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Stress and Immune Responses IV. Adrenal Involvement in the Alteration of Antibody Responses in Restraint-Stressed Mice
Stress IV.
Adrenal
and
Involvement
Responses Tsutomu
Immune in the
of
Alteration
in Restraint-Stressed
OKIMURA*,
Masami
OGAWA,
and Yasuo Department
Responses
Immunochemistry,
Faculty
Tsushima-naka Accepted
Mice
Toshihiko
YAMAUCHI
SASAKI*
of Pharmaceutical 1 -1,
of Antibody
Okayama
March
Sciences, 700,
Okayama
University,
Japan
6, 1986
Abstract-We investigated the correlation between restraint stress-induced alter ation of antibody responses and adrenal hormones. Adrenalectomy (Adx) blocked both the suppression of antibody response against T cell-dependent (TD) antigen and the enhancement of that against T cell-independent (TI) antigen in stressed mice. Adx also inhibited the atrophy of both thymus and spleen caused by restraint. Pre-treatment of metyrapone, an inhibitor of adrenocortical steroid biosynthesis, had an effect that was similar to, but far weaker than that of Adx on stressed mice. The pre-administration of phenoxybenzamine, an a-adrenergic blocking agent, to mice prevented both the inhibition of antibody response to TD antigen and the decrease in spleen cell number of restrained mice. A similar effect was observed in mice pre-treated with 6-hydroxydopamine, an adrenergic neuron degenerating agent. However, no effects of these two agents were observed on the enhancement of antibody response to TI antigen. The suppressive effect of the antibody response to TD antigen was augmented by the pre-administration of propranolol, a e3-adrenergic blocking agent. These results suggest that the suppression of the function of T cells in restrained mice are attributed to the released adrenocortical and adrenal medullary hormones and activated sympathetic nervous system and that the enhance ment of B cell function is due to the adrenocortical hormones. We showed previously that restraint stress altered lymphocyte functions in mice (1, 2). There are considerable evidences for the modulation by corticosteroids (3-6). These observations support the hypothesis that adrenocortical hormones are the primary mediator of stress effects on the immune system (7-9). However, it is suggested that a variety of other hormonal and neuro secretory systems may be involved in the stress-induced modulation of immunity (10 13). * Present Division, Tamatsukuri Japan
address:
Research
Morishita 1 -chome,
Jintan
and Co.,
Higashi-ku,
Development Ltd., Osaka
1-30, 540,
In order to clarify whether restraint-induced modulation of antibody response is mediated through the hypothalamic-pituitary adrenal system, we examined antibody responses of restraint-stressed mice pre-administered with a or a-adrenergic blocking agents, adre nergic neuron degenerating agent or inhibitor of adrenocortical steroid biosynthesis. Anti body responses were also examined in adrenalectomized and stressed mice. In the present paper, we described the involvement of both adrenocortical and adrenal medullary hormones in the alteration of lymphocyte functions.
Materials and Methods Animals: Female BALB/c mice, all 8 weeks old, were obtained from Japan Charles
River Breeding Laboratories (Tokyo, Japan) and were used at 10-15 weeks of age. Chemicals, culture media and antigen: Chemicals used in the present work were purchased from the following sources; phenoxybenzamine (Tokyo Chemical In dustry, Tokyo, Japan), propranolol (Imperial Chemical Industries, Ltd., England), me tyrapone (Aldrich Chemical Company, Inc. Milwaukee, WI), 6-hydroxydopamine (6 OHDA) (Sigma Chemical Company, St. Louis, M0). Eagle's minimus essential medium was purchased from Nissui Phar maceutical Company (Tokyo, Japan). Sheep red blood cells (SRBC) were obtained from Nishinippon Sheep Farm (Fukuyama, Japan) as a suspension in Alserver's solution. Trinitrophenylated lipopolysaccharide (TNP LPS) was prepared by reacting trinitrobenzene sulfonate (Tokyo Kasei, Tokyo) with LPS by the procedure of Rittenberg and Amkraut (14). LPS was the phenol-water extracts of Escherichia coli 055:B5 (Difco Laboratories, Detroit, MI). Immunization of antigens and assay of antibody forming cells: Mice were im munized with 1 X108 SRBC intravenously, or 10 i g TN P-LPS intraperitoneally. Four days later, antibody synthesis was assayed by enumerating the number of hemolytic plaque-forming cells (PFC) by the method of Jerne and Nordin (15). Lightly conjugated TNP-SRBC were prepared as described by Rittenberg and Pratt (16) for detecting anti TNP PFC. Stress procedure: Experimental mice were fixed in restraint cages for 12 hr a day at night (21:00-9:00) and placed in home cages for the remaining 12 hr with food and water ad libitum. The restraint cages were prepared according to the literature (17). Control animals were maintained in home cages from which food and water were removed for the period of stress loading of the counterparts. Usually mice were loaded with restraint stress for 2 consecutive days. The estimation of corticosterone concen trations: Plasma corticosterone concen trations were determined by a fluorometric method, essentially as described by Imura (18). Adrenalectomy: Adrenalectomy was car
ried out by resecting bilateral adrenals according to Kumagai's method (19). Statistics: Statistical significance was calculated by Student's t-test. Differences were considered to be significant when the probability (P) value was <0.05. Results Influence of adrenalectomy on the modulation of antibody response in restraint-stressed mice: In order to clarify whether the change of antibody responses in stressed mice is due to adrenal hormones, we examined the responses of adrenalec tomized mice. Plasma corticosterone con centrations in control and stressed mice under the sham -adrenalectomization or adre nalectomization are shown in Fig. 1. Im
Fig. 1. Plasma corticosterone levels after restraint stress in mice. BAL B/c mice were adrenalectomized 3 days before the initiation of stress loading. were fixed for 12 hr a day for 2 consecutive Immediately after the stress loading, blood collected from these mice by decapitation. results represent the mean±S.D. Significant difference from the control group: *P<0.05, **P<0.01.
