Association of an interleukin 1 alpha polymorphism with Alzheimer's disease [see Comments]
Retrospective epidemiologic studies suggest that individuals exposed to anti-inflammatory agents such as nonsteroidal anti-inflammatory drugs have a lower probability of developing AD as well as an older age at onset for the illness. Neuroinflammation may play an important role in the pathogenesis of AD. Interleukin 1 (IL-1), a potent proinflammatory cytokine, is colocalized immunohistochemically to neuritic plaques, a requisite neuropathologic feature for AD. A polymorphism in the 5'-flanking regulatory region at -889 of the IL-1 alpha gene (a C-to-T transition designated as IL-1A[-889] allele 2) may cause an overexpression of IL-1 alpha, a finding shown to be associated with inflammatory diseases. The IL-1A(-889) allele 2 polymorphism may be associated with AD pathogenesis. A total of 259 patients with AD and 192 nondemented control subjects were included from two different centers (Indianapolis, IN, and Munich, Germany). Genotyping for APOE alleles and IL-1A(-889) allele 2 was performed by PCR-based amplification followed by restrictive endonuclease digestion. Statistical analyses were conducted by center-, gender group-, and age group-stratified Mantel-Haenszel odds ratios, CI, and p values. The allele frequency of IL-1A(-889) allele 2 was 46% in clinically diagnosed patients with probable AD versus 34% in control subjects from the combined centers. The authors found an increased risk for AD with an estimated Mantel-Haenszel odds ratio of 1.68 (95% CI 1.1 to 2.6; p = 0.022) for heterozygous carriers and 7.2 (95% CI 2.0 to 24.5; p = 0.003) for individuals homozygous for IL-1A(-889) allele 2. They found no evidence for an interaction between the IL-1A and the apoE epsilon 4 polymorphisms (carriers and homozygotes), age, or gender with regard to conferred risk. The data strongly support an association between the IL-1A(-889) allele 2, especially in homozygotes, and later-onset AD.
Association of an interleukin 1␣
polymorphism with Alzheimer’s disease
Y. Du, PhD; R.C. Dodel, MD; B.J. Eastwood, PhD; K.R. Bales, MSc; F. Gao, MD; F. Lohmu¨ller, BSc;
U. Mu¨ller, MD; A. Kurz, MD; R. Zimmer, MD; R.M. Evans, MD; A. Hake, MD; T. Gasser, MD;
W.H. Oertel, MD; W.S.T. Griffin, PhD; S.M. Paul, MD; and M.R. Farlow, MD
Article abstract—Background: Retrospective epidemiologic studies suggest that individuals exposed to anti-
inflammatory agents such as nonsteroidal anti-inflammatory drugs have a lower probability of developing AD as well as
an older age at onset for the illness. Neuroinflammation may play an important role in the pathogenesis of AD.
Interleukin 1 (IL-1), a potent proinflammatory cytokine, is colocalized immunohistochemically to neuritic plaques, a
requisite neuropathologic feature for AD. A polymorphism in the 5⬘-flanking regulatory region at ⫺889 of the IL-1␣gene
(a C-to-T transition designated as IL-1A[⫺889] allele 2) may cause an overexpression of IL-1␣, a finding shown to be
associated with inflammatory diseases. The IL-1A(⫺889) allele 2 polymorphism may be associated with AD pathogenesis.
Methods: A total of 259 patients with AD and 192 nondemented control subjects were included from two different centers
(Indianapolis, IN, and Munich, Germany). Genotyping for APOE alleles and IL-1A(⫺889) allele 2 was performed by
PCR-based amplification followed by restrictive endonuclease digestion. Statistical analyses were conducted by center-,
gender group-, and age group–stratified Mantel–Haenszel odds ratios, CI, and pvalues. Results: The allele frequency of
IL-1A(⫺889) allele 2 was 46% in clinically diagnosed patients with probable AD versus 34% in control subjects from the
combined centers. Conclusion: The authors found an increased risk for AD with an estimated Mantel–Haenszel odds ratio
of 1.68 (95% CI 1.1 to 2.6; p⫽0.022) for heterozygous carriers and 7.2 (95% CI 2.0 to 24.5; p⫽0.003) for individuals
homozygous for IL-1A(⫺889) allele 2. They found no evidence for an interaction between the IL-1A and the apoE ⑀4
polymorphisms (carriers and homozygotes), age, or gender with regard to conferred risk. The data strongly support an
association between the IL-1A(⫺889) allele 2, especially in homozygotes, and later-onset AD.
