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Th2 balance toward Th2-dominant immunity in mice
Immunology Letters 62 (1998) 39 – 43
The restraint stress drives a shift in Th1/Th2 balance toward Th2-dominant immunity in mice Kenji Iwakabe a, Masako Shimada a, Akio Ohta a, Takashi Yahata a, Yasushi Ohmi a, Sonoko Habu a, Takashi Nishimura a,b,* b
a Department of Immunology, Tokai Uni6ersity School of Medicine, Bohseidai, Isehara 259 -11, Japan Research Center for Genetic Engineering and Cell Transplantation, Tokai Uni6ersity School of Medicine, Bohseidai, Isehara 259 -11, Japan
Received 8 January 1998; received in revised form 11 February 1998; accepted 12 February 1998
Abstract When mice were physically restrained in 50-ml tubes for 24 h, a marked decrease of NK activity was demonstrated in parallel with the elevation of serum corticosterone levels. The release of mice from restraint stress resulted in the recovery of NK activity, with a decrease of serum corticosterone levels within 48 h. Using this stress model, we also investigated the influence of restraint stress on mouse Th1/Th2 balance. Consistent with the decrease of NK activity, IFN-g production of mouse spleen cells greatly reduced after suffering from restraint stress. In contrast, the IL-4 producing ability of spleen cells was not so much affected by restraint stress. These results initially indicated that stress may induce the skewing of the Th1/Th2 balance toward Th2-dominant immunity, which stimulates the occurrence of infectious diseases and allergic disorders. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Restraint stress; Th1/Th2 balance; NK; Cytokine
1. Introduction A large body of research in recent decades has suggested that stress may be associated with the onset, course, and outcome of physical illness [1 – 3]. Stress-induced immunologic alterations have been considered as major cause of increased risk for immune-related diseases, such as cancer, autoimmune disorders, and infection [4,5]. In general, stressors trigger physiological responses involving the hypothalamic-pituitary-adrenal axis (HPA) and autonomic nervous system via the release of corticosteroid hormones and catecholamines, respectively [6–8]. These nervous and endocrine systems are closely associated with immune systems and thus the stressor induces the alternation of host immune responses [9,10]. * Corresponding author. Tel.: +81 463 931121; fax: + 81 463 942976. 0165-2478/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0165-2478(98)00021-2
It has been reported that stress generally induced the suppression of cellular immune responses, but resulted in no effect or rather increase of humoral immunity [9,11]. However, little has been investigated about the precise mechanisms underlying this stress-induced phenomena. Recently, it has been demonstrated that cellular immunity and humoral immunity is regulated by distinct subsets of helper T-cells [12]. The former response is regulated by Th1 cells, which can produce IL-2, IFN-g and lymphotoxin. The latter is controlled by Th2 cells, which can produce IL-4, IL-5, IL-6 and IL-10. Because the glucocorticoids, which is elevated by physical stress, suppress Th1 cells but stimulate Th2 cells [13,14], it is possible to speculate that stress may influence immune responses via the modulation of the Th1/Th2 balance. As well as stress, the imbalance of Th1/Th2 immunity has been demonstrated to be associated with increased risk for various immune diseases [15,16]. Therefore, it is important to investigate the effect of stress on the Th1/Th2 balance.
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K. Iwakabe et al. / Immunology Letters 62 (1998) 39–43
In the present paper, we found that restraint stress strongly inhibited IFN-g production in parallel with the suppression of NK activity, while it did not affect the production of Th2 cytokine, IL-4 to the same degree. Thus, these data initially showed direct evidence that restraint stress caused a shift in Th1/Th2 balance toward Th2-dominant immunity, which might accelerate the onset of infectious diseases and allergic disorders.
2.6. Cytokine production Spleen cells (5 × 105 per well) were stimulated with concanavalin A (Con A) (2.5 mg/ml) for 36 h in Falcon 96-well plate. After incubation, culture supernatants were harvested and their IFN-g and IL-4 activity was determined by ELISA kits (Amersham, Tokyo, Japan) [19].
