Download Mediated by Heat Shock Factor-1 (HSF1) Protective Stress

Physiological Fever Temperature Induces a
Protective Stress Response in T Lymphocytes
Mediated by Heat Shock Factor-1 (HSF1)
This information is current as
of June 18, 2017.
Patience Murapa, Siva Gandhapudi, Hollie S. Skaggs, Kevin
D. Sarge and Jerold G. Woodward
J Immunol 2007; 179:8305-8312; ;
doi: 10.4049/jimmunol.179.12.8305
http://www.jimmunol.org/content/179/12/8305
Subscription
Permissions
Email Alerts
This article cites 52 articles, 27 of which you can access for free at:
http://www.jimmunol.org/content/179/12/8305.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
References
The Journal of Immunology
Physiological Fever Temperature Induces a Protective Stress
Response in T Lymphocytes Mediated by Heat Shock
Factor-1 (HSF1)1
Patience Murapa,* Siva Gandhapudi,* Hollie S. Skaggs,† Kevin D. Sarge,†
and Jerold G. Woodward2*
T
he febrile response is an adaptive physiological response
in animals that is well conserved throughout evolution. In
mammals, the febrile response involves an increase in cytokines and acute phase proteins as well as activation of the central
nervous and endocrine systems, all of which participate in the elevation of core body temperature. In ectotherms, elevation of core
body temperature is achieved by moving to warmer environments
(1). In both endotherms and ectotherms, survival after experimental infection is reduced drastically if temperature elevation is inhibited, indicating that temperature elevation is critical for survival
from pathogens (2– 4). How temperature elevation translates into
increased resistance to infection is poorly understood. It is generally accepted that temperature elevation acts to enhance the host
immune response rather than pathogen growth or survival, and a
large number of studies generally support this (reviewed in Refs.
2 and 4). The best examples of fever-enhancing effects have been
in the innate immune system (5–10). The effects of fever temperature on the adaptive immune system are less clear. Some studies
have shown increases in T or B cell responses whereas other stud-
*Department of Microbiology, Immunology and Molecular Genetics, and †Department of Molecular and Cellular Biochemistry, University of Kentucky Medical Center, Lexington, KY 40536
Received for publication October 27, 2006. Accepted for publication October
14, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants EY014060 (to
J.G.W.), GM61053 (to K.D.S.), and GM64606 (to K.D.S.). H.S.S. was supported by
National Institutes of Health Training Grant T32ES007266.
2
Address correspondence and reprint requests to Dr. Jerold G. Woodward, Department of Microbiology, Immunology and Molecular Genetics, MN426, 800 Rose
Street, University of Kentucky Medical Center, Lexington, KY 40536. E-mail address: [email protected]
www.jimmunol.org
ies have shown no change or reduced responses (2, 11–16). Because of the variations in experimental systems and the complex
cellular interactions involved, it has been difficult to study temperature effects at the mechanistic level. Thus, our current knowledge of how fever temperature affects immune cell function is still
very limited.
The cellular response to increased temperature has been studied
extensively. Exposure of cells to elevated temperatures, typically
42°C, causes rapid induction of heat shock proteins that serve a
variety of important protective roles for the cell (17–19). This is
typified by the classical heat shock response in which cells exposed to 42°C are found to be protected from the lethal effects of
a subsequent 45°C exposure. This effect is primarily mediated by
the transcription factor known as heat shock factor-1 (HSF1)3 (20,
21). In response to elevated temperature or other stressors, cytoplasmic monomeric HSF1 trimerizes and translocates to the nucleus where it binds to heat shock-responsive elements found in
numerous heat-inducible genes, in particular heat shock protein
(HSP) genes (reviewed in Ref. 22). The temperature at which
HSF1 becomes activated in different cell types has been studied
extensively using EMSAs to measure activated HSF1 binding to
heat shock-responsive elements. These cell types include cell lines
such as HeLa and K562 (23–25) and tissues such as liver (26),
lung, kidney, and heart (27). In all of these cases, activation of
HSF1 is not apparent until the cells or tissue are warmed to 42°C,
a temperature at the very high end of the physiological fever range.
When physiological fever temperatures are used, little or no HSF1
activation in these tissues is apparent. One notable exception to
3
Abbreviations used in this paper: HSF1, heat shock factor-1; DCFH-DA, 2⬘,7⬘dichlorodihydrofluorescein diacetate; HSP, heat shock protein; HSP70i, inducible
HSP70; ROS, reactive oxygen species.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Heat shock factor-1 (HSF1) is a transcription factor that serves as the major temperature-inducible sensor for eukaryotic cells.
In most cell types, HSF1 becomes activated to the DNA binding form at 42°C and mediates the classical heat shock response,
protecting the cells from subsequent lethal temperatures. We have recently demonstrated that HSF1 is activated at a lower
temperature in T lymphocytes than in most other cell types (39°C vs 42°C), within the physiological range of fever. In this study,
we show that T cell activation at fever temperatures not only activates HSF1 but induces the up-regulation of the HSF1 protein
and the HSF1-regulated protein, HSP70i. T cells from HSF1 knockout mice proliferate normally under optimal conditions but are
impaired in proliferation at physiological fever temperatures and low CO2 concentrations, conditions that do not impair wild-type
T cells. This defect in proliferation appears to be mediated by a block in the G1/S transition of the cell cycle and is independent
of HSP70. Elevated temperature and low CO2 concentrations resulted in a dramatic reduction of the intracellular reactive oxygen
species (ROS) levels in both normal and knockout T cells. Wild-type T cells were able to restore ROS levels to normal within 5 h,
whereas HSF1ⴚ/ⴚ T cells were not. These results suggest that the proliferation defect seen in T cells from HSF1ⴚ/ⴚ mice at fever
temperatures was because of dysregulated ROS levels and that HSF1 is important in maintaining ROS homeostasis and cell cycle
progression under the stressful conditions encountered during fever. The Journal of Immunology, 2007, 179: 8305– 8312.
8306
suggesting that the inability of HSF1⫺/⫺ T cells to proliferate at
fever temperatures is because of depressed ROS levels. Finally,
this proliferation defect in HSF1⫺/⫺ T cells was not because of a
lack of HSP70i expression. These results suggest that HSF1 plays
a key role in facilitating the proliferation of T cells and maintaining ROS homeostasis at fever temperatures.
Materials and Methods
Mice
HSF1⫺/⫺ mice on the 129 ⫻ BALB/c mixed background were colonized
from breeding pairs provided by I. Benjamin (University of Utah, Salt Lake
City, UT). Mice were maintained by breeding heterozygotes. Homozygous
HSF1⫺/⫺ mice were obtained by intercrossing heterozygous males and
females or homozygous males and heterozygous females and selecting
HSF1 ⫺/⫺ mice by PCR. Heterozygous or wild-type littermates were used
as controls. HSP70⫺/⫺ mice on a B6 ⫻ 129 mixed background (B6;129S5Hspa1atm1Dix Hsp1btm1Dix/UCD) were obtained from the Mutant
Mouse Regional Resource Center (University of California, Davis, CA).
These mice are deleted for both hsp1a and hsp1b genes encoding the inducible HSP70i proteins (42). Homozygous knockout mice were obtained
by intercrossing heterozygotes or homozygotes and selecting by PCR.