Various genetic linkage analyses and association
studies of AD have identified one well established
and several potential genetic risk factors that cause
or predispose carriers to late-onset AD. Mutations in
the genes encoding the ␤-amyloid precursor protein
(APP), presenilin-1, and presenilin-2 cause early-
onset AD, but they occur very rarely in the general
Susceptibility polymorphisms associated
with the risk to develop AD for the far more common
late-onset form of the illness include APOE ⑀4,
receptor-related protein (LRP),
an intron of presenilin-1,
as well as a polymorphism
within the APOE promoter region.
polymorphism within the regulatory region of the
interleukin 6 (IL-6) gene, which has been demon-
strated to downregulate blood levels of the IL-6 pro-
tein, has been suggested to be “protective,” reducing
the probability for developing AD. Of the three
APOE alleles, the ⑀4 allele appears to be a signifi-
cant risk factor for AD. However, this allele is nei-
ther necessary nor sufficient to cause disease, as
many ⑀4/⑀4 individuals well into their nineties ap-
pear to be disease-free. Together, these data suggest
the presence of additional genetic or other risk fac-
tors that, either alone or in concert with ⑀4, alter the
risk and the age at onset for AD.
An increasing number of studies have suggested
See also page 464
From the Departments of Pharmacology and Toxicology (Drs. Du and Gao) and Neurology (Drs. Evans, Hake, and Farlow), Indiana University School of
Medicine; Neuroscience Discovery Research (K.R. Bales and Dr. Paul) and Department of Statistical and Mathematical Sciences (Dr. Eastwood), Lilly
Research Laboratories, Eli Lilly and Company, Indianapolis, IN; Geriatric and Mental Health Research Education and Clinical Centers (Dr. Griffin),
McClellan Memorial Veterans Affairs Medical Center, Little Rock, AR; Department of Neurology (Dr. Dodel, F. Lohmu¨ller, and Dr. Oertel), Philipps
University, Marburg, Germany; Department of Psychiatry (Drs. Kurz and Zimmer), Technical University, Munich, Germany; Department of Neurology (Dr.
Gasser), Ludwig Maximilians University, Munich, Germany; and Department of Human Genetics (Dr. Mu¨ller), University of Giessen, Germany.
Drs. Du and Dodel contributed equally to this article.
Supported in part by NIH P30AG10133 (Indiana authors) and NIH P01AG12411 (WSTG) and the National Alzheimer’s Disease Cell Repository.
Received February 29, 2000. Accepted in final form April 26, 2000.
Address correspondence and reprint requests to Dr. M. Farlow, Department of Neurology, Indiana University School of Medicine, Indianapolis, IN 46202;
480 Copyright © 2000 by AAN Enterprises, Inc.
that inflammatory processes take part in the
pathogenetic cascade of events that leads to AD
This has been supported by the
following observations: 1) activated astrocytes and
microglia are intimately associated with extracellular
; 2) complement proteins and
acute-phase proteins such as ACT, C-reactive protein,
-macro-globulin, and amyloid p-component are im-
munohistochemically detected in senile plaques
and 3) epidemiologic studies have demonstrated that
the use of anti-inflammatory agents, in particular
nonsteroid anti-inflammatory drugs, is associated
with delayed onset or slower progression of disease.
Induction of neuroinflammation involves the activa-
tion of astrocytes and microglial cells followed by the
subsequent expression of a number of cytokines. Ac-
tivated microglia producing proinflammatory cyto-
kines such as interleukin 1␣(IL-1␣) and interleukin
have been found in areas sur-
rounding extracellular amyloid plaques. Because
there are no signs of acute infection in the brain of
patients with AD, chronic production of these cyto-
kines may initiate a chain reaction that leads to
enhanced release of neurotoxic cytokines, amyloid
deposition, and subsequent neuritic amyloid plaque
The IL-1 family consists of at least three structur-
ally related polypeptides (IL-1␣, IL-1␤, and IL-1 re-
ceptor antagonist), each of which is encoded by a
separate gene located in a cluster on the long arm of
Several reports have tried to corre-
late polymorphisms of the IL-1␣gene with the sever-
ity or outcome of inflammatory diseases. For
example, an IL-1␣polymorphism, IL-1A(⫺889) allele
2, has been associated with juvenile rheumatoid ar-
and with AD.