2.7. Mixed lymphocyte reaction 2. Materials and methods
2.1. Mice BALB/c mice and C57BL/6 mice were purchased from Charles River, Japan, (Yokohama 222, Japan). All mice were female and used when they were 8–10 weeks old.
2.2. Restraint stress protocol According to the methods of Bonneau et al. [17], mice were physically restrained in a 50-ml Falcon tube in which various holes with a diameter of 0.4 cm had been drilled. The mice were first wetted with water and restrained in a 50-ml tube for various times without supplying food and water. The control mice were left in usual animal cages for the same time without food and water. The smaller mice, younger than 8 weeks old, were not suitable for this restraint stress experiment using the 50-ml tube.
2.3. Cytotoxicity assay NK activity was determined by 4-h 51Cr-release assay using YAC-1 cells as target cells. The % cytotoxicity and lytic units (LU/106) were calculated as described previously [18].
2.4. Measurement of serum corticosterone le6els The serum corticosterone levels of control or restraint mice was determined using Immuchem™ Double Antibody Corticosterone Ria Kit for Rat and Mice (ICN Biomedicals, CA 92626) according to the manufacturer’s instructions.
2.5. Flow cytometry The analysis of cell surface markers was carried out by FACScan using the Consort 30 program. The detailed procedure for staining and sorting was described in a previous paper [19]. All FITC or PE-conjugated mAbs used in this paper was purchased from PharMingen (San Diego, CA).
BALB/c spleen cells (5 × 106 cells/well) were stimulated with mitomycin C-treated C57BL/6 mouse spleen cells for 4 days. After the culture, CTL activity against C57BL/6-derived MBL-2 lymphoma cells was determined by 4-h 51Cr-release assay. The IFN-g activity of culture supernatants during mixed lymphocyte reaction (MLR) was also examined by ELISA kits.
3. Results and discussion Restraint stress was used to examine the influence of stress on the immune system. Consistent with previous reports [20], the acute stress (24-h restraint stress) caused the elevation of serum corticosterone levels (Fig. 1A). The NK activity against YAC-1 cells was also markedly suppressed by the acute stress in contrast to a recent report by Mizobe et al. [20] (Fig. 1A). This discrepancy may be because of the differential method for restraint stress. The restraint stress using the 50-ml tube appeared to be a more severe stressor for mice compared with the previous stress protocol using a wire net stress cage. As shown in Fig. 1B, the reduced NK activity recovered within 48 h after the release of the mice from restraint stress, in parallel with the decrease of serum corticosterone levels. Therefore, this restraint stress appeared to suppress NK activity through the induction of the elevated levels of endogenous gluococorticoids. Using C57BL/6 mouse strain expressing NK1.1 marker, it was also demonstrated that the percentage of NK cells in the spleen did not significantly change before (4.7893.2%) and after (5.2791.5%) the restraint stress. In accord with the finding that glucocorticoids could induce the apoptosis of thymocytes [21], the restraint stress caused a marked reduction of the number of thymocytes and spleen cells (Fig. 2A). However, the percentage of CD4 + CD8 + T-cells was not reduced by restraint stress (Fig. 2B). Moreover, spleen cells obtained from stressed mice showed the same levels of proliferative responses to Con A as those from control mice (Fig. 2C). Recently, however, glucocorticoids have demonstrated to reveal the differential effect against Th1 and Th2 immunity [13,14], which has a pivotal role in
K. Iwakabe et al. / Immunology Letters 62 (1998) 39–43
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Fig. 1. Reduction of NK activity by restraint stress. (A) C57BL/6 mice were restrained in a 50-ml tube for various times. Thereafter, their NK activity against YAC-1 cells () and the elevation of serum corticosterone levels was determined by the method described in Section 2. For measurement of NK activity, whole spleen cells were used in this experiment. (B) Mice were first restrained in a 50-ml tube for 24 h and released from the stress. Then, NK activity against YAC-1 cells () and the elevation of serum corticosterone levels () was determined various times after the release from the stress. For the measurement of NK activity, nylon-passed spleen cells were used in this experiment. The data represent mean9 S.E. of five mice. The suppression of NK activity by the stress was also demonstrated in BALB/c strain mice.