Wild-type littermates were used as controls. Mice were maintained under
specific pathogen-free conditions at the Division of Laboratory Animal
Resources, University of Kentucky Medical Center (Lexington, KY). All
animal experiments have been reviewed and approved by the University of
Kentucky Institutional Animal Care and Use Committee.
Lymphocyte cultures
T cells were purified from spleen and lymph nodes using anti-CD3 magnetic beads and the autoMACS (Miltenyi Biotec). For proliferation assays,
2 ⫻ 105 T cells were cultured in RPMI 1640 medium supplemented with
10% FBS, 1 mM glutamine, 1 mM sodium pyruvate, 1⫻ MEM nonessential amino acids, 50 ␮M 2-ME (except where indicated), 100 U/ml penicillin, and 100 U/ml streptomycin (complete medium), alone or with plate
bound anti-CD3, Con A (2 ␮g/ml), or PMA/ionomycin for 3– 4 days at the
indicated temperature. [3H]Thymidine was added during the last 18 h and
proliferation was determined by scintillation counting. For CFSE experiments, purified T cells were first labeled with CFSE as previously described (43) and cultured at 1 ⫻ 106 cells per well in 24-well plates, coated
or not coated with anti-CD3 at the indicated temperature and CO2 concentration. Cells were harvested, stained with anti-CD4 or anti-CD8, and analyzed on a FACScan flow cytometer. For cell cycle analysis, cells were
harvested from 24-well plates, fixed, permeabilized, and stained with propidium iodide to determine DNA content. RNA synthesis was determined
by the addition of [3H]uridine for 18 h. Cell viability was determined by
trypan blue exclusion.
Western blot analysis
CD4⫹ T cells were purified by using anti-CD4 magnetic beads and
autoMACS (Miltenyi) according the manufacturer’s directions. Purified
CD4⫹ T cells were incubated in complete medium in the presence or
absence of anti-CD3 for 6 h at either 37°C or 39.5°C.
Cells were extracted for 10 min at 4°C in 5 volumes of buffer C (25%
(v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 20 mM
HEPES (pH 7.9), 1 mM PMSF, and 1 mM DTT) and centrifuged at 13,000
rpm at 4°C. Five microliters of the supernatant was boiled for 5 min in the
presence of 1⫻ SDS-PAGE loading buffer and electrophoresed on an 8%
SDS-polyacrylamide gel for 1 h at 180 V. The proteins were then transferred to nitrocellulose and probed with polyclonal anti-HSF1 (K.D.S.),
polyclonal anti-HSP70i (SPA-812; Stressgen), or monoclonal anti-␤-actin
(clone AC-15; Sigma-Aldrich). The membrane was washed three times for
5 min each with 0.1% Tween 20 and PBS. Detection was performed with
the appropriate secondary Abs coupled to HRP (Zymed Laboratories) followed by ECL (Femto System; Pierce) detection.
Measurement of intracellular pH
Purified T cells were cultured at the different temperatures and CO2 levels
as described. At 48 and 72 h of culture, the intracellular pH of the cells was
determined. The cells were harvested, washed in 1⫻ PBS, and counted.
Cells (2 ⫻ 105) from each sample were aliquoted into fresh 5-ml polypropylene tubes. SNARF 1 (10 ␮M) (catalog no. C1272; Molecular Probes)
was loaded into the cells in PBS and incubated for 30 min at 37°C. pH
standards (pH 6.5–7.8) were made in a high K⫹ buffer (120 mM KCl, 30
mM NaCl, 0.5 mM MgSO4, 1 mM CaCl, 1 mM NaHPO3, 5 mM glucose,
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
this is the male germ cell. The temperature of the testes is tightly
regulated at 30°C, and HSF1 activation in the male germ cell occurs at a temperature of 35°C (26, 28). Nonetheless, even in the
testes neighboring interstitial somatic cells show a 42°C activation
temperature for HSF1 (20). Because of this, HSF1 activation has
largely been viewed as a protective mechanism in response to severe thermal stress, but not fever (29 –31). However, we recently
demonstrated that in T lymphocytes HSF1 becomes activated at
39°C, well within the physiological range of fever (27). This lower
threshold for HSF1 activation does not occur in B lymphocytes,
which show the more typical 42°C activation threshold. Thus, the
only somatic cell type to date showing HSF1 activation at fever
temperature is the T lymphocyte. This led us to hypothesize that
HSF1 serves a protective and/or function-enhancing role in T cells
at fever temperatures (27).
One of the most widely studied genes controlled by HSF1 is the
inducible form of HSP70 (HSP70i). HSP70i has been shown to be
regulated by HSF1 whereas a related constitutive form of HSP70
(HSC70) is not (20). HSP70 has numerous effects on the immune
system. As a chaperone, HSP70 binds a wide variety of self and
foreign peptides, resulting in greatly enhanced uptake of Ags by
APC and enhanced cross-presentation (32–34). HSP70 also directly activates APC, possibly by direct interaction with TLRs (32,
35–37). Fever temperatures cause the up-regulation of HSP70i in
a variety of leukocytes including T cells, leading several groups to
propose that fever temperatures provide an innate “danger” signal
to the immune system (8, 9, 27, 38). These inducible effects can
largely be attributed to HSP70I, because HSC70 is constitutively
expressed in all cell types including T cells (38). Because HSP70i
appears to be strictly regulated by HSF1, these latter studies support the idea that HSF1 plays an important role in the enhancement
of immune responses at fever temperatures. However, the role of
HSF1 in T cell function at fever temperatures has not been
examined.
Recently, HSF1⫺/⫺ mice have provided additional insight into
the biological role of HSF1. HSF1⫺/⫺ mice generally survive to
adulthood, but there is some embryonic lethality mediated by defects in the chorioallantoic placenta that is dependent on the background genotype (39). HSF1⫺/⫺ females are also sterile because of
a requirement for maternal HSF1 in early postfertilization development of the zygote (40). Cells from HSF1⫺/⫺ mice lack the
classical heat shock response, underscoring the key role of HSF1
as the major temperature-inducible transcription factor in cells
(20). Adult HSF1⫺/⫺ mice have normal frequencies of T and B
cells and normal T cell proliferative responses following immunization with nominal Ag (34). However, they show an impairment
in IgG1 and IgG2a Ab in response to immunization (41). HSF1⫺/⫺
mice are also deficient in a variety of HSP70-mediated immunologic effects, primarily at the level of the APC (9, 34). However,
there have been no reports demonstrating a role for HSF1 in T
cells.
In this study we show that T cells from HSF1⫺/⫺ mice were
profoundly deficient in proliferation and cell cycle progression at
fever temperatures. The inhibitory effect of elevated temperature
on proliferation was more pronounced at low CO2 concentrations
and appeared to be due to a block between the G1 and S phase of
the cell cycle. The modulating effect of CO2 led us to investigate
intracellular reactive oxygen species (ROS) levels in T cells at
fever temperatures. Surprisingly, we found that fever temperature
significantly lowered ROS levels in T cells, and the levels were
further reduced in low CO2 environments. T cells normally increase their ROS levels upon activation, and wild-type T cells
could overcome this temperature-induced depression in ROS. In
contrast, HSF1⫺/⫺ T cells were unable to recover from this effect,
HSF1, ROS, AND T CELL PROLIFERATION
The Journal of Immunology
8307
FIGURE 1. Fever temperatures induce Hsp70 and HSF1 protein expression. Purified CD4⫹ T cells from either HSF1⫹/⫹ or HSF1⫺/⫺ mice were
cultured for 6 h in medium alone or with plate-bound anti-CD3 (␣-CD3) at
either 37°C or 39.5°C. Shown are Western blots of cell extracts probed
with anti-HSP70i, anti-HSF1, or anti-actin Ab as a loading control.
