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1 Department of Hygiene, ABSTRACT Kizaki, Takako, Tomomi Ookawara, Tetsuya Izawa, Junichi
Nagasawa, Shukoh Haga, Zsolt Radák, and Hideki Ohno.
Relationship between cold tolerance and generation of suppressor
macrophages during acute cold stress. J. Appl.
Physiol. 83(4): 1116-1122, 1997.
glucocorticoid; immunosuppression; cold stress; cold acclimation; brown adipose tissue
INTRODUCTION STRESS, which is generally defined as a state of
altered homeostasis resulting from an external or an internal challenge
(i.e., a stressor), is characterized by activation of both the
hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous
system (SNS). The resulting neurochemical changes have been
demonstrated to affect immune function both directly and indirectly
(20). Indeed, environmental and psychological stresses reduce
cell-mediated immunity, which may in turn lead to high susceptibility
of infection and facilitation of tumor growth or metastasis (10, 17,
24). Although most studies have shown stress-induced immunosuppressive
effects (21, 26), others have found no changes or even an enhancement
of the immune response after stressor application (15).
Such controversial results may be attributable not only to the stressor
characteristics but also to complicated responses of the neuroendocrine
system to stress exposure. For example, under prolonged or chronic
stress, excessive actions of products of the HPA axis and SNS can lead to alterations in the physiological response to acute stress in many
body systems (6). Therefore, depending on length of exposure, the
nature of the stressor, and the timing of stress application, immune
functions are suppressed, enhanced, or remain unchanged.
Environmental temperature is known to modify the immune capacity of a
number of animal species. For instance, cold-exposed animals showed
qualitative and quantitative differences in the response to infections
from various microorganisms (9, 22). In our previous study (14), we
showed that the proliferative responses to concanavalin A (Con A) in
spleen cells from mice exposed to 5°C for 24 h (acute cold stress)
were significantly lower than those from control mice and that the
depressed Con A responses were attributable to the suppressive
regulation by adherent cells (representative of cells of
monocyte/macrophage lineage). Furthermore, we have recently
demonstrated that acute cold stress markedly increases suppressor
peritoneal macrophages, which strikingly suppress Con A responses
of spleen cells from control mice by releasing nitric oxide
(12, 13). The suppressor macrophages were characterized by the
expression of large numbers of type II/III receptor to the
crystalizable fragment (Fc) portion of immunoglobulin (Ig) G
(MAC-1+Fc Because environmental stressors, including cold stress, are prevalent
and sometimes unavoidable, their harmful influence should be understood
so that treatment modalities can be developed. Thus we were interested
in the differences of physiological responses to acute cold stress
between naive and cold-acclimated mice. The present study was
undertaken to investigate whether the improved cold tolerance by
exposure to chronic cold stress prevented the depression of immune
responses by an acute cold stress and focused in particular on the
generation of suppressor macrophages.
METHODS
Acute cold
stress induces suppressor macrophages expressing large numbers of
receptors to the crystallizable fragment (Fc) portion of
immunoglobulin G
(MAC-1+Fc
RII/IIIbright
cells), resulting in the immunosuppression of splenocyte mitogenesis. The generation of
MAC-1+FcRII/IIIbright
cells is mediated by the action of glucocorticoids (GCs) through the
GC-receptor. In the present study, the generation of
MAC-1+FcRII/IIIbright
cells in peritoneal exudate cells was closely related to the decrease
of rectal temperature during 3-day exposure to 5°C. We next
investigated the effects of improved cold tolerance on the generation
of
MAC-1+FcRII/IIIbright
cells during acute cold stress. Mice were adapted to cold by exposure
to 5°C for 3 wk (cold-acclimated mice) and then reexposed to
5°C for 3 h (acute cold stress) after living at 25°C for 24 h.
The rectal temperature of cold-acclimated mice was not decreased by the
acute cold stress. In addition, the proportion of
MAC-1+FcRII/IIIbright
cells in peritoneal exudate cell population from cold-acclimated mice
was unaffected by the acute cold stress. The cold acclimation significantly attenuated the increases in serum corticosterone levels
and the expression of the GC-receptor mRNA on peritoneal exudate cells
in response to acute cold stress. These results suggest that the
altered GC response to acute cold stress by the improvement of cold
tolerance inhibits the generation of suppressor macrophages during
acute cold stress.
RII/IIIbright
cells) (13).