In addition, a composite
genotype comprising IL-1A(⫺889) allele 2 plus an
IL-1␤polymorphism (a C-to-T transition at position
3,953) has been reported to be over-represented in
patients with periodontitis
as well as AD.
report an association of IL-1A(⫺889) allele 2 poly-
morphism in a relatively large case control cohort of
patients with AD and nondemented elderly control
Methods. Study population. We enrolled 451 individu-
als who were consecutively recruited in outpatient clinics
for cognitive disorders from the Indiana AD Center, Indi-
ana University School of Medicine, Indianapolis, IN (IU),
and the Department of Psychiatry, Technische Universi-
ta¨ t, Munich, Germany (MU). The IU group consisted of 78
AD patients (mean age 72.5 ⫾9.6 years; range 50 to 94
years; 43 women, 35 men) and 71 unrelated control sub-
jects (mean age 72.5 ⫾9.1 years; range 51 to 94 years; 38
women, 33 men). The MU group consisted of 181 AD pa-
tients (mean age 70.9 ⫾9.8 years; range 50 to 95 years;
112 women, 69 men) and 121 unrelated control individuals
(mean age 69.0 ⫾10.8 years; range 50 to 95 years; 84
women, 37 men). Patients and control subjects with a fam-
ily history of autosomal dominant dementia were not in-
cluded in this study. Normal control subjects within the IU
group had normal physical and brief neurologic examina-
tions and normal range scores on the Consortium to Estab-
lish a Registry for AD (CERAD) neuropsychologic test
The MU control group consisted of subjects scor-
ing 28 or higher on the German version of the Mini-Mental
State Examination test. Patients of the combined control
group also had no history or evidence of neurologic disease
with potential to affect cognition and no deficits in their
ability to perform activities of daily living. All AD patients
had a clinical examination, including neurologic and neu-
ropsychologic evaluations, to document deficits in cognition
and activities of daily living and laboratory studies to ex-
clude reversible causes of dementia. All patients met
ICD-10 criteria for dementia as well as National Institute
of Neurological and Communicative Disorders and
Stroke–AD and Related Disorders Association criteria for
probable or possible AD.
Only patients and control subjects who were 50 years or
older were included. All individuals (or, for significantly
cognitively impaired individuals, their legal guardian or
caregiver with power of attorney) had given written in-
Genotyping of IL-1A(⫺889) and APOE. Genomic DNA
was extracted from blood samples according to standard
protocols. Genotyping of the APOE alleles and of IL-
1A(⫺889) allele 2 was performed, utilizing a PCR-based
amplification followed by restrictive endonuclease diges-
Restriction fragments were resolved on 4% (for
APOE allele) or 2% (for IL-1A[⫺889] allele 2) agarose gels
with 1 mg/mL ethidium bromide for 30 minutes at 200 V
and directly detected under ultraviolet light.
Statistical analysis. Age and gender. The difference
in age between AD and control individuals was assessed by
analysis of variance (ANOVA) in the IU and MU samples.
The two sample sets were analyzed independently, and a
statistical analysis indicated that they were appropriate to
be combined. The difference in gender distribution was
test in the IU and MU samples. The test
statistics were matched to the study design for each center.
Odds ratios. Patients and control subjects were subdi-
vided into the following age groups: ⬍55, 55 to 64, 65 to 74,
75 to 84, and 85⫹. All analyses of odds ratios was con-
ducted controlling for center-, age group-, and gender
group–stratified Mantel–Haenszel odds ratios, CIs, and
hypothesis tests. Additionally, odds ratios for the IL-
1A(⫺889) allele 2 were also controlled by APOE allele sta-
tus and vice versa. The interaction between the APOE ⑀4
allele and the IL-1A(⫺889) allele 2 was assessed by logistic
regression in both populations.
Results. The distributions of the IL-1A(⫺889) allele 2
and the APOE ⑀4 allele in AD and control individuals are
presented in table 1. The genotype distributions for both
genes were in Hardy–Weinberg equilibrium for both AD
and control populations. Our data confirm previous obser-
vations on the association of ⑀4 and AD.
odds ratios for being affected as a function of carrying at
least one or two IL-1A(⫺889) allele 2 polymorphisms in the
entire population were 1.68 (95% CI 1.1 to 2.6; p⫽0.022)
and 7.2 (95% CI 2.0 to 24.5; p⫽0.003) when compared
with those in subjects not having the IL-1A(⫺889) allele 2
polymorphism (table 2). In older patients (⬎60 years old),
the results for heterozygous and homozygous carriers of
this allele were similar (odds ratio 1.75, 95% CI 1.1 to 2.6,
p⫽0.02; odds ratio 8.7, 95% CI 1.8 to 42.5, p⫽0.007). We
August (2 of 2) 2000 NEUROLOGY 55 481
found no interaction between IL-1A(⫺889) allele 2 and age
(p⫽0.48), gender ( p⫽0.30), or APOE ⑀4(p⫽0.64).