ROS measurements
Purified T cells were activated with plate-bound anti-CD3 at the indicated
temperatures and CO2 concentrations for 2 or 5 h. 2⬘,7⬘-Dichlorodihydrofluorescein diacetate (DCFH-DA) (catalog no. D399 (H2DCFHDA);
Invitrogen Life Technologies) was added to the cells at a final concentration of 10 mM 30 min before harvest. Cells were harvested, washed once
in PBS, and immediately analyzed by flow cytometry. ROS levels were
measured as fluorescence intensity in the FL1-H channel, gating on viable
cells.
Statistical analysis
Results are expressed as mean values ⫾ SD. Comparisons were made using
a paired t test. Statistical indications in the figures are defined as follows:
ⴱ, p ⱕ 0.01; ⴱⴱ, p ⱕ 0.005; and ⴱⴱⴱ, p ⱕ 0.001. All experiments were
repeated at least three times.
Results
Physiological fever temperatures and T cell activation
up-regulate and activate HSF1 in T lymphocytes
Our previous studies demonstrated that HSF1 is activated to the
DNA binding form in T cells at physiological fever temperatures
(39°C) in contrast to 42°C for B cells and other nonlymphoid cells
(27). To further understand the role of HSF1 in T cells, we examined the expression of HSF1 protein and the product of an HSF1responsive gene, HSP70i, in T cells by Western blotting (Fig. 1).
This method detects total HSF1 protein in cells regardless of
whether it is active or not, whereas the presence of HSP70i is an
indication of transcriptionally active HSF1. HSF1 protein was constitutively expressed but inactive in resting T cells, as revealed by
the lack of HSP70i. After incubation of resting T cells at 39.5°C,
HSF1 protein levels were slightly reduced but HSF1 activation
was apparent through the induction of HSP70i. The reduction in
HSF1 protein levels was most likely because of its activation, localization in the nucleus, and binding to nuclear material. In contrast, anti-CD3 activation of T cells resulted in up-regulation of
HSF1 protein and HSF1 transcriptional activity. This effect was
observed at 37°C but was much more pronounced at 39°C. No
HSF1 protein was detectable in T cells from HSF1⫺/⫺ mice. Importantly, there was no HSP70i induced in T cells from HSF1⫺/⫺
FIGURE 2. T cells from HSF1⫺/⫺ mice fail to proliferate at fever temperatures and low CO2 concentrations. Purified CD3⫹ T cells from either
HSF1⫹/⫹ or HSF1⫺/⫺ mice were cultured for 4 days at either 37°C or
39.5°C in the presence of the indicated concentrations of plate-bound antiCD3 (A and B), anti-CD3 (␣-CD3) and anti-CD28 (␣-CD28) (C and D), 3
days with Con-A (E), or 3 days with PMA/ionomycin (unpurified spleen
and LN cells) (F). Purified CD3⫹ T cells were cultured at different temperatures (Temp) and CO2 concentrations as indicated (G and H) with
plate-bound anti-CD3. Data are expressed as cpm of [3H]thymidine deoxyribose (H3 TdR) incorporated during the last 18 h of culture. pb, Platebound; sol, soluble (C and D).
mice, indicating that temperature- and activation-induced HSP70i
expression is totally dependent on HSF1. Thus, T cell activation,
even at 37°C, results in the up-regulation of HSF1 protein and
conversion to an active transcription factor, and this is further enhanced at fever temperatures. Previous studies have documented
the induction of HSP70i in lymphocytes at febrile temperatures (8,
38). Our results further demonstrate that this induction is entirely
dependent on HSF1 and that HSF1 activation occurs in stimulated
T cells even at 37°C.
HSF1 is required for optimal T cell proliferation under
conditions of elevated temperature in the fever range and low
CO2 concentrations
Because HSF1 is strongly activated in T cells at physiological
fever temperatures, and given the critical role of T cell function
during fever, we hypothesized that HSF1 plays an important role
in either the protection of T cells and/or the enhancement of T cell
function at fever temperatures. To test this, T cells from HSF1⫹/⫹
or HSF1⫺/⫺ mice were stimulated in vitro with anti-CD3, antiCD3 plus anti-CD28, Con-A, or PMA/ionomycin at either 37°C or
39.5°C (Fig. 2, A–F). For all four stimuli, HSF1⫹/⫹ T cells proliferated extensively at both 37°C and 39.5°C. Occasionally we
noted some reduction in proliferation of HSF1⫹/⫹ T cells at 39°C
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
10 mM HEPES, and 10 mM PIPES). After staining, cells were washed
once and resuspended in PBS. Cells for the standard curve were resuspended in the pH standards containing 20 ␮m of nigericin (Catalog no.
N1495; Molecular Probes) to equilibrate intracellular pH with the extracellular medium. The cells were analyzed at 37°C using a MoFlo flow
cytometer and the fluorescence intensity ratios of two wavelengths, ␭580/
␭640 (FL3/FL2), were calculated. A value of pH for the samples was determined using a linear regression equation derived from the standard
curve.
8308
HSF1⫺/⫺ T cells fail to undergo significant cell division at
elevated temperature and low CO2
Because our thymidine incorporation experiments only measured
DNA synthesis during the last 18 h of culture, the above experiments do not reveal the cell division history or the viability of the
HSF1⫺/⫺ T cells. To further examine the mechanism of this reduction in proliferation, purified CD3⫹ T cells were labeled with
CFSE, stimulated with plate-bound anti-CD3 for 3 days at various
temperatures and CO2 concentrations, and analyzed for CFSE dye
dilution by flow cytometry. Cells from HSF1⫹/⫹ mice showed
extensive cell division when stimulated at 37°C and 7% CO2, 37°C
and 3% CO2, or 39.5°C and 3% CO2. In contrast, T cells from
HSF1⫺/⫺ mice showed extensive cell division at 37°C and 7% or
3% CO2, but only minimal cell division at 39.5°C and 3% CO2
(Fig. 3). Thus, the block in thymidine incorporation measured in
the experiments of Fig. 2 appears to be due to a lack of cell divi-
FIGURE 3. CFSE analysis reveals a global inhibition in cell division in
T cells from HSF1⫺/⫺ mice at fever temperatures and low CO2 concentrations. Purified T cells from HSF1⫹/⫹ or HSF1⫺/⫺ mice were labeled
with CFSE and stimulated with anti-CD3 at the indicated temperature and
CO2 concentrations. After 3 days, cells were harvested, counted, and
stained with anti-CD3 and the CFSE fluorescence was analyzed within the
gated CD3⫹ population. Histograms are from a representative HSF1⫹/⫹
and two HSF1⫺/⫺ mice. These experiments have been repeated five times
with identical results. Markers M1 and M2 represent undivided and divided
cells, respectively, and the numbers shown represent the percentage of total
CD3⫹ cells within each marked region.
sion rather than an enhanced apoptosis of cells that had already
proliferated. When CD4⫹ or CD8⫹ T cells from HSF1⫺/⫺ mice
were gated separately, neither population showed significant CFSE
dilution, indicating that the proliferation defect affects both cell
populations (data not shown). Collectively, these data indicate that
HSF1 is critical for both CD4 and CD8 T cells to undergo cell
division at fever temperatures.
