vis, Danielle Hijdra and Jan Broess for technical and laboratory assistance.
Disclosure
None.
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