Lowered Temperature Set Point for Activation of the Cellular
Stress Response in T-lymphocytes*
Lisa Q.
Gothard
,
Marvin E.
Ruffner§,
Jerold G.
Woodward§,
Ok-Kyong
Park-Sarge¶, and
Kevin D.
Sarge
From the Departments of
Molecular and Cellular
Biochemistry, § Microbiology, Immunology, and Molecular
Genetics, and ¶ Physiology, University of Kentucky, Chandler
Medical Center, Lexington, Kentucky 40536-0294
Received for publication, September 13, 2002, and in revised form, December 17, 2002
 |
ABSTRACT |
The induction of heat shock protein gene
expression in response to stress is critical for the ability of
organisms to cope with and survive exposure to these stresses.
However, most studies on HSF1-mediated induction of hsp70
gene expression have utilized immortalized cell lines and
temperatures above the physiologically relevant range. For these
reasons much less is known about the heat shock response as it occurs
in mammalian cells within tissues in the intact organism. To gain
insight into this area we determined the temperature thresholds for
activation of HSF1 DNA binding in different mouse tissues. We have
found that HSF1 DNA binding activity and hsp70 synthesis are
induced in spleen cells at significantly lower temperatures relative to
cells of other tissues, with a temperature threshold for activation
(39 °C) that is within the physiological range for fever.
Furthermore, we found that the lowered temperature set point for
induction of the stress response in spleen is specific to T-lymphocytes
residing within this tissue and is not exhibited by B-lymphocytes. This
lowered threshold is also observed in T-lymphocytes isolated from lymph
nodes, suggesting that it is a general property of T-lymphocytes, and
is seen in different mouse strains. Fever is an early event in the
immune response to infection, and thus activation of the cellular
stress response in T-lymphocytes by fever temperatures could serve as a
way to give these cells enough time to express hsps in anticipation of
their function in the coming immune response. The induced hsps likely
protect these cells from the stressful conditions that can exist during
the immune response, for example increasing their protection against
stress-induced apoptosis.
 |
INTRODUCTION |
When cells are exposed to elevated temperature, they respond by
rapidly increasing the expression of heat shock proteins (hsps), which
act to protect essential cellular functions from the adverse effects of
increased temperature (1-5). This phenomenon, known as the cellular
stress response, is mediated by a transcriptional regulatory protein
called heat shock factor 1 (HSF1),1 which exhibits
heat-inducible DNA binding activity. Upon exposure of cells to elevated
temperature, HSF1 is converted from an inactive monomeric form to a
trimeric DNA-binding form, which then interacts with specific sequences
in the promoters of hsp genes and induces their transcription
(5-11).
Fever represents a physiological example of elevated temperature in an
organism. Our previous studies analyzing HSF1 activation in a limited
set of mouse tissues in response to whole body hyperthermia at
different temperatures revealed that the threshold temperature for HSF1
activation can vary between tissues. The temperature threshold for
activation of HSF1 DNA binding in male germ cells is 35 °C,
consistent with the known temperature sensitivity of this cell type
(12). However, in the somatic testis cell types and liver cells of
these heat-treated mice, HSF1 DNA binding was not activated until a
temperature of ~42 °C was reached (12, 13). This is well above the
temperature of most fevers, leading us to question whether any cell
types in mammalian species exhibit HSF1 activation at lower
temperatures (e.g. 39-40 °C) more consistent with the
typical fever.
To test this we repeated our experiment and analyzed HSF1 activation
temperature thresholds in a larger group of mouse tissues, including
lung, spleen, kidney, and heart. Similar to previous results on liver
and somatic testis cells, lung, pancreas, and heart exhibited an HSF1
activation threshold of ~42 °C. However, HSF1 DNA binding was
activated in spleen beginning at a temperature of 39 °C, well within
the range of normal febrile temperatures. This was associated with
induction of hsp70 expression in this same temperature range. Two major
cell types present in spleen are B-cells and T-cells. Interestingly,
the lowered temperature threshold for HSF1 activation is exhibited by
T-cells but not B-cells, the latter exhibiting activation at
~42 °C. T-lymphocytes isolated from lymph nodes also show this
lowered threshold. Since appearance of fever is one of the first events
associated with infection, activation of the cellular stress response
in T-cells at febrile temperatures could serve to ratchet up the level
of stress protection in these cells in anticipation of the coming immune response, ensuring their ability to function under the stressful conditions that can exist during this response.