Mice days. was The
of 4 animals. Sham-operated
mediately after the restraint stress, mice with intact adrenals had approximately two times more plasma corticosterone than non stressed control animals. Adrenalectomy completely blocked the stress-induced increase in plasma cor ticosterone in restrained mice. Then, by using these animals, a relationship between adrenal hormones and stress effect on anti body responses was assessed. As shown in Fig. 2, the stress-induced suppression of anti-SRBC PFC response was not observed in adrenalectomized mice. Moreover, adre nalectomy suppressed the increase of number of anti-TNP-LPS PFC/106 spleen cells to the control level in stressed mice. Adrenalectomy also inhibited the atrophy of both the thymus and spleen caused by restraint (Table 1). Influence of adrenocortical hormones on the modulation of antibody responses by
Fig. 2. BALB/c
Influence of adrenalectomy mice were adrenalectomized
restraint stress: Metyrapone competes with deoxycorticosterone for the same binding site on cytochrome P-450, thus inhibiting the biogenesis of corticosterone (20). Then, methyrapone permitted us to study the effects of adrenocorticosteroid hormones in the presence of intact adrenal gland. Therefore, adrenal catecholamine secretion remained intact. In this experiment, metyrapone (100 mg/kg) was orally administered two times a day (morning and evening) from 2 days before the start of stress loading during the stress period. As seen in Fig. 3, the pre treatment of metyrapone partially blocked the suppression of anti-SRBC PFC response in restrained mice. On the other hand, the anti TNP-LPS PFC response in stressed mice was suppressed clearly. Metyrapone blocked neither the involution of the thymus nor the decrease in the spleen cell number (data not shown).
on the modulation of antibody 3 days before the initiation
responses n restraint-stressed mice. of stress loading. These mice were
fixed for 12 hr a day for 2 consecutive days and then immunized with SRBC or TNP-LPS. after immunization, the number of anti-SRBC or anti-TNP PFC in the spleen was determined.
Four days The results
represent
O.01.
the mean±S.D.
of 4 animals.
Significant
difference
from each
control
group:
**P
Table
1.
Influence
of
adrenalectomy
stressed
mice
Fig. 3.
Effect of metyrapone
on
the
on the modulation
number
of
of antibody
spleen
cells
responses
and
thymus
weight
in restraint-stressed
in
restraint
mice. BALB/c
mice were orally administered with 100 mg/kg metyrapone 2 times daily from 2 days before stress loading during the stress period. These mice were fixed for 1 2 hr a day for 2 consecutive days and then immunized with SRBC or TNP-LPS. Four days after immunization, the number of anti-SRBC or anti-TNP PFC in the spleen from each
was determined. The results control group: **P<0.01 .
represent
Influence of adrenal medullary hormones on the modulation of antibody responses in stressed mice: It has been known that there are two major types of receptors (a and ;3) through which adrenal medullary hormones affect the activity of effector cells (21). In order to clarify a possible correlation between
the mean±S.D.
of 4 animals.
Significant
difference
adrenal medullary hormones and stress effects, we assessed the effect of a and 13 adrenergic blocking agents on the modu lation of immune responses caused by restraint stress. At first, mice were orally administered with phenoxybenzamine, an a blocking agent, at a dose of 100 mg/kg two
times per day (morning and evening) starting 2 days before stress loading during the stress period. As shown in Fig. 4, phenoxy benzamine prevented the suppression of PFC response to SRBC in stressed mice. On the other hand, the response to TN P
Fig. 4.
