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Sci. Med. J., July - October 2008; 20(3-4): 1-9
ISSN 1110 - 5607
Original
Article
ESCME
Hearing Loss: Immunological Aspects
Suzan Muhammad Farouk1, Helmy El Gazzar2, Nevein A. Omran2, Eman
Abdel Badeea3
1
Clinical Pathology Benha Teaching Hospital, 2Clinical Pathology Department,
Audiology Department, Hearing and Speech Institute
3
ABSTRACT
Background: Autoimmune inner ear disease (AIED) consists of hearing loss and/or dizziness which is
caused by antibodies or immune cells attacking the inner ear. AIED may be the result of either organspecific disease or systemic disorder. In the vestibule-auditory sensorineural hearing loss (SNHL) there
should be auto-antibodies or evidence of cell mediated immune responses directed against inner ear
antigens. Autoimmune SNHL is a clinical syndrome that typically produces a rapidly progressive hearing
loss. There still exists some controversy in designating the disease “autoimmune”, as the microorganism
that triggers the immune response has not yet been identified. There have been sporadic reports of
sudden or rapidly progressive hearing losses that have been found to be immune-mediated or in the
setting of other systemic autoimmune disease. The identity of a highly relevant inner-ear self antigen is
still required. In recent years, the role of anti-heat shock protein 70 “anti-HSP70”, which is present in the
outer hair cells of inner ear, has been suggested which also can predict corticosteroid responsiveness.
Aim of the Work: The present study aimed to delineate to what extent SNHL is due to autoimmune
malfunction. Also, evaluate the role of the serum antibodies to anti-HSP70 as a marker for autoimmune
SNHL.
Patients and Methods: A total of 45 patients were included in this study, they were complaining of
sudden or rapidly progressive SNHL and presented within two weeks from time of onset of hearing
loss. In addition to 15 healthy, age and sex matched controls were also included. All participants were
subjected to audiological examination, laboratory investigations (CRP, RF, ANA and ASMA) and
detection of anti- anti-HSP70.
Results: Pure tone audiometric test provided that of the patient group 14 (31.1%) had moderate hearing
loss, 16 (35.6%) had severe hearing loss and 15 (33.3%) had profound hearing loss. No significant
differences were noted as regards serum levels of CRP, RF, ANA, and ASMA in patients group as
compared to controls. Anti-HSP70 antibodies were detected in 60% of patients’ serum (27/45), while
the percentage of patients with positive CRP, RF, ANA and ASMA were 17.8%, 15.6%, 4.4% and 8.9%,
respectively. In anti-HSP70 positive patients, there was a highly statistically significant elevation for
ASMA in comparison to anti-HSP70 negative patients. No significant relation was determined between
the results of the hearing tests and positive anti-HSP70.
Conclusion: Anti-HSP70 can be used to prove the humoral immune response of inner ear components
in patients with SNHL and also for early diagnosis and its presence correlates to the disease activity.
Other immunological investigations as ASMA and ANA are recommended when general autoimmune
disorders are suspected.
Key Words: Hearing Loss, Anti-HSP70, AIED.
Corresponding Author: Dr. Helmy El Gazzar, Clinical Pathology Department, Hearing and Speech
Institute, Giza, Egypt, e-mail: [email protected], Mobile: 0101676395.
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Hearing Loss: Immunological Aspects
an autoimmune etiology, as it could be reversed or
stabilized by immunosuppressant drugs. McCabe
(1979) proposed that a diagnosis of immune inner
ear disease may be made on the basis of clinical
symptoms, laboratory tests and a positive response
to immunosuppressant medication. In 1984,
McCabe expanded this concept to include the
vestibular system and established the designation
AIED. There still exists some controversy in
designating the disease “autoimmune”, as the
causative agent that triggers the immune response
has not yet been identified (Furohashi, et al.
2002).
INTRODUCTION
Immunological mechanisms play an etiological
role in the diseases of the inner ear, despite
the fact that this organ is enclosed within a
bony capsule and lack of lymphatic drainage.
Autoimmune inner ear disease “AIED” may
be in the form of hearing loss and/or dizziness
which is caused by antibodies or immune cells
attacking the inner ear. AIED may be the result of
either organ-specific disease or systemic disorder
(Matt, 2006). In organ specific autoimmunity, the
immune reaction is localized to the inner ear only.
In the case of vestibule-auditory autoimmunity
there should be auto-antibodies or evidence of
cell mediated immune responses (lymphocyte
transformation, lymphokine production, or
delayed hypersensitivity) directed against inner
ear antigens (cochlear, vestibular, or both)
(Harris, 1998). On the other hand, an autoimmune
disease in other parts of the body can cause or
be associated with AIED. Systemic autoimmune
diseases coexist in 15-30% of patients with AIED
(Bovo, et al. 2006).