immunoregulation. Therefore, it is possible to speculate that restraint stress may affect the Th1/Th2 balance. To evaluate the issues, the effect of restraint stress on the production of IFN-g (Th1 cytokine) and IL-4 (Th2 cytokine) was examined using Con A-activated spleen cells. As clearly demonstrated in Fig. 3, control mice, which were left in the usual cage without food and water, showed high levels of IFN-g production when their spleen cells were stimulated with Con A for 36 h. In contrast, the IFN-g production of Con A-stimulated spleen cells was strongly reduced by restraint stress (Fig. 3A). Surprisingly, however, IL-4 production of
Con A-stimulated spleen cells was not so much affected by restraint stress (Fig. 3B). In a previous report, Ramı´rez et al. [14] demonstrated that a prolonged treatment of CD4 + T-cells in vitro with glucocorticoids, and IL-4, resulted in the enhanced IL-4 production of purified CD4 + T-cells. However, we could not always detect the augmentation of the IL-4 producingability of T-cells by stress-induced corticosterone. This discrepancy may be because of the different conditions for corticosterone-treatment between their in vitro study and our in vivo study. Thus, restraint stress did not cause the augmented IL-4 production, but it
Fig. 2. The effect of restraint stress on cell numbers, subpopulations and function of T-cells. BALB/c mice were restrained in a 50-ml tube for 24 h. (A) The effect of restraint stress on the number of cell in thymus, spleen and lymph nodes was determined by counting cell number under microscope: % of control = number of cells in restrained mice /number of cells in control mice × 100. (B), The percentage of CD4 + and CD8 + T-cells in thymus (a, b) and in spleen (c, d) was determined by FACScan before (a, c) and after (b, d) suffering with restraint stress. (C) The effect of restraint stress on splenic T-cell response to Con A was determined using control () or restrained mice (). The proliferative response was determined by pulsing cells with [3H]thymidine for 4 h before harvesting them. The data showed the mean 9 S.E. of five mice.
K. Iwakabe et al. / Immunology Letters 62 (1998) 39–43
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Fig. 3. Restraint stress inhibited IFN-g production but not IL-4 production in spleen. Spleen cells were prepared from BALB/c mice various times after the initiation of restraint stress and they were stimulated with Con A (2.5 mg/ml) for 36 h. Then, IFN-g (A) and IL-4 (B) activity in the culture supernatants were determined using ELISA kits. The data showed the mean 9 S.E. of five mice.
strongly modulated the Th1/Th2 balance through the marked decrease of IFN-g production as summarized in Table 1. If the initial Th1/Th2 ratio was designated as 1.00, its ratio was dramatically decreased, indicating that the balance was tilted to Th2 side. As demonstrated previously [22], IL-4 is greatly dominated against IFN-g , and the addition of small amounts of IL-4 during the differentiation of Th1 or Th2 cells resulted in suppression of Th1 development and augmentation of Th2 differentiation even when enough levels of endogenous IFN-g was produced. Therefore, our finding that restraint stress caused about 90% inhibition of IFN-g production but not IL-4 production (6.5% inhibition), strongly suggested that the stress caused a shift in Th1/Th2 balance toward Th2-dominant immunity. This supposition may be supported by the previous evidence that stress generally inhibited cellular immunity but not humoral immunity [9,11]. Moreover, we have already confirmed that restraint stress mice showed reduced IL-12-responsiveness in
Fig. 4. Restraint stress inhibited CTL generation in parallel with the decreased IFN-g production in MLR. BALB/c spleen cells were prepared from control (untreated) or restrained mice (restraint stress) and they (5 × 106) were cultured with mitomycin C-treated C57BL/6 mouse spleen cells (2.5 ×106 cells) for 4 days. After the culture, the cytotoxic activity against MBL-2 was determined by 4h-51Cr-release assay ( ). IFN-g activity in 4-day culture supernatants was determined by ELISA kits (a).
