Production of tumor necrosis factor in cells of hibernating ground squirrels Citellus Undulatus during annual cycle

Life Sciences 67 (2000) 1073Ð1080

Production of tumor necrosis factor in cells of hibernating ground squirrels Citellus Undulatus during annual cycle Elena G. Novoselova*, Stella G. Kolaeva, Vera R. Makar, Tatiyna A. Agaphonova Institute of Cell Biophysics, Russian Academy of Sciences, Laboratory of Biophysics Reception, Laboratory of Hypometabolic States, Pushchino, Moscow Region, 142290, Russia

Abstract TNF production has been studied in peritoneal macrophages and splenic T cells of Arctic Yakutian ground squirrel (Citellus Undulatus Pallas) in hibernating and awake animals in winter and in prehibernating autumn as well as in active euthermic spring-summer animals. A high level of TNF production in macrophages of ground squirrel is observed over the active period and during arousals in winter. There are no signiÞcant season variations in TNF production in splenic T lymphocytes of ground squirrels. This suggests the major role of activated macrophages in the arousals of hibernating animals. T lymphocyte proliferation in ground squirrels in the active period is higher than in winter, and the most signiÞcant seasonal variations are found in T cell mitogenic response, which increases in springsummer period. Evidence is presented that functional activity of macrophages of squirrel in autumn has much in common with that in winter rather than in spring-summer period. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Hibernating squirrels; Macrophages; T cells; TNF production; Proliferation

Introduction In autumn-winter, blood cells and lymphocytes from other tissues of hibernating animals are known to undergo the considerable changes: small lymphocyte and B cell counts in lymph nodes decrease, whereas splenic T lymphocyte number increases (1). The macrophage count in lymph nodes increases in autumn and remains at a high level throughout the hibernation period (2). In winter, a low blood leukocyte count is observed (1). The thymus of hibernating mammals undergoes conspicuous winter alterations, which are considered as an functional involution present well before the animals enter winter hiberna-

* Corresponding author. Fax: 7-0967-790-509. E-mail address: [email protected] (E.G. Novoselova) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 9 8 -6

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tion. Indeed, the yearly involution of the thymus in three species of hibernators, woodchuck Marmota Monax (3) and ground squirrels Citellus citellus (4,5) and Citellus erythrogenus Br (6), starts in summer, in the normothermal period. During hibernation, the thymus lymphoid tissue is replaced by the adipose fatty tissue, resulting in complete thymus destruction. It is noteworthy that the thymus involution also occurs in winter active hibernating animals (3). The seasonal profound functional involutive process of the thymus can not be compared with that observed in general adaptation syndrome of non-hibernating animals. Functional changes in the immune system of hibernating mammals have been studied less extensively. However, based on the available data, the seasonal dynamics can be represented as follows: a decrease in functional activity in autumn before hibernation, its signiÞcant depression during the hibernation period, and activation after arousal (7,8Ð11). The activity of cytotoxic splenic T lymphocytes in C. citellus ground squirrels is maximum in summer, decreases in the autumn normothermal period, and, falls to a minimum level during hibernation (4). The dynamics of seasonal changes in the cytotoxic activity of macrophages remains poorly studied. There are few data on the immune status of hibernating animals during cyclic short arousals in winter when the organism undergoes severe physiological alterations. This study was primarily concerned with the secretion of tumor necrosis factor (TNF) by two main producers of this cytokine, T lymphocytes and macrophages. TNF serves not only to remove foreign substances (viruses, bacteria, and tumor cells) from the body but also acts as a regulator of synthesis of all known cytokines (12). In addition, we studied seasonal changes both in spontaneous and mitogen-dependent proliferative activity of splenic T lymphocytes and T cell proliferation during periodical arousals of ground squirrels from deep hibernation.

