Influence of the elevated ambient temperature on immunoglobulin G and immunoglobulin G subclasses in sera of Wistar rats

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.


Introduction
as well as ihe increased spontaneous lymphoprolireraiion (10. 11).There are reports with special reference lo humoral immu-Wc 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 re-spon^ to toxoplasma (7), and tetanus toxoid (4).Functional suit of Earth global wanning, which cause increa.sedmorbidity compensation when only one ecological factor was changed in and nwrtality of population (1, 2).Therefore, the detailed ex-the 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 neuroendo-ceming 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 equi-classes 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 du-lgG2c 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.

Methods
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 (2x1.5x3m) 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.

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).

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)(27)(28)(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.

30 Fig. 2 -
Fig. 2 -Changes in concentration of immunoglobulin Gl in serum of rats during acclimation.Results are expressed as Mean ± SD.

Fig. 1 -
Fig. 1 -Changes in concentration of immunoglobulin G in serum of rats during acclimation.Results are expressed as Mean±SD*p<0.05.