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Effects of footshock stress upon spleen and peripheral blood lymphocyte mitogenic responses in rats with lesions of the paraventricular nuclei
Journal of Neuroimmunology
ELSEVIER
Journal of Neuroimmunology 53 (1994) 39-46
Effects of footshock stress upon spleen and peripheral blood lymphocyte mitogenic responses in rats with lesions of the paraventricular nuclei Michael A. Pezzone a,., Janos Dohanics b, Bruce S. Rabin
a
a Department of Pathology, Brain, Behavior and Immunity Center, 200 Lothrop Street, Pittsburgh, PA 15213-2582, USA b Department of Medicine, University of Pittsburgh School of Medicine Pittsburgh, PA 15213, USA Received 26 October 1993; revised received 25 February 1994; accepted 18 April 1994
Abstract
To assess the role of the hypothalamic paraventricular nucleus (PVN) in mediating stressor-induced immune alterations, male Lewis rats were subjected to a 1-h session of intermittent footshock stress or home cage conditions 6 days after receiving bilateral or sham PVN lesions. Splenic and peripheral blood lymphocyte proliferative responses to the non-specific mitogens, concanavalin A (ConA) and phytohemagglutinin (PHA), were subsequently measured as were plasma corticosterone levels. In sham-operated rats, footshock markedly elevated plasma corticosterone levels and concurrently suppressed the proliferative responses of peripheral blood and splenic lymphocytes. In PVN-lesioned rats, however, the shock-induced suppression of lymphocyte proliferation in the peripheral blood and the elevation of plasma corticosterone were significantly attenuated, while lymphocyte proliferation in the spleen was suppressed below the level of the sham-treated animals. Thus, by utilizing ablation studies, we have determined that the PVN may play a direct role in the alteration of lymphocyte function during stress, and an intact PVN buffers the effect of stress on the responsiveness of spleen lymphocytes to non-specific mitogens.
Key words: Stress; Glucocorticoid; Lymphocyte proliferation; Hypothalamic-pituitary-adrenal axis; Paraventricular nucleus; Neuroimmunomodulation
I. Introduction
Stress is capable of altering the function of the immune system (Rabin et al., 1989, 1990; Kiecolt-Glaser and Glaser, 1991). Although the definitive pathways mediating these immune alterations have not been fully characterized, involvement of the sympathetic nervous system (SNS) and the hypothalamic-pituitaryadrenal (HPA) axis has been strongly suggested (Rabin et al., 1989). Through direct innervation of lymphoid tissue, as well as through endocrine mechanisms, the central nervous system (CNS) may exert its influences upon cells of the immune system (Felten et al., 1985). By acting as a central coordinator of an integrated stress response, the paraventricular nucleus (PVN) of the hypothalamus may be responsible for mediating many of the observed physiological effects of stress.
* Corresponding author. Phone (412) 647 3787; Fax (412) 647 7741. 0165-5728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 5 7 2 8 ( 9 4 ) 0 0 0 6 2 - S
Possessing neuronal projections that can influence both pituitary hormone secretion (Wiegand and Price, 1980) and preganglionic neurons of the sympathetic and parasympathetic divisions of the autonomic nervous system (Saper et al., 1976), the PVN is capable of initiating a stress response characterized by concomitant activation of the H P A axis and the SNS. Attempting to characterize the role of the PVN during stress, we have previously used c-Fos induction as a marker of neuronal activity in rats following exposure to footshock and conditioned, immunomodulating stimuli (Pezzone et al., 1992). Our studies showed that c-Fos immunoreactivity was strongly expressed in cells of the medial parvocellular division of the PVN and in cells bordering the posterior magnocellular division of the PVN following administration of the immune-modulating stimuli. Moreover, because many of the activated PVN neurons also exhibited corticotropin-releasing hormone (CRH) immunoreactivity, a role of CRH in stressor-induced immune alterations was further substantiated (Pezzone et al., 1992).
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M.A. Pezzone et al. /Journal of Neuroimmunology 53 (1994) 39-46
To further investigate the immunomodulatory role of the PVN, splenic and peripheral blood lymphocyte mitogenic responses and plasma corticosterone levels were measured following acute footshock stress in male Lewis rats subjected to bilateral or sham PVN lesions. Because PVN lesions cause complete disappearance of CRH-immunoreactive fibers from the median eminence (Tilders et al., 1982; Antoni et al., 1984; Bruhn et al., 1984) and block the adrenocorticotropic hormone (ACTH) (Makara et al., 1981; Bruhn et al., 1984; Darlington et al., 1988) and corticosterone responses to stress (Makara et al., 1981; Tilders et al., 1982), the immunological effects of footshock stress were consequently studied without activation of the H P A axis by the PVN.