Methods Animals Ground squirrels Citellus undulatus of both sexes were trapped in the Republic of Sacha of the Russian Federation in August. They were kept in individual cages at 228C under natural photoperiod and supplied with standard rodent chow and water ad libitum. On November 1 all the animals were placed into hibernaculum maintained at 58C. They were used during each of the followings periods: (1), in period of prehibernation (October): (2) in period of winter hibernation (January-February). No animal was used as hibernator unless it previously completed at least two-three hibernation bout. The time of artiÞcial arousal was approached to naturally occurring interbout on. (3), in spring-summer period (March-June). Winter bouts of hibernation were monitored by using sawdust to inspect each animal at least three times daily. Winter hibernating animals were divided into three groups. Group 1, hibernating squirrels (the middle of bouts, body temperature of torpid animals was 4Ð58C. Group 2, the animals were transferred to warm room (T-228C, for 1.5Ð2 hours) before to spontaneous arousal. These animals were sacriÞced as body temperature approached 26Ð288C. Group 3, euthermic winter animals between bouts (1 day after artiÞcial arousal, body temperature was 358C). The colonic temperature were measured with TEM-1 electrothermometer (Russia).

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Blood cell count Red blood cells and leukocytes were counted using cytometer. The differential leukocyte count was carried out according the conventional hospital method after Giemsa-RomanowskyÕs solution staining. Preparation of peritoneal macrophages Tissue and cells were isolated immediately after decapitation. All measurements were performed for each individual animal. Squirrel peritoneal macrophages were obtained from a mixed white-cell population collected by peritoneal lavage. Cells were washed three times by centrifugation at 1503g, 48C, 6 min with serum-free RPMI 1640 (Sigma, USA) supplemented with 2 mM glutamine (Sigma, USA), 100 mg/ml streptomycin, and 531025 M b-mercaptoethanol (Sigma, USA), and were counted after trypan blue and eosin exclusion. Peritoneal cells were seeded into ßat-bottomed culture dishes and were allowed to adhere. After plating in 24-well culture plates for 1 h at 378C in a humidiÞed atmosphere containing 5% CO2 in air, the cultures were washed carefully with jets of medium to remove nonadhering cells. The remaining cells formed a conßuent monolayer (0.53106/ml) and contained .95% macrophages as determined earlier (13). The morphology of monolayer was examined by light microscopy using an inverted microscope. The incubation was continued for 24 h at 378C , in RPMI 1640 medium containing 10% fetal calf serum, 1% of 1M HEPES solution, 2 mM glutamine, 0.5% gentamicin. At the end of the incubation, the cell-free supernatant was harvested and stored at 2208C until assayed for TNF activity. In some experiments, the cell suspension was subjected after incubation to three cycles of freeze-thawing. The TNF present in this lysate was cell-associated plus secreted. Preparation of splenic T lymphocytes Spleens were removed aseptically and ground in glass homogenizers in EagleÕs or 199 medium (Sigma, USA) containing 1% of 1M HEPES solution, 0.5% gentamicin and 10% bovine serum. Erythrocytes were lysed by treatment with Tris-buffered ammonium chloride (0.01 M Tris-HCl with 0.15 M NaClÑ0.83% NH4Cl, 9:1) and the cells were washed two times. Then the cells were resuspended in RPMI-1640 medium supplemented with 1% of 1M HEPES buffer, 0.5% gentamicin, and 5% fetal calf serum (FCS) to a Þnal concentration of 73106 cell/ml. Splenic B and T lymphocyte populations were fractionated by panning (14). Plastic cell culture ßasks were coated with the afÞnity-puriÞed rabbit immunoglobulin (Ig) (Sigma, USA) with speciÞcity for mouse Ig at a concentration of 200 mg/ml in 7 ml of phosphate buffer saline (PBS). After incubation overnight at 48C, the ßasks were washed three times with the medium and allowed to stay for at least 30 minutes with RPMI 1640 medium. Then 73106 spleen cells in the RPMI 1640 medium were placed into the ßasks in 7 to 8 ml aliquots. Flasks were incubated at 48C for 60 min with gentle stirring to redistribute cells every 30 min. Nonadherent cells were carefully poured off, washed once and resuspended into RPMI-1640 medium supplemented with 1% of 1M HEPES buffer, 0.5% gentamicin, 2 mM L-glutamine, 531025 M b-mercaptoethanol, and 5% FCS.