Although auto-antibodies and auto-reactive T
cells have been implicated in the pathogenesis of
SNHL, the identity of a highly relevant inner-ear
self antigen is still required. In recent years, the
role of Anti-HSP70, present in the outer hair cells
of inner ear, has been suggested which can predict
corticosteroid responsiveness (Hain, 2003).
AIM OF THE WORK
Our objective was to delineate to what extent
the SNHL is due to autoimmune malfunction as
well evaluate the role of the serum anti-HSP70 as
a marker for autoimmune SNHL.
Autoimmune SNHL is a clinical syndrome that
typically produces a rapidly progressive hearing
loss. It is defined as greater than 30 decibels (dB)
hearing reduction, over at least 3 contiguous
frequencies, occur over 72 hours or less. It occurs
most frequently in the 30 to 60 year age group
and affects males and females equally (Timothy
and Hain, 2007).
PATIENTS AND METHODS
A prospective study was conducted on 45
patients (24 males and 21 females), whose ages
ranged from 20 to 60 years. They were selected
from the audiology outpatient clinics of Hearing
and Speech Institute during the period from
October 2005 to December 2006. All cases
complained of sudden progressive hearing loss
with no previous history of ear disease and were
presented within two weeks from onset of the
disease. They were neurologically free with
normal middle ear function. In addition, Fifteen
healthy individuals (6 males and 9 females), whose
ages ranged from 23 to 50 years were included as a
control group. They were volunteers from patient
The hearing loss is usually unilateral and is
often accompanied by tinnitus, vertigo, or both.
The amount of hearing loss may vary from mild
to severe and may involve different parts of
the hearing frequency range. Hearing loss may
be temporary or permanent (Rambold et al.,
2005). Lehnhardt (1958) first introduced the
possibility that some of these cases of rapidly
progressing SNHL caused by an immune process
in the cochlea. Schiff and Brown (1974) further
hypothesized that the process was most likely of
2
Farouk et al.
relatives or subjects coming for regular cheek
up. All controls were having normal peripheral
hearing with no medical or ENT complaint. All
cases and controls were subjected to full history
taking and general and otoscopic examination.
provided from Orgentic Diagnostika, NY, U.S.A
for ASMA and Immco Diagnostic, NY U.S.A. for
ANA, respectively.
Anti-HSP70 antibodies were detected by
Western blot immunoassay. Kit was provided
from Otoimmune Diagnostic a division of
IMMCO Daignostic, NY, U.S.A.
Pure tone audiometry was done which
included air conduction for octave frequencies
of (250-8000Hz) and bone conduction for octave
frequencies of (500-4000Hz) in sound treated
room using two channel clinical audiometer
Interacoustics model AC40 calibrated according
to ANSI (1969). The air conduction stimulus
was delivered via supraaural headphones (TDH
49),the bone conduction stimulus was delivered
via bone vibrator B71.
In this technique, microwells were coated with
anti-nuclear antigen for ANA and extractable
nuclear antigen for ASMA, followed by blocking
the unreacted sites to reduce non-specific
binding. Controls, calibrator and patient serum
samples were incubated in the antigen coated
wells which allow specific antibodies present in
the serum to bind. Unbound-antibodies and other
serum proteins were removed by washing the
microwells. Bound antibodies were detected by an
enzyme labeled anti-human IgG conjugate added
to the wells. Unbound conjugates were removed
by washing. Specific enzyme substrates were then
added to the wells and the presence of antibodies
was detected by colour changes produced by the
conversion of the substrate. The reaction was
stopped and the intensity of the colour changes
were proportional to the concentration of the
antibodies, the absorbance of each microwell was
read at 405 nm for ANA and 450 nm for ASMA
using microplate reader against the reagent blank
set at zero absorbance. The concentrations were
calculated using computer software capable of
generating a four parameter algorithm.
Speech audiometry was performed including
speech reception threshold (SRT) using Arabic
spondee words and speech discrimination score
(SD%) using Arabic phonetically balanced
words.
Immittancemetry including tympanometry
and acoustic reflex threshold measurement using
clinical immittancemeter (Interacoustics model
AZ 26), was also preformed.
More advanced audiological tests (Auditory
brain stem evoked potentials and transient evoked
otoacoustic emission) were done when needed in
some cases to exclude retrocochlear lesion.