The proliferation defect in HSF1⫺/⫺ T cells is due to a block
between the G1 and S phase of the cell cycle
The previous CFSE experiments suggest that the majority of T
cells from HSF1⫺/⫺ mice fail to enter into cell division when
cultured under nonpermissive conditions. To further determine the
phase of the cell cycle at which these cells were blocked, purified
T cells were activated with plate-bound anti-CD3 under permissive
or nonpermissive conditions for 48 h, stained for DNA content
with propidium iodide, and analyzed for the percentage of cells in
either G0/G1 or S/G2/M phases of the cell cycle by flow cytometry.
Roughly 50% of the T cells from HSF1⫹/⫹ mice had entered into
the S/G2/M phase of the cell cycle after 48 h of activation at either
37°C and 7% CO2 or 39.5°C and 3% CO2. In contrast, T cells from
HSF1⫺/⫺ mice showed normal entry into the S phase of the cell
cycle at 37°C and 7% CO2 but minimal entry at 39.5°C and 3%
CO2 (Fig. 4A). To measure the transition from the G0 to the G1
phase of the cell cycle, incorporation of [3H]uridine was measured
as an indication of RNA synthesis in the first 24 h of activation
(Fig. 4B). Both HSF1⫹/⫹ and HSF1⫺/⫺ T cells showed high and
comparable levels of uridine incorporation at 37°C and 7% CO2.
Although uridine incorporation in T cells from both HSF1⫹/⫹ and
HSF1⫺/⫺ mice was somewhat diminished at 39.5°C/3% CO2,
these differences were not statistically significant. Furthermore, 24
h activated T cells from HSF1⫺/⫺ showed an increase in cell size
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
(Fig. 2, A and B), but the vast majority of experiments revealed
little or no significant differences (Fig. 2, C–H, and data not
shown). More importantly, even when reductions in thymidine incorporation levels were noted as in Fig. 2, A and B, the levels were
still robust, indicating strong proliferation. In contrast, T cells from
HSF1⫺/⫺ mice showed a normal level of proliferation at 37°C but
a dramatic reduction in proliferation at 39.5°C. In many cases proliferation was almost completely shut down, whereas in others the
reduction was highly significant. This effect was observed over a
wide range of anti-CD3 and anti-CD28 concentrations and under
optimal T cell stimulation conditions. Superoptimal anti-CD28
stimulation may partially reverse this effect, but this was not consistently observed. The block in proliferation of HSF1⫺/⫺ T cells
at 39.5°C was also observed after stimulation with Con A or PMA/
ionomycin (Fig. 2, E and F). Because PMA/ionomycin stimulation
bypasses the proximal TCR signal transduction machinery, the
block in proliferation must occur downstream from these events.
Furthermore, because these experiments were performed with purified T cells, this effect is T cell intrinsic.
During the course of our experiments we noticed that the 39.5°C
incubator had been running at a lower CO2 percentage than the
37°C incubator (3 vs 5%, respectively) due to the fact that the
higher temperature setting altered the calibration of the CO2 sensor. Given that wild-type cells still proliferated normally at 39.5°C
and 3% CO2, this effect was selective for the HSF1 ⫺/⫺ T cells. We
therefore performed a series of experiments to determine the effect
of CO2 concentration on cell proliferation of HSF1⫺/⫺ T cells. A
series of incubators were set up at either 37°C or 39.5°C and CO2
concentrations of 8, 5, or 3%, carefully calibrated using the Fryrite
CO2 sensor. Purified T cells from either wild-type or HSF1⫺/⫺
mice were then stimulated with plate-bound anti-CD3 under different temperatures and CO2 conditions. Wild-type T cells proliferated well at 39.5°C and all CO2 concentrations. In contrast, T
cells from HSF1⫺/⫺ mice proliferated well at 39.5°C and 8 or 5%
CO2, but failed to proliferate at 39.5°C and 3% CO2 (Fig. 2E). In
a separate experiment, purified T cells from HSF1⫺/⫺ mice proliferated well at 37°C and 7% CO2 or 37°C and 3% CO2, but not
at 39.5°C and 3%CO2. T cells from heterozygous HSF1⫹/⫺ mice
proliferated normally at all temperatures and CO2 concentrations,
indicating that this phenotype requires a homozygous HSF1 mutation (Fig. 2F). Thus, the proliferation defect observed in
HSF1⫺/⫺ T cells was only observed under conditions of elevated
temperature in the fever range and low CO2 concentrations, conditions that do not impair T cells from wild-type or HSF1 heterozygous mice. These data demonstrate that HSF1 and/or downstream target genes play a protective role in facilitating T cell
proliferation at fever temperatures.
HSF1, ROS, AND T CELL PROLIFERATION
The Journal of Immunology
comparable to that of their wild-type counterparts (data not
shown). These data indicate that T cells from HSF1⫺/⫺ mice were
not impaired in their early activation from the G0 to the G1 phase
of the cell cycle. Cell viability measurements during the first 24 h
of culture showed no significant reduction in viability in the
HSF1⫺/⫺ T cells cultured at 39.5°C and 3% CO2 (Fig. 4C), indicating that these cells were not undergoing apoptosis. To determine the reversibility of the HSF1⫺/⫺ T cell proliferation defect at
different time points, T cells from HSF1⫹/⫹ or HSF1⫺/⫺ mice
were cultured at either 37°C and 7% CO2 or 39.5°C and 3% CO2
for the entire 4 days or with switching between temperatures at
various time points. As previously shown, T cells from HSF1⫹/⫹
mice proliferated well at either temperature or combination
thereof. Also as expected, T cells from HSF1⫺/⫺ mice proliferated
well when cultured for all 4 days at 37°C and 7% CO2 but poorly
when cultured for all 4 days at 39.5°C and 3% CO2. When T cells
from HSF1⫺/⫺ mice were cultured at 37°C and 7% CO2 for the
first 24 h and then switched to 39.5°C and 3% CO2, proliferation was still almost completely inhibited, whereas switching
these cells after 48 h allowed completely normal proliferation.
In contrast, when T cells from HSF1⫺/⫺ mice were cultured at
39.5°C and 3% CO2 for the first 24 h and then switched to 37°C
and 7% CO2, substantial proliferation occurred at ⬃50% of the
level seen when these cells were cultured for all 4 days at 37°C.
These results point to a critical period between 24 and 48 h
when the block in proliferation occurs in HSF1⫺/⫺ T cells.
Collectively, these results point to a block in the G1/S phase
transition in the cell cycle in HSF1⫺/⫺ T cells when activated
at 39.5°C and 3%CO2.