Finally, we examined the joint distribution of IL-
1A(⫺889) allele 2 and APOE ⑀4 allele in the entire data
set. Our data are consistent with previous observations in
which increased allele frequencies of the APOE ⑀4 allele
are associated with AD. Logistic regression analyses ad-
justed for the effect of APOE ⑀4 on risk for AD for the two
different centers indicated overall that there was no signif-
icant interaction between IL-1A(⫺889) allele 2 and APOE
⑀4 allele ( p⫽0.584).
Discussion. Our data suggest that a polymor-
phism of the IL-1␣gene (IL-1A[⫺889] allele 2) in-
creases the risk for developing AD. In a large group
of AD patients and control subjects that included 451
individuals, the odds ratios in patients with one or
two copies of IL-1A(⫺889) allele 2 showed a strong
gene dosage effect, similar to the increased risk of
developing late-onset AD seen with increased num-
bers of APOE ⑀4 allele. The risk to develop AD as a
function of carrying at least one IL-1A(⫺889) allele 2
is significant and similar to those calculated for
other polymorphisms, including ACT and LRP.
strong correlation between homozygosity for IL-
1A(⫺889) allele 2 and AD (odds ratio 7.2) suggests
the IL-1A polymorphism may be another particularly
strong risk gene for AD, like APOE.
IL-1A(⫺889) allele 2 polymorphism is located in
the promoter region of the IL-1␣gene, which may
influence the expression of the IL-1␣protein. The
IL-1 protein induces the expression and processing of
APP, resulting in the increased production of se-
creted APP and further activation of microglia and
overexpression of IL-1.
The ␤-amyloid peptide, at
least in vitro, perpetuates an environment in which
microglial/astroglial activation is potentiated, lead-
ing to additional increases in IL-1␣/␤protein synthe-
sis, activating the classic complement pathway, and
thus amplifying an autocrine/paracrine neuroinflam-
matory cycle. Secreted APP itself is able to activate
microglia, enhancing the production of inducible ni-
tric oxide synthase and IL-1␣/␤, both of which, alone
or together, further amplify the cytokine cascade.
Several investigators have demonstrated that activa-
tion of astrocytes by IL-1 results in an upregulation
of APOE and ACT,
with increased levels of both
favoring the extracellular deposition of ␤-amyloid
peptide into protease-resistant plaques. Elevated tis-
sue levels of the IL-1 protein and increased numbers
of activated IL-1-immunoreactive astrocytes are
found in AD brain.
Elevated IL-1 has been found in
young AD patients and Down syndrome patients,
suggesting that IL-1 overexpression, and thus ampli-
fication of a cytokine cycle, may be a prodromal
event occurring long before the onset of clinical
Additionally, IL-1 stimulates S100␤,an
astrocyte-derived protein encoded by a gene on chro-
that, when overexpressed in the brain
of transgenic mice, results in neuropathologic
changes such as astrocytosis.
The IL-1A(⫺889) allele 2 polymorphism may be a
strong risk factor in development of AD. The contro-
versy surrounding whether or not the absolute level
of IL-1␣protein increases in CSF or plasma along
with its increase in brain tissue levels in AD patients
may be due to the large diversity of populations in-
vestigated thus far. The genotyping data from this
work should allow us to identify potential subpopula-
tions of AD patients (carriers of IL-1A[⫺889] allele 2,
for example) in which elevated levels of circulating
cytokines in CSF may be more evident. If true, iden-
tification of AD subpopulation(s) may further enhance
accumulating data suggesting that neuroinflamma-
Table 1 Number and frequency of IL-1A(⫺889) (as percentages) and frequency of alleles (as percentages) in the combined cohort of AD
patients and in the combined cohort of controls
1/1 1/2 2/2 1/1 1/2 2/2
n 126 62 3 141 97 21
Genotype frequency 65.6 32.8 1.6 54.4 37.5 8.1
APOE /2 /3 /4 /2 /3 /4
⑀2 0.5 18.8 1.6 0 8.9 1.5
⑀3 18.8 59.9 17.2 8.9 34.4 44.0
⑀4 1.6 17.2 2.1 1.5 44.0 11.2
In the table, 1 is wild type (C at the ⫺889 of IL-1␣gene) and 2 is a polymorphism (T at the ⫺889 of IL-1␣gene).
Table 2 Mantel-Haenszel odds ratio estimates for effect of
IL-1A(⫺889) carrier, IL-1A(⫺889) homozygote, and
risk for AD for entire sample
Factor OR 95% CI pValue
IL-1A(⫺889) carrier 1.68 1.1–2.6 0.02
IL-1A(⫺889) homozygote 7.2 1.9–27.6 0.004
APOE ⑀4 5.1 3.3–7.9 0.001
482 NEUROLOGY 55 August (2 of 2) 2000
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