I.M. Rea et al. / Experimental Gerontology 36 (2001) 341±352

Experimental Gerontology 36 (2001) 341±352

341

www.elsevier.nl/locate/expgero

Serum heat shock protein and anti-heat shock protein
antibody levels in aging
I.M. Rea a,*, S. McNerlan a, A.G. Pockley b
a

Department of Geriatric Medicine, The Queen's University of Belfast, Whitla Medical Building,
97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK
b
Division of Clinical Sciences (NGH), Clinical Sciences Centre (University of Shef®eld),
Northern General Hospital, Herries Road, Shef®eld S5 7AU, UK
Received 10 April 2000; received in revised form 25 October 2000; accepted 30 October 2000

Abstract
We have previously reported the presence of Hsp60 and Hsp70 in the peripheral circulation of
normal individuals. Given that the capacity to generate stress proteins declines with age, this study
measured Hsp60 and Hsp70 levels in the sera of 60 individuals aged between 20 and 96 years. Levels
of anti-human Hsp60, anti-human Hsp70 and anti-mycobacterial Hsp65 antibody were also
measured. Senieur-approximated elderly subjects were well and randomly selected from the Belfast
Elderly Longitudinal Free-living Aging STudy (BELFAST). Samples from younger individuals were
obtained from the Northern Ireland Blood Transfusion Service. Hsp60, anti-Hsp60, anti-Hsp70 and
anti-mycobacterial Hsp65 antibodies were detected in all samples, whereas Hsp70 was detectable in
only 46 of the samples analysed (77%). Regression analysis revealed a progressive decline in Hsp60
(759 ng/ml ,40 years; 294 ng/ml $90 years) and Hsp70 (400 ng/ml ,40 years; 20 ng/ml $90
years) levels with age whereas no relationship was apparent for anti-Hsp60 and Hsp65 antibody
levels. Hsp70 antibody levels tended to increase with age (115 U/ml ,40 years; 191 U/ml $90
years). This study in Senieur-approximated subjects demonstrates an apparent decrease in Hsp60 and
Hsp70 with increasing age that does not appear to be related to anti-heat shock protein antibody
status. These ®ndings support in vitro work that demonstrates an age-related reduced ability to
respond to stress. Further studies are required to understand the basis for declining serum Hsp60
and Hsp70 levels in aging and to elucidate their origin and role in the maintenance of homeostasis
and resistance to environmental challenges. q 2001 Elsevier Science Inc. All rights reserved.
Keywords: Heat shock proteins; Aging; Serum; Enzyme immunoassay

* Corresponding author. Tel.: 144-1232-272-156; fax: 144-1232-325-839.
E-mail address: i.rea@qub.ac.uk (I.M. Rea).
0531-5565/01/$ - see front matter q 2001 Elsevier Science Inc. All rights reserved.
PII: S 0531-556 5(00)00215-1

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I.M. Rea et al. / Experimental Gerontology 36 (2001) 341±352

