Am J Respir Crit Care Med
Internet address: www.atsjournals.org
Vol 162. pp 755–758, 2000
Increased Risk of Fibrosing Alveolitis Associated with
Interleukin-1 Receptor Antagonist and Tumor Necrosis
MOIRA WHYTE, RICHARD HUBBARD, RICCARDO MELICONI, MICHELLE WHIDBORNE, VANESSA EATON,
COLIN BINGLE, JANINE TIMMS, GORDON DUFF, ANDREA FACCHINI, ANGELA PACILLI, MARIO FABBRI,
IAN HALL, JOHN BRITTON, IAN JOHNSTON, and FRANCESCO DI GIOVINE
Division of Molecular and Genetic Medicine, University of Sheffield, Sheffield, and Department of Respiratory Medicine, University of Nottingham,
Nottingham, United Kingdom; and Policlinico S. Orsola and Istituti Ortopedici Rizzoli, University of Bologna, Bologna, Italy
Fibrosing alveolitis (FA) is characterized by persistent inflammation
and elevated production of tumor necrosis factor-alpha (TNF-
interleukin-1 beta (IL-1
), and interleukin-1 receptor antagonist (IL-
1ra) in the lung. Single base variations at position
1ra gene (IL-1
RN) and position -308 in the TNF-
overrepresented in other chronic inflammatory disease populations.
We have tested the hypothesis that predisposition to FA may also
be influenced by these polymorphisms by genotyping 88 cases and
matched controls from England and 61 cases and 103 unmatched
controls from Italy. The rarer allele for IL-1
nated allele 2 in each case. For IL-1
the relative odds of FA were increased in homozygous subjects by
an odds ratio (OR) of 10.2 (95% confidence intervals [CI], 1.26 to
0.03) and for carriers by an OR of 1.85 (95% CI, 0.94 to
0.075). In the Italian population, the risk of FA was in-
creased, in IL-1RN allele 2 homozygotes (OR, 2.54; 95% CI, 0.68 to
0.2) and in carriers (OR 2.40; 95% CI, 1.26 to 4.60; p
0.008). Carriage of TNF-A allele 2 was also associated with increased
risk of FA in the English (OR, 1.85; 95% CI, 0.94 to 3.63; p
and Italian (OR, 2.50; 95% CI, 1.14 to 5.47; p
These data suggest IL-1RN (
2018) allele 2 and TNF-
confer increased risk of developing FA and, therefore, that unop-
and/or excessive TNF-
role in this condition.
2018 in the IL-
RN and TNF-A was desig-
RN allele 2, in the English group,
A (-308) allele 2
may play a pathophysiologic
The pathology of fibrosing alveolitis (FA ) is characterized by
persistent alveolar inflammation and interstitial pulmonary fi-
brosis, processes mediated by proinflammatory and profibrotic
cytokines (1). A lthough the basis of an individual’s susceptibil-
ity to fibrosing alveolitis is unknown, a genetic component is
suggested by approximately 3% of cases of cryptogenic fibros-
ing alveolitis (CFA ) being familial, with pathology that is indis-
tinguishable from nonfamilial forms (2).
A number of cytokine gene polymorphisms have been de-
scribed within the interleukin-1 (IL-1) gene cluster on chromo-
some 2 (3) and in the TNF-
(4), which are associated with susceptibility to inflammatory,
A) on chromosome 6
autoimmune, and infectious diseases. The three known IL-1
genes are arranged in close proximity on chromosome 2q13
(3). The IL-1
A and IL-1B genes encode agonist proteins, the
proinflammatory cytokines IL-1
roles in inflammation and innate immunity. The third known
gene in this cluster (IL-1
RN) produces a related protein, the
IL-1 receptor antagonist (IL-1ra), which binds the IL-1 signal-
ing receptor (IL-1R Type 1) but does not elicit a response (5).
A single base variation occurs at position
(6), with the rarer allele, [IL-1RN
plicated in inflammatory diseases; for example, coronary ar-
tery disease (7). A polymorphism in the TNF locus has been
identified in the promoter region of TNF-
A (-308) allele 2] implicated in inflammatory dis-
eases, including asthma (9) and chronic bronchitis (10).
We hypothesized that susceptibility to FA may be deter-
mined by the occurrence of these disease-associated alleles,
2018) allele 2 and TNF-
tested this hypothesis in two independent FA case-control co-
horts, one English and one Italian.