terms of NK activation when compared with control mice (data not shown). Although polyclonal T-cell activation was not suppressed by restraint stress (Fig. 2), alloantigen-specific CTL generation by MLR was strongly inhibited by restraint stress in accord with the reduction of IFN-g production (Fig. 4). This result suggested that stress might become the cause of the increased susceptibility against infectious diseases via the reduction of CTL generation. The data also indicated acute elevation of serum corticosterone levels was enough to suppress CTL generation from spleen cells. In contrast to our present data, it was known that the continuous infusion of glucocorticoids from the osmotic pump was not
Table 1 The effect of restraint stress on Th1/Th2 balance Experiment no.
1 2 3 a
Restraint stress micea
Control mice
Th1/Th2 ratioc
IFN-g (ng/ml)b
IL-4 (pg/ml)
IFN-g (ng/ml)
IL-4 (pg/ml)
124.79 31 141.09 11 47.09 11
177.0 9 28 155.39 2 95.79 26
14.192 13.7 9 2 9.3 9 2
143.2 92 189.3 922 82.3 913
11/80.9 (0.14) 9.7/121 (0.08) 19.8/85.9 (0.23)
BALB/c mice were restrained in a 50-ml tube for 24 h. IFN-g and IL-4 activity was determined by ELISA kits. The data represent mean 95 mice. c The IFN-g and IL-4 producing ability in control mice was designated as 100%. Then, the % of control of both IFN-g and IL-4 producing ability in restraint stress mice was first calculated. The Th1/Th2 ratio was calculated by the following formula; Th1/Th2 ratio =% of control of IFN-g producing ability in mice/% of control of IL-4 producing ability in mice. From this formula, the Th1/Th2 ratio in control mice is designated as 1 (100/100). b
K. Iwakabe et al. / Immunology Letters 62 (1998) 39–43
effective on the inhibition of CTL generation [23]. However, in their infusion system, serum glucocorticoids levels increased at only 1 day after infusion and it decreased to near normal levels when the mice were sacrificed. Therefore, the serum corticosterone levels at the time when the mice were sacrificed might be an important factor to demonstrate the suppressive effect of stress-induced corticosterone on the CTL generation. As discussed in Figs. 1 and 4, NK activity and CTL activity appeared to decrease in parallel with the elevation of serum corticosterone levels. However, it still remained unclear whether the stress-induced decrease of NK activity, IFN-g production and CTL activity shown here is totally dependent on serum corticosterone levels until we demonstrate the blocking effect of anti corticosterone antibody or adrenalectomy. However, previous reports which demonstrated that stress-induced suppression of immunity was also induced in an adrenal-independent manner [17,20,24], strongly suggested that a variety of other hormones, neurosecretory products and cytokine may be involved in such stress-related modulation of immunity in addition to adrenal systems. In recent decades, there has been an increase in sensitivity and prevalence of allergic disorders such as atopic dermatitis in developed countries [25 – 27]. Although many investigators have been focused on the increased air pollution, increased indoor exposure to dust mite antigen, dietary changes and the diminished exposure to infection as causes for the increased allergic disorders [28,29]. However, in this paper, we further propose that a stressful life, which is one of the important problems in modern life [30], may become one of the causes for the increase of allergic disorders through its effect on Th1/Th2 balance regulating IgE production [31].
Acknowledgements The authors would like to thank Drs Michiko Kobayashi and Steven H. Herrmann for their kind gift of IL-12. This work was supported in part by a grant-inaid for The Science Frontier Program of MESSC, a grant-in-aid for scientific research on priority areas from MESC, and a grant-in-aid from the Ministry of Health and Welfare for Cancer Control, and by a grant-in-aid for the IL-12 project of Tokai University School of Medicine.