 |
EXPERIMENTAL PROCEDURES |
Experimental Animals and Whole-body Hyperthermia--
C3H/HeNCr
male mice were obtained from NCI (Charles River) and maintained under a
controlled light cycle (14 h light:10 h dark). These studies were
conducted in accordance with the National Institutes of Health Guide
for the Care and Use of Laboratory Animals and University of Kentucky
Guidelines. For whole-body hyperthermia, 2-month-old mice were placed
in modified Falcon tubes in water baths of various temperatures for 60 min. Core body temperatures taken using a Digisense thermocouple
(Cole-Parmer 8528-20) showed that core body temperatures reached the
temperature of the water bath within 15 min and then remained at that
temperature for the rest of the treatment period. Following incubation,
mice were sacrificed by cervical dislocation, and tissues were rapidly frozen on dry ice and stored at
80 °C until use. To control for restraint-induced stress, we also examined tissues from mice that were
placed in the modified Falcon tube but remained at room temperature for
60 min.
Isolation of T-lymphocytes and B-lymphocytes--
For isolation
of T-lymphocytes, mice were sacrificed by cervical dislocation and the
spleen removed. A spleen cell suspension was made in RPMI medium + 5% fetal calf serum (FCS) using a Stomacher 80 for 60 s on high
speed, and the cells were collected by centrifugation at 3,000 × g for 10 min. Red cells were lysed by incubation in Tris-NH4Cl for 4 min on ice, after which 9× volume of
fresh medium was added. Following centrifugation, the cell
pellet was washed with medium and resuspended in 4 ml of
medium (1 ml/spleen). The spleen cell suspension was passed over
a nylon wool column. Non-adherent cells were collected and incubated
with
-
antibody for 30 min on ice, then complement was added and
the cells incubated at 37 °C for 45 min. T-lymphocytes were pelleted
and washed three times with fresh medium. For isolation of
B-lymphocytes (14), a single cell suspension was prepared from spleens
in 5% FCS/Hanks' balanced salt solution (FCS/BSS) using a Stomacher
80. After centrifugation, cells were washed with FCS/BSS and pelleted,
then resuspended in 10 ml of FCS/BSS. T-cells were removed by treating
with monoclonal antibody
-Thy-1.2,
-CD4, and complement. After
washing twice with FCS/BSS, cells were centrifuged on Percoll
gradients. The resting B-cell layer was placed in RPMI medium
containing 5% FCS. Isolated T-cells and B-cells were aliquotted and
incubated at various temperatures for 60 min. Cells were pelleted,
washed with phosphate-buffered saline, and frozen.
CD4+ T-cells from spleen and lymph node of BALB/c mice were
purified using Dynabeads (Dynal Inc., Lake success, NY) according to
the manufacturer's protocol. Briefly, spleen and LN cells were
isolated separately from BALB/c mice, and spleen cells were depleted of red blood cells by ammonium chloride lysis. Cells were incubated on ice
in biotinylated anti-CD4 monoclonal antibody (L3T4, Pharmingen) followed by the addition of streptavidin-Dynabeads (M-280).
CD4+ T-cells were isolated, washed by magnetic separation,
and lysed in lysis buffer. Flow cytometry revealed that the cells were
greater than 95% pure CD4+ T-cells (data not shown).
Native Gel Mobility Shift Analysis--
Protein extracts were
prepared and gel mobility shift analysis performed as described
previously (15). For identification of DNA binding activity, extract
from purified T-lymphocytes incubated at 39 °C (10 µg) was mixed
with 1 µl of a 1:50 dilution of polyclonal antibodies specific to
HSF1 or HSF2 polypeptides (15) and incubated at 25 °C for 10 min
before subjecting to gel shift analysis.