, with well-known
2018 in IL-1 RN
2018) allele 2], being im-
A (8) and the rarer
A (-308) allele 2, and have
The English case-control sample comprised 88 consecutively re-
cruited cases and age- and sex-matched controls recruited from the
same primary care physician lists as the index cases, and who were
part of a larger case-control study previously reported from Notting-
ham, England (11). A lthough the original study included more than
200 cases, blood for DNA extraction was stored only from later stages
of the study. Of the 88 available cases, 51 were prevalent at the begin-
ning of recruitment into the study, and 37 were incident during an 18
mo recruitment period. The Italian cohort comprised 61 cases col-
lected from the Istituto di Patologia Medica of the University of Bolo-
gna, Italy, from a study described previously (12), and 103 controls
obtained by taking 414 consecutive ethnically matched, healthy blood
donors from the Bologna area and, blinded to genotype, taking the
upper quartile of age distribution (
priate age distribution for the controls.
FA was diagnosed either from an open lung biopsy or by the pres-
ence of basal inspiratory pulmonary crackles, bilateral interstitial lung
shadowing on chest radiograph, and restrictive lung function (%FEV
70% together with FV C or D
absence of restrictive lung function patients were included only if there
were pathognomonic changes of FA on a high-resolution computed to-
mography (HRCT) scan and if there was no evidence of another pa-
renchymal process caused by occupational or domestic exposures, sar-
coid, bronchiectasis, or connective tissue disease (11). Of the English
cases, 25 of 88 had undergone lung biopsy and a further 42 of 88 had
had a HRCT scan. FA was diagnosed by open lung biopsy in 11 of 61
54 yr of age) to obtain an appro-
80% of predicted). In the
Supported by a Project Grant from the Medical Research Council to the Univer-
sity of Nottingham, a Project Grant from the Special Trustees of the Former
United Sheffield Hospitals, and grants from the Ministero dell’Universita e della
Ricerca Scientifica e Tecnologica (MURST) and Ricerca Corrente Istituti Ortope-
Correspondence and requests for reprints should be addressed to Professor
Moira Whyte, Respiratory Medicine, Division of Molecular and Genetic Medicine,
University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, UK. E-mail:
Received in original form September 14, 1999 and in revised form December 16, 1999)
AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1622000
Italian cases, 56 of 61 cases had had HRCT scans and all patients ful-
filled the exclusion criteria as previously detailed (11, 12).
Ethics approval for the English study was granted by the Notting-
ham City Hospital Medical Ethics Committee, for the Italian study by
the Institutional Review Board of the University Hospital, Bologna,
and for the genetic analyses by the South Sheffield Research Ethics
2018 in exon 2 of the IL-1
phism is a single base variation (G/A ) at -308 in the promoter region
(8). Genotyping was performed blind to case status either by restric-
tion-enzyme digest of PCR products, as previously described (6, 8), or
by TaqMan allelic discrimination (13). TaqMan probes were pur-
chased from A BI-PE (Warrington, UK) and were as follows: for IL-
2018) Probe 1: 5
FA M)A A CCA A CT A GT TGC TGG
A TA CTT GCA A G(
TA MRA )-3
A CT A GT TGC CGG A TA CTT GCA A G(
-A A G TTC TGG GGG A CA CA G GA A G-3
-A CG GGC A A A GTG A CG TGA TG-3
308) Probe 1: 5
TET) CC CCG TCC CCA TGC CC
TA MRA )-3
Probe 2: 5
FA M) A C CCC GTC CTC A TG CCC
TA MRA )-3
Forward Primer: 5
TGT GTG T-3
Reverse Primer: 5
CA A TA G GTT-3
. The English samples were genotyped by the
PCR-RFLP method and the Italian samples by the TaqMan method;
10% of samples were selected at random and tested by both tech-
niques as quality-control. Genotype frequencies in patients and con-
trol subjects in both populations were not significantly different from
those predicted under Hardy Weinberg equilibrium.
2018) polymorphism is a single base variation (C/T) at
RN gene (6). The TNF-A (-308) polymor-
Probe 2: 5
TET)A A CCA
TA MRA )-3
. For TNF-
-GGC CA C TGA CTG A TT
-CA A A A G A A A TGG A GG
Demographic details were compared between and within case-control
populations by Z test and chi-square tests as appropriate and odds ra-
tios (ORs) estimated by logistic regression using STA TA (version 5)
software. ORs for the matched case-control study were estimated by
conditional logistic regression, comparing carriers of allele 2 for IL-
2018) or TNF-A (
308), in total or as 1,2 heterozygotes or 2,2
homozygotes, with 1,1 homozygotes. A similar analysis was per-
formed for the unmatched case-control study (Italian) using logistic
regression. Multiplicative terms were used to look for evidence of in-
teraction between genotype effects within populations, comparing
carriage of allele 2 for either gene with 1,1 homozygotes, and to look
for effect modification by age or sex. To estimate a summary OR for
RN and TNF-A effects, across both study populations and com-
bining matched and unmatched data, we used a fixed effects meta-
analysis method to derive a weighted average of the log odds, the
weight being the inverse of the variance of the log OD. The signifi-
cance test for the summary OD was a Z test calculated as the log of
the summary OD divided by its standard error (14).