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Proliferative test T cells were cultured for up to 3 days in the presence of mitogen. Cultures containing 0,53106 cells/0.2 ml for each animal were prepared in quadruplicate in 96-well microtiter plates and incubated at 378C in 5% CO2 atmosphere for 3 days. In parallel, 5 mg/ml Con A (Pharmacia, Sweden) was added to cell cultures in quadruplicate. Cultures were pulsed with 2 mCi/ml 3H-thymidine and harvested 24 h later onto GFC glass Þbber Þlters for liquid scintillation spectroscopy (LS-6500 scintillation counter, Beckman). The standard deviation in experiments of 3H-TdR uptake was ,15% of the mean quadruplicate cultures. TNF bioassay TNF was assayed in macrophages and T cell culture by the procedure described earlier (15). Brießy, 33104 murine L-929 cells in 100 ml of RPMI containing 10% fetal calf serum were placed into wells of 96-well microtiter plates and incubated for 24 h at 378C in a humidiÞed atmosphere containing 5% CO2. Actinomycin D (Sigma) at a Þnal concentration of 1 mg/ml was added prior to sample dilutions (100 ml). The cells were incubated at 378C for 24 h, and then extensively washed in DMEM or 199 medium. The remaining adherent cells were stained for 10 min with 0.05% crystal violet (Sigma ) in 2% ethanol, washed with PBS and air dried. To solubilize the stained cells, 0.1 ml of 1% SDS was added to each well. The absorbance of each well was read on a microplate reader at 546 nm. The amounts of TNF in test lysates were calculated on the basis of cytotoxicity measured in the presence of various dilutions of recombinant human TNF-a (Sigma, USA) as a positive control. The percentage of cytotoxicity was deÞned as the relative L-929 cytotoxicity absorbance of the tested sample versus the absorbance of the control (medium only) wells. % cytotoxicity 5 (1 2 absorbance samples/absorbance control) 3 100 Assays of 24 h culture lysates were done in 12 wells/animal. To conÞrm that L-929 cells were lysed by TNF, parallel cultures were incubated with antibodies speciÞc for TNF (Sigma). The anti-TNF-a antibody was not directly cytotoxic for L-929 cells. The lower limit of detection by such bioassays is 0.5 pg/ml. Statistical analysis Statistical signiÞcance was calculated by StudentÕs t-test. Results Immune functions of cells of ground squirrels during deep hibernation (JanuaryÐFebruary) Experiments were carried out in the middle of the bout in three groups of animals. Table 1 (1Ð3 columns) shows changes in TNF production in cells from ground squirrels at different body temperatures. An increase in the body temperature of ground squirrels to 26Ð288C induces an abrupt increase in TNF production in macrophages. The arousal to the active state (body temperature 358C) does not stimulate a further increase in macrophage activity. A different situation is observed for TNF-producing activity of T lymphocytes (Table 1). The tran-

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Table 1 TNF production in the cells of ground squirrels during annual cycle JanuaryÐFebruary Macrophages T cells

48C

278C

358C

March

April

May

June

October

2563.1 2162.8

79*66.8 2363.1

80*69.0 2562.9

8267.1* 2062.1

8069.3* 2261.9

7568.4* 2062.4

7068.1* 2162.2

1860.9 1561.2

Data are expressed as TNF concentration, pg/ml, and each value is the mean 6 SD from four squirrels. All measures were provided for each individual squirrel in 12 wells/animal. Body temperature in active animals (March, April, May, June, and October) was about 378C. * SigniÞcantly different from hibernating winter animals (body temperature 48C), p,0.001.