1. Introduction
HEAT SHOCK or stress proteins belong to several families based on molecular weights
and are among the most phylogenetically conserved proteins. They exhibit a range of
intracellular functions including the stabilisation and protection of newly synthesised
proteins (Hightower, 1991). In addition to being constitutively expressed, stress proteins
are induced in response to biological (infection, certain cytokines) and physicochemical
(heat, toxic substances) factors (Kiessling et al., 1991).
Although typically regarded as being intracellular, it is now apparent that heat shock
proteins can be released from cultured cells (Hightower and Guidon, 1989; Child et al.,
1995; Bassan et al., 1998; Liao et al., 2000) and circulating Hsp60 and Hsp70 has been
identi®ed in groups of normal individuals (Pockley et al., 1998, 1999; Xu et al., 2000). The
capacity of cells to respond to stress and synthesise heat shock proteins is reduced with age
(Faassen et al., 1989; Fargnoli et al., 1990; Nitta et al., 1994; Heydari et al., 1995;
Pahlavani et al., 1995) and this study determined whether the age-related decline in the
capacity to generate stress responses as demonstrated in vitro are re¯ected by changes in
circulating heat shock protein levels in vivo. In that regard, Hsp60 and Hsp70 levels in the
serum of 60 apparently well subjects ranging from less than 40 to over 90 years old were
measured by enzyme immunoassay, as were anti-human Hsp60 (hHsp60), anti-human
Hsp70 (hHsp70) and anti-mycobacterial 65 kDa protein (mHsp65) antibody levels.
2. Subjects, materials and methods
2.1. Subjects
Elderly subjects were randomly selected from the previously described Belfast Elderly
Longitudinal Free-living Aging STudy (BELFAST; (Rea et al., 2000)). Volunteers in the
older age groups were assessed at home by medical and functional history, and their
mental competence was evaluated using the Mini Mental State Examination (MMSE;
(Folstein et al., 1975)). Subjects who were overtly unwell, gave a history of cancer,
were taking immune modulating drugs or who scored less than 28 on the MMSE (normal
range, 28±30) were excluded. Elderly subjects participating in the study were therefore
apparently well, living in their own homes, cognitively intact and mobile and met the
clinical (Lighhart et al., 1984), but not necessarily the biochemical criteria for Senieur
status over which there has been debate (Reibnegger et al., 1988). Younger subjects were
randomly selected volunteers from transfusion donor sessions at the National Blood
Service, Belfast City Hospital, Northern Ireland. Permission for the study was obtained
from the Ethical Committee of the Queen's University of Belfast. EDTA anti-coagulated
blood was collected in the morning and returned to the laboratory within 90 min.
2.2. Enzyme immunoassays for Hsp60 and Hsp70
Serum Hsp60 and Hsp70 levels were determined as described (Pockley et al., 1998,
1999). Microtiter plates were coated with a murine monoclonal antibody (mAb) to human
Hsp60 (clone LK1) or Hsp70 (clone C92F3A-5; StressGen Biotechnologies Corp.,

I.M. Rea et al. / Experimental Gerontology 36 (2001) 341±352

343

Table 1
Composition of age groups
Age groups

Total

, 40

40±69

70±89

$ 90

Males
Females

31
29

7
6

11
5

8
12

5
6

Total

60

13

16

20

11

Victoria, Canada). Plates were washed and non-speci®c binding sites were blocked with
bovine serum albumin (Sigma Chemical Company Limited, Poole, UK). One hundred
microlitres of recombinant human Hsp60 (0±2500 ng/ml), recombinant human Hsp70 (0±
20,000 ng/ml; StressGen) or samples (typically 1:5 dilution) were added and the plates
incubated for 1 h at room temperature. Bound heat shock protein was detected using rabbit
polyclonal anti-Hsp60 or anti-Hsp70 antibody (StressGen) followed by alkaline phosphatase (AP)-conjugated murine anti-rabbit immunoglobulin mAb. p-nitro phenyl phosphate
(p-NPP) substrate was added and the resulting absorbance was measured at 405 nm. The
concentration of heat shock proteins was determined from the standard dose±response
curves using ASSAYZAP data analysis software (BIOSOFT, Cambridge, UK)
2.3. Anti-Hsp60, -Hsp70 and Hsp65 antibody levels
Anti-heat shock protein antibody levels were also determined as described (Pockley et
al., 1998, 1999). Microtitre plates were coated with recombinant human Hsp60 (hHsp60),
recombinant human Hsp70 (hHsp70; StressGen) or recombinant M.bovis Hsp65
(mHsp65), blocked and washed as described above. Recombinant mycobacterial Hsp65
was obtained from Dr M. Singh, UNDP/World Bank/WHO Special Program for Research
and Training in Tropical Diseases. Test serum (typically 1/100 dilution) was added and
Table 2
Subject characteristics (n/a, sample not available for analysis)
Age group (years)
, 40
Haemoglobin, gm/dl a
Total leukocytes, £ 10 9/l
Neutrophil count, £ 10 9/l
CD3 1/CD25 1, £ 10 9/l
Systolic blood pressure, mm Hg
Diastolic blood pressure, mm Hg
Cholesterol, mmol/l
Urea, mmol/l
Glucose, mmol/l
Albumin, mmol/l
a

