Acute restraint stress enhances calcium mobilization and proliferative response in splenic lymphocytes from mice

Abstract

Calcium (Ca²⁺ ) plays an essential role in lymphocyte activation and maturation. Acute and chronic stress has been shown to modulate the lymphocyte immune response; but the relationship between cytosolic free Ca²⁺ concentration ([Ca²⁺ ]_i) and the immune response in lymphocytes following exposure to stress has not been examined. In the present study, we investigated the effects of acute restraint stress on [Ca²⁺ ]_i and the proliferation of splenic lymphocytes from mice. We observed that 2 h of restraint significantly increased plasma corticosterone levels in mice. On examining [Ca²⁺ ]_i and the proliferation ex vivo of splenic lymphocytes isolated from restraint-stressed mice using fura-2 and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, respectively, we found that acute restraint stress caused a significant increase in resting [Ca²⁺ ]_i and significantly enhanced the ability of concanavalin A (Con A; a T-cell-selective mitogen) to increase [Ca²⁺ ]_i but not that of lipopolysaccharide (LPS; a B-cell-selective mitogen). In addition, acute restraint stress significantly enhanced Con A-stimulated but not LPS-stimulated lymphocyte proliferation. Overall, there was a positive correlation between [Ca²⁺ ]_i and T-cell proliferation following acute restraint stress. The enhancements of [Ca²⁺ ]_i and T-cell proliferation were completely suppressed by verapamil (a Ca²⁺ channel blocker). These results suggest that acute restraint stress enhances Con A-stimulated T-cell proliferation by increasing [Ca²⁺ ]_i via stimulation of Ca²⁺ entry.

• Acute restraint stress
• concanavalin A
• cytosolic free Ca²⁺
• lipopolysaccharide
• lymphocyte proliferation
• MTT assay

Introduction

Stress has long been known to alter the behavior and homeostatic state of animals including endocrine and immunological functions. The impact of stress exposure on the development of an immune response depends on a variety of factors, such as the duration and the type of the stressor (McEwen 1998). For example, acute stress has been reported to enhance immune function, whereas chronic stress has been shown to suppress it (Millan et al. 1996; Dhabhar and McEwen 1997; Dhabhar and McEwen 1999).

Several studies in laboratory animals have revealed that stress causes changes in cellular and humoral immunity. Exposure of rodents to acute stress increases mitogen-induced T-cell proliferation (Lysle et al. 1990; Bauer et al. 2001), the T-cell-dependent delayed-type hypersensitivity response (Dhabhar and McEwen 1996), and both T-cell-dependent and -independent antibody production (Okimura et al. 1986; Millan et al. 1996). Other authors, however, have reported a decrease in the response to T-cell-dependent antigens following acute stress (footshock), depending on its severity and the amount of time between immunization and application of the stress (Laudenslager et al. 1988; Zalcman et al. 1988; Zalcman and Anisman 1993; Fleshner et al. 1996; Millan et al. 1996). On the other hand, chronic stress exposure disrupts cellular immunity (Kusnecov and Rabin 1993; Dhabhar and McEwen 1997), reduces mitogen-induced T-cell proliferation (Silberman et al. 2002), causes a poor T-cell-dependent antibody response (Fukui et al. 1997; Tournier et al. 2001), and disrupts cytokine secretion (Zhang et al. 1998). However, the mechanisms by which these alterations occur have not been fully elucidated.

The spleen is primarily involved in dealing with blood-borne pathogens, such as encapsulated bacteria (Mond et al. 1995). The spleen cell population is heterogeneous, consisting mostly of lymphocytes, i.e. T- and B-cells (Arora et al. 1981), which play an important role in cellular and humoral immunity, respectively.

Calcium (Ca²⁺ ) plays an essential role in lymphocyte activation and maturation. Cross-linking of antigen or Fc receptors on T- and B-cells rapidly increases the cytosolic free Ca²⁺ concentration ([Ca²⁺ ]_i) (Braun et al. 1979; van Leeuwen and Samelson 1999), which activates many signaling factors, including nuclear factor of activated T cells (NFAT), nuclear factor-kB (NF-kB) and c-Jun NH₂-terminal kinase-1 (JNK1) (Dolmetsch et al. 1997; Feske et al. 2001). These factors, in turn, regulate the expression of several inducible genes that mediate diverse genetic programs, including effector immune function and cell proliferation, differentiation and death (Teague et al. 1999).