Effect of phenoxybenzamine
BALB/c mice were orally administered before stress loading during the stress
on the modulation
LPS in stressed mice was not influenced by phenoxybenzamine. Phenoxybenzamine par tially (50%) blocked the decrease in spleen cell number, but not the involution of the thymus (Table 2). Effect
of
of antibody
propranolol,
responses
a i9-blocking
in restraint-stressed
agent,
mice.
with 100 mg/kg phenoxybenzamine 2 times daily from 2 days period. These mice were fixed for 12 hr a day for 2 consecutive
days and then immunized with SRBC or TNP-LPS. Four days after immunization, the number of anti SRBC or anti-TNP PFC in the spleen was determined. The results represent the mean+S.D. of 4 animals. Significant difference from each control group: *P-'0.05, **P<0.01. Significant difference from the non-treated
Table stressed
2.
stressed
Effect mice
of
group:
$P<0.05.
phenoxybenzamine
on
the
number
of
spleen
cells
and
thymus
weight
in
restraint
Fig. 5. Effect of propranolol mice were intraperitoneally stress
loading
during
on the modulation administered with
the stress
period.
These
of antibody response 10 mg/kg propranolol mice were
in restraint-stressed 2 times daily from
mice. BALB/c 2 days before
fixed for 12 hr a day for 2 consecutive
days
and then immunized with SRBC or TNP-LPS. Four days after immunization, the number of anti-SRBC or anti-TNP PFC in the spleen was determined. The results represent the mean±S.D. of 4 animals. Significant difference from each control group **P<0.01. Significant difference from the non-treated control group: ~P<0.05. Significant difference from the non-treated stressed group: §P<0.05.
was examined in the next experiment. Propranolol was intraperitoneally admin istered two times per day (morning and evening) from 2 days before stress loading during the stress period. As depicted in Fig. 5, propranolol-treated restrained mice showed more suppressed response to SRBC than non treated stressed mice. On the other hand, propranolol did not affect the antibody response to TNP-LPS in either control or stressed mice. The dose of 100 mg/kg 6-OHDA, an adrenergic neuron degenerating agent, was intraperitoneally medicated two times, 1 day and 5 days before stress loading. Figure 6 shows the results. Suppressed anti-SRBC PFC response of restrained mice was recovered partially by 6-OHDA. However, 6-OHDA did not affect the PFC response to TNP-LPS in restrained mice. Furthermore, the involution of the thymus and the
decrease in the number of spleen cells were not prevented by 6-OHDA. These results (Figs. 4, 5 and 6) were suggestive of the involvement of the adrenal medullary hor mones and sympathetic nervous system in the suppression of T cell-mediated response. Discussion Adrenalectomy completely prevented the changes of antibody responses induced by the restraint stress, i.e., the suppression of the PFC response to TD antigen and the increase of number of PFC/106 spleen cells to TI antigen. Corticosteroids have long been considered to be the primary mediator of stress-induced modulation of immunity (7 9). There are a few, but reliable reports about the relationship between stress-induced modulation of immune functions and adrenal medullary hormones (9, 22, 23). In order to clarify the mechanisms of the stress-induced
Fig. 6. stressed
Effect of pre-administration mice. BALB/c mice were
1 day and 5 days
before
stress
of 6-OHDA on the modulation of antibody response in restraint intraperitoneally administered with 100 mg/kg 6-OHDA 2 times,
loading.
These
mice were
fixed for 12 hr a day for 2 consecutive
days
and then immunized with SRBC or TNP-LPS. Four days after immunization, the number of anti-SRBC or anti-TNP PFC in the spleen was determined. The results represent the mean+S.D. of 4 animals. Significant difference from each control group: *P<0.05, **P<0.01. Significant difference from the non-treated
stressed
group:
~P<0.05.