Ten ml venous blood were collected from each
individual in a plain tube, allowed to clot and
centrifuged to get serum that was stored at –20°C
as aliquots until use. The serum from each subject
was collected individually. All samples were
subjected to some non-specific immunological
tests: CRP, RF, ANA and ASMA, as well as
detection of anti-HSP70. Determination of CRP
and RF was performed by turbidimetric assay
with kits provided from Human, Germany.
Quantitative measurement of serum ASMA
and ANA was performed by ELISA with kits
In the detection of anti HSP70, antigen was
purified from bovine kidney cell line. Purified
HSP70 antigen was mixed with a MW marker
of 61kDa and subjected to polyacrylamide gel
electrophoresis and transferred to a polyvinylidene
fluoride (PVDF) membrane. The membranes
were cut into 3 mm x 5 cm strips using blunt
forceps, the required number of strips was placed
and labeled side up into individual wells of the
assay tray soaked in blocking-diluent (1 ml / strip)
to block non-specific binding sites and incubated
3
Hearing Loss: Immunological Aspects
at least 30 minutes. Then 10 μl of positive and
negative controls and patient samples were
added into appropriate wells, incubated 60 min
at room temperature with shaking thus antibodies
specifically bind to the HSP70 protein on the strip.
All strips were washed triple, each for 5 min with
gentle agitation by washing buffer. One ml/strip
conjugate A was added followed by incubation
for 30 minutes at room temperature with shaking.
All strips were washed triple as above, then 1 ml
/ well conjugate B was added and incubated for
30 min at room temperature with shaking. After
that all strips were washed as above and 1 ml
of a precipitable substrate was added into each
strip. Strips were incubated with gentle shaking
for 20 min and were let to dry for 15-20 min in
the dark at room temperature. Finally, all strips
were removed form assay tray and were placed
gently onto absorbent filter paper and were let to
dry for 15-20 minutes. HSP70 antibodies positive
reactions appear as a blue-violet band at 70 KD.
Check for a crisp band on the test strip that
aligns with 70 kDa band on the positive strip.
Positive reactions can occur in varying intensities
from weak to strong. Weak reactions should be
compared with baseline reaction intensities at the
corresponding position on the negative control
strip.
Statistical analysis: was done using SPSS
system version 11.5 in form of Student “t”
test, Chi-squire (X2) and distribution and
multiregression analysis. Results were considered
not significant if P > 0.05, significant if P < 0.05
and highly significant if P < 0.001.
RESULTS
Pure tone audiometric test results revealed
that 14 (31.1%) of the patient group had
moderate hearing loss, 16 patients (35.6%) had
sever hearing loss and 15 patients (33.3%) had
profound hearing loss. No statistically significant
difference was found between numbers of patient
in different degrees of hearing loss.
The strips were blotted with three proteins
of MW 61, 70 and 72 kDa. The 61 kDa protein
served as MW marker, the 70 kDa band composed
of inducible HSP70 protein and 72 kDa bands
consisted of an unrelated protein co-purified with
the HSP70 protein. Three steps to read the results
were described as follows:
•
Hold test strip between the positive and
negative control reactions on the provided,
laminated control card and align test
strip using the 61 kDa MW marker as
the reference point. The control strips are
mounted on gridlines to facilitate accurate
alignment of the test strips with the control
strips.
•
Compare reaction of test strip with
those of the controls on either side. Use
of a magnifying glass facilitate proper
alignment of test strips along the MW
marker and assist in observation of weak
reactions.
Males were more in number than females with
statistically insignificant difference.
The frequency and the percentage of the
positive immunological parameters in patients
group are shown in Table-1. A total of 27 (out of
45) patients had positive anti-HSP70 representing
60%, 8 patients were positive for CRP (17.8%),
while 7 patients showed positive RF (15.6%). As
regards ANA and ASMA, only 2 and 4 patients
were positive which represent 4.4% and 8.9%,
respectively.
No significant differences were noticed in the
values of CRP, RF, ANA and ASMA of patients
and controls, (Data are not shown).
4
Farouk et al.
In positive anti-HSP70 antibodies, 9 patients had
moderate hearing loss (HL), 8 had severe HL and
10 had profound HL. While among the negative
anti-HSP70 group, 5 patients had moderate HL,
8 had severe HL and 5 had profound HL. These
differences were statistically (by Chi-square tests)
not significant (P >0.05, data are not shown).
Table 1: Frequency and percentage of positive immunological
parameters in the patients group.
Parameters
Frequency
Percentage
CRP (>5 mg/l)
8
RF (>8 mg/l)
7
15.6 %
ANA (>1 EU/ml)
2
4.4 %
ASMA (>25 EU/ml)
4
8.9 %
Anti-HSP70 (Positive)
27
60 %
17.8 %
Table 3: Comparison between anti-HSP70 +ve and antiHSP70 -ve groups as regards immunological parameters.