FIGURE 5. The intracellular pH of HSF1⫺/⫺ or HSF1⫹/⫹ T cells remains within normal levels at 3% CO2. A, Intracellular pH (pHi) standards
made by incubating T cells in buffers of known pH and equilibrating extracellular and intracellular pH with nigericin. Cells were then harvested
and stained with the intracellular pH dye SNARF-1. A pH standard curve
was generated by plotting pH vs FL3/FL2 ratios. B, Purified T cells from
HSF1⫺/⫺ or HSF1⫹/⫹ mice were stimulated with anti-CD3 and cultured at
37°C with 5% CO2 or 39.5°C with 3% CO2 for 3 days. The FL3/FL2 ratios
were plotted over time to insure stability of the measurement. The numbers
above the graph represent pH values calculated using the linear equation
generated in the standard curve.
T cells from HSF1⫺/⫺ mice show a dysregulation in levels
of ROS
To account for the effect of low CO2 concentrations on the proliferation defect of HSF1⫺/⫺ T cells, we considered two possible
explanations. First, because the CO2 percentage regulates the bicarbonate buffer system in the medium, the lower CO2 could raise
the pH of the medium and possibly the intracellular pH, thus exacerbating the effect of temperature on proliferation of the
HSF1⫺/⫺ T cells. Second, dissolved CO2 levels can affect ROS
levels in cells, potentially altering cell signaling pathways (44, 45).
Several experiments were performed to evaluate the effect of 3%
CO2 on the pH of the anti-CD3-stimulated cultures. As expected,
medium alone had a higher pH at 3% CO2 than at 5% (pH 7.6 vs
7.3). However, when the pH of the cultures containing metabolizing cells was measured, the pH at both CO2 concentrations was
similar at ⬃7.2 (data not shown). To determine whether the intracellular pH was altered at the lower CO2 concentration, we used a
flow cytometric method to measure the intracellular pH of the
activated T cells (46). Purified CD3⫹ T cells were activated for 48
or 72 h at either 5 or 3% CO2 and then stained with SNARF-1 to
measure intracellular pH. Contrary to expectations, T cells from
both wild-type and HSF1⫺/⫺ mice had a slightly lower intracellular pH at 39.5°C and 3% CO2 compared with cells cultured at
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 4. The block in proliferation of T cells from HSF1⫺/⫺ mice at
fever temperatures occurs at the G1/S phase transition of the cell cycle. A,
Purified T cells from HSF1⫺/⫺ or HSF1⫹/⫹ mice were cultured at either
37°C/5% CO2 or 39.5°C/3% CO2 with plate-bound anti-CD3. The cells
were harvested after 48 h and intracellular DNA was stained with propidium iodide. The percentage of cells in each phase of the cell cycle was
quantitated by flow cytometry and analysis using ModFit. The bar graphs
show the average percentages of three mice ⫾ SD. This experiment was
repeated at least three times with the same result. B, To measure RNA
synthesis, [3H]uridine deoxyribose (3H UdR) was added during the last
12 h of a 24-h culture of purified T cells from HSF1⫹/⫹ or HSF1⫺/⫺ mice
at either 37°C and 5% CO2 or 39.5°C and 3% CO2. Data are expressed as
cpm ⫾ SD of triplicate cultures. C, Purified CD3⫹ T cells from HSF1⫹/⫹
or HSF1⫺/⫺ mice were stimulated with anti-CD3 for 24 h and the percentage of viable cells was determined by trypan blue dye exclusion. D, Individual cultures of purified CD3⫹ T cells were stimulated with anti-CD3 for
4 days at either 37°C and 5% CO2, 39.5°C and 3% CO2, or combinations
thereof on different days as indicated. [3H]thymidine (3H TdR) was added
for the final 18 h of culture. Data represent the mean cpm ⫾ SD of triplicate
wells.
8309
8310
37°C and 5% CO2, but still at a level well within physiological
intracellular pH levels (Fig. 5). Finally, using a bicarbonate-free
medium in which pH is independent of the CO2 concentration, the
low CO2 effect was still observed in the HSF1⫺/⫺ T cells (data not
shown). Thus, it is unlikely that a change in intracellular pH was
the cause of the proliferation defect of HSF1⫺/⫺ cells at 39.5°C
and 3% CO2. Because CO2 concentrations also have the potential
to alter the redox status of cells (44), we evaluated the effect of
temperature and CO2 concentrations on the intracellular ROS levels in T cells using the ROS-sensitive fluorochrome DCFH-DA
and flow cytometry. DCFH-DA is relatively nonfluorescent unless
oxidized by ROS and thus the level of fluorescence is proportional
to the total level of intracellular ROS. Studies using DCFH-DA
and other redox assays have established that T cells undergo a
significant increase in ROS upon activation and, if this increase is
inhibited by antioxidants, T cell proliferation is blocked (reviewed
in Ref. 47). To our surprise, we found that elevated temperature
alone resulted in a decrease of the total intracellular ROS levels
within 2 h in both wild-type and HSF1⫺/⫺ T cells, stimulated or
unstimulated (Fig. 6). This decrease was further exacerbated at low
CO2 concentrations. By 5 h, the ROS levels of the unstimulated
wild-type and knockout T cells cultured at 39.5°C and 5% CO2
returned to the level of the cells cultured at 37°C, while the ROS
levels of the cells cultured at 39.5°C and 3% CO2 did not. In the
stimulated cultures, the ROS levels of wild-type cells cultured at
both 39.5°C and 5% CO2 and 39.5° and 3% CO2 returned to the
levels seen in 37°C cultured cells, whereas the ROS levels of
HSF1⫺/⫺ T cells only returned to normal in the 37°C and 5% CO2
cultures. Thus, elevated temperature and low CO2 concentrations
result in reduced intracellular ROS levels in T cells. Wild-type T
cells can compensate for this, whereas T cells lacking HSF1 cannot. The fact that ROS levels are inhibited under the very same
conditions that result in a block in proliferation in HSF1⫺/⫺ T cells
suggests that ROS dysregulation is at the heart of the proliferation
defect in HSF1⫺/⫺ cells at fever temperature.
The proliferation defect of HSF1⫺/⫺ cells is independent of
HSP70
Our own data in T cells (Fig. 1) and other studies in non-T cells
(20) clearly show that HSP70i is largely, if not entirely, dependent
FIGURE 7. HSP70⫺/⫺ T cells are not impaired in proliferation at
39.5°C and low CO2. CD3⫹ purified T cells were isolated from spleen and
lymph nodes of HSP70⫺/⫺ or HSF1⫺/⫺ mice and stimulated with anti-CD3
for 3 days. Cells were cultured at 37°C and 7% (37/7), 37°C and 3% (37/3),
39.5°C and 5% CO2 (39.5/5), or 39.5°C and 3% CO2 (39.5/3). Data are
expressed as cpm of [3H]thymidine deoxyribose (H3 TdR) incorporated
during the last 18 h of cell culture.
on HSF1. Furthermore, HSP70 has been shown to have a wide
range of protective effects on cellular function, as well as immunostimulatory effects (32). To determine whether a lack of HSP70i
was responsible for the proliferation defect observed in HSF1⫺/⫺
mice, we tested the proliferation of T cells from HSP70⫺/⫺ mice
at permissive and nonpermissive conditions (Fig. 7). As previously
shown, proliferation of T cells from two different HSF1⫺/⫺ mice
was significantly impaired at 39.5°C and 3% CO2 compared with
37°C and 3 or 7% CO2. In this experiment, proliferation of
HSF1⫺/⫺ cells was also impaired at 39.5°C and 5% CO2 (Fig. 7),
indicating that the requirement for low CO2 is not absolute and that
elevated temperature is perhaps the dominant effect. In contrast, T
cells from HSP70⫺/⫺ mice proliferated equally at both temperatures and CO2 concentrations (Fig. 7). These results indicate that
an absence of HSP70i is not responsible for the HSF1⫺/⫺ T cell
proliferation defect observed at fever temperatures.