modulation, we separately investigated the effects of adrenocortical and adrenal medullary hormones. Pre-administration of metyrapone partially blocked the suppression of PFC response to TD antigen, but on the contrary, it inhibited the increase of number of PFC/106 spleen cells to TI antigen. We have previously demonstrated that both helper T cell and suppressor T cell activities were suppressed, but B cell activities were rather enhanced in splenocytes from restrained mice (24). In this study, the number of PFC/spleen to TNP-LPS was not increased by restraint stress. However, it would be due to the atrophy of the spleen caused by the stress. So we think that the increase of number of PFC/106 spleen cells to TNP-LPS by restraint stress indicates the enhancement of anti-TN P LPS PFC response. Therefore, our results presented in this article suggest that corticosteroids suppress T cell
activities but augment B cell activity. As noted before, corticosteroids, have been known to exert immuno-suppressive effects (3-6). However, these results were obtained when corticosteroids at several orders of magnitude higher than the physiological con centrations were exogeneously injected into animals. So, little is known about the action of endogeneous corticosteroids on the immune system. Recently, it has been demon strated that corticosteroids at physiological levels rather augment the antibody pro duction (25-27). On the other hand, adminis tration of metyrapone could not block either the involution of the thymus or the decrease in the number of splenocytes. We propose three possible reasons why this agent can not block these events: 1) Metyrapone could not completely inhibit the synthesis of cor ticosteroids. 2) Original corticosteroids syn thesized before administration of metyrapone
might have an effect. 3) Some factors other than corticosteroids were involved in these effects. Explanation 2) or 3) seem the most likely to us because we could not prevent the involution of these tissues in spite of the administration of higher doses of metyrapone (data not shown). The suppression of the PFC response to TD antigen was blocked by the pre-adminis tration of phenoxybenzamine, an a-adrener gic blocking agent, in restrained mice. It is interesting to note that phenoxybenza mine also blocked the decrease in the number of splenocytes. The prevention of stress induced suppression of the response was observed in mice treated with adrenergic neuron degenerating agent. This agent, however, did not prevent the decrease in the number of splenocytes. We suggest from these results that epinephrine or norepine phrine secreted from the adrenal mudulla decreases the number of splenocytes and that these adrenal medullary hormones and an activated sympathetic nervous system would suppress the T cell functions by acting on a-receptors in restraint-stressed mice. Pre-administration of a (3-blocker showed the suppression of the antibody response to TD antigen in both non-stressed control and stressed mice. It has been reported that E3 stimulant promotes the in vitro antibody responses (28, 29). Therefore, the obser vation in the experiment using a l3-blocker would be attributed to the direct suppressive effect of (3-blocker on immunocompetent cells. In this article, we could not clarify what degree adrenal medullary hormones and the sympathetic nervous system are involved in the alteration of antibody response. However, we conclude that the suppression of PFC response to TD antigen in restraint-stressed mice would be due to the released adrenal medullary and adrenocortical hormones and an activated sympathetic nervous system and that the enhancement of PFC response to TI antigen in these animals would be due to the adrenocortical hormones. Acknowledgment: We wish to thank Dr. I. Yamamoto, Professor and Dr. H. Ohmori, Assist. Prof. (Department of Immunochemistry, Okayama
University) for helpful discussions. References 1 Okimura, T. and Nigo, Y.: Stress and immune responses. I. Suppression of T cell function in restraint-stressed mice. Japan. J. Pharmacol. 40, 505-511 (1986) 2 Okimura, T., Ogawa, M. and Yamauchi, T.: Stress and immune responses. III. Effect of restraint stress on delayed type hypersensitivity (DTH) response, natural killer (NK) activity and phagocytosis in mice. Japan. J. Pharmacol. 41, 229-235 (1986) 3 Fauci, A.S. and Dale, D. C.: Effect of in vivo hydrocortisone on subpopulations of human lymphocytes. J. Clin. Invest. 53, 240-246 (1974) 4 Claman, H.N.: How corticosteroids work. J. Allergy Clin. Immunol. 55, 145-151 (1975) 5 Fauci, A.S.: Mechanism of the immunosup pressive and anti-inflammatory effects of gluco corticosteroids. J. Immunopharmacol. 1, 1-25 (1978-1979) 6 Guyre, P.M., Bodwell, J.E. and Munck, A.: Glucocorticoid action on lymphoid tissue and the immune system; Physiologic and therapeutic implications. Proc. Clin. Biol. Res. 142, 181-194 (1984) 7 Gisler, R.H., Bussard, A.E., Mazie, J.C. and Hess, R.: Hormonal regulation of the immune response. II. Influence of pituitary and adrenal activity cn immune responsiveness in vitro. Cell. 8 Gisler, R.H.: Stress and hormonal regulation of the immune response in mice. Psychother. Psychosom. 23, 197-208 (1974) 9 Solomon, G.F.: Psychoneuroendocrinological effects on the immune response. Annu. Rev. Microbiol. 35, 155-184 (1981) 10 Blecha, F., Kelley, K.W. and Scatterlee, D.C.: Adrenal involvement in the expression of delayed-type hypersensitivity to SRBC and contact sensitivity to DNFB in stressed mice. Proc. Soc. Exp. Biol. Med. 169, 247-252 (1982) 11 Keller, S.E., Weiss, J.M., Schleifer, S.J., Miller, N.E. and Stein, M.: Stress-induced suppression of immunity in adrenalectomized rats. Science 221, 1301-1304 (1983) 12 Boranic, M., Pericic, D., Radacic, M., Poljakblazi, M., Sverko, V. and Miljenovic. G.:
13
Shavit, R.P.