Demographic information for the two study populations and
pulmonary function parameters for the FA cases are shown in
Table 1. There were no significant demographic differences
between or within the English and Italian cases and controls,
but the Italian cases had significantly lower values for FV C
than the English cases (p
tribution and frequency of IL-1
308) genotypes in FA cases and controls are shown in Table
2. The frequency of IL-1
RN allele 2 was increased in the En-
glish FA cases, of whom 44.3% carried allele 2 compared with
31.8% of controls (Table 2). Most of these subjects were het-
erozygous for the mutation, but of the 12 who were homozy-
gous for allele 2, 10 were cases. The OD for FA in those ho-
mozygous for IL-1
2018) allele 2 was 10.2 (95% CI, 1.26
to 81.4; p
0.03), for heterozygotes it was 1.43 (95% CI, 0.70
to 2.92; p
0.3), and for carriage of allele 2 compared with 1,1
homozygotes it was 1.85 (95% CI, 1.15 to 3.06; p
0.0001, Z test). The dis-
2018) and TNF-A
ble 3). In the Italian cohort the frequency of the IL-1
2018) allele 2 in the patients with FA was 57.4% compared
with 36.0% of control subjects (Table 2) and the OD for FA
for homozygotes was 2.54 (95% CI, 0.68 to 9.5; p
heterozygotes it was 2.38 (95% CI, 1.21 to 4.67; p
for carriage of allele 2 versus 1,1 homozygotes it was 2.40
(95% CI, 1.26 to 4.60; p
0.008) (Table 3). Estimated ODs
obtained by combining these estimates across the two studies
were 3.77 (95% CI, 1.24 to 11.5; p
gotes, 1.87 (95% CI, 1.15 to 3.06; p
and 2.12 (95% CI, 1.33 to 3.38; p
2 compared with 1,1 homozygotes. In view of previously de-
scribed linkage disequilibrium within the IL-1 gene cluster and
the association of IL-1
B gene polymorphisms with other in-
flammatory diseases, we subsequently examined two informa-
tive polymorphisms at positions
gene (3). There was no significant difference in the genotype
distributions at these loci between patients and control sub-
jects in either population (data not shown).
Carriage of TNF-
308) allele 2 was also increased in
both case populations, occurring in 39.8% of the English cases
compared with 27.3% of controls and 34.4% of Italian cases
compared with 15.6% of controls (Table 2). Few homozygotes
were identified for TNF-
A (-308), none in the Italian cohort
and only three patients and two control subjects in the English
cohort. For carriage of allele 2 versus 1,1 homozygotes, the
OD for FA was 1.85 (95% CI, 0.94 to 3.63; p
English cohort and 2.50 (95% CI, 1.14 to 5.47; p
the Italian cohort (Table 3). The combined estimated OD for
carriage of allele 2 was 2.10 (95% CI, 1.26 to 3.50; p
0.001) for carriage of allele
0.01) for allele 2 homozy-
0.006) for heterozygotes,
3954 in the IL-1B
0.075) in the
CHARACTERISITICS OF CASES AND CONTROLS*
Male sex, %
Current or former
FVC, % pred
DLCO, % pred
68 ? 10
68 ? 10
62 ? 13
60 ? 6
80 ? 22
50 ? 17
65 ? 19
36 ? 18
* Values shown are mean ? SD.
† Age refers to age at diagnosis.
‡ Not available.
PREVALENCE AND FREQUENCY (AS %) OF IL-1RN (?2018) AND
OF TNF-A (?308) GENOTYPES IN 88 FA CASES AND AGE- AND
SEX-MATCHED CONTROLS FROM AN ENGLISH COHORT AND
IN 61 FA CASES AND 103 ETHNICALLY MATCHED BLOOD
DONOR CONTROLS FROM AN ITALIAN COHORT
5 (4.9) 103
0 (0) 103
There was no evidence of interaction between genotypes or
effect modification because of age or sex for either IL-1RN
(?2018) or TNF-A (?308) allele 2 in either population.
The results of this study suggest carriage of allele 2 for IL-1RN
(?2018) or TNF-A (?308) is significantly associated with in-
creased risk of development of FA . This effect was observed
in two independent populations, one English and one Italian.
A s far as we are aware, this is the first demonstration of ge-
netic factors that may contribute to individual susceptibility to
FA . Our sample sizes were determined by the numbers of sam-
ples available from cases of FA in existing archives rather than
by estimates of numbers required to achieve statistical power,
and samples were drawn from matched and unmatched study
designs. We were therefore unable to pool data from these stud-
ies directly, but we were able to produce a single best estimate
of combined OD by a simplified meta-analysis technique. Cases
included in the study were pulmonary physician-diagnosed
and fulfilled normal clinical criteria for the diagnosis of FA ,
with the great majority having had a HRCT scan and biopsy
information available in a quarter of cases. Misclassification of
case and control status cannot be completely excluded but
would, if anything, dilute the observed genotypic associations.