In aged mice, both resting and mitogen-stimulated [Ca²⁺ ]_i are significantly lower in T-cells from those exposed to chronic overcrowding stress than in T-cells from those not subjected to such stress (Csermely et al. 1995). Sei et al. (1991) observed that acute restraint stress suppresses mitogen-stimulated Ca²⁺ mobilization in CD4⁺ T-cells but enhances Ca²⁺ mobilization in CD8⁺ T-cells. They also noted that chronic restraint stress suppresses mitogen-stimulated Ca²⁺ mobilization in CD4⁺ T-cells and B-cells (Sei et al. 1991). Since immunofluorescence was the method used in this research to distinguish patterns of Ca²⁺ mobilization within a heterogeneous cell population (i.e. labeled with various monoclonal antibodies), accurate estimations of [Ca²⁺ ]_i were not performed for pure mouse splenic lymphocytes, and to our knowledge, the effect of acute stress on [Ca²⁺ ]_i in lymphocytes has not yet been investigated, nor has the relationship between [Ca²⁺ ]_i and the immune response in lymphocytes after acute stress exposure. Therefore, in the present study, we investigated the effects of acute restraint stress on resting and mitogen-stimulated [Ca²⁺ ]_i and proliferation of splenic lymphocytes from mice.

Materials and methods

Chemicals

Concanavalin A (Con A), Tris (hydroxymethyl) aminomethane and corticosterone were purchased from Wako Pure Chemicals (Osaka, Japan). Fura-2-AM and HEPES were supplied by Dojindo Laboratories (Kumamoto, Japan) and lipopolysaccharide (LPS), verapamil hydrochloride and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) by Sigma (St Louis, MO, USA). Dulbecco's modified eagle medium (DMEM) was supplied by Invitrogen (Carlsbad, CA, USA) and fetal bovine serum by Cambrex Bio Science (Walkersville, MD, USA). Other chemicals were of reagent grade or the highest quality available.

Media

The standard incubation medium for the measurement of [Ca²⁺ ]_i contained the following (in mM): NaCl 125, KCl 5, NaH₂PO₄ 1.2, MgCl₂ 1.2, NaHCO₃ 5, glucose 6, CaCl₂ 1 and HEPES 25, with the pH adjusted to 7.4. DMEM containing 12 mM HEPES, 100 U/ml penicillin G potassium and 100 μg/ml streptomycin sulfate was used for cell culture.

Animals

Male ddY strain mice (6 weeks of age) were purchased from Japan SLC (Shizuoka, Japan). Animals were housed in standard light conditions (lights on from 6:00 am to 6:00 pm) and temperature (22 ± 2°C). Food and water were provided ad libitum. Mice were killed by decapitation after experiment. All procedures pertaining to the care and use of experimental animals were approved by the Animal Research Committee, Obihiro University, and conducted in accordance with the Declaration of Helsinki and/or the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the United States National Institutes of Health.

Stress protocol

Mice (13 weeks of age) were randomly assigned to one of two experimental groups: nonstressed group and acute restraint-stressed group. Mice were exposed to acute restraint stress as described by Sei et al. (1991) and Silberman et al. (2003), which involved placing them in a well-ventilated polypropylene tube (40 mm diameter × 90 mm length) for 2 h starting at 10:00 am. The mice were kept in their home cages for 15 min following the stress and then sacrificed. The nonstressed control animals were left undisturbed in their home cages prior to sacrifice. Those exposed to restraint stress were not physically compressed and did not experience pain.

Corticosterone determination

To avoid fluctuations in plasma corticosterone levels due to circadian rhythms, mice were all decapitated at 12:15 pm on the day of sacrifice. Trunk blood was collected in heparinized tubes, centrifuged and separated plasma frozen at − 80°C until analysis. Total plasma corticosterone concentrations were measured as described by Glick et al. (1964). Briefly, 900 μl of isooctane was added to 200 μl of each plasma sample with thorough mixing. After centrifugation at 300g for 5 min, the top isooctane later was discarded. Following the addition of 900 μl of chloroform, each sample was mixed well and centrifuged at 300g for 5 min. The top aqueous layer was discarded, 800 μl of the chloroform was removed and the sample was added to 320 μl of acid-alcohol solution (65% H₂SO₄ and 35% ethanol), and this was mixed well. After centrifugation at 300g for 5 min, the bottom acid layer from each sample was transferred to a cuvette. The intensity of fluorescence of the samples was measured using a model FP-770 spectrophotofluorometer (Jasco, Hachioji, Tokyo, Japan) with excitation at 350 nm and measurement of emission at 520 nm. Corticosterone concentrations were calculated from a standard curve and expressed in ng/ml. All samples were measured in a single assay and intra-assay coefficient of variation was 7%.