Y., and
Lewis, Leibesking,
J.W.,
Terman, J.C.:
Opioid
G.Y.,
Gale,
peptides
mediate the suppressive effect of stress on natural killer cell cytotoxicity. Science 223, 188 190 (1984) 14 Rittenberg, M.B. and Amkraut, A.A.: Immu nogenicity of trinitrophenyl-hemocyanin: Pro duction of primary and secondary anti-hapten precipitins. J. Immunol. 97, 421-430 (1966) 15 Jerne, N.K. and Nordin, A.A.: Plaque formation in agar by single antibody producing cell. Science 140, 405 (1963) 16 Rittenberg, M.B. and Pratt, K.L.: Anti-trini trophenyl (TNP) plaque assay. Primary response of BALB/c mice to soluble and particulate
23 Besedovsky, H., Rey, A.D., Sorkin, E., Prada, M.D., Burri, R. and Honegger, C.: The immune response evokes changes in brain noradrenergic neurons. Science 221, 564-566 (1983) 24 Okimura, T., Satomi-Sasaki, Y. and Ohkuma, S.: Stress and immune responses. II. Identification of stress-sensitive cells in murine spleen cells. Japan. J. Pharmacol. 40, 513-525 (1986) 25 Fauci, A.S., Pratt, K.R. and Whalen, G.: Activation of human B lymphocytes. IV. Regu
17 Yano, S. and Harada, M.: A method for the production of stress erosion in the mouse stomach and related pharmacological studies. Japan. J. Pharmacol. 23, 57-64 (1973) 18 Imura, H.: A fluorometric method for the deter mination of plasma and adrenal corticosterone. Naikahokan 9,130-136 (1962) (Abs. in English) 19 Kumagai, A.: Hypofunction in adrenal. In Methods in Medical Chemistry 6B, Edited by Kumahara, Y., p. 40-42, Nakayama Shoten, Tokyo (1972) (in Japanese) , 0 Williamson, D.G. and Odonnell, V.J.: The
26 Calvano, S.E., Mark, D.A., Good, R.A. and Fernandes, G.: In vitro assessment of immune function in adrenalectomized rats. Immuno pharmacology 4, 291-302 (1982) 27 Knapp, W., Berger, R. and Posch, B.: Regulation of pokeweed mitogen-induced human B cell differentiation; Effects of hydrocortisone. Im munopharmacology 5, 1-10 (1982) 28 Burchiel, S.W. and Melmon, K.L.: Augmentation of the in vitro humoral immune response by pharmacologic agents. I. An explanation for the differential enhancement of humoral immunity via agents that elevate c-AMP. Immuno pharmacology 1, 137-150 (1979) 29 Sherman, N.A., Smith, R.S. and Middleton, E., Jr.: Effect of adrenergic compounds, amino phylline and hydrocortisone, on in vitro immuno globulin synthesis by normal human peripheral lymphocytes. J. Allergy Clin. Immunol. 52, 13 22 (1972)
21 Ahlquist, R.P.: A study of adrenotropic receptors. Am. J. Physiol. 153, 586-600 (1948) 22 Joasoo, A. and McKenzie, J.M.: Stress and the immune response in rats. Int. Arch. Allergy Appl. Immunol. 50, 659-663 (1976)
Adrenal
and
Involvement
Responses Tsutomu
Immune in the
of
Alteration
in Restraint-Stressed
OKIMURA*,
Masami
OGAWA,
and Yasuo Department
Responses
Immunochemistry,
Faculty
Tsushima-naka Accepted
Mice
Toshihiko
YAMAUCHI
SASAKI*
of Pharmaceutical 1 -1,
of Antibody
Okayama
March
Sciences, 700,
Okayama
University,
Japan
6, 1986
Abstract-We investigated the correlation between restraint stress-induced alter ation of antibody responses and adrenal hormones. Adrenalectomy (Adx) blocked both the suppression of antibody response against T cell-dependent (TD) antigen and the enhancement of that against T cell-independent (TI) antigen in stressed mice. Adx also inhibited the atrophy of both thymus and spleen caused by restraint. Pre-treatment of metyrapone, an inhibitor of adrenocortical steroid biosynthesis, had an effect that was similar to, but far weaker than that of Adx on stressed mice. The pre-administration of phenoxybenzamine, an a-adrenergic blocking agent, to mice prevented both the inhibition of antibody response to TD antigen and the decrease in spleen cell number of restrained mice. A similar effect was observed in mice pre-treated with 6-hydroxydopamine, an adrenergic neuron degenerating agent. However, no effects of these two agents were observed on the enhancement of antibody response to TI antigen. The suppressive effect of the antibody response to TD antigen was augmented by the pre-administration of propranolol, a e3-adrenergic blocking agent. These results suggest that the suppression of the function of T cells in restrained mice are attributed to the released adrenocortical and adrenal medullary hormones and activated sympathetic nervous system and that the enhance ment of B cell function is due to the adrenocortical hormones. We showed previously that restraint stress altered lymphocyte functions in mice (1, 2). There are considerable evidences for the modulation by corticosteroids (3-6). These observations support the hypothesis that adrenocortical hormones are the primary mediator of stress effects on the immune system (7-9). However, it is suggested that a variety of other hormonal and neuro secretory systems may be involved in the stress-induced modulation of immunity (10 13). * Present Division, Tamatsukuri Japan
address:
Research
Morishita 1 -chome,
Jintan
and Co.,
Higashi-ku,
Development Ltd., Osaka
1-30, 540,
In order to clarify whether restraint-induced modulation of antibody response is mediated through the hypothalamic-pituitary adrenal system, we examined antibody responses of restraint-stressed mice pre-administered with a or a-adrenergic blocking agents, adre nergic neuron degenerating agent or inhibitor of adrenocortical steroid biosynthesis. Anti body responses were also examined in adrenalectomized and stressed mice. In the present paper, we described the involvement of both adrenocortical and adrenal medullary hormones in the alteration of lymphocyte functions.