Discussion
This study reveals an important new function for HSF1 in T lymphocytes. T cells from HSF1⫺/⫺ mice show a profound defect in
proliferation at fever range temperatures that is exacerbated by low
CO2 concentrations. This defect is T cell intrinsic and does not
affect early T cell activation events but rather manifests as a block/
delay in the G1/S phase transition of the cell cycle. These conditions of elevated temperature and low CO2 did not affect proliferation of HSF1⫹/⫹ or HSF1⫹/⫺ T cells. Thus, HSF1 appears to
regulate a set of genes that play an important role in T cell cycle
progression at fever temperatures. This effect of HSF1 in T cells is
in agreement with a variety of studies showing an important role
for HSF1 in cell cycle progression in other cell types. In yeast,
HSF plays a critical role in cell cycle progression, partly through
the induction of HSP90 (48). Similar to our results in T cells, in the
absence of functional HSF, yeast cell cycle progression is temperature sensitive and blocked at the G2/M phase (48). Likewise, deletion of HSF1 and HSF3 in a chicken B cell line resulted in a
temperature-sensitive block in G1/S and G2 phases of the cell cycle. As in yeast, the block was attributed to a lack of HSP90 in the
mutant cells with Cdc2 as one of the molecular targets (49). Thus,
there appears to be an evolutionarily conserved fundamental role
for HSF1 in the stabilization of cell cycle progression at elevated
temperatures. We have not yet examined the role of HSP90 in
HSF1⫺/⫺ T cells but have confirmed that the block in cell cycle
progression is not because of a lack of HSP70i expression. Thus,
it is possible that HSP90 is a critical HSF1 target gene controlling
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 6. Reduced intracellular ROS levels in activated HSF⫺/⫺ T
cells. Purified T cells from HSF1⫹/⫹ or HSF1⫺/⫺ mice were activated with
plate-bound anti-CD3 at the indicated temperatures and CO2 concentrations for either 2 or 5 h. DCFH-DA (10 ␮M) was added to the cultures 30
min before the harvest. Following activation, cells were harvested, washed,
and analyzed immediately using a flow cytometer. The overall intracellular
redox levels were measured as the fluorescence of DCFH-DA plotted as a
histogram. All treatments were made in duplicate and the experiments have
been repeated three times.
HSF1, ROS, AND T CELL PROLIFERATION
The Journal of Immunology
Surprisingly, fever range temperature and low CO2 synergized
to lower ROS levels in T cells early on in their activation, suggesting a causal role of low ROS in the proliferation block of
HSF1⫺/⫺ T cells. This appears to be at odds with a conventional
view of oxidative stress resulting from an increase in ROS species.
However, this view is currently changing with the increasing
awareness that ROS play a critical role in normal signal transduction and cell cycle progression (47, 51). Thus, in cells undergoing
activation and entry into cell cycle, such as T cells, an increase in
ROS is an obligatory part of cell division, and if the ROS is inhibited with antioxidants, cell division is blocked (47, 51, 52). We
showed that fever range temperature and low CO2 lowered ROS
levels in both HSF1⫹/⫹ and HSF1⫺/⫺ T cells. However, only
HSF1⫹/⫹ and not HSF1⫺/⫺ T cells were able to recover normal
ROS levels after 5 h of activation. These results suggest that HSF1
plays a role in regulating ROS levels during T cell activation.
Recently, such a role for HSF1 has been demonstrated in mouse
cardiac muscle. Although in this model HSF1 deficiency resulted
in elevated ROS levels, the mechanism was shown to involve
changes in the levels of glutathione, the major ROS buffer in cells.
Thus, it is possible that HSF1 is important in maintaining ROS
homeostasis by preventing either abnormally high or low ROS
levels.
Our previous results showing HSF1 activation in T cells at fever
temperatures led us to hypothesize that HSF1 either protects T
cells or enhances the T cell response during fever. Our current
results suggest that HSF1 is critical for optimum T cell clonal
expansion during fever. The implication of this finding is that fever
range temperature is inherently detrimental to cell division and that
HSF1, through the induction of HSP and other genes, acts to facilitate cell cycle progression Our data suggest that ROS dysregulation may be one factor affecting optimum cell cycle progression
during fever, but many more studies are needed to understand exactly how fever range temperature affects cell division. Likewise,
it will be important to identify the target genes induced by HSF1
that confer this protection. Our data rule out HSP70i as playing a
role in cell cycle protection, but HSP90 and HSP25 remain strong
candidates. In contrast, HSP70i is strongly induced in activated T
cells in an HSF1 dependent manner. Given that HSP70i augments
both Ag presentation and costimulatory functions (8, 9, 32–37),
HSF1 is therefore involved in more than cell cycle protection.
Thus, we conclude that HSF1 is important for both the protection
of T cells as well as the enhancement of immune responses at fever
temperatures.
Acknowledgments
We acknowledge the technical assistance of Julia Jones, Marvin Ruffner,
Martin Ward, and Zachary Threlkeld. We also thank Ivor Benjamin for
providing the HSF1⫺/⫺ mice.
Disclosures
The authors have no financial conflict of interest.
References
1. Vaughn, L. K., H. A. Bernheim, and M. J. Kluger. 1974. Fever in the lizard
Dipsosaurus dorsalis. Nature 252: 473– 474.
2. Roberts, N. J., Jr. 1991. Impact of temperature elevation on immunologic defenses. Rev. Infect. Dis. 13: 462– 472.
3. Kluger, M. J., D. H. Ringler, and M. R. Anver. 1975. Fever and survival. Science
188: 166 –168.
4. Kluger, M. J., W. Kozak, C. A. Conn, L. R. Leon, and D. Soszynski. 1998. Role
of fever in disease. Ann. NY Acad. Sci. 856: 224 –233.
5. Shen, R. N., L. Lu, P. Young, H. Shidnia, N. B. Hornback, and H. E. Broxmeyer.
1994. Influence of elevated temperature on natural killer cell activity, lymphokine-activated killer cell activity and lectin-dependent cytotoxicity of human umbilical cord blood and adult blood cells. Int. J. Radiat. Oncol. Biol. Phys. 29:
821– 826.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
cell cycle progression in T cells at fever temperatures, and we are
currently examining this possibility.