RII/III monoclonal antibody (MAb) (2.4G2, rat
IgG2b, American Type Culture Collection, Rockville, MD) and fluorescein
isothiocyanate-labeled goat anti-rat Ig antibody (Caltag Laboratories,
South San Francisco, CA). Rat myeloma protein (rat IgG2b, Serotec,
Oxford, England) was used as isotypic control antibodies. After
extensive washing, the cells were treated with phycoerythrin
(PE)-conjugated anti-MAC-1 MAb (Caltag Laboratories). After each step,
the cells were extensively washed with phosphate-buffered saline
containing 0.1% bovine serum albumin (Sigma Chemical, St. Louis, MO)
and 0.1% NaN3 to reduce nonspecific staining.
-actin or with 30 cycles for glucocortisoid (GC)
receptor. PCR products were separated by electrophoresis on 4%
acrylamide gel and were visualized by ultraviolet illumination after
being stained with ethidium bromide.
Statistical analysis.
All data are expressed as means ± SE. When only two means were
compared, Student's t-test for
unpaired samples was used. For more than two groups, the statistical
significance of the data was assessed by analysis of variance. When
significant differences were found, individual comparisons were made
between groups by using the t
statistic and adjusting the critical value according to the Bonferroni
method (3). Differences were considered significant at
P < 0.05.
RESULTS
RII/IIIbright
cells during cold stress.
Figure 1 shows the effect of cold stress on
the rectal temperature in naive mice. Within 1 h of cold stress, the
rectal temperature was significantly decreased. The lowest rectal
temperature was observed in mice at 24 h of cold exposure. Thereafter,
the rectal temperature was increased and returned to its normal levels
after 72 h of cold stress.
, n = 8) or 5°C (
, n = 7). Rectal temperatures were measured by thermistor thermometer inserted 4 cm into
rectum at indicated time. Results are expressed as means ± SE.
* Significantly lower than control value at same duration of
exposure, P < 0.01.
We then examined the effect of cold stress on the populations of peritoneal exudate cells. Figure 2A shows the data comparing typical profiles of two-color immunofluorescence staining with MAbs specific to MAC-1 and Fc
RII/III
on peritoneal cells from three to five mice each. Two distinct cell
populations
(MAC-1+FcRII/IIIbright
cells and
MAC-1+FcRII/IIIdull
cells) could be seen in control mice and in mice exposed to the different periods of cold stress. It is apparent from Fig.
2B that the
MAC-1+ cells bearing higher
amounts of the FcRII/III molecule increased significantly in the peritoneal exudate cells from mice cond stressed for 3 h. The proportion of
MAC-1+FcRII/IIIbright
cells was markedly increased in peritoneal exudate cells at 24 h of
cold stress and decreased thereafter. As summarized in Fig. 2C, the highest proportion of
MAC-1+FcRII/IIIbright
cells was demonstrated in peritoneal exudate cells at 24-h cold stress.
These results suggested that the highest expression of FcRII/III on the peritoneal exudate cells was
induced when the effect of low temperature reached its peak.
RII/III in peritoneal
exudate cells was analyzed by flow cytometry. Cells (1 × 106) were stained with
anti-FcRII/III monoclonal antibody (MAb) followed by
fluorescein isothiocyanate (FITC)-anti-rat immunoglobulin (Ig), then
reacted with phycoerythrin (PE)-anti-MAC-1 MAb.
A: two-color flow cytometric analysis
of MAC-1 and FcRII/III on peritoneal exudate cells.
B: single histogram of
FcRII/III on MAC-1+ peritoneal exudate cells.
C:
%FcRII/IIIbright
cells (mean ± SE) in peritoneal exudate cells from 4 or 5 mice. * Significantly higher than 0-h value;
P < 0.01.
BAT weight and cold-tolerance test. The time course of BAT weight in terms of weight per unit body weight is illustrated in Fig. 3. The significant increase of the BAT weight was observed in mice on day 3 of cold exposure. The BAT weight of cold-acclimated mice was markedly higher than that of control mice, suggesting the improvement of cold tolerance. Cold tolerance of the cold-acclimated mice to an acute cold stress was then examined. The cold-acclimated mice were reared at 25°C for 24 h before the reexposure to acute cold stress (5°C for 3 h). Figure 4 shows the effect of acute cold stress on the rectal temperature in control and cold-acclimated mice. The rectal temperature in control mice decreased significantly during 3 h of acute cold stress, whereas the cold stress did not affect the rectal temperature of cold-acclimated mice. The finding suggested that the increased capacity of BAT developed by cold exposure for 3 wk is sufficient to maintain the rectal temperature in response to 3 h of cold stress.
,
n = 3) or 5°C (,
n = 4) and killed after indicated
period of cold exposure. Interscapular BAT was removed and weighed.
Results are expressed as means ± SE. Error bars are too small to be
distinguishable in the figure. * Significantly higher than
control value at same duration of exposure,
P < 0.01.