Materials and Methods Animals: Female BALB/c mice, all 8 weeks old, were obtained from Japan Charles
River Breeding Laboratories (Tokyo, Japan) and were used at 10-15 weeks of age. Chemicals, culture media and antigen: Chemicals used in the present work were purchased from the following sources; phenoxybenzamine (Tokyo Chemical In dustry, Tokyo, Japan), propranolol (Imperial Chemical Industries, Ltd., England), me tyrapone (Aldrich Chemical Company, Inc. Milwaukee, WI), 6-hydroxydopamine (6 OHDA) (Sigma Chemical Company, St. Louis, M0). Eagle's minimus essential medium was purchased from Nissui Phar maceutical Company (Tokyo, Japan). Sheep red blood cells (SRBC) were obtained from Nishinippon Sheep Farm (Fukuyama, Japan) as a suspension in Alserver's solution. Trinitrophenylated lipopolysaccharide (TNP LPS) was prepared by reacting trinitrobenzene sulfonate (Tokyo Kasei, Tokyo) with LPS by the procedure of Rittenberg and Amkraut (14). LPS was the phenol-water extracts of Escherichia coli 055:B5 (Difco Laboratories, Detroit, MI). Immunization of antigens and assay of antibody forming cells: Mice were im munized with 1 X108 SRBC intravenously, or 10 i g TN P-LPS intraperitoneally. Four days later, antibody synthesis was assayed by enumerating the number of hemolytic plaque-forming cells (PFC) by the method of Jerne and Nordin (15). Lightly conjugated TNP-SRBC were prepared as described by Rittenberg and Pratt (16) for detecting anti TNP PFC. Stress procedure: Experimental mice were fixed in restraint cages for 12 hr a day at night (21:00-9:00) and placed in home cages for the remaining 12 hr with food and water ad libitum. The restraint cages were prepared according to the literature (17). Control animals were maintained in home cages from which food and water were removed for the period of stress loading of the counterparts. Usually mice were loaded with restraint stress for 2 consecutive days. The estimation of corticosterone concen trations: Plasma corticosterone concen trations were determined by a fluorometric method, essentially as described by Imura (18). Adrenalectomy: Adrenalectomy was car
ried out by resecting bilateral adrenals according to Kumagai's method (19). Statistics: Statistical significance was calculated by Student's t-test. Differences were considered to be significant when the probability (P) value was <0.05. Results Influence of adrenalectomy on the modulation of antibody response in restraint-stressed mice: In order to clarify whether the change of antibody responses in stressed mice is due to adrenal hormones, we examined the responses of adrenalec tomized mice. Plasma corticosterone con centrations in control and stressed mice under the sham -adrenalectomization or adre nalectomization are shown in Fig. 1. Im
Fig. 1. Plasma corticosterone levels after restraint stress in mice. BAL B/c mice were adrenalectomized 3 days before the initiation of stress loading. were fixed for 12 hr a day for 2 consecutive Immediately after the stress loading, blood collected from these mice by decapitation. results represent the mean±S.D. Significant difference from the control group: *P<0.05, **P<0.01.
Mice days. was The
of 4 animals. Sham-operated
mediately after the restraint stress, mice with intact adrenals had approximately two times more plasma corticosterone than non stressed control animals. Adrenalectomy completely blocked the stress-induced increase in plasma cor ticosterone in restrained mice. Then, by using these animals, a relationship between adrenal hormones and stress effect on anti body responses was assessed. As shown in Fig. 2, the stress-induced suppression of anti-SRBC PFC response was not observed in adrenalectomized mice. Moreover, adre nalectomy suppressed the increase of number of anti-TNP-LPS PFC/106 spleen cells to the control level in stressed mice. Adrenalectomy also inhibited the atrophy of both the thymus and spleen caused by restraint (Table 1). Influence of adrenocortical hormones on the modulation of antibody responses by
Fig. 2. BALB/c
Influence of adrenalectomy mice were adrenalectomized
restraint stress: Metyrapone competes with deoxycorticosterone for the same binding site on cytochrome P-450, thus inhibiting the biogenesis of corticosterone (20). Then, methyrapone permitted us to study the effects of adrenocorticosteroid hormones in the presence of intact adrenal gland. Therefore, adrenal catecholamine secretion remained intact. In this experiment, metyrapone (100 mg/kg) was orally administered two times a day (morning and evening) from 2 days before the start of stress loading during the stress period. As seen in Fig. 3, the pre treatment of metyrapone partially blocked the suppression of anti-SRBC PFC response in restrained mice. On the other hand, the anti TNP-LPS PFC response in stressed mice was suppressed clearly. Metyrapone blocked neither the involution of the thymus nor the decrease in the spleen cell number (data not shown).