40±69

70±89

$ 90

Anova

12.97 (2.76)
5.99 (1.68)
3.22 (1.03)
0.31 (0.18)
n/a
n/a
n/a
n/a
n/a
n/a

14.32 (1.07)
5.77 (1.65)
3.49 (1.22)
0.25 (0.12)
n/a
n/a
n/a
n/a
n/a
n/a

13.27 (1.43)
6.53 (1.78)
4.07 (1.58)
0.22 (0.15)
141.3 (15.2)
85.2 (9.0)
5.61 (1.47)
7.91 (3.1)
5.21 (0.97)
40.2 (2.33)

13.45 (1.78)
6.16 (1.35)
3.50 (0.83)
0.19 (0.11)
131.8 (12.9)
83.6 (6.0)
5.05 (0.31)
6.21 (1.57)
4.47 (0.63)
40.4 (1.69)

p  0.16
p  0.51
p  0.21
p  0.17
p  0.08
p  0.6
p  0.31
p  0.15
p  0.07
p  0.85

Data presented as means with standard deviations in brackets.

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I.M. Rea et al. / Experimental Gerontology 36 (2001) 341±352

bound antibodies were detected using AP-conjugated goat anti-human IgA/G/M
polyclonal antibody followed by p-NPP substrate (Sigma).
All sera were pre-screened and samples having a high level of the respective heat shock
protein antibody were identi®ed. These samples were assigned a concentration of
1000 arbitrary units/ml (Aunits/ml) and were used to generate standard dose±response
curves from which antibody levels in sera were determined.
2.4. Statistical methods
Data were analysed using SPSS 10.0 for Windows. Heat shock protein and heat shock
protein antibody level data demonstrated a skewed distribution and the data in Table 3 are
therefore presented as medians and ®rst and third quartiles (Q25 and Q75). Four age groups
were de®ned: ,40, 40±69, 70±89, and $90 years and the composition of these groups and
their characteristics are detailed in Tables 1 and 2. Comparisons in heat shock protein and
heat shock protein antibody levels between the ,40 and $90 age groups were performed
on logarithmically transformed data using an independent-samples t-test. The strength of
relationships between variables was evaluated using the Pearson correlation and linear
regression analysis of log-transformed data. A p-value ,0.05 was considered to indicate
statistically signi®cant differences/relationships in all cases.
3. Results
3.1. Subject characteristics
The ages and sex of the subjects in the four age groups are shown in Table 1.
Haemoglobin, white cell and neutrophil count and the activation subset CD3 1CD25 1,
which is associated with immune activation, were measured in the four subject groups and
there were no signi®cant differences in in¯ammatory markers between the four groups
(Table 2). There were no differences in systolic and diastolic blood pressure, cholesterol,
urea, glucose and albumin, between the older age groups (79±89 and .90 years) and
values were within normal laboratory range. This information was not available for the
younger groups as these individuals were recruited from the donor sessions at the National
Blood Service, Belfast. However, these subjects would not have been permitted to donate
blood had there been any evidence of recent infection, anaemia or serious illness, or had
they been taking any medication.
Given that higher autoimmune reactions in the elderly age group may have affected the
measured parameters, an autoimmune screen of the older subjects including anti-nuclear
antibody, anti-double stranded DNA, p ANCA, RA latex and anti-cardiolipin antibodies
was performed. Six subjects in the 70±89 and $90 years age-groups showed slightly
positive ANA (data not shown), however, overall the autoimmune screen gave no support
for the belief that signi®cant autoimmune disease increases with age, at least in this
Senieur-approximated study group. There was no association with increased heat shock
protein antibody levels in those instances where a positive autoimmune screen was present
(data not shown).
In addition, it could be suggested that a less rigorously selected older population may

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345

Table 3
Serum heat shock protein and heat shock protein antibody levels by age group (**p  0.02, *p  0.03, ns Ð nonsigni®cant versus ,40 age group (independent-samples t-test on log transformed data))
Age group (years)
, 40
Hsp60, ng/ml a
Hsp70, ng/ml
Positive samples b
Anti-hHsp60, U/ml
Anti-hHsp70, U/ml
Anti-mHsp65, U/ml