sition of animals from the torpid (48C) to the intermediate state (26Ð288C) and then to the active state (358C) induces no marked changes in T cell TNF production. Thus, the measurements of TNF production revealed considerable differences in the functional state of two cell populations upon the transition from 48C to normal temperature. It is likely that phagocytes are more responsive to variations in environmental conditions compared with T lymphocytes, whose ability to produce TNF remained practically unchanged in three states tested. In addition to the estimation of the TNF-producing capacity of T lymphocytes, we studied T cell proliferation (spontaneous and Con A-stimulated) at different stages of artiÞcially evoked arousals during deep hibernation. As shown in Table 2, the level of T cell proliferation is higher in arousal ground squirrels as compared with that in torpid animals. However, there is no signiÞcant difference in the level of 3H-thymidine incorporation into lymphocytes between aroused ground squirrels (26Ð288C) and active (358C) squirrels. It should be noted that the raise of DNA synthesis was lower than that of protein synthesis during arousal of C. Undulatus (16). The results indicate that the red cell count during hibernation increases compared with the summer period (Table 3). During short arousals in winter, the erythrocyte number rapidly falls but remains higher than in summer. In these experiments, we also determined peripheral blood erythrocyte and leukocyte numbers in ground squirrels during hibernation and compared them with the cell counts in active ground squirrels in summer (July). The blood leukocyte count during deep hibernation (48C) was found to be lower compared with that in active summer animals (Table 3). However, upon arousal of animals (268C), the blood leukocyte Table 2 Splenic T cell proliferation of ground squirrels during annual cycle JanuaryÐFebruary 48C Without Con A Plus Con A

278C

2.060.3 3.060.4 3.2604 4.8*60.5

358C

March

April

May

June

October

3.4*60.4 5.6*60.4

3.060.1 6.160.2*

3.260.4 5.960.6*

2.960.3 8.661.0*

3.560.4* 9.060.8*

0.660.05* 1.260.1*

Data are expressed as 3H-TdR uptake in T lymphocytes (cpm/0.5?105 cells) 3 1023 and are mean 6 SD from four animals. All measures were provided for each individual squirrel in 12 wells/animal. Body temperature in active animals (March, April, May, June, and October) was about 378C. * SigniÞcantly different from 3H-thymidine incorporation during hibernation (48C), p,0.05.

1078 E.G. Novoselova et al. / Life Sciences 67 (2000) 1073Ð1080 . Table 3 Blood cell number from squirrels in JanuaryÐFebruary (48C, 26Ð278C, 358C body temperatures) and in July Blood cells Erythrocytesa Leukocytesb Lymphocytesb Neutrophilsb

48C

26Ð278C

358C

July

6.0060.41 1320697 530648 (40.263.4) 370641 (28.062.5)

5.2860.53 23306176* 9096123* (39.064.2) 676672* (29.062.3)

4.3860.36* 48006358* 21206214* (43.863.9) 1390695* (29.063.0)

3.3560.37* 62006489* 21706234* (35.063.8) 35346298* (57.064.9*)

Data are expressed: a total cell number in 1 mm331026; b total cell number in 1 mm3; percent to total leukocyte number is shown in parentheses. All measures were provided for each individual squirrel; for each animal $300 cells were counted. Each value is average mean 6 SD from four animals. * SigniÞcantly different from cell count during hibernation (48C): p,0.05.

count rapidly increases; in awaken ground squirrels (358C), the blood leukocyte count is even higher although it still does not reach the level registered in summer. The absolute neutrophil number also increases with an increase in body temperature, but their percent count in blood does not change and accounts for about 30% of the total leukocytes number, which is substantially lesser than in summer. During hibernation, the lymphocytes/neutrophils ratio is higher than unity, whereas, in summer, the neutrophil count becomes higher, reaching 57% of the total leukocyte number. As the body temperature increases and animals begin to arouse, the absolute lymphocyte count increases; however, their percent level does not change signiÞcantly. Immune functions of cells of ground squirrels during the spring-summer active period (March-June) The level of TNF production in peritoneal macrophages and splenic T cells of squirrels does not change signiÞcantly over the active period within four months (Table 1). The results show that the two different TNF-producing cell populations markedly differ in their secretory potential in the active spring-summer period. TNF production in macrophages of active animals is higher than during hibernation, and TNF production in T cells remains at a stable low level. T lymphocyte proliferation in ground squirrels in the active period is higher than in winter (Table 2); however, the most pronounced seasonal differences are observed in Con Astimulated T cell proliferation, which substantially increases in spring and summer. Immune functions of cells of ground squirrels in autumn (October) The regularities that were revealed in the cellular immunity of ground squirrels in autumn transition period indicated that the functional activity and cell responses of animals have much in common with those in winter rather than in the spring-summer period (Tables 1 and 2). Thus, TNF production in macrophages of autumn squirrels is approximately equal to that in winter hibernating animals and, in some cases, even lower; whereas the production of this lymphokin is high during arousals in winter and during the active spring-summer period. In addition, as shown in Table 2, T cells of active ground squirrels in October show a very low proliferative activity (the level of 3H-thymidine incorporation in this period was the lowest).