on the modulation of antibody 3 days before the initiation
responses n restraint-stressed mice. of stress loading. These mice were
fixed for 12 hr a day for 2 consecutive days and then immunized with SRBC or TNP-LPS. after immunization, the number of anti-SRBC or anti-TNP PFC in the spleen was determined.
Four days The results
represent
O.01.
the mean±S.D.
of 4 animals.
Significant
difference
from each
control
group:
**P
Table
1.
Influence
of
adrenalectomy
stressed
mice
Fig. 3.
Effect of metyrapone
on
the
on the modulation
number
of
of antibody
spleen
cells
responses
and
thymus
weight
in restraint-stressed
in
restraint
mice. BALB/c
mice were orally administered with 100 mg/kg metyrapone 2 times daily from 2 days before stress loading during the stress period. These mice were fixed for 1 2 hr a day for 2 consecutive days and then immunized with SRBC or TNP-LPS. Four days after immunization, the number of anti-SRBC or anti-TNP PFC in the spleen from each
was determined. The results control group: **P<0.01 .
represent
Influence of adrenal medullary hormones on the modulation of antibody responses in stressed mice: It has been known that there are two major types of receptors (a and ;3) through which adrenal medullary hormones affect the activity of effector cells (21). In order to clarify a possible correlation between
the mean±S.D.
of 4 animals.
Significant
difference
adrenal medullary hormones and stress effects, we assessed the effect of a and 13 adrenergic blocking agents on the modu lation of immune responses caused by restraint stress. At first, mice were orally administered with phenoxybenzamine, an a blocking agent, at a dose of 100 mg/kg two
times per day (morning and evening) starting 2 days before stress loading during the stress period. As shown in Fig. 4, phenoxy benzamine prevented the suppression of PFC response to SRBC in stressed mice. On the other hand, the response to TN P
Fig. 4.
Effect of phenoxybenzamine
BALB/c mice were orally administered before stress loading during the stress
on the modulation
LPS in stressed mice was not influenced by phenoxybenzamine. Phenoxybenzamine par tially (50%) blocked the decrease in spleen cell number, but not the involution of the thymus (Table 2). Effect
of
of antibody
propranolol,
responses
a i9-blocking
in restraint-stressed
agent,
mice.
with 100 mg/kg phenoxybenzamine 2 times daily from 2 days period. These mice were fixed for 12 hr a day for 2 consecutive
days and then immunized with SRBC or TNP-LPS. Four days after immunization, the number of anti SRBC or anti-TNP PFC in the spleen was determined. The results represent the mean+S.D. of 4 animals. Significant difference from each control group: *P-'0.05, **P<0.01. Significant difference from the non-treated
Table stressed
2.
stressed
Effect mice
of
group:
$P<0.05.
phenoxybenzamine
on
the
number
of
spleen
cells
and
thymus
weight
in
restraint
Fig. 5. Effect of propranolol mice were intraperitoneally stress
loading
during
on the modulation administered with
the stress
period.
These
of antibody response 10 mg/kg propranolol mice were
in restraint-stressed 2 times daily from
mice. BALB/c 2 days before
fixed for 12 hr a day for 2 consecutive
days
and then immunized with SRBC or TNP-LPS. Four days after immunization, the number of anti-SRBC or anti-TNP PFC in the spleen was determined. The results represent the mean±S.D. of 4 animals. Significant difference from each control group **P<0.01. Significant difference from the non-treated control group: ~P<0.05. Significant difference from the non-treated stressed group: §P<0.05.
