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BQJHOCAHHTETCKH n P E M E f l
CipaHa 657
UDC: 612.017.1:57.04
O R I G I N A L
A R T I C L E S
Influence of the elevated ambient
temperature on immunoglobulin G and
immunoglobulin G subclasses in sera of
Wistar rats
Maja Jurhar-Pavlova*, Alcksandar Petlichkovski*, Dcjan Trajkov*, Olivija
LCrinska-MIadenovska*, Todor Arsov*, Ana Strezova*, Suzana Dinevska-Kjovkarova*, Slavcho Milev*, Mirko Spiroski*
Medical Faculty, *lnsiituie of Immunobiology and Human Genetics. Faculty of Natural
Sciences, 'Depurtmcnl ut' Physiology and Bicx:hciiii:^iry, Skopje, Republic of Macedonia
The aim of our research was to examine changes in the immune system of the rats influenced by (he. elevated ambient temperature. Male Wistar rats were divided, into 2
groups and housed at 20 ± 2° C (n=64, control group) and 35 ± 1° C (n=74, experimental group), during precise timing of 1, 4. 7. 14. 21. and 30 days. All the animals
were given food and water ad libitum, and were lighted during 12 hours per day. We
have measured igG. IgGl. lfiG2a. lgG2b and lgG2c. Tlie obtained results .showed significant elevation in the level of IgG after 4 and 7 days (+32%), lgG2a after 7th
f+SS%;. I4th and 2lnd day (+110%). lgG2b after 14 days (+60%) at 35 ± 1° C compared with the control group at 20 ± 2° C. fgGl level was not affected and lgG2c
showed significant decrease after 21st day at 35 ± 1° C. In conclusion, during the elevated ambient temperature the immune system is activated as one of the regulation
mechanisms in homeostasis and survival of the population.
Key
words:
temperature; acclinialization; immune system; immunoglobulins; immunoglobulin G.
Introduction
as well as ihe increased spontaneous lymphoprolireraiion (10.
11). There are reports with special reference lo humoral immuWc are the witnesses of seasonal heat waves (temperature
nity against specific antigens that clarify niore efficient reof 32° C and above. lasting for more than three days) as a respon^ to toxoplasma (7), and tetanus toxoid (4). Functional
suit of Earth global wanning, which cause increa.sed morbidity
compensation when only one ecological factor was changed in
and nwrtality of population (1, 2). Therefore, the detailed exthe controlled laboratory chamber for the period from few days
amination of the elevated ambient temperature effects on many
lo few weeks, was named acclimation by Eagan and Folk (15,
functions in the human organism is necessary. The actual
16). Available literature data gave no clear explanation conmodel of homeostatic functioning is composed of neuroendoceming in vivo changes in immunoglobulin levels during excrine system, immune system, ajid environment. A single
position to the elevated ambient temperature long enough to
change within these systems has the influence on homeostasis.
achieve acclimation. Bazin et al. defined immunoglobulin
and induces changes in order to establish nonpathologic equiclasses IgM. IgA. IgG. and IgG subclasses in semm of Wistar
librium (3). Emerging body of evidence has confinned that the
rats (17. IS). We found no literature daia that clarified variaelevated ambient temperature affects the immune system.
tion in the concentration of IgG. IgGl, IgG2a, lgG2b. and
Gianges are polymorphic and depend on the intensity and dulgG2c in Wistar rats due to ambient temperature. The aim of
ration of the exposition, species, gender and aging (4-13). The
this study was to investigate in vivo changes in the concentradecreased body weight (14). decreased relative thymus mass.
tion of IgG. and IgG subclasses In serum of Wistar rats housed
and leucopenia in the Wistar rats have already been reported. at 35 ±1 " C during 1. 4. 7. 14. 21, and 30 days, and to com-
Jurhar-Pavlova M, et al. Vojnosanit Pregl 2003; 60(6): 657-661.
658
BOJHOCAHHTETCKH
pare them with the respective values found in the control group
of the rats housed at 20 ± 2° C.
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Values for IgGl did not show significant difference
among experimental and control groups (Fig. 2).