,
n = 5) and cold-acclimated (,
n = 5) mice were exposed to 5°C.
Rectal temperatures were measured by thermistor thermometer inserted 4 cm into rectum at indicated time. Results are expressed as means ± SE. Error bars are too small to be distinguishable in the figure.
* Significantly lower than 0 h value,
P < 0.01.
Effects of cold acclimation on the generation of MAC-1+Fc
RII/IIIbright
cells by acute cold stress.
To investigate the effects of the improvement of cold tolerance on the
immune system, we investigated the generation of
MAC-1+FcRII/IIIbright
cells in peritoneal exudate cells by acute cold stress in
cold-acclimated mice. As illustrated in Fig.
5A, before
the acute cold exposure, the peritoneal exudate cell populations of
cold-acclimated mice appeared to be almost the same as those of control
mice. The proportion of
MAC-1+FcRII/IIIbright
cells in peritoneal exudate cells from control mice was significantly increased by 3 h of cold stress (Fig. 5,
A and
B). The proportion of
MAC-1+FcRII/IIIbright
cells, on the other hand, did not increase in cold-acclimated mice with
exposure to 3 h of cold stress. These findings are more clearly
demonstrated in Fig. 5.
RII/IIIbright
cells in peritoneal exudate cells by acute cold stress. Control
(n = 5) and cold-acclimated
(n = 5) mice were exposed to 5°C
for 3 h, and peritoneal exudate cells were harvested. Expression of MAC-1 and FcRII/III on peritoneal exudate cells was
analyzed by flow cytometry. Cells (1 × 106) were stained with
anti-FcRII/III MAb followed by FITC-anti-rat Ig and
then reacted with PE-anti-MAC-1 MAb.
A: 2-color flow-cytometric analysis of
MAC-1 and FcRII on peritoneal exudate cells.
B: single histogram of
FcRII/III on
MAC-1+ peritoneal exudate cells.
C: mean
%FcRIIbright
cells in peritoneal exudate cells with SE.
* P < 0.01, stressed vs.
unstressed controls.
Effects of cold acclimation on GC responses to acute cold stress. As illustrated in Fig. 6, basal serum corticosterone concentrations of cold-acclimated mice were almost the same as those of control mice. Serum corticosterone concentrations of control mice increased markedly during 3 h of acute cold stress. On the other hand, the acute cold stress did not affect serum corticosterone concentrations of cold-acclimated mice.
Effects of cold acclimation on GC-receptor mRNA expression on peritoneal exudate cells by acute cold stress. GC-receptor mRNA was not detected in whole peritoneal cells from control mice but was detected in those from acute cold-stressed mice (Fig. 7). The expression of GC-receptor mRNA was observed in peritoneal exudate cells from cold-acclimated mice, but the expression was lower than in those cells from acute cold-stressed control mice. Although the expression level of
-actin
mRNA was almost the same among all samples analyzed, the expression of GC-receptor mRNA in peritoneal exudate cells was unaffected by acute
cold stress in cold-acclimatized mice.
-actin mRNA in
peritoneal exudate cells from control mice, acute cold-stressed control
mice, cold-acclimated mice, and cold-acclimated, acute cold-stressed
mice were analyzed by reverse transcription-polymerase chain reaction.
bp, Base pairs.
DISCUSSION
Cold exposure stimulates heat production by means of nonshivering as well as shivering thermogenesis. A number of studies have now established that metabolic acclimation to cold is characterized by an enhanced nonshivering thermogenesis as a more efficient means of heat acquisition than shivering. BAT is a principal energy source of nonshivering thermogenesis through the presence of a tissue-specific uncoupling protein that is located in the inner mitochondrial membrane. During chronic exposure to cold, BAT mass increases, and this in turn enhances nonshivering thermogenesis (25). In the present study, the BAT mass increased significantly in mice exposed to cold for 3 days compared with the BAT mass in mice reared at 25°C. Meanwhile, the rectal temperature of naive mice exposed to cold was decreased during the initial 24 h, increased thereafter, and was completely restored to the normal temperature on day 3 of cold exposure. Thus the increased mass of BAT on day 3 of cold exposure was accompanied by an increase in thermogenesis. This increase in BAT mass may contribute to the restoration of the rectal temperature during acute cold stress.
In our previous study (13), we demonstrated that
MAC-1+FcRIIbright
cells suppressed Con A responses of spleen cells from control mice.