RT-PCR Analysis--
Total RNA was prepared from mouse tissues
by homogenization in guanidinium isothiocyanate and centrifugation
through 5.7 M CsCl. Reverse transcription and PCR were
performed as described previously (16). Two oligonucleotide primers
(5'-ATCACCATCACCAACGACAAGG-3' and 5'-TGCCCAAGCAGCTATCAAGTGC-3') were
used to amplify a 497-bp product from mouse HSP72 cDNA (17). A
104-bp fragment of mouse ribosomal protein S16 cDNA was amplified
as an internal control (5'-TCCAAGGGTCCGCTGCAGTC-3' and
5'-CGTTCACCTTGATGAGCCCATT-3') (18). Amplification was carried out for
22, 24, or 26 cycles using an annealing temperature of 65 °C. The
intensity of bands was quantified with a Amersham Biosciences
PhosphorImager, and the levels of hsp72 product were compared after
normalization to S16 internal control. The quantitative nature of the
RT-PCR assay was determined by analyzing PCR products from reactions utilizing the various PCR cycle numbers (22, 24, and 26 cycles), which
showed that the RT-PCR assay is linear with respect to input template
cDNA over at least 26 cycles of amplification. The data shown in
Fig. 2 are from PCR reactions employing 24 cycles.
 |
RESULTS |
To determine whether there was any variation in the temperature
set point for induction of the cellular stress response between different somatic cell types, we first compared the temperature profiles for activation of HSF DNA binding in cells of various mouse
tissues. Mice were subjected to whole-body hyperthermia at various
temperatures from 38 to 42 °C for 60 min. Measurements indicated
that core body temperatures of the animals reached the temperature of
the water bath within 15 min and thereafter stabilized at that
temperature for the duration of the treatment. Following the
treatments, tissues were collected and analyzed by gel shift assay
employing a specific HSF-binding oligonucleotide probe. Most of the
tissues tested, including heart, liver, and kidney, displayed an HSF
activation profile very similar to that displayed by lung (Fig.
1A). In these tissues,
significant HSF activation is not observed until temperatures of
41 °C are reached, with higher levels observed in animals treated at
42 °C. In contrast, spleen displayed an HSF activation profile that
is shifted to significantly lower temperatures relative to the other
tissues (Fig. 1B). High levels of HSF DNA binding activity
are observed in spleens of animals treated at a temperature of
39 °C, with similar levels present at 40 and 41 °C and
diminishing levels at 42 °C.

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Fig. 1.
Lowered temperature threshold for activation
of HSF DNA binding in spleen cells. A and B,
C3H/HeNcr mice were subjected to whole-body hyperthermia at various
temperatures for 60 min, and then protein extracts prepared from lung
(A) and spleen (B) tissues of these mice were
analyzed by gel shift assay using a specific 32P-labeled
HSF-binding oligonucleotide. Temperatures at which mice were incubated
are indicated at the top of the panels. Results for control
animals, which were treated exactly as the experimental animals except
that they were not subjected to heat treatment, are shown in the lane
marked C.
|
|
To verify that the HSF DNA binding activity induced in spleen at the
lowered temperature threshold of 39 °C does in fact mediate a
productive cellular stress response, we performed RT-PCR analysis to
measure levels of hsp70 mRNA in total RNA preparations of lung and
spleen of animals treated at the various temperatures. To allow a good
comparison to the HSF DNA-binding profiles in each tissue, the samples
of tissue used for the RT-PCR analysis were from the same animals used
for the gel shift analysis whose results are shown in Fig. 1. hsp70
amplification products were quantitated and normalized to S16 ribosomal
protein mRNA internal controls to facilitate accurate comparison of
hsp70 mRNA levels between tissue samples. Very little induction of
hsp70 mRNA is observed in lung at temperatures below 41 °C, with
maximal induction at 42 °C (Fig. 2,
A and C). However, significant induction of hsp70 mRNA is first observed in spleen at the temperature of 39 °C
(8-fold), with similar levels at 40 °C (8.6-fold) and slightly
higher levels at 41 °C (14.6-fold) and 42 °C (23-fold) (Fig. 2,
B and C).

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Fig. 2.
Induction of hsp synthesis at lower
temperatures in spleen cells. A and B, mice
were subjected to whole body hyperthermia at various temperatures from
37 to 42 °C for 60 min, and then the levels of hsp72 mRNA in
lung (A) and spleen (B) were determined by
performing RT-PCR analysis on total RNA isolated from each tissue,
using PCR primers specific to the heat-inducible hsp72 mRNA (17).