40±69

70±89

$ 90

759 [239±1356]
400 [60±1520]
10/13; 77%
482 [427±603]
115 [102±144]
119 [48±267]

383 [145±777]
80 [40±315]
14/16; 88%
439 [370±491]
143 [130±172]
201 [142±291]

221 [49±547]
50 [0±270]
14/20; 70%
389 [234±548]
232 [134±269]
138 [107±226]

294 ns [117±361]
20** [0±245]
8/11; 73%
577 ns [486±783]
191 ns [146±267]
268* [181±507]

a

Data are presented as medians with inter-quartile ranges in brackets.
All 60 samples contained detectable levels of Hsp60 and anti-hHsp60, anti-hHsp70 and anti-mHsp65 antibodies. The numbers of samples with detectable levels of Hsp70 is indicated.
b

include subjects with possible impaired renal function, in which case renal factors may
contribute to an increased heat shock protein antibody levels. However, urea levels in our
population group were within the normal range even for the oldest age groups (Table 2).
3.2. Serum Hsp60 and Hsp70 levels and age
Hsp60 was detected in all 60 of the serum samples analysed (Table 3). Compared to the
,40 age group, Hsp60 levels were lower in the $90 age group, although this failed to
reach statistical signi®cance p  0:058; Table 3). An age-related decline in Hsp60 levels
was indicated by correlation/regression analysis (Fig. 1). Circulating Hsp70 was also
detected, although in only 46 of the 60 samples assayed (77%). Hsp70 levels were
4.50

p=0.006
4.00

Log Hsp60

3.50
3.00
2.50
2.00
1.50
1.00
20

30

40

50

60

70

80

90

100

Age (years)
Fig. 1. Regression plot of Hsp60 levels in the peripheral circulation and age. Data are log values.

346

I.M. Rea et al. / Experimental Gerontology 36 (2001) 341±352

p=0.029

4.50
4.00

Log Hsp70

3.50
3.00
2.50
2.00
1.50
1.00
20

30

40

50

60

70

80

90

100

Age (years)
Fig. 2. Regression plot of Hsp70 levels in the peripheral circulation and age. Data are log values.

signi®cantly lower in the $90 age group and declining levels were signi®cantly associated
with increasing age (Table 3; Fig. 2).
3.3. Serum anti-heat shock protein antibodies and age
Anti-heat shock protein antibodies were detected in all individuals. Anti-hHsp60 levels
showed no signi®cant age-related change or gender difference (Table 3; Fig. 3). Although
regression analysis indicated no signi®cant relationship between anti-mHsp65 antibody
levels and age in the entire subject group, levels in the $90 age group were signi®cantly
higher than those in the ,40 years age group (Table 3). Regression analysis revealed a
signi®cant increase in anti-hHsp70 antibody levels with age (Fig. 3), however, this
increase was not of statistical signi®cance when the ,40 and $90 age groups were
directly compared (Table 3). These ®ndings exclude the possibility that reduced serum
levels of Hsp60 and Hsp70 in older subjects was due to an increase in the levels of their
respective antibodies.
Hsp60/anti-Hsp60 antibody levels and Hsp70/Hsp70 antibody levels appeared to be
related, however, this relationship was not statistically signi®cant for the latter (Fig. 4).
4. Discussion
Recent studies have identi®ed Hsp60 and Hsp70 in the circulation of normal
individuals (Pockley et al., 1998; Pockley et al., 1999; Xu et al., 2000) and indicated that an elevated level of circulating heat shock proteins is associated with
cardiovascular disease (Pockley et al., 2000; Wright et al., 2000; Xu et al., 2000).
This study has identi®ed an apparent decrease in serum Hsp60 and Hsp70 levels
with increasing age. Although a number of groups have reported the release of heat

I.M. Rea et al. / Experimental Gerontology 36 (2001) 341±352

347

Log anti-hHsp60

4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
20 30 40 50 60 70 80 90 100

Age (years)

Log anti-hHsp70

4.50

p=0.017

4.00
3.50
3.00
2.50
2.00
1.50
1.00
20 30 40 50 60 70 80 90 100

Age (years)