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The restraint stress drives a shift in Th1/Th2 balance toward Th2-dominant immunity in mice Kenji Iwakabe a, Masako Shimada a, Akio Ohta a, Takashi Yahata a, Yasushi Ohmi a, Sonoko Habu a, Takashi Nishimura a,b,* b
a Department of Immunology, Tokai Uni6ersity School of Medicine, Bohseidai, Isehara 259 -11, Japan Research Center for Genetic Engineering and Cell Transplantation, Tokai Uni6ersity School of Medicine, Bohseidai, Isehara 259 -11, Japan
Received 8 January 1998; received in revised form 11 February 1998; accepted 12 February 1998
Abstract When mice were physically restrained in 50-ml tubes for 24 h, a marked decrease of NK activity was demonstrated in parallel with the elevation of serum corticosterone levels. The release of mice from restraint stress resulted in the recovery of NK activity, with a decrease of serum corticosterone levels within 48 h. Using this stress model, we also investigated the influence of restraint stress on mouse Th1/Th2 balance. Consistent with the decrease of NK activity, IFN-g production of mouse spleen cells greatly reduced after suffering from restraint stress. In contrast, the IL-4 producing ability of spleen cells was not so much affected by restraint stress. These results initially indicated that stress may induce the skewing of the Th1/Th2 balance toward Th2-dominant immunity, which stimulates the occurrence of infectious diseases and allergic disorders. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Restraint stress; Th1/Th2 balance; NK; Cytokine
1. Introduction A large body of research in recent decades has suggested that stress may be associated with the onset, course, and outcome of physical illness [1 – 3]. Stress-induced immunologic alterations have been considered as major cause of increased risk for immune-related diseases, such as cancer, autoimmune disorders, and infection [4,5]. In general, stressors trigger physiological responses involving the hypothalamic-pituitary-adrenal axis (HPA) and autonomic nervous system via the release of corticosteroid hormones and catecholamines, respectively [6–8]. These nervous and endocrine systems are closely associated with immune systems and thus the stressor induces the alternation of host immune responses [9,10]. * Corresponding author. Tel.: +81 463 931121; fax: + 81 463 942976. 0165-2478/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0165-2478(98)00021-2
It has been reported that stress generally induced the suppression of cellular immune responses, but resulted in no effect or rather increase of humoral immunity [9,11]. However, little has been investigated about the precise mechanisms underlying this stress-induced phenomena. Recently, it has been demonstrated that cellular immunity and humoral immunity is regulated by distinct subsets of helper T-cells [12]. The former response is regulated by Th1 cells, which can produce IL-2, IFN-g and lymphotoxin. The latter is controlled by Th2 cells, which can produce IL-4, IL-5, IL-6 and IL-10. Because the glucocorticoids, which is elevated by physical stress, suppress Th1 cells but stimulate Th2 cells [13,14], it is possible to speculate that stress may influence immune responses via the modulation of the Th1/Th2 balance. As well as stress, the imbalance of Th1/Th2 immunity has been demonstrated to be associated with increased risk for various immune diseases [15,16]. Therefore, it is important to investigate the effect of stress on the Th1/Th2 balance.
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K. Iwakabe et al. / Immunology Letters 62 (1998) 39–43
In the present paper, we found that restraint stress strongly inhibited IFN-g production in parallel with the suppression of NK activity, while it did not affect the production of Th2 cytokine, IL-4 to the same degree. Thus, these data initially showed direct evidence that restraint stress caused a shift in Th1/Th2 balance toward Th2-dominant immunity, which might accelerate the onset of infectious diseases and allergic disorders.
2.6. Cytokine production Spleen cells (5 × 105 per well) were stimulated with concanavalin A (Con A) (2.5 mg/ml) for 36 h in Falcon 96-well plate. After incubation, culture supernatants were harvested and their IFN-g and IL-4 activity was determined by ELISA kits (Amersham, Tokyo, Japan) [19].