Primers specific to the mRNA for the S16 ribosomal protein were
included as an internal control (18). C, quantitation of
hsp72 mRNA levels induced in lung and spleen following whole body
hyperthermia. PCR bands corresponding to hsp72, and S16 mRNAs shown
in A and B were quantitated using a Amersham
Biosciences PhosphorImager. Values obtained for hsp72 mRNA
levels were normalized to levels of S16 mRNA in each sample and
plotted as fold induction relative to values observed for control
animals (shown in lane marked C), which were treated exactly
as the experimental animals except that they were not subjected to
heat treatment.
|
|
We next determined whether the lowered temperature set point we
observed for HSF activation in spleen was a general property of spleen
cell types or whether it was specific to one or more of the cell types
contained within this tissue. T-lymphocytes and B-lymphocytes were
isolated from spleens of C3H/HeNCr mice, heated in vitro at
various temperatures, and then extracts made from these cells were
subjected to gel shift analysis. Significant HSF DNA binding is
observed in B-lymphocytes only at temperatures of 41 °C or higher
(Fig. 3A). This temperature
profile for activation of HSF DNA binding is very similar to that
observed for cells of most somatic tissues (e.g. Fig.
1A). In contrast, purified T-lymphocytes exhibit a profile
of HSF activation that is shifted to significantly lower temperatures
relative to B-lymphocytes (Fig. 3B). High levels of HSF DNA
binding activity are induced in these cells by treatment at 39 °C,
with slightly higher levels induced at 40 °C and decreasing levels
at 41 °C.

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Fig. 3.
Lowered HSF activation temperature in spleen
is specific to T-lymphocytes. A and B,
B-lymphocytes (A) and T-lymphocytes (B) were
isolated from spleens of C3H/HeNCr mice, incubated in vitro
at various temperatures from 37 to 42 °C for 60 min, and then
extracts of these cells were subjected to gel shift analysis using a
specific 32P-labeled HSF-binding oligonucleotide.
Temperatures at which cells were incubated are indicated at
top of panel.
|
|
To determine whether this lowered temperature threshold of HSF
activation in T-lymphocytes could be specific to this strain of mice,
we repeated this experiment using CD4+ T-lymphocytes
isolated from spleens of BALB/c mice. The results (Fig.
4A) show a similar profile of
HSF activation at temperatures in the range of 39-41 °C, indicating
that reduced HSF activation temperature in splenic T-lymphocytes is
likely a general property and not mouse strain-dependent.
Another possibility is that this temperature profile may be a property
unique to T-lymphocytes found in the spleen, not shared by
T-lymphocytes found outside this tissue. This possibility was tested by
examining HSF activation temperature of CD4+ T-lymphocytes
isolated from lymph nodes. As shown in Fig. 4B, the HSF
activation temperature profile of these cells is very similar to that
observed for cells isolated from spleen (Figs. 3B and
4A), suggesting that the phenomenon of lowered HSF
activation temperature is a general property of T-lymphocytes.

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Fig. 4.
Lowered HSF activation temperature of
T-lymphocytes is not mouse strain-dependent and is also
observed in T-lymphocytes isolated from lymph nodes. A
and B, CD4+ T-lymphocytes were isolated from
spleens (A) or lymph nodes (B) of BALB/c mice, incubated
in vitro at various temperatures from 37 to 42 °C for 60 min, and then extracts of these cells were subjected to gel shift
analysis using a specific 32P-labeled HSF-binding
oligonucleotide. Temperatures at which cells were incubated are
indicated at top of panel.
|
|
Our previous studies showed that mouse cells express two different
members of an HSF protein family, HSF1 and HSF2, and demonstrated that
the DNA binding activity of HSF1 is heat-inducible while that of HSF2
is not (37). In most cell types studied to date, HSF1 DNA binding is
activated only at temperatures of 41 °C and higher. Fig.
5 shows that the DNA binding activity
induced in purified T-lymphocytes by heat treatment at 39 °C is
composed of HSF1 and not HSF2, as antibodies specific to HSF1 perturb
this activity while antibodies to HSF2 do not. These results indicate that some mechanism specific to T-lymphocytes functions to lower the
temperature set point for activation of HSF1 DNA binding in this cell
type relative to other cell types.

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Fig. 5.