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Thus, it can be concluded that the functional activity of immunocompetent cells of hibernators in autumn falls to a level close to or lower that registered in winter. Discussion Before proceeding to the analysis of the results, it is pertinent to note some features in the application of hibernating animals and the problems encountered in the study of cellular immunity. First, considering that all analyses of cell functional activity were carried out in vitro at 378C, a question arises as to how the results of measurements reßect the real situation, when animals are under hypothermic conditions. It is common knowledge that both the number of cell mitoses in different tissues and the rate of DNA synthesis in vitro decrease during hibernation (17Ð19). The study of DNA synthesis in lymphocytes isolated from the spleen and thymus of hibernating animals and cultured at 378C showed that the incorporation of 3Hthymidine in cells of hibernating animals was lower than in cells of active winter animals (13). These experiments indicate that, in hibernating animals, the suppression of metabolism, which is inherent in these animals during hibernation, is determined not only by hypothermia. Based on the analysis of seasonal changes in cellular immunity determined by TNF production, we suppose that the two cell populations are to a different degree responsible for the regulation of TNF production in ground squirrel. It is known that the main producers of TNF are macrophages, which secrete TNF-a, and T lymphocytes, which produce small amounts of TNF-b (lymphotoxin). These two proteins possess a similar afÞnity for TNF-sensitive receptors, and their physiological functions also coincide; therefore, TNF-a and TNF-b are usually recognised not by properties but by their origin (macrophages or T cells). The pool of TNF synthesized by cells is not entirely secreted outside; a part of the pool remains within the cell and cytoplasm membrane. By immunochemical methods and cytoßuorimetry, in vivo TNF localized not to all resident macrophages and circulating T lymphocytes but only to some activated cells (12). This is consistent with the fact that the TNF concentration in mammalian lymphoid tissues is very low (10). As we measured the total TNF pool present in the cell and the extracellular space, it can be considered that we tested the total level of the lymphokin independently of the dynamics of its secretion. In this case, it is important because the rates of accumulation and secretion of the lymphotoxin are lower than those of TNF-a, which is essentially produced by macrophages (20). We registered a substantial activation of TNF production in peritoneal macrophages of ground squirrels during arousals in winter and in active period, while the level of TNF production in T cells throughout the year was unchanged. This suggests that the TNF metabolism in hibernating ground squirrels is accomplished by a common regulatory system, and the major contribution is made by activated macrophages and not T cells. The results suggest that the arousal of ground squirrels leads to a strong activation of macrophages and leaves the rather conservative system of TNF production in T lymphocytes unchanged. The low level of peripheral blood lymphocyte count in winter hibernating ground squirrels compared with spring-summer animals has been reported earlier (1). The rapid increase in the leukocyte count during arousal is known to be due to the cells entrance into blood from the organs where they were deposited during hibernation. One of these organs is the spleen, since its weight decreases 2Ð2.5 times, irrespective of the way the animals aroused from hibernation (2). The high level of the erythrocytes count during hi-

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bernation can be explained by a decrease in the volume of blood of torpid animals. In addition, we found a low level of the erythrocyte number during arousals of ground squirrels in winter. The physiological signiÞcance of the redistribution of lymphocytes upon the transition from hibernation to activity seems to be very important. The possibility to deposit cells and to release them rapidly into blood upon arousal is the unique mechanism of adaptation of the immune system of hibernators. Although all measurements were made in vitro, it can be concluded that lymphoid cells of hibernating squirrel have a substantial activation potential. We believe that a rapid realisation of this potential through the rise of TNF production in macrophages is an important factor that secures the adaptation of the organism to sharp functional changes in physiological status during short arousals in winter. Acknowledgments This work was supported by the Russian Foundation For Basic Research (Leading Scientific Schools, Grant 96-15-97787), Russian Foundation For Basic Research (Grant 98-04-49178). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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