Our original studies showed that HSF1 was activated to the
transcriptionally active form in T cells at fever temperatures. However, those studies were done using resting T cells (27). In the
present study, we showed that HSF1 became transcriptionally active (as assessed by the expression of HSP70i) even at 37°C upon
T cell activation. Activation of T cells at fever range temperatures
resulted in enhanced expression of HSF1 and enhanced transcriptional activation of target genes. Our studies of T cell activation in
vitro show “normal” proliferation of HSF1⫺/⫺ T cells at 37°C,
indicating that HSF1 is dispensable for T cell proliferation in vitro
at normal temperatures. However, we have recently found that in
vivo T cell expansion in response to staphylococcal enterotoxin B
administration is blunted in HSF1⫺/⫺ mice in the absence of fever
(S. Gandhapudi and J. Woodward, manuscript in preparation).
Thus, HSF1 has demonstrable effects in T cells even at normal
temperatures and may play a role in T cell clonal expansion in
vivo, even in the absence of obvious “stress.”
Indeed, recent data challenge a conventional view of HSF1 as a
strictly stress-inducible transcription factor. It is becoming apparent that HSF1 plays important roles in a number of cellular functions not normally associated with stress. Although HSF1⫺/⫺ mice
are viable, they exhibit a number of phenotypic effects. The mice
are often smaller than their wild-type littermates and embryonic
lethality of HSF1⫺/⫺ embryos is common, particularly as the mice
are backcrossed onto a pure inbred strain, indicating a role for
HSF1 in normal development (39) (J. G. Woodward, unpublished
observations). Also, HSF1⫺/⫺ females are sterile because of a requirement for maternal HSF1 in the postfertilization embryo, presumably to facilitate optimal early cell division of the embryo (40).
HSF1 also appears to be important in regulating redox homeostasis
in mouse cardiac muscle under normal physiological conditions in
vivo (50). Thus, there are likely a host of more subtle stressors
encountered in the in vivo environment for which HSF1 plays an
important role. During a typical immune response, the T cell is
likely to encounter many different potential stressors, including
fever range temperatures, changes in ROS levels, changes in O2
and CO2 levels, changes in osmolarity, and exposure to cytokines.
Therefore, it is likely that HSF1 plays an important role in optimizing T cell cycle progression during immune responses in vivo.
In vitro, increasing the temperature to the fever range and lowering
the CO2 concentration created a set of conditions that resulted in our
ability to visualize a proliferation defect in HSF1⫺/⫺ T cells. These
conditions did not affect HSF1⫹/⫹ or HSF1⫹/⫺ mice. The critical
question is, how do these conditions compare with those encountered by T cells in vivo? Certainly, fever range temperature is
encountered by T cells in vivo during the proliferation phase in
response to many viruses and bacteria. The local concentration of
CO2 in blood and tissues can also vary, although there are limited
data available allowing direct comparison of incubator CO2 levels
with the partial pressures of CO2 encountered in blood, lymph, and
tissues. Our data ruled out pH as an explanation for the low CO2
effect but supported a role for ROS changes as a possible explanation. Thus, given the myriad of differences between in vitro culture conditions and in vivo physiology, there is no compelling
evidence that 5% CO2 in an incubator is any more physiologically
relevant than 3% CO2. In fact, the critical in vivo conditions that
affect T cell proliferation may be ROS levels and not necessarily
CO2 levels. Thus, we feel it is likely that similar conditions may be
encountered by T cells during immune responses in vivo, and recent experiments showing reduced T cell expansion in vivo in
HSF1⫺/⫺ mice support this idea (S. Gandhapudi and J. Woodward, manuscript in preparation).
8311
8312
29. Sorger, P. K. 1991. Heat shock factor and the heat shock response. Cell 65:
363–366.
30. Morimoto, R. I., K. D. Sarge, and K. Abravaya. 1992. Transcriptional regulation
of heat shock genes: a paradigm for inducible genomic responses. J. Biol. Chem.
267: 21987–21990.
31. Pirkkala, L., P. Nykanen, and L. Sistonen. 2001. Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. FASEB J.
15: 1118 –1131.
32. Srivastava, P. 2002. Roles of heat-shock proteins in innate and adaptive immunity. Nat. Rev. Immunol. 2: 185–194.
33. Millar, D. G., K. M. Garza, B. Odermatt, A. R. Elford, N. Ono, Z. Li, and
P. S. Ohashi. 2003. Hsp70 promotes antigen-presenting cell function and converts
T-cell tolerance to autoimmunity in vivo. Nat. Med. 9: 1469 –1476.
34. Zheng, H., and Z. Li. 2004. Cutting edge: cross-presentation of cell-associated
antigens to MHC class I molecule is regulated by a major transcription factor for
heat shock proteins. J. Immunol. 173: 5929 –5933.
35. Vabulas, R. M., P. Ahmad-Nejad, S. Ghose, C. J. Kirschning, R. D. Issels, and
H. Wagner. 2002. HSP70 as endogenous stimulus of the toll/interleukin-1 receptor signal pathway. J. Biol. Chem. 277: 15107–15112.
36. Vabulas, R. M., H. Wagner, and H. Schild. 2002. Heat shock proteins as ligands
of toll-like receptors. Curr. Top. Microbiol. Immunol. 270: 169 –184.
37. Quintana, F. J., and I. R. Cohen. 2005. Heat shock proteins as endogenous adjuvants in sterile and septic inflammation. J. Immunol. 175: 2777–2782.
38. Di, Y. P., E. A. Repasky, and J. R. Subjeck. 1997. Distribution of HSP70, protein
kinase C, and spectrin is altered in lymphocytes during a fever-like hyperthermia
exposure. J. Cell. Physiol. 172: 44 –54.
39. Xiao, X., X. Zuo, A. A. Davis, D. R. McMillan, B. B. Curry, J. A. Richardson,
and I. J. Benjamin. 1999. HSF1 is required for extra-embryonic development,
postnatal growth and protection during inflammatory responses in mice. EMBO
J. 18: 5943–5952.
40. Christians, E., A. A. Davis, S. D. Thomas, and I. J. Benjamin. 2000. Maternal
effect of Hsf1 on reproductive success. Nature 407: 693– 694.
41. Inouye, S., H. Izu, E. Takaki, H. Suzuki, M. Shirai, Y. Yokota, H. Ichikawa,
M. Fujimoto, and A. Nakai. 2004. Impaired IgG production in mice deficient for
heat shock transcription factor 1. J. Biol. Chem. 279: 38701–38709.
42. Hampton, C. R., A. Shimamoto, C. L. Rothnie, J. Griscavage-Ennis, A. Chong,
D. J. Dix, E. D. Verrier, and T. H. Pohlman. 2003. HSP70.1 and -70.3 are required for late-phase protection induced by ischemic preconditioning of mouse
hearts. Am. J. Physiol. 285: H866 –H874.
43. Egan, R. M., C. Yorkey, R. Black, W. K. Loh, J. L. Stevens, E. Storozynsky,
E. M. Lord, J. G. Frelinger, and J. G. Woodward. 2000. In vivo behavior of
peptide-specific T cells during mucosal tolerance induction: antigen introduced
through the mucosa of the conjunctiva elicits prolonged antigen-specific T cell
priming followed by anergy. J. Immunol. 164: 4543– 4550.
44. Vesela, A., and J. Wilhelm. 2002. The role of carbon dioxide in free radical
reactions of the organism. Physiol. Res. 51: 335–339.
45. Arbogast, S., and M. B. Reid. 2004. Oxidant activity in skeletal muscle fibers is
influenced by temperature, CO2 level, and muscle-derived nitric oxide.