Cell suspensions

Spleens were isolated from the control and stressed mice after decapitation for corticosterone measurement and passed through a 1-mm metal mesh. The spleen cells were suspended in standard medium or DMEM and then filtered through a 10-μm nylon mesh. Next, the cells were treated with pH 7.65 buffer (17 mM Tris–HCl and 0.83% ammonium chloride) to lyse erythrocytes, after which the cells were washed three times in standard medium or DMEM. For [Ca²⁺ ]_i measurement or further cell culture, the cells were resuspended in standard medium or DMEM supplemented with 10% heat-inactivated fetal calf serum, respectively. Cell viability was greater than 80% as determined by the trypan blue exclusion test, which is consistent with that for human lymphocytes as reported by Falciola et al. (1994).

[Ca²⁺ ]_i determination

Intracellular [Ca²⁺ ] concentrations were measured by monitoring the intensity of fura-2 fluorescence as described previously (Satoh et al. 2003). Briefly, a spleen cell suspension (10⁷ cells/ml) was incubated for 30 min at 37°C with 5 μM fura-2-AM dissolved in dimethyl sulfoxide. The cells were washed three times and then resuspended in 5 ml of standard medium. Aliquots (0.5 ml) of the cell suspension were placed in cuvettes and incubated for 10 min before addition of mitogen. Measurement of fluorescence was initiated 4 min before addition of mitogen and continued for 8 min thereafter. Samples in the cuvette were maintained at 37°C and mixed with a magnetic stirrer. Fura-2 fluorescence was measured with a model CAF-100 spectrofluorometer (Jasco) using the ratio mode. Excitation wavelengths were 340 and 380 nm, and the emission wavelength was 500 nm. [Ca²⁺ ]_i was calculated according to the method of Grynkiewicz et al. (1985).

Proliferation assay

Proliferation was determined in 24-well plates containing 1-ml aliquots of spleen cells (4 × 10⁶ cells per well) in DMEM supplemented with 10% heat-inactivated fetal calf serum. Wells were adjusted to 1, 2, or 4 μg/ml of Con A or 5, 10, or 25 μg/ml of LPS by adding 0.5 ml of the respective test solution. Control cultures received 0.5 ml of culture medium without Con A or LPS. The cells were then cultured for 42 h at 37°C in an atmosphere containing 5% CO₂. Mitogenic activity was measured by mitochondrial conversion of MTT detected at 570 nm as described by Mosmann (1983) using a model Ubest-35 spectrophotometer (Jasco).

Statistical analysis

All data are expressed as the means ± SEM. Student's t-test for unpaired values was used to determine the level of significance for normally distributed data. Mitogen-stimulated [Ca²⁺ ]_i and proliferation data were analyzed by two-way analysis of variance, using a repeated measures design. The main factors were “group” (control and acute restraint-stressed mice) and “mitogen concentration”. Tukey's HSD test was used for post-hoc analysis. Differences between means were considered significant at p < 0.05.

Results

Plasma corticosterone concentration

We measured plasma corticosterone concentrations under basal conditions and after acute restraint stress, which significantly increased corticosterone level. The plasma corticosterone concentrations in nonstressed and stressed mice were 217 ± 23 and 320 ± 25 ng/ml, respectively (t(1, 10) = 3.015, p < 0.05). The corticosterone level in the nonstressed control group was consistent with values reported by Silberman et al. (2002, 2003, 2004) and Stark et al. (2001).

Resting [Ca²⁺ ]_i

We also measured resting [Ca²⁺ ]_i in the mouse splenic lymphocytes under basal conditions and after exposure to acute restraint stress, which produced a significant increase in resting [Ca²⁺ ]_i. The resting [Ca²⁺ ]_i levels in nonstressed and stressed mice were 75 ± 2.6 and 87 ± 3.6 nM, respectively (t(1, 23) = 2.713, p < 0.05). The resting level in the nonstressed control group was consistent with that reported by Grinstein and Dixon (1989).