was examined in the next experiment. Propranolol was intraperitoneally admin istered two times per day (morning and evening) from 2 days before stress loading during the stress period. As depicted in Fig. 5, propranolol-treated restrained mice showed more suppressed response to SRBC than non treated stressed mice. On the other hand, propranolol did not affect the antibody response to TNP-LPS in either control or stressed mice. The dose of 100 mg/kg 6-OHDA, an adrenergic neuron degenerating agent, was intraperitoneally medicated two times, 1 day and 5 days before stress loading. Figure 6 shows the results. Suppressed anti-SRBC PFC response of restrained mice was recovered partially by 6-OHDA. However, 6-OHDA did not affect the PFC response to TNP-LPS in restrained mice. Furthermore, the involution of the thymus and the
decrease in the number of spleen cells were not prevented by 6-OHDA. These results (Figs. 4, 5 and 6) were suggestive of the involvement of the adrenal medullary hor mones and sympathetic nervous system in the suppression of T cell-mediated response. Discussion Adrenalectomy completely prevented the changes of antibody responses induced by the restraint stress, i.e., the suppression of the PFC response to TD antigen and the increase of number of PFC/106 spleen cells to TI antigen. Corticosteroids have long been considered to be the primary mediator of stress-induced modulation of immunity (7 9). There are a few, but reliable reports about the relationship between stress-induced modulation of immune functions and adrenal medullary hormones (9, 22, 23). In order to clarify the mechanisms of the stress-induced
Fig. 6. stressed
Effect of pre-administration mice. BALB/c mice were
1 day and 5 days
before
stress
of 6-OHDA on the modulation of antibody response in restraint intraperitoneally administered with 100 mg/kg 6-OHDA 2 times,
loading.
These
mice were
fixed for 12 hr a day for 2 consecutive
days
and then immunized with SRBC or TNP-LPS. Four days after immunization, the number of anti-SRBC or anti-TNP PFC in the spleen was determined. The results represent the mean+S.D. of 4 animals. Significant difference from each control group: *P<0.05, **P<0.01. Significant difference from the non-treated
stressed
group:
~P<0.05.
modulation, we separately investigated the effects of adrenocortical and adrenal medullary hormones. Pre-administration of metyrapone partially blocked the suppression of PFC response to TD antigen, but on the contrary, it inhibited the increase of number of PFC/106 spleen cells to TI antigen. We have previously demonstrated that both helper T cell and suppressor T cell activities were suppressed, but B cell activities were rather enhanced in splenocytes from restrained mice (24). In this study, the number of PFC/spleen to TNP-LPS was not increased by restraint stress. However, it would be due to the atrophy of the spleen caused by the stress. So we think that the increase of number of PFC/106 spleen cells to TNP-LPS by restraint stress indicates the enhancement of anti-TN P LPS PFC response. Therefore, our results presented in this article suggest that corticosteroids suppress T cell
activities but augment B cell activity. As noted before, corticosteroids, have been known to exert immuno-suppressive effects (3-6). However, these results were obtained when corticosteroids at several orders of magnitude higher than the physiological con centrations were exogeneously injected into animals. So, little is known about the action of endogeneous corticosteroids on the immune system. Recently, it has been demon strated that corticosteroids at physiological levels rather augment the antibody pro duction (25-27). On the other hand, adminis tration of metyrapone could not block either the involution of the thymus or the decrease in the number of splenocytes. We propose three possible reasons why this agent can not block these events: 1) Metyrapone could not completely inhibit the synthesis of cor ticosteroids. 2) Original corticosteroids syn thesized before administration of metyrapone
might have an effect. 3) Some factors other than corticosteroids were involved in these effects. Explanation 2) or 3) seem the most likely to us because we could not prevent the involution of these tissues in spite of the administration of higher doses of metyrapone (data not shown). The suppression of the PFC response to TD antigen was blocked by the pre-adminis tration of phenoxybenzamine, an a-adrener gic blocking agent, in restrained mice. It is interesting to note that phenoxybenza mine also blocked the decrease in the number of splenocytes. The prevention of stress induced suppression of the response was observed in mice treated with adrenergic neuron degenerating agent. This agent, however, did not prevent the decrease in the number of splenocytes. We suggest from these results that epinephrine or norepine phrine secreted from the adrenal mudulla decreases the number of splenocytes and that these adrenal medullary hormones and an activated sympathetic nervous system would suppress the T cell functions by acting on a-receptors in restraint-stressed mice. Pre-administration of a (3-blocker showed the suppression of the antibody response to TD antigen in both non-stressed control and stressed mice. It has been reported that E3 stimulant promotes the in vitro antibody responses (28, 29). Therefore, the obser vation in the experiment using a l3-blocker would be attributed to the direct suppressive effect of (3-blocker on immunocompetent cells. In this article, we could not clarify what degree adrenal medullary hormones and the sympathetic nervous system are involved in the alteration of antibody response. However, we conclude that the suppression of PFC response to TD antigen in restraint-stressed mice would be due to the released adrenal medullary and adrenocortical hormones and an activated sympathetic nervous system and that the enhancement of PFC response to TI antigen in these animals would be due to the adrenocortical hormones. Acknowledgment: We wish to thank Dr. I. Yamamoto, Professor and Dr. H. Ohmori, Assist. Prof. (Department of Immunochemistry, Okayama
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