The essential role of cytokines in mediating both inflamma-
tory and fibrotic processes in the lung is well established (1).
IL-1 and TNF-?, key proximal cytokines, are produced pre-
dominantly by alveolar macrophages and can, in turn, stimu-
late the production of other proinflammatory and profibrotic
cytokines. IL-1ra, the naturally occurring antagonist of IL-1,
counteracts the proinflammatory functions of IL-1 and the
balance between IL-1 and IL-1ra is seen as a crucial ratio in
destructive inflammatory disease (5). IL-1ra production in
lung tissue of patients with FA has been localized to alveolar
macrophages (15), hyperplastic type II pulmonary epithelial
cells, and fibroblasts (16). A lveolar macrophages from pa-
tients with FA produce higher levels of IL-1ra in vitro than do
those from healthy control subjects (15) and increases in IL-
1ra levels have been documented in bronchoalveolar lavage
fluid from patients with FA (16). There is evidence for a pro-
tective role of exogenous IL-1ra in a number of animal models
of acute lung injury. Overexpression of IL-1ra, targeted to dis-
tal airway epithelium under the transcriptional control of the
surfactant protein-C gene promoter, has recently been shown
to give partial protection from IL-1? induced airway inflam-
mation and injury (17). Moreover, Piguet and colleagues (18)
have shown that exogenous administration of IL-1ra via an in-
traperitoneal pump can partially reverse changes of pulmo-
nary fibrosis and to a lesser extent BA L cellularity in bleomy-
cin-induced fibrosis in mice.
The functional effects of IL-1RN (?2018) allele 2 are un-
certain. There is evidence for the association of allele 2 both
with reduced IL-1ra protein production in inflammatory
bowel disease samples ex vivo (19) and with increased IL-1ra
production upon stimulation of peripheral blood monocytes in
vitro (20). Such effects may well be stimulus- and cell-type
specific, and further study is clearly required. However, the as-
sociation of IL-1RN allele 2 with a number of other inflamma-
tory diseases (3, 7), in addition to FA , implies a proinflamma-
tory effect of IL-1RN allele 2.
TNF-? has also been implicated in the pathogenesis of FA ,
with increased expression of TNF-? protein in lung tissue of pa-
tients with FA (21). The murine TNF-? gene has been overex-
pressed under the control of the human surfactant protein SP-C
promoter in transgenic mice, and the pulmonary pathology
showed a striking resemblance to human FA , with a leukocytic
alveolitis, type II cell hyperplasia and extensive fibrosis (22).
The TNF-A (?308) allele 2 promoter polymorphism has previ-
ously been associated with a number of inflammatory lung dis-
eases, including asthma (9) and chronic bronchitis (10), and it
was recently shown to be a much more powerful transcriptional
activator of the TNF-A gene than the wild-type allele 1 (23).
Identification and understanding of the role of genetic risk
factors for FA is currently at a very early stage of development,
but it may lead to significant therapeutic opportunities in the
management of patients with FA , and to the identification of
those who are at increased risk of developing pulmonary fibro-
sis in the future. The latter category may include those in high-
risk occupations, those with other diseases associated with an
increased risk of development of pulmonary fibrosis such as
rheumatoid arthritis or systemic sclerosis, and those exposed to
other recognized risk factors for pulmonary fibrosis such as
amiodarone or bleomycin therapy. Insights into the pathophys-
iology of FA gained from genetic studies are likely to be partic-
ularly important, by identifying key molecular regulators of the
proinflammatory and profibrotic processes in the lung and thus
potential therapeutic targets at a molecular level. Both the ge-
netic associations identified in this study are thus of potential
pathophysiologic and therapeutic relevance to the understand-
ing of a progressive and currently untreatable disease.
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IL-1RN (?2018) AND TNF-A (?308) GENOTYPE DISTRIBUTION ANALYSIS
AND RISK OF FIBROSING ALVEOLITIS
OR 95% CI p Value OR 95% CIp Value OR95% CIp Value
1.26 to 81.4
0.68 to 9.50
0.70 to 2.92
1.21 to 4.67
0.94 to 3.63
1.26 to 4.60
0.31 to 12.10.5 1.84
0.92 to 3.67
1.14 to 5.47
0.94 to 3.63
1.14 to 5.47
* Significant odds ratio (p ? 0.05).
† Significant odds ratio (p ? 0.01).
‡ No Italian homozygotes.
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