Mitogen-stimulated [Ca²⁺ ]_i

Next, we examined the changes in [Ca²⁺ ]_i induced by T- or B-cell selective mitogens in splenic lymphocytes from nonstressed and acute restraint-stressed mice. Treatment with either mitogen caused a gradual increase in [Ca²⁺ ]_i that plateaued within 8 min (Figures 1(a) and 2(a)). Con A (1–4 μg/ml), the T-cell selective mitogen, caused a dose-dependent increase in [Ca²⁺ ]_i in the splenic lymphocytes (F(2, 39) = 138.81, p < 0.0001, Figure 1(b)). This increase in [Ca²⁺ ]_i was enhanced significantly by exposure of the mice to acute restraint stress (F(1, 39) = 36.41, p < 0.0001, Figure 1(a),(b)). Enhancement was significant at 1 μM (p < 0.01), 2 μM (p < 0.01) and 4 μM (p < 0.05) of Con A (Figure 1(b)). However, no significant interaction between group and mitogen concentration was observed. Pretreatment of splenic lymphocytes with verapamil (a Ca²⁺ channel blocker; 100 μM) abolished Con A-induced increases in [Ca²⁺ ]_i for both nonstressed and stressed mice (Figure 1(b)). The inhibitory effect of verapamil in nonstressed mice agreed with the finding for human T lymphocytes reported by Nakabayashi et al. (1992).

Figure 1 (a) Time course of changes in [Ca^{2 +} ]_i in response to Con A (2 μg/ml) in splenic lymphocytes from control and acute restraint-stressed mice; (b) effects of Con A and Con A plus verapamil on [Ca^{2 +} ]_i in splenic lymphocytes from control (n = 7 per group) and acute restraint-stressed (n = 9 per group) mice. Fura-2-loaded lymphocytes were incubated for 8 min with or without Con A. Verapamil was added 10 min before exposure to Con A. The net Δ[Ca^{2 +} ]_i is the difference in the increase in [Ca^{2 +} ]_i 8 min after addition of Con A or Con A plus verapamil, vs. the vehicle. Data are presented as group means ± SEM. Statistically significant differences from control are indicated as: ^*P < 0.05, ^**P < 0.01.

Figure 2 (a) Time course of changes in [Ca^{2 +} ]_i in response to LPS (25 μg/ml) in splenic lymphocytes from control and acute restraint-stressed mice; (b) effect of LPS on [Ca^{2 +} ]_i in splenic lymphocytes from control (n = 6 per group) and acute restraint-stressed (n = 7 per group) mice. Fura-2-loaded lymphocytes were incubated for 8 min with or without LPS. The net Δ[Ca^{2 +} ]_i is the difference in the increase in [Ca^{2 +} ]_i 8 min after addition of LPS vs. vehicle. Data are presented as group means ± SEM.

LPS (5–25 μg/ml), the B-cell selective mitogen, also caused a dose-dependent increase in [Ca²⁺ ]_i in lymphocytes from nonstressed and acute restraint-stressed mice (F(2, 32) = 24.17, p < 0.0001, Figure 2(b)), but exposure to acute restraint stress failed to enhance the LPS-induced increase in [Ca²⁺ ]_i (Figure 2(a),(b)). Moreover, no significant interaction between group and mitogen concentration was observed.

Mitogen-stimulated proliferation

We further examined the proliferative responses to T-cell- or B-cell-selective mitogens in splenic lymphocytes derived from nonstressed and acute restraint-stressed mice. Con A (1–4 μg/ml) caused a dose-dependent increase in the proliferation of lymphocytes from nonstressed and acute restraint-stressed mice (F(3, 48) = 22.35, p < 0.0001, Figure 3). The increases in lymphocyte proliferation were enhanced significantly by exposure of mice to acute restraint stress (F(1, 48) = 15.72, p < 0.001, Figure 3). Enhancement was significant at 1 μM (p < 0.05), 2 μM (p < 0.01) and 4 μM (p < 0.01) of Con A (Figure 3). However, no significant interaction between group and mitogen concentration was observed. Pretreatment of splenic lymphocytes with verapamil (100 μM) completely suppressed Con A-stimulated lymphocyte proliferation in nonstressed and stressed mice (Figure 3). The inhibitory effect of verapamil in nonstressed mice agreed with the findings for human T lymphocytes reported by Birx et al. (1984) and Nakabayashi et al. (1992). At the concentration used in the present study, it had no effect on cell viability as assessed by the trypan blue exclusion test (data not shown).