Methods
IgGl mgfl)
The experiment comprised 2-months-otd male Wistar rats
(154± 18 g). from the Institute of Immunobiology and Human
Genetics, Faculty of Medicine, Skopje. Commercial chow
(Manufactory for animal food Radobor - Bitola). and fresh
water were provided ad libitum. A tolal of 138 rats were randomly divided into two groups, Control group (n=64) housed
at the ambient temperature of 20 ± 2° C, and experimental
group (n=74) kept in hot chamber (2x1.5x3 m) at 35 ± 1° C,
and relative humidity of 30-40%. Animals were maintained on
12:12 hrs light-dark cycle. Six phases of acclimatory periods
were defined depending on the duration of heat exposition:
first phase lasted 1 day, second - 4 days, and consequently 7,
14,21 day, the last phase in the duration of 30 days. According
to this different duration of the exposition, the rats from each
group were subdivided inio 6 subgroups (n=9-I4). At the end
of each acclimatory phase the rats were sacrificed precisely at
9-10 AM, in order to avoid circadian variations in examined
parameters (19). Animals were sacrificed under ether anesthesia, and blood was collected from abdominal aorta. Serum
(3000 rpm/10 min) was kepi at -20° C. Radial immunodiffusion plates (20) (ICN Immunobiologicals, Costa Mesa, CA
92623) were used for the determination of immunoglobulin G,
IgGl, IgG2a, IgG2b, and IgG2c concentration. Using Student's t-test showed statistical significance of ihe observed differences between the analyzed groups. Values of p<0.05 were
considered as statistically significant.
1800
I
T
1000
-r
T
T
I
4
1
X
7
14
21
30
Days of acclimation
Fig. 2 - Changes in concentration of immunoglobulin Gl in
serum of rats during acclimation. Results are expressed as
Mean ± SD.
In rats exposed to 35 ± P C the IgG2a concentration
was significantly increased (from +88% to +110%) on the 7th
(p<0.05), I4th (p<0.05), and 21st (p<0.01) day, in comparison with the concentration in rats kept at 20 ± 2° C (Fig. 3).
l9G2a (fng/l)
—•—20*2°C
9000
-a-35±rc
**
6000
3000
0
•
1
T
Results
We have monitored changes in the concentration of
IgG, IgGl, IgG2a, IgG2b, and IgG2c in serum of rats from
six subgroups housed at 35 ± 1° C, and compared them with
the respective subgroups kept at 20 ± 2° C.
The IgG concentration was elevated on the 4ih day,
remained increased until the 21st day, and then declined on
the 30th day. Statistically significant difference (+32%;
p<0.05) compared to the control group was observed on the
4th and 7th day (Fig. 1).
35±rc
• - -20±2°C-I
2600 -
U
t1
M
Days of acclimation
Fig. 3 - Changes in concentration of immunoglobulin G2a
in serum of rats during acclimation. Description in Figure 1.
*p<0.05, **p<0.01.
Concentration of IgG2b was significantly increased on
the 14th day (+60%; p<0.05) (Fig. 4), whereas the concentration of IgG2c was significantly lower (-32%; p<0.05) on
the 21st day at 35 ± 1° C in comparison with the values
found in rats kept at 20 ± 2° C (Fig. 5).
lgG2b (rng/l)
—•—20±2'=C —O—35±rC
20000
T
15000
10000
5000 ^
i
1
4
TI
7
*
•
14
21
i
30
Days of acclimation
Fig. 1 - Changes in concentration of immunoglobulin G in
serum of rats during acclimation. Results are expressed as
Mean±SD*p<0.05.
Fig. 4 - Changes in concentration of immunoglobulin G2b
in serum of rats during acclimation. Description in Figure 1.
•p<0.05.
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BOJHOCAHHTETCKH
Fig. 5 - Changes in concentration of immunoglobulin G2c
in serum of rats during acclimation. Description in Figure 1.
*p<0.05.
Discussion
The processes of acclimation in the changed living environment trigger complex mechanisms and regulatory
molecules (cytokines. neuropeptides) that modulate the immune response.
Our results showed that the immune system was activated. We have mentioned reports referring to more efficient response to various antigens (tetanus toxoid, toxoplasmosis, sheep erythrocytes) in the conditions of the elevated ambient temperature. If there is no exogenous antigen,
it is assumed that ambient temperature act as a nonspecific
activator. Following the seasonal variations in the immune
system of wild raLs, Lochmiler (11) noticed increased
spontaneous lymphoproliferation in August. Wang (21), reported that changes in lymphocytes at the elevated temperature were similar to those in antigen challenged lymphocytes. He observed the protein kinase distribution and
activity in T lymphocytes from peripheral blood of BALB/C
mice kept in hot chambers. Until recently, relative presence or
activation of ThI and Th2 was thought to have the regulatory
effect on the immune behavior. Balance of Thl and Th2 cytokine profile was considered as a basis of immune system
homeostasis. Nowadays, specialized subsets of regulatory T
cells, as well as their cytokines (ILIO and TGF), are held to
be responsible for the immune system balance (22). In the experiments dating back to 1988, Eden et al, (23) showed that
hsps triggered regulatory T cells (24, 25).