Log anti-mHsp65

4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
20 30 40 50 60 70 80 90 100

Age (years)
Fig. 3. Regression plots of anti-hHsp60 (upper), anti-hHsp70 (middle) and anti-mHsp65 (lower) antibody levels
in the peripheral circulation and age. Data are log values.

shock proteins from intact cells (Hightower and Guidon, 1989; Child et al., 1995;
Bassan et al., 1998; Liao et al., 2000), the source of in vivo circulating heat shock
proteins is also currently unknown. Cultured human peripheral blood mononuclear
cells do not release heat shock proteins (Pockley et al., 1999), whereas vascular

348

I.M. Rea et al. / Experimental Gerontology 36 (2001) 341±352

4.00

p=0.025
Log anti-hHsp60

3.50
3.00
2.50
2.00
1.50
1.00
1.00

1.50

2.00

2.50

3.00

3.50

4.00

Log Hsp60
3.00

Log anti-hHsp70

2.50

2.00

1.50

1.00
1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

Log Hsp70
Fig. 4. Regression plots for log Hsp60 (upper panel) and Hsp70 (lower panel) concentrations with levels of their
respective antibody. The Kendall correlation suggested a relationship between Hsp60 and anti-Hsp60 antibody
levels, although this was not of statistical signi®cance (p  0.07).

smooth muscle cells can release heat shock proteins in response to oxidative stress
(Liao et al., 2000). It therefore seems likely that there are different sources of, and
conditions for heat shock protein release (Marienthoz et al., 1997).
The physiological role(s) of circulating heat shock proteins have also yet to be de®ned.
They may be cytoprotective, as Hsp70 enhances the survival of cultured arterial smooth
muscle cells (Johnson et al., 1990; Johnson and Tytell, 1993) and protects cells from
apoptosis via a cytochrome c-mediated mechanism (Mosser et al., 2000). Heat shock
protein reactive T cells are present in the peripheral circulation (Munk et al., 1989;
Cohen, 1996; Ramage et al., 1997; Ramage et al., 1999) and self-heat shock protein

I.M. Rea et al. / Experimental Gerontology 36 (2001) 341±352

349

responsiveness can down-regulate autoimmune responses (Anderton et al., 1995;
Anderton and van Eden, 1996), suggesting that self-reactivity to these proteins may be
involved in the regulation of potentially harmful pro-in¯ammatory responses. However,
no association between heat shock proteins and autoimmune reactivity in the Senieurapproximated group was observed in this study.
The reason for the progressive decline in serum Hsp60 and Hsp70 levels with aging is
not clear. It may re¯ect a reduced capacity to synthesise these proteins, which would be
consistent with in vitro ®ndings that Hsp70 expression in heat-stressed lung cells (Fargnoli
et al., 1990), hepatocytes and liver (Heydari et al., 1995; Hall et al., 2000), splenocytes
(Pahlavani et al., 1995), myocardium (Gray et al., 2000) and mononuclear cells is reduced
with increasing age (Richardson and Holbrook, 1996), as is the induction of Hsp70
expression in response to ischaemia (Nitta et al., 1994) and mitogenic stimulation (Faassen
et al., 1989). Hsp70 gene expression declines during normal aging in human retina
(Bernstein et al., 2000) and heat shock-induced Hsp70 expression is decreased in
senescent and late passage cells, both of which suggest that the process of aging itself
may be associated with reduced Hsp70 production (Liu et al., 1989; Luce and Cristofalo,
1992; Effros et al., 1994).
If this were the case, suggested mechanisms for an attenuated stress response in aging
might include a reduced availability of Heat Shock Factor (HSF), which binds to Heat
Shock Element (HSE) prior to transcription (Richardson and Holbrook, 1996). Alternatively, there may be an age-associated increase in abnormal or denatured proteins that
could interfere with HSF binding to HSE (Munro and Pelham, 1985) or decrease in the
oligomerisation of HSF that is essential for binding to HSE. Certainly, work in Drosophila
supports the latter proposition (Gershon, 1998).
A previous study has reported an increase in the titres of anti-Hsp65 antibodies in
control subjects and patients having had a thrombotic stroke or transient ischaemic attack
in the age groups 40±49, 50±59, 60±69, 70±79 years and concluded that heat shock
protein antibody levels might be a marker of age (Mantle et al., 1995). In part, our ®ndings
con®rm this conclusion in that anti-mHsp65 antibody levels in the $90 age group were
signi®cantly higher than those in the ,40 years age group. However, a comparison of antiHsp65 antibody levels in all subjects over 70 with those in subjects ,40 in our study
revealed no signi®cant difference. Direct comparisons between the two studies are dif®cult, as our study did not include suf®cient patients in the respective age ranges, nor are the
two study groups directly comparable on the basis of selection and other factors. For
instance, nonagenarians in the BELFAST study have lower homocysteine levels than
similarly selected 70±89 year olds (Rea et al., 2000), suggesting that they have a different
atherosclerotic risk pro®le to those in the Mantle group that included subjects with
vascular disease.
The co-existence of heat shock proteins and anti-heat shock protein antibodies in serum
suggests that immune complexes may be present in the circulation. This observation poses
two key questions. Firstly, to what extent will immune complexes in¯uence the assays
used in this study and secondly, might immune complexes be removed from the
circulation.
The possibility that immune complexes interfere with the assays used in this study
cannot be excluded at the present time. Although antibodies to oxidised low-density