Upon the result of anti-HSP70, our patients were
divided into two groups positive group (n=27)
and negative group (n=18). Table (2) shows the
percentage of positive studied immunological
parameters in patients with positive anti heat shock
protein antibodies (27 patients), CRP (22%), RF
(11%), ANA (3.7%) and ASMA (14.8%). In
Table (3), the comparative analysis of CRP, RF,
ANA and ASMA in positive and negative HSP70
groups revealed a highly statistically significant
elevation for ASMA in anti-HSP70 positive
patients (P <0.001).
Frequency
Percentage
(CRP (>5 mg/l
6
% 22
(RF (>8 mg/l
3
% 11
(ANA (>1 EU/ml
1
% 3.7
(ASMA (>25 EU/ml
4
% 14.8
Anti-HSP70
negative group
Mean ± SD
CRP
(mg/l)
2.18 ± 4.96
2.44 ± 4.06
:P > 0.05
N.S
RF
(mg/l)
2.43 ± 5.85
2.78 ± 6.28
P > 0.05
N.S
ASMA
(Eu/ml)
9.10 ± 14.11
6.32 7.57±
P <0.001
H.S
ANA
(Eu/ml)
0.28 ± 0.35
0.45 ± 0.30
P > 0.05
N.S
P-value
P > 0.05: not significant (N.S), P < 0.05: significant,
P < 0.001: highly significant (H.S).
DISCUSSION
The importance of diagnosing an immunemediated inner-ear disorder is highlighted in the
context of being one of few forms of treatable
inner-ear disorder with a good response to
immunosuppressive therapy (Agrup and Luxon,
2006). The diagnosis is based on history, findings
on physical examination, laboratory investigations
and the results of hearing and vestibular tests
(Hain, 2003).
Table 2: Frequency and percentage of positive immunological
parameters in positive anti-HSP70 group (n=27).
Parameters
Anti-HSP70
positive group
Mean ± SD
Parameters
Our goal was to investigate the humoral
immune response to inner ear components in
patients with SNHL and to determine whether
these immunological tests can play a role for
early diagnosis of AIED and what are the most
beneficial tests.
In a comparison between the previous 2 groups
as regards the gender, there was no significant
difference between them (p>0.05, data are not
shown).
In the present study, some general
immunological laboratory investigations namely
5
Hearing Loss: Immunological Aspects
CRP, RF, ANA and ASMA, moreover, one
specific test (anti-HSP70) were carried out on
45 patients having SNHL, as well as 15 healthy
subjects were included as a control group.
et al. (2002) and Mafong et al. (2002). In the
present study positive ANA cases represented
only 4.4 % of the total number of patients, while
in Garcia Berrocal et al. (2002) study ANA
positive cases were found in 34.4% of patients
and Mafong et al. (2002) investigated children
with SNHL, ANA was present in 25% of them.
This difference can be explained by the diverse
clinical forms of the disease which may include
other systemic autoimmune diseases in the work
of Garcia Berrocal et al. (2002) and by the
limited age group in the study of Mafong et al.
(2002) which concentrated on 114 children with
no adult cases.
It was evident from results that patients with
idiopathic SNHL can present with any degree of
hearing loss as there was no significant increase
in number of patients in any specific degree of
hearing loss.
It was found that the percentage of male to
female patients was 53.3% to 47.7 %, respectively.
These results are in agreement with Hain (2003),
who found that AIED affects males and females
equally. On the contrary, Harris and Ryan, (1995)
found in their study that 65 % of AIED patients
were female. Rahman et al. (2001) reported also
that more women than men have AIED. They
explained this finding by the fact that, 75% of
all autoimmune diseases occur in women. The
difference between the results of these two studies
on one hand and our study on the other hand may
be attributed in part to the negligence of females
in our area to seek medical advice (as all subjects
in our study were collected from out patient
clinic of Hearing and Speech Institute, Imbaba,
Giza which is located in one of the most famous
rural areas in Egypt). In addition, the majority of
patients in this study did not suffer from systemic
autoimmune diseases hence there is no reason to
correlate between sex and percentage of affection
as in the studies of Harris and Ryan (1995) and
Rahman et al. (2001).
As regards ASMA, the percentage of positive
ASMA was 8.9 %, with highly statistically
significant difference between patients with antiHSP70 positive and negative groups. Our results
are in agreement with Yoshida et al. (1996)
providing a relatively high incidence for ASMA
in the patients with SNHL.