2.7. Mixed lymphocyte reaction 2. Materials and methods
2.1. Mice BALB/c mice and C57BL/6 mice were purchased from Charles River, Japan, (Yokohama 222, Japan). All mice were female and used when they were 8–10 weeks old.
2.2. Restraint stress protocol According to the methods of Bonneau et al. [17], mice were physically restrained in a 50-ml Falcon tube in which various holes with a diameter of 0.4 cm had been drilled. The mice were first wetted with water and restrained in a 50-ml tube for various times without supplying food and water. The control mice were left in usual animal cages for the same time without food and water. The smaller mice, younger than 8 weeks old, were not suitable for this restraint stress experiment using the 50-ml tube.
2.3. Cytotoxicity assay NK activity was determined by 4-h 51Cr-release assay using YAC-1 cells as target cells. The % cytotoxicity and lytic units (LU/106) were calculated as described previously [18].
2.4. Measurement of serum corticosterone le6els The serum corticosterone levels of control or restraint mice was determined using Immuchem™ Double Antibody Corticosterone Ria Kit for Rat and Mice (ICN Biomedicals, CA 92626) according to the manufacturer’s instructions.
2.5. Flow cytometry The analysis of cell surface markers was carried out by FACScan using the Consort 30 program. The detailed procedure for staining and sorting was described in a previous paper [19]. All FITC or PE-conjugated mAbs used in this paper was purchased from PharMingen (San Diego, CA).
BALB/c spleen cells (5 × 106 cells/well) were stimulated with mitomycin C-treated C57BL/6 mouse spleen cells for 4 days. After the culture, CTL activity against C57BL/6-derived MBL-2 lymphoma cells was determined by 4-h 51Cr-release assay. The IFN-g activity of culture supernatants during mixed lymphocyte reaction (MLR) was also examined by ELISA kits.
3. Results and discussion Restraint stress was used to examine the influence of stress on the immune system. Consistent with previous reports [20], the acute stress (24-h restraint stress) caused the elevation of serum corticosterone levels (Fig. 1A). The NK activity against YAC-1 cells was also markedly suppressed by the acute stress in contrast to a recent report by Mizobe et al. [20] (Fig. 1A). This discrepancy may be because of the differential method for restraint stress. The restraint stress using the 50-ml tube appeared to be a more severe stressor for mice compared with the previous stress protocol using a wire net stress cage. As shown in Fig. 1B, the reduced NK activity recovered within 48 h after the release of the mice from restraint stress, in parallel with the decrease of serum corticosterone levels. Therefore, this restraint stress appeared to suppress NK activity through the induction of the elevated levels of endogenous gluococorticoids. Using C57BL/6 mouse strain expressing NK1.1 marker, it was also demonstrated that the percentage of NK cells in the spleen did not significantly change before (4.7893.2%) and after (5.2791.5%) the restraint stress. In accord with the finding that glucocorticoids could induce the apoptosis of thymocytes [21], the restraint stress caused a marked reduction of the number of thymocytes and spleen cells (Fig. 2A). However, the percentage of CD4 + CD8 + T-cells was not reduced by restraint stress (Fig. 2B). Moreover, spleen cells obtained from stressed mice showed the same levels of proliferative responses to Con A as those from control mice (Fig. 2C). Recently, however, glucocorticoids have demonstrated to reveal the differential effect against Th1 and Th2 immunity [13,14], which has a pivotal role in
K. Iwakabe et al. / Immunology Letters 62 (1998) 39–43
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Fig. 1. Reduction of NK activity by restraint stress. (A) C57BL/6 mice were restrained in a 50-ml tube for various times. Thereafter, their NK activity against YAC-1 cells () and the elevation of serum corticosterone levels was determined by the method described in Section 2. For measurement of NK activity, whole spleen cells were used in this experiment. (B) Mice were first restrained in a 50-ml tube for 24 h and released from the stress. Then, NK activity against YAC-1 cells () and the elevation of serum corticosterone levels () was determined various times after the release from the stress. For the measurement of NK activity, nylon-passed spleen cells were used in this experiment. The data represent mean9 S.E. of five mice. The suppression of NK activity by the stress was also demonstrated in BALB/c strain mice.