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I.M. Rea et al. / Experimental Gerontology 36 (2001) 341±352

lipoproteins (LDL) can exist in both the free form and as antigen±antibody
complexes (Mironova et al., 1997), immune complexes have been shown to interfere
with assays for anti-oxidised LDL antibody levels (Lopes-Virella et al., 1999).
However, soluble antigen±antibody complexes are relatively unstable and easily
dissociate into smaller complexes and free antigen and free antibody in vitro, the
dynamic equilibrium between which depends on antibody af®nity, the molar ratio of
antibody to antigen and the absolute concentration of the reagents (Knutson et al.,
1979). It is therefore possible that the presence of high af®nity monoclonal antibodies in the assay system accentuates the instability and promotes the dissociation
of immune complexes. Further studies in which immune complexes are precipitated
from serum using polyethylene glycol and the levels of antigen and antibody in the
non-precipitated fraction and the precipitated fraction after dissociation are required
(Mironova et al., 1997; Lopes-Virella et al., 1999).
Although heat shock proteins and their respective antibodies may form immune
complexes in vivo, their clearance depends on solubility, which in turn is in¯uenced by
the relative proportions of antigen and antibody, and the valency of the antigen (Van Es et
al., 1979). Certainly the deposition of immune complexes does not appear to be a problem
as we have no evidence that impaired renal function is a contributory factor across the age
groups included in this study.
Notwithstanding the complex relationship between circulating heat shock proteins
and heat shock protein antibodies and the potential in¯uence that immune
complexes may have on measured levels of these analytes, the substantial change
in Hsp60 and Hsp70 levels with increasing age remains an important ®nding that
warrants further investigation.
In summary, increased age is associated with a reduced capacity to maintain
homeostasis in all physiological systems and it may be that there is also a parallel and
progressive decline in the ability to produce heat shock proteins. If this is so, it could
contribute to the increased susceptibility to environmental challenges and the more
prevalent morbidity and mortality seen in aged individuals (Richardson and Holbrook,
1996). This study supports such a proposition, although the mechanisms underlying
declining levels of circulating heat shock proteins with age and its consequence on the
aging process have yet to be de®ned. Further work aimed at providing insight into the agerelated changes in heat shock protein levels and the role of these circulating proteins in
health and disease is undoubtedly required.
Acknowledgements
We are grateful to our elderly subjects, our General Practitioner colleagues, Dr C.
Baraucha, the Northern Ireland Blood Transfusion Service and to our dedicated research
nurse Anne Murphy for their willing help with this research project. We also thank Joey
Shepherd for her technical assistance. This study received ®nancial support from the
Department of Health and Social Services (Northern Ireland), the Wellcome Trust
(IMR; grant 045519) and the UNDP/World Bank/WHO Special Program for Research
and Training in Tropical Diseases (TDR).

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