Am. J. Physiol. 287: R698 –R705.
46. Wieder, E. D., H. Hang, and M. H. Fox. 1993. Measurement of intracellular pH
using flow cytometry with carboxy-SNARF-1. Cytometry 14: 916 –921.
47. Williams, M. S., and J. Kwon. 2004. T cell receptor stimulation, reactive oxygen
species, and cell signaling. Free Radical Biol. Med. 37: 1144 –1151.
48. Morano, K. A., N. Santoro, K. A. Koch, and D. J. Thiele. 1999. A trans-activation
domain in yeast heat shock transcription factor is essential for cell cycle progression during stress. Mol. Cell. Biol. 19: 402– 411.
49. Nakai, A., and T. Ishikawa. 2001. Cell cycle transition under stress conditions
controlled by vertebrate heat shock factors. EMBO J. 20: 2885–2895.
50. Yan, L. J., E. S. Christians, L. Liu, X. Xiao, R. S. Sohal, and I. J. Benjamin. 2002.
Mouse heat shock transcription factor 1 deficiency alters cardiac redox homeostasis and increases mitochondrial oxidative damage. EMBO J. 21: 5164 –5172.
51. Havens, C. G., A. Ho, N. Yoshioka, and S. F. Dowdy. 2006. Regulation of late
G1/S phase transition and APC Cdh1 by reactive oxygen species. Mol. Cell. Biol.
26: 4701– 4711.
52. Jackson, S. H., S. Devadas, J. Kwon, L. A. Pinto, and M. S. Williams. 2004. T
cells express a phagocyte-type NADPH oxidase that is activated after T cell
receptor stimulation. Nat. Immunol. 5: 818 – 827.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
6. Rice, P., E. Martin, J. R. He, M. Frank, L. DeTolla, L. Hester, T. O’Neill,
C. Manka, I. Benjamin, A. Nagarsekar, et al. 2005. Febrile-range hyperthermia
augments neutrophil accumulation and enhances lung injury in experimental
gram-negative bacterial pneumonia. J. Immunol. 174: 3676 –3685.
7. Chen, Q., D. T. Fisher, K. A. Clancy, J. M. Gauguet, W. C. Wang, E. Unger,
S. Rose-John, U. H. von Andrian, H. Baumann, and S. S. Evans. 2006. Feverrange thermal stress promotes lymphocyte trafficking across high endothelial
venules via an interleukin 6 trans-signaling mechanism. Nat. Immunol. 7:
1299 –1308.
8. Basu, S., and P. K. Srivastava. 2003. Fever-like temperature induces maturation
of dendritic cells through induction of hsp90. Int. Immunol. 15: 1053–1061.
9. Ostberg, J. R., and E. A. Repasky. 2006. Emerging evidence indicates that physiologically relevant thermal stress regulates dendritic cell function. Cancer Immunol. Immunother. 55: 292–298.
10. Zheng, H., I. J. Benjamin, S. Basu, and Z. Li. 2003. Heat shock factor 1-independent activation of dendritic cells by heat shock: implication for the uncoupling
of heat-mediated immunoregulation from the heat shock response. Eur. J. Immunol. 33: 1754 –1762.
11. Hanson, D. F. 1993. Fever and the immune response: the effects of physiological
temperatures on primary murine splenic T-cell responses in vitro. J. Immunol.
151: 436 – 448.
12. Jampel, H. D., G. W. Duff, R. K. Gershon, E. Atkins, and S. K. Durum. 1983.
Fever and immunoregulation, III: hyperthermia augments the primary in vitro
humoral immune response. J. Exp. Med. 157: 1229 –1238.
13. Saririan, K., and D. A. Nickerson. 1982. Enhancement of murine in vitro antibody formation by hyperthermia: enhancement of murine in vitro antibody formation by hyperthermia. Cell. Immunol. 74: 306 –312.
14. Smith, J. B., R. P. Knowlton, and S. S. Agarwal. 1978. Human lymphocyte
responses are enhanced by culture at 40°C. J. Immunol. 121: 691– 694.
15. Mullbacher, A. 1984. Hyperthermia and the generation and activity of murine
influenza-immune cytotoxic T cells in vitro. J. Virol. 52: 928 –931.
16. Huang, Y. H., A. Haegerstrand, and J. Frostegard. 1996. Effects of in vitro hyperthermia on proliferative responses and lymphocyte activity. Clin. Exp. Immunol. 103: 61– 66.
17. Kregel, K. C. 2002. Heat shock proteins: modifying factors in physiological stress
responses and acquired thermotolerance. J. Appl. Physiol. 92: 2177–2186.
18. Travers, K. J., C. K. Patil, and J. S. Weissman. 2001. Functional genomic approaches to understanding molecular chaperones and stress responses. Adv. Protein Chem. 59: 345–390.
19. Flanagan, S. W., A. J. Ryan, C. V. Gisolfi, and P. L. Moseley. 1995. Tissuespecific HSP70 response in animals undergoing heat stress. Am. J. Physiol. 268:
R28 –R32.
20. McMillan, D. R., X. Xiao, L. Shao, K. Graves, and I. J. Benjamin. 1998. Targeted
disruption of heat shock transcription factor 1 abolishes thermotolerance and
protection against heat-inducible apoptosis. J. Biol. Chem. 273: 7523–7528.
21. Sarge, K. D., V. Zimarino, K. Holm, C. Wu, and R. I. Morimoto. 1991. Cloning
and characterization of two mouse heat shock factors with distinct inducible and
constitutive DNA-binding ability. Genes Dev. 5: 1902–1911.
22. Wu, C. 1995. Heat shock transcription factors: structure and regulation. Annu.
Rev. Cell. Dev. Biol. 11: 441– 469.
23. Baler, R., G. Dahl, and R. Voellmy. 1993. Activation of human heat shock genes
is accompanied by oligomerization, modification, and rapid translocation of heat
shock transcription factor HSF1. Mol. Cell. Biol. 13: 2486 –2496.
24. Lee, B. S., J. Chen, C. Angelidis, D. A. Jurivich, and R. I. Morimoto. 1995.
Pharmacological modulation of heat shock factor 1 by antiinflammatory drugs
results in protection against stress-induced cellular damage. Proc. Natl. Acad. Sci.
USA 92: 7207–7211.
25. Sarge, K. D., S. P. Murphy, and R. I. Morimoto. 1993. Activation of heat shock
gene transcription by heat shock factor 1 involves oligomerization, acquisition of
DNA-binding activity, and nuclear localization and can occur in the absence of
stress. Mol. Cell. Biol. 13: 1392–1407.
26. Sarge, K. D., A. E. Bray, and M. L. Goodson. 1995. Altered stress response in
testis. Nature 374: 126.
27. Gothard, L. Q., M. E. Ruffner, J. G. Woodward, O. K. Park-Sarge, and
K. D. Sarge. 2003. Lowered temperature set point for activation of the cellular
stress response in T-lymphocytes. J. Biol. Chem. 278: 9322–9326.
28. Sarge, K. D. 1995. Male germ cell-specific alteration in temperature set point of
the cellular stress response. J. Biol. Chem. 270: 18745–18748.
HSF1, ROS, AND T CELL PROLIFERATION