immunoregulation. Therefore, it is possible to speculate that restraint stress may affect the Th1/Th2 balance. To evaluate the issues, the effect of restraint stress on the production of IFN-g (Th1 cytokine) and IL-4 (Th2 cytokine) was examined using Con A-activated spleen cells. As clearly demonstrated in Fig. 3, control mice, which were left in the usual cage without food and water, showed high levels of IFN-g production when their spleen cells were stimulated with Con A for 36 h. In contrast, the IFN-g production of Con A-stimulated spleen cells was strongly reduced by restraint stress (Fig. 3A). Surprisingly, however, IL-4 production of
Con A-stimulated spleen cells was not so much affected by restraint stress (Fig. 3B). In a previous report, Ramı´rez et al. [14] demonstrated that a prolonged treatment of CD4 + T-cells in vitro with glucocorticoids, and IL-4, resulted in the enhanced IL-4 production of purified CD4 + T-cells. However, we could not always detect the augmentation of the IL-4 producingability of T-cells by stress-induced corticosterone. This discrepancy may be because of the different conditions for corticosterone-treatment between their in vitro study and our in vivo study. Thus, restraint stress did not cause the augmented IL-4 production, but it
Fig. 2. The effect of restraint stress on cell numbers, subpopulations and function of T-cells. BALB/c mice were restrained in a 50-ml tube for 24 h. (A) The effect of restraint stress on the number of cell in thymus, spleen and lymph nodes was determined by counting cell number under microscope: % of control = number of cells in restrained mice /number of cells in control mice × 100. (B), The percentage of CD4 + and CD8 + T-cells in thymus (a, b) and in spleen (c, d) was determined by FACScan before (a, c) and after (b, d) suffering with restraint stress. (C) The effect of restraint stress on splenic T-cell response to Con A was determined using control () or restrained mice (). The proliferative response was determined by pulsing cells with [3H]thymidine for 4 h before harvesting them. The data showed the mean 9 S.E. of five mice.
K. Iwakabe et al. / Immunology Letters 62 (1998) 39–43
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Fig. 3. Restraint stress inhibited IFN-g production but not IL-4 production in spleen. Spleen cells were prepared from BALB/c mice various times after the initiation of restraint stress and they were stimulated with Con A (2.5 mg/ml) for 36 h. Then, IFN-g (A) and IL-4 (B) activity in the culture supernatants were determined using ELISA kits. The data showed the mean 9 S.E. of five mice.
strongly modulated the Th1/Th2 balance through the marked decrease of IFN-g production as summarized in Table 1. If the initial Th1/Th2 ratio was designated as 1.00, its ratio was dramatically decreased, indicating that the balance was tilted to Th2 side. As demonstrated previously [22], IL-4 is greatly dominated against IFN-g , and the addition of small amounts of IL-4 during the differentiation of Th1 or Th2 cells resulted in suppression of Th1 development and augmentation of Th2 differentiation even when enough levels of endogenous IFN-g was produced. Therefore, our finding that restraint stress caused about 90% inhibition of IFN-g production but not IL-4 production (6.5% inhibition), strongly suggested that the stress caused a shift in Th1/Th2 balance toward Th2-dominant immunity. This supposition may be supported by the previous evidence that stress generally inhibited cellular immunity but not humoral immunity [9,11]. Moreover, we have already confirmed that restraint stress mice showed reduced IL-12-responsiveness in
Fig. 4. Restraint stress inhibited CTL generation in parallel with the decreased IFN-g production in MLR. BALB/c spleen cells were prepared from control (untreated) or restrained mice (restraint stress) and they (5 × 106) were cultured with mitomycin C-treated C57BL/6 mouse spleen cells (2.5 ×106 cells) for 4 days. After the culture, the cytotoxic activity against MBL-2 was determined by 4h-51Cr-release assay ( ). IFN-g activity in 4-day culture supernatants was determined by ELISA kits (a).