2. Materials and methods
2.1. Experimental animals Male rats of the Lewis strain, 65 days old and 250-300 g in weight were purchased from Harlan Sprague Dawley (Indianapolis, IN). The animals were individually housed in hanging wire cages under a 12-h day-night cycle (lights on at 0700) of artificial illumination and ad lib access to food and water. Upon arrival in the animal housing facility, rats were given a 2-week acclimatization to the new surroundings. Furthermore, the animals were gently handled approximately 2 m i n / d a y for 5 consecutive days during their first week in the new colony room.
2.3. Shock apparatus Eight identical rodent chambers ( B R S / L V E ) measuring 27 x 30 X 24 cm, served as the shock apparatuses. The chambers had clear Plexiglas front, top, and back panels, stainless steel side panels, and a grid floor consisting of stainless steel bars 0.48 cm in diameter, spaced 1.9 cm apart. Chamber illumination was provided by a houselight externally mounted on the wall of each chamber and operated at 28 V, dc. The grid floor of each chamber was connected through timer circuitry to the output of a shock generator and scrambler ( B R S / L V E Models SG 903 and SC 922) to provide a 5.0-s, 1.6-mA, footshock. The chambers were individually enclosed in identical sound-attenuating cubicles, 42 x 56 x 41 cm, with an ambient sound level of 72 dB provided by operating the cubicle's ventilating fan at 110 V, ac.
2.4. Parameters of shock Six days following surgery (an adequate period of time to permit physiological recovery from surgery and to minimize neuronal regeneration), PVN- and shamlesioned rats were exposed to either one session of footshock or home cage conditions. Shocked animals received 16 presentations of a 5-s, 1.6-mA footshock that was delivered once every 4 min; hence, one daily shock session consisted of 16 shocks and lasted 64 rain. Immediately following the last footshock, the shocked animals were sacrificed and prepared for specimen collection. Likewise, the control animals were transported from their home cages to the procedure room and sacrificed at the same time as the shocked animals.
2.2. Paraventricular nucleus lesions 2.5. Specimen collection Rats were anesthetized with methoxyflurane (Metafane) (Pitman-Moore) and mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) with toothbar 6.5 mm below the interaural line. Lesions of the paraventricular nucleus of the hypothalamus (PVN) were performed as previously described (Makara et al., 1986) using a rotating triangular knife with a shape similar to the coronal cross section of the PVN. The knife was lowered into the brain 2.0 mm behind the bregma in the midline with the blade pointing caudally until the tip of the wire touched the floor of the skull; the knife was then rotated 360 ° in both directions and removed, producing a lesion in the shape of an inverted cone. In sham-operated rats, the knife was lowered but not rotated. Upon sacrificing the rats at the conclusion of the study, histological verification of the lesions was determined. Brains were fixed in 10% paraformaldehyde, cut into 40-/zm serial sections using a Reichert freezing microtome, and were stained with Cresyl violet.
Each rat was rapidly sacrificed by cervical dislocation using a clamp. The animal was placed on its back and sprayed with alcohol, and a mid-abdominal incision was made to expose the abdominal aorta. Within 1 min, blood was collected into a 5-ml heparinized syringe fitted with a 21-gauge needle. Approximately 1.5 ml of this blood was placed into an Eppendorf microcentrifuge tube and kept on ice for later corticosterone measurement. From the same animal, the spleen was then extracted and placed into a 15-ml polypropylene centrifuge tube, containing 7 ml of RPMI 1640 tissue culture medium supplemented with 10 mM Hepes, 2 mM glutamine, and 50 ~g gentamicin/ml. After all specimens were collected, the samples of heparinized blood collected for corticosterone measurement were centrifuged at 12000 rpm for 3 min in a microcentrifuge. The plasma layer was removed and frozen at -70°C. The remainder of the blood and spleen were later processed for lymphocyte mitogenic proliferation.