Heat shock protein, present in the outer hair
cells of inner ear, is believed to protect cells
by dissolving and refolding the misfolded or
denatured protein and is induced by various
forms of stress including heat, ischemia and toxic
agents. However, this antigen is also recognized
in brain, intestine, liver, kidney and muscle (Oh
et al. 2000).
Antibodies against HSP70 were used to prove
the humoral immune response of inner ear
components in patients with SNHL and also used
for early diagnosis and its presence correlates
with disease activity and responsiveness to
corticosteroid treatment (Naumann, 2001).
Mc Cabe (1989) proposed the usage of ANA
and RF as a screening panel for AIED in high risk
patients. As regards to RF in our study, 7 out of
45 patients (15.6 % ) were positive, which is very
comparable with the percentage of positive RF in
the study of Toubi et al. (2004) (6 out of 51 with
percentage of 12 %).
In the present study, 60 % of our patients
suffered from SNHL were found to be positive
for anti-HSP70 (27 out of 45), whereas none of
control group was positive. Our results confirmed
the results of Harris and Sharp (1990), Moscicki
et al. (1994) and Disher et al. (1997) who found
For ANA, the results are controversial between
the present work and those of Garcia Berrocal
6
Farouk et al.
that the sera of about 50% of patients with SNHL
reacted with 68-kDa protein constituent of inner
ear extract.
in 67 patients with SNHL and age-sex matched
20 normal controls by highly sensitive ELISA
test and confirmed with WB immunoassay.
They found that serum anti-HSP70 levels were
significantly higher in the patients with SNHL
than those of normal controls by ELISA test (P
< 0.001) and their presence were confirmed as
strong bands in the WB assay. Our results are in
agreement with Tebo et al. (2007) and Bonaguri
et al. (2007),who used the WB assay to assess
IgG antibody responses to the recombinant
human HSP70, revealing that 33% of patients
with idiopathic SNHL had positive result in
comparison controls.
Also, Hirose et al. (1999) recommended
the Western blot (WB) assay as the best for
testing anti-HSP70 predicting corticosteroid
responsiveness on their studied 82 SNHL patients,
that were evaluated with review of clinical
history, laboratory tests, audiograms, response to
therapy and final diagnoses including the results
of ESR, CRP and the WB assay for anti-HSP70.
Despite its low sensitivity (42%)), the specificity
was 90% and the positive predictive value of this
test was excellent at 91%. The ESR was as good
as the CRP in detecting acute-phase reactants.
Finally, we tried to find a correlation among
all the studied parameters in our study. Upon the
positivity of anti-HSP70, the patients group was
divided into two groups. As regards the relation
between anti-HSP70 (positive and negative
groups) and other general immunological
parameters, ASMA was the only parameter that
showed a highly significant elevation in positive
group. This means that positive ASMA in patients
with sudden hearing loss is suggestive of positive
anti-HSP70, while no statistically significant
differences were noticed between both groups as
regards the other parameters.
Accordingly, Naumann, (2001) studied the
presence of antibodies to inner ear components
by immuno-blotting extracted bovine inner ear
proteins in patients with hearing impairment.
Forty percent of the patients with progressive
SNHL showed reactivity against a 68-kDa
protein extracted from bovine inner ear, 10%
with Meniere’s disease showed reactivity against
this protein. Approximately 6% of the patients
with otosclerosis showed reactivity to inner ear
proteins. Our positive cases represented about
46% of all patients with HL. The slight difference
between results of Naumann (2001) and ours
may be due to the origin of HSP70. In our study,
HSP70 antigen was obtained from the bovine
kidney cell line, while in Naumann’s study 68kDa protein was extracted from bovine inner ear.
The sensitivity of anti-HSP70 antigen from the
bovine kidney cell line is more than that of 68kDa protein from bovine inner ear.
In conclusion, the results of the present study
indicate that CRP and RF can be used as a screening
pannel for AIED in high risk patients. AntiHSP70 can be used to prove the humoral immune
response of inner ear components in patients with
SNHL. Other immunological investigations as
ASMA and ANA are recommended when general
autoimmune disorders are suspected.
Also, Zeitoun et al. (2005) studied 63 patients
with rapidly progressive SNHL of unknown
cause suggestive of autoimmune SNHL for the
presence of anti-HSP70 by WB. More than half of
the patients (59%) had antibodies to HSP70. Park
et al. (2006) estimated serum anti-HSP70 levels
Further investigations on a large scale are
needed to evaluate the role of other antibodies
as anti-phospholipids antibodies and antiendothelial antibodies to confirm the autoimmune
pathogenesis of inner ear diseases.
7
Hearing Loss: Immunological Aspects
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