terms of NK activation when compared with control mice (data not shown). Although polyclonal T-cell activation was not suppressed by restraint stress (Fig. 2), alloantigen-specific CTL generation by MLR was strongly inhibited by restraint stress in accord with the reduction of IFN-g production (Fig. 4). This result suggested that stress might become the cause of the increased susceptibility against infectious diseases via the reduction of CTL generation. The data also indicated acute elevation of serum corticosterone levels was enough to suppress CTL generation from spleen cells. In contrast to our present data, it was known that the continuous infusion of glucocorticoids from the osmotic pump was not
Table 1 The effect of restraint stress on Th1/Th2 balance Experiment no.
1 2 3 a
Restraint stress micea
Control mice
Th1/Th2 ratioc
IFN-g (ng/ml)b
IL-4 (pg/ml)
IFN-g (ng/ml)
IL-4 (pg/ml)
124.79 31 141.09 11 47.09 11
177.0 9 28 155.39 2 95.79 26
14.192 13.7 9 2 9.3 9 2
143.2 92 189.3 922 82.3 913
11/80.9 (0.14) 9.7/121 (0.08) 19.8/85.9 (0.23)
BALB/c mice were restrained in a 50-ml tube for 24 h. IFN-g and IL-4 activity was determined by ELISA kits. The data represent mean 95 mice. c The IFN-g and IL-4 producing ability in control mice was designated as 100%. Then, the % of control of both IFN-g and IL-4 producing ability in restraint stress mice was first calculated. The Th1/Th2 ratio was calculated by the following formula; Th1/Th2 ratio =% of control of IFN-g producing ability in mice/% of control of IL-4 producing ability in mice. From this formula, the Th1/Th2 ratio in control mice is designated as 1 (100/100). b
K. Iwakabe et al. / Immunology Letters 62 (1998) 39–43
effective on the inhibition of CTL generation [23]. However, in their infusion system, serum glucocorticoids levels increased at only 1 day after infusion and it decreased to near normal levels when the mice were sacrificed. Therefore, the serum corticosterone levels at the time when the mice were sacrificed might be an important factor to demonstrate the suppressive effect of stress-induced corticosterone on the CTL generation. As discussed in Figs. 1 and 4, NK activity and CTL activity appeared to decrease in parallel with the elevation of serum corticosterone levels. However, it still remained unclear whether the stress-induced decrease of NK activity, IFN-g production and CTL activity shown here is totally dependent on serum corticosterone levels until we demonstrate the blocking effect of anti corticosterone antibody or adrenalectomy. However, previous reports which demonstrated that stress-induced suppression of immunity was also induced in an adrenal-independent manner [17,20,24], strongly suggested that a variety of other hormones, neurosecretory products and cytokine may be involved in such stress-related modulation of immunity in addition to adrenal systems. In recent decades, there has been an increase in sensitivity and prevalence of allergic disorders such as atopic dermatitis in developed countries [25 – 27]. Although many investigators have been focused on the increased air pollution, increased indoor exposure to dust mite antigen, dietary changes and the diminished exposure to infection as causes for the increased allergic disorders [28,29]. However, in this paper, we further propose that a stressful life, which is one of the important problems in modern life [30], may become one of the causes for the increase of allergic disorders through its effect on Th1/Th2 balance regulating IgE production [31].
Acknowledgements The authors would like to thank Drs Michiko Kobayashi and Steven H. Herrmann for their kind gift of IL-12. This work was supported in part by a grant-inaid for The Science Frontier Program of MESSC, a grant-in-aid for scientific research on priority areas from MESC, and a grant-in-aid from the Ministry of Health and Welfare for Cancer Control, and by a grant-in-aid for the IL-12 project of Tokai University School of Medicine.
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