It is known that heat shock proteins are induced by
heat (26-29). Amphetamine induced hyperthermia results in
the increased level of both hsp70 and hsp90 in hepatocytes
of Wistar rats (30). Hsps are remarkably immunogenic, despite their high degree of evolutionary conservation. Dominant immunoglobulin class is IgG in humoral immune re-
sponse of mice immunized with Hsp70 (31). Prakken et al
(32), revealed that immunization of rats with hsp70 led to a
higher expression of Th2 cytokines profile (IL-10 and IL-4), and consequently higher amount of IgG2a. Our results
are in accordance with these reports, and partly explain significantly elevated IgG concentration on the 4th and 7th day
in sera of Wistar rats exposed to 35 ± 1° C. Analyzing the
immunoglobuin G subclas.ses, we noticed that IgG2a subclass was mostly affected by the elevated ambient temperature. Initial elevation of IgG2a was on the 4th day, but statistically significant difference comparing to the control group
values was achieved on the 7th, 14'^ and 21st day. The level
of Hsp60 was elevated under hyperthermic condition (28).
This protein induced the secretion of cytokines of Thl profile
(IFN-y) in raLs. Some studies also revealed that elevated ambient temperature caused the increase of IFN-y secreting cells
(33). In rats IFN-v stimulated the production of IgG2b (34,
35). This contributed to our results and offered an explanation
why the concentration of IgG2b was increased in serum of
our heat acclimated rats, in comparison with the respected
values of the control group (20 ± 2°).
It is very difficult to predict the dominant immunoglobulin subclass in the immune response to different antigens (T dependent, and/or T independent) in rats (36). Depending on the given adjuvant, the dose antigen, and the
used carrier, the immune response is quite different against
the same antigen or hapten. In vivo it also depends on the
cytokine profile of the microenvironment. Our experiment
did not demonstrate the efficiency of the immune response
to antigen challenge. However, it was evident that elevated
ambient temperature provoked time-dependent changes in
the concentration of different IgG subclasses in Wistar rats.
A mixed Thl/Th2 pattern was observed. Changes were noticed from the 4th lo 21st day. The examined parameters
were normalized on the 30th day.
Conclusion
In conclusion, during the exposition to the elevated
ambient temperature, there was an increased concentration
of IgG2aon the 7th, 14th and 21st day, IgG2b on the I4th
day, while the concentration of IgG2c was decreased on
the 21st day. The values for IgGl were not significantly
changed. On the 30th day there was no significant difference in the concentration of IgG and igG subclasses
among the examined groups and we could have assumed
that the triggered homeostatic mechanisms achieved acclimation.
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The paper was received on February 17,2003.
Ap st rakt
Jurhar-Pavlova M, PetliCkovski A, Trajkov D, Efinska-Mladenovska O, Arsov T,
Strezova A, Dinevska-Kjovkarova S, Mitev S, Spiroski M. Vojnosanit Pregl 2003;
60(6): 657-661.
UTICAJ POVI§ENE S P O L J A S N J E TEMPERATURE NA IMUNOGLOBULIN G I
PODGRUPE IMUNOGLOBULINA G U SERUMU PACOVA SOJA WISTAR
Cilj ovog ispitivanja bio je da se utvrde promene u imunskom sistemu pacova pod
ulicajem povigene spoljaSnje temperature. Pacovi soja Wistar muSkog pola podeljeni su u dve grupe. Kontroina grupa (n=64) drzana je na 20 ± 2° C, a eksperimenlna (n=74) na 35±1°C u trajanju od 1, 4. 7, 14, 21 i 30 dana. Uspostavljen je
svetlosni ciklus od 12/12 iasova i zivotinjama je davana hrana i voda ad libitum.
Merena je koncentraciju imunoglobulina G, lgG1, lgG2a, lgG2b i lgG2c. Rezultati
ispitivanja pokazali su statistitki zna6ajno poviSene koncentracije IgG fietvrtog i
sedmog dana, lgG2a sedmog, 14. i 21. dana, a lgG2b ietrnaestog dana u eksperi me ntnoj. u odnosu na kontroinu grupu. Izmedu ispitivanih grupa nije bilo znatajnih raziika u koncentraciji IgGi, dok je koncentracija lgG2c bila znaCajno niza
dvadesetprvog dana na poviSenoj spoljaSnjoj temperaturi. Moze se zakljutiti da je u
usiovima poviSene spoljaSnje temperature aktivisan imunski sistem kao jedan od
kljuCnIh regulatora u oCuvanju homeostaze i opstanka populacije.
Kl]u£ne
re£l:
temperatura; akiimatizacija; imunski sistem;
imunoglobulini; igG.
Correspondence to: Maja Jurhar-Pavlova, Medicinski fakultet. Institut za imunobiologiju i humanu genetiku; 1109 Skopje,
PP 60, "50 divizija" br. 6, Makedonija. Tel. +389 2110 556, E-matl: jurharm@yahoo.com