HSF activated at lower temperature threshold
in T-cells is composed of HSF1. Extracts of purified T-lymphocytes
incubated at 39 °C were subjected to gel shift analysis in the
absence (No Ab) or presence of polyclonal antibodies
specific to HSF1 ( -HSF1) or HSF2 ( -HSF2).
The positions of HSF DNA-binding activity (HSF), nonspecific
DNA binding activity (NS), and free probe (F) are
indicated.
|
|
 |
DISCUSSION |
Our results show that when an organism experiences fever-like
temperatures, T-lymphocytes exhibit HSF1 activation while other cell
types, including B-lymphocytes, do not. We do not know the specific
function of activation of the cellular stress response in T-cells at
fever temperatures. However, in light of the known cytoprotective
functions of hsps, one possibility is that hsp expression at fever
temperatures provides T-lymphocytes with an enhanced ability to
withstand the adverse cellular environments that can exist during an
immune response. In support of this possibility, previous studies
showed that elevation of hsp70 expression in T-cells by transfection
decreases stress-induced apoptosis, and thus one possibility is that
the mechanism we have uncovered could serve to protect T-cells against
apoptosis during an immune response when they are often exposed to
stressful environments (19, 20). hsps also play essential roles as
molecular chaperones in the folding, assembly, and transport of newly
translated proteins (1-5). Therefore, another possibility is that
T-lymphocytes require higher levels of hsps to handle the large
increase in protein synthesis that occurs in those T-cells that become
activated and undergo rapid proliferation. Up-regulation of hsps could
also be necessary to chaperone the receptors that function in signaling events between T-lymphocytes and other cells of the immune system (21,
22).
One or more of the mechanisms above could explain previous
observations that fever temperatures enhance a number of important functional properties of T-lymphocytes, including mitogen- and cytokine-induced proliferation and cell-mediated responses (23-30). On
a higher level, these mechanisms could contribute at least partially to
the long known observation that the presence of fever increases the
ability of organisms to mount an immune response (Refs. 31-38 and
reviewed in Refs. 39-42). Therefore, fever-induced hsp expression in
T-lymphocytes may represent an important molecular mechanism by which
fever acts to boost the immune response of an organism.
These results also indicate that cell
type-dependent regulation of the temperature set point for
activation of the cellular response may be a more widespread phenomenon
than was previously thought. Our previous studies showed that male germ
cell types of mouse testis activate HSF1 DNA binding at a threshold
temperature of 35 °C, a temperature 7 °C lower than the
temperature at which HSF1 is activated in cell types such as liver
cells (42 °C) (12, 13). These studies also revealed that somatic
cell types present in the testis do not share the lowered HSF
activation temperature exhibited by the male germ cell types of this
tissue, and instead display an HSF temperature set point (42 °C)
that is identical to that of other somatic cell types such as liver. On
the basis of these results, we had hypothesized that alteration in the
temperature set point for the cellular stress response is a property
unique to male germ cell types and that all somatic cell types would exhibit the "normal" set point of 42 °C. However, the results presented in this paper show unequivocally that this is not the case
and that the temperature set point for inducible hsp expression can
also be differentially regulated in somatic cell types. Indeed, another
study found that HSF1 DNA binding activity is induced in cells of the
nervous system of rabbit, particularly the cerebellum, at temperatures
in the fever range (43). The goal of future studies is to elucidate the
mechanism(s) that regulate cell type-dependent differences
in HSF activation temperature, which will also increase our
understanding of the fundamental mechanism of heat-induced HSF
activation itself.
 |
ACKNOWLEDGEMENTS |
We thank Charles Snow and Donald Cohen
for assistance with B-cell and T-cell isolation procedures and
Katherine Cullen and Michael Goodson for assistance and
valuable comments during these experiments.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
(NIH) Grants HD32008 and GM61053 and March of Dimes Award 5-FY95-0009 and by Reproductive Sciences Training Grant (NIH Award T32-HD07436) (to
L. Q. G.) at the University of Kentucky.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
859-323-5777; Fax: 859-323-1037; E-mail: kdsarge@uky.edu.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M209412200
 |
ABBREVIATIONS |
The abbreviations used are:
HSF, heat
shock factor;
FCS, fetal calf serum;
BSS, Hanks' balanced salt
solution;
RT, reverse transcription.
 |
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