Figure 3 Proliferative effects of Con A and Con A plus verapamil on splenic lymphocytes from control (n = 7 per group) and acute restraint-stressed (n = 7 per group) mice. Lymphocytes were stimulated for 42 h with Con A or Con A plus verapamil, and proliferation was measured by MTT assay. Verapamil was added 10 min before exposure to Con A. Data are presented as group means ± SEM. Statistically significant differences vs. control are indicated as: ^*P < 0.05, ^**P < 0.01. OD₅₇₀, optical density at 570 nm.

Though LPS (5–25 μg/ml) also caused a dose-dependent increase in the proliferation of lymphocytes from nonstressed and acute restraint-stressed mice (F(3, 48) = 25.53, p < 0.0001, Figure 4), exposure to acute restraint stress failed to enhance the LPS-stimulated proliferation (Figure 4). Moreover, no significant interaction between group and mitogen concentration was observed. The dose-response curves of Con A- and LPS-stimulated proliferation in nonstressed mouse lymphocytes were similar to those reported by Lysle et al. (1990), Mosmann (1983) and Silberman et al. (2002). Finally, the proliferation of resting lymphocytes was not affected by acute restraint stress.

Figure 4 Proliferative effect of LPS on splenic lymphocytes from control (n = 7 per group) and acute restraint-stressed (n = 7 per group) mice. Lymphocytes were stimulated for 42 h with various concentrations of LPS, and proliferation was measured by MTT assay. Data are presented as group means ± SEM. OD₅₇₀, optical density at 570 nm.

Discussion

In the present study, we observed that acute restraint stress significantly increases the plasma corticosterone concentration in ddY strain male mice. Physiological responses induced by stress involve activation of both the hypothalamic-pituitary-adrenal (HPA) axis and autonomic nervous system, with consequent increments in plasma glucocorticoids and catecholamines, respectively (Kvetnansky et al. 1971; Keim and Sigg 1976). Therefore, the acute restraint conditions in the present study were sufficient to induce a stress response in the mice and may have also increased plasma adrenaline/noradrenaline concentrations.

We demonstrated for the first time that acute restraint stress significantly increases the resting [Ca²⁺ ]_i in lymphocytes. Three transport systems, i.e. the Ca²⁺ -ATPase of the plasma membrane, the Ca²⁺ -ATPase of the endoplasmic reticulum, and the Na⁺/Ca²⁺ exchanger, act in concert to regulate Ca²⁺ homeostasis in lymphocytes (Balasubramanyam et al. 1993; Lewis 2001), and we assume that one, two or all of these transport systems was/were modified by the acute restraint stress. To our knowledge, however, there is no report describing that glucocorticoid or adrenaline/noradrenaline modify calcium fluxes in lymphocytes.

Con A and LPS increase [Ca²⁺ ]_i in T- and B-cells, respectively (Tsien et al. 1982; Rosoff and Cantley 1985; Proust et al. 1987). We demonstrated for the first time that prior acute restraint stress enhances a Con A-induced but not a LPS-induced increase in [Ca²⁺ ]_i in mouse splenic lymphocytes, which indicates that acute restraint stress enhances the increase in [Ca²⁺ ]_i in T-cells in particular.

Proust et al. (1987) and Tsien et al. (1982) reported that an increase in [Ca²⁺ ]_i in mouse T-cells induced by Con A was largely inhibited by removal of extracellular Ca²⁺ . We obtained the same result (data not shown). The finding that verapamil abolished Con A-induced increases in [Ca²⁺ ]_i in lymphocytes from both nonstressed and acute restraint-stressed mice in the present study suggests that acute restraint stress enhances the Con A-induced increase in [Ca²⁺ ]_i by stimulating Ca²⁺ entry via a verapamil-sensitive pathway. However, Velbinger et al. (2000) observed that acute immobilization stress did not influence a phytohemagglutinin (PHA)-induced increase in [Ca²⁺ ]_i in splenic T-lymphocytes of rats. This disagreement with our finding could be due to various differences between the studies, such as rodent species, sex, stress conditions, housing conditions and light–dark cycle.