This suggests that the acute cold stress generates suppressor macrophages that may cause immune suppression. We also showed that the
MAC-1+FcRIIbright
cells were at functionally high levels (13). Thus it appears that
peritoneal exudate cells are activated in some way after acute exposure
to cold. The observation that activated macrophages (MAC-1+FcRIIbright
cells) function as suppressor cells is congruent with the concept that
activated macrophages are more suppressive than their resident or
nonactivated counterparts (1, 2, 18, 19, 23). On the other hand, during
the course of inflammatory reactions induced by infectious processes or
by autoimmune responses, activated macrophage-derived cytokines, such
as interleukin (IL)-1, IL-6, and interferon-
, affect
the brain by activating the HPA axis and inducing fever, slow-wave
sleep, and decreased appetites (4, 7, 16). Therefore, cells in
monocyte/macrophage lineage appear to have a profound effect on host
resistance, survival, and a variety of physiological responses to
infection. In addition, the recent study by Hofman and Hinton
(8) indicated that the pyrogenic effects of IL-1 in rats
are mediated centrally and are caused by the sympathetic activation of
thermogenesis in BAT, thereby resulting in a rise in the metabolic
rate. Furthermore, Burysek et al. (5) have suggested a dual effect of
IL-1 on BAT: one mediated centrally through sympathetic innervation and the other peripherally by direct interaction with adipocytes. As shown
in Fig. 2, the proportion of
MAC-1+FcRIIbright
cells in peritoneal exudate cells rapidly increased at an early stage
of cold exposure when the rectal temperature was lowered, and the
proportion decreased on day 3 when
normothermia was achieved. Although any conclusion is speculative, it
seems that neuroendocrine pathways activated by the response to cold
stress may result in macrophage activation and cytokine production that
may result in thermogenesis. Our data could be interpreted to suggest
that activated macrophages are important in early hyperthermia, whereas hyperthermia after day 3 may be
mediated by increased BAT mass.
In any event, the generation of
MAC-1+FcRII/IIIbright
cells in peritoneal exudate cells by acute cold stress appeared to be
closely related to the decrease of rectal temperature. Using
cold-acclimated mice, we then investigated whether improved cold
tolerance would inhibit the immunomodulation by acute cold stress. BAT
mass was greatly increased during chronic exposure to cold. The acute
cold stress test clearly demonstrated improved cold tolerance in
cold-acclimated mice. When cold-acclimated mice were reexposed to 3 h
of cold stress after living at 25°C for 24 h, the rectal
temperature did not change substantially, whereas the rectal
temperature of control mice decreased significantly. The increased mass
of BAT obtained by exposure to cold for 3 wk seemed to be sufficient to
keep the rectal temperature constant. Cold acclimation did not affect
the basal percentage of
FcRII/III+ macrophages
compared with nonacclimated mice. The proportion of
MAC-1+FcRII/IIIbright
cells in peritoneal exudate cells from cold-acclimated mice, unlike
those in unacclimated controls, was unaffected by 3 h of acute cold
stress. Thus these results suggest that improved cold tolerance inhibits not only the decrease of body temperature but also
the generation of suppressor macrophages during acute cold stress.
As already stated, stress, which is broadly defined as the response of
an organism to stimulation or change, is characterized by activation of
both the autonomic nervous system and the HPA axis. In our previous
study, we demonstrated that the generation of the
MAC-1+FcRII/IIIbright
cells during acute cold stress is mediated to a greater or lesser degree by increased GC levels after the activation of the HPA axis
(13). Thus we investigated effects of acute cold stress on serum
corticosterone concentrations of cold-acclimated mice. Cold acclimation
did not affect basal corticosterone levels but did attenuate the
corticosterone response to acute cold stress compared with
nonacclimated controls. In addition, cold acclimation attenuated the
increase in the GC mRNA expression in peritoneal exudate cells caused
by acute cold stress. These observations, coupled with our previous
findings that adrenalectomy and administration of the GC antagonist
RU-38486 can block suppressor macrophage generation in unacclimated,
acute cold-stressed mice (13), suggest that attenuated GC responses to
acute cold stress in cold-acclimated mice may be the mechanism
responsible for the lack of
MAC-1+FcRII/IIIbright
cell generation during acute cold stress. The critical mechanisms that
regulate the cellular immune responses characterized in the present
report and the role of FcRII/III
macrophages remain to be elucidated. Further studies will be needed to
clarify the immunological or nonimmunological roles of cells of
monocyte or macrophage lineage in the response to acute cold stress.
ACKNOWLEDGEMENTS
The authors thank M. Segawa for excellent technical assistance.
FOOTNOTES
Address for reprint requests: T. Kizaki, Dept. of Hygiene, National Defense Medical College, 3-2, Namiki, Tokorozawa 359, Japan.
Received 23 January 1997; accepted in final form 9 June 1997.
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ABSTRACT