Lysle et al. (1990) and Silberman et al. (2002) observed that Con A and LPS stimulate the proliferation of mouse splenic lymphocytes, more specifically the proliferation of T- and B-cells, respectively. In the present study, we observed that acute restraint stress enhances Con A-stimulated but not LPS-stimulated lymphocyte proliferation, indicating that acute restraint stress further enhances T-cell proliferation. Bauer et al. (2001) and Lysle et al. (1990) also reported that exposure of rodents to acute stress increases mitogen-stimulated T-cell proliferation.

Con A and LPS mobilize Ca²⁺ by activating T- and B-cell receptors, respectively (Tsien et al. 1982; Rosoff and Cantley 1985; Proust et al. 1987) and an increase in [Ca²⁺ ]_i constitutes an essential triggering signal for T- and B-cell proliferation (Klaus et al. 1986; Crabtree 1989). In the present study, there was a positive correlation between [Ca²⁺ ]_i and T-cell proliferation following acute restraint stress, and verapamil completely suppressed the proliferative response to Con A in lymphocytes from both nonstressed and acute restraint-stressed mice. These results suggest that acute restraint stress enhances Con A-stimulated T-cell proliferation by increasing [Ca²⁺ ]_i via stimulation of Ca²⁺ entry. Verapamil also significantly suppresses interleukin-2 (IL-2; a cytokine essential for T-cell proliferation) release by human peripheral T-cells (Drach et al. 1996). However, since an increase in [Ca²⁺ ]_i is essential for IL-2 production by T-cells (Negulescu et al. 1994), it is unlikely that verapamil inhibits T-cell proliferation by this means.

Exposure to acute footshock stress has been reported to enhance the induction phase of the immune response (Wood et al. 1993). In addition, Millan et al. (1996) found that short-term restraint stress (2 h on two consecutive days) enhances the primary serum T-cell-dependent antibody response to sheep red blood cells. Other authors, however, have reported a decreased response to a T-cell-dependent antigen following acute exposure to footshock (Laudenslager et al. 1988; Fleshner et al. 1996). These contradictory results can be explained by dependence of the response of the immune system to stress on several factors, including the intensity and/or the duration of the stress, the antigenic challenge, and how much time has passed between the application of the stressor and immunization (Zalcman et al. 1988; Zalcman and Anisman 1993; Millan et al. 1996).

Bauer et al. (2001) suggested that increased splenic lymphocyte proliferation following acute restraint stress exposure might have important biological implications. Since the spleen is primarily involved in dealing with blood-borne pathogens, such as encapsulated bacteria (Mond et al. 1995), stress-induced activation of splenocyte function could enhance protection against blood-borne pathogens.

In the present study, an increase in splenic lymphocyte proliferation caused by Con A was enhanced by the exposure of mice to acute restraint stress. Bauer et al. (2001) observed that acute restraint stress increases PHA-stimulated splenic lymphocyte proliferation and suppresses peripheral blood lymphocyte (PBL) proliferation in rats, and studies prior to theirs had demonstrated that footshock and immobilization stress decrease PBL proliferation in rats (Rinner et al. 1992; Shurin et al. 1994). Taken together with our finding that exposure to acute restraint stress enhances Con A-induced splenic lymphocyte proliferation in mice, this suggests that stress-induced immune changes are compartment-specific.

In conclusion, the present study demonstrates that acute restraint stress enhances Con A-stimulated T-cell proliferation by increasing [Ca²⁺ ]_i via stimulation of Ca²⁺ entry. The influences of stress on immunity are mediated not only by glucocorticoids but also by catecholamines, endogenous opioids and pituitary hormones such as growth hormone (Dantzer and Kelley 1989). These substances could also play a role in mediating the effects of stress on lymphocytes. Additional studies are now needed to further clarify how acute restraint stress enhances Ca²⁺ mobilization and T-cell proliferation.

Acknowledgements

This study was supported by grants from the 21st Century COE Program (A-1) of the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Research Foundation of Obihiro University of Agriculture and Veterinary Medicine.