M.4. Pezzone et al. / Journal of Neuroimmunology 53 (1994) 39-46 2.6. Lymphocyte mitogenic assay
Mitogen stimulation assays were performed as previously described (Lysle et al., 1987) on whole blood and splenic lymphocytes by using the non-specific T-cell mitogens, concanavalin A (ConA) (Difco) and phytohemagglutinin (PHA) type HA-16 (Wellcome). Spleens were dissociated into a single-cell suspension using sterile frosted glass slides. Splenic leukocytes were counted using a Coulter counter (Model ZBI) and diluted to 5 x 106 leukocytes/ml in supplemented RPMI 1640 with 10% fetal calf serum (FCS) (Difco) without further washing. Whole blood leukocytes were counted using a Unopette and hemocytometer and diluted 1 : 10 with supplemented media containing heparin, 5 units/ml. ConA was prepared in supplemented RPMI 1640 at concentrations of 1.0, 5.0, and 10/.~g/ml, and 100 /xl was added in triplicate to the wells of a 96-well, fiat-bottom, microtiter plate (Costar No. 3596). PHA was prepared in supplemented RPMI-1640 at concentrations of 1.0 and 10 p.g/ml, and 100 /~1 was added in triplicate to the wells of a 96-well, fiat-bottom, microtiter plate. To background culture wells, 100 /zl of supplemented RPMI 1640 was added. Then, 100 /zl of the adjusted cell suspension was added to each well, and the plates were incubated at 37°C in a humidified incubator with 5% CO 2. Preliminary investigations showed that ConA at a final concentration of 2.5-5.0 /xg/ml provided optimal lymphocyte stimulation, while PHA provided optimal stimulation at a final concentration of 5 /zg/ml. The spleen cultures were pulsed with 1 /xCi [3H]thymidine (sp. act. 6.7 Ci/mM; Dupont-New England Nuclear) in 50 /~1 of supplemented RPMI 1640 during the last 5 h of a 48-h incubation. The cultures were harvested onto glass filter paper using a Skatron Cell Harvester (Skatron Inc.), and the incorporation of [3H]thymidine was determined with a liquid scintillation counter (Packard Model 1500) and was expressed as counts per min (cpm). The blood leukocyte cultures were incubated for a total of 96 h and were pulsed with [3H]thymidine (1 /~Ci/well) during the last 18 h of the incubation. The blood cultures were harvested and counted in the same manner as the spleen cultures. The average cpm/well for the blood cultures were normalized to 105 leukocytes/well. 2. 7. Plasma corticosterone assay
Plasma corticosterone concentrations were determined by a previously described competitive proteinbinding radioassay, sensitive to 0.2 /zg/dl (Murphy, 1967). 2.8. Data analysis
The data presented in this study were obtained from three identical experiments, each with 3-6 rats per
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group. The four groups studied included: (i) nonshocked rats with sham lesions ( - / S H A M ) (n = 13); (ii) non-shocked rats with PVN lesions ( - / L E S I O N ) (n = 13); (iii) shocked rats with sham lesions ( + / S H A M ) (n = 12); and (iv) shocked rats with PVN lesions ( + / L E S I O N ) (n = 10). The total subject population (N) was 48. Plasma corticosterone levels and the means of the triplicate counts per min for the peripheral blood and splenic mitogenic assays were separately analyzed using a standard two-two-three analysis of variance (ANOVA). One factor consisted of stress, a session of footshock or home cage conditions. The second factor was surgery, PVN lesion or sham lesion, and the third factor was repetition, one, two or three. In the event of a significant main effect, the group means were subjected to multiple post-hoc comparisons using the Neuman-Keuls test. Because the mitogen concentrations used in this study produced the same overall effects in the lymphocyte stimulation assays, only data from mitogen concentrations producing optimal lymphocyte stimulation are presented.
3. Results
3.1. P V N histology
Histological assessment of the sectioned brains was utilized to ensure completeness of the PVN lesions. The very discrete, rotating knife used to destroy the PVN, however, occasionally damaged surrounding structures, such as the anterior commissure, the anterior commissural nucleus, and the periventricular hypothalamic nucleus. In rats receiving PVN lesions, additional damage to the above structures caused no apparent difference in their corticosterone and immune responses when compared to rats of the same treatment group with lesions limited solely to the PVN (data not shown). 3.2. Plasma corticosterone
The results of the plasma corticosterone analysis are depicted in Fig. 1. ANOVA of the corticosterone data revealed significant main effects of stress (F{1,36} = 260.4, P < 0.0001), surgery (F{1,36} = 108.2, P < 0.0001), and repetition (F{2,36} = 4.54, P < 0.05). In addition, there were significant 2-way interactions between stress and lesion (F{1,36} = 115.4, P < 0.0001) and between lesion and repetition (F{2,36} = 5.37, P < 0.01). Post hoc multiple comparisons of the group means revealed highly significant elevations of plasma corticosterone in the shock/sham and shock/lesion treatment groups relative to the home cage/sham and home cage/lesion groups (P < 0.01). Furthermore, the shock-induced elevation of plasma corticosterone was much greater in the sham-operated animals compared
M.A. Pezzone et al. /Journal of Neuroimmunology 53 (1994) 39-46
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-/SHAM -/LESION +/SHAM +/LESION Fig. 1. Corticosterone responses to footshock stress and home cage conditions in PVN- and sham-lesioned rats. The data in the figure represent the plasma levels of corticosterone (p.g/dl) ( + S E ) for 10-13 rats per group. Non-shocked ( - ) animals with sham ( - / S H A M ) or PVN lesions ( - / L E S I O N ) had plasma corticosterone concentrations that were significantly lower than the shocked ( + ) animals. Lesions of the PVN significantly reduced the levels of plasma corticosterone in shocked animals ( + / L E S I O N ) in comparison to shocked animals receiving only sham PVN lesions ( + / S H A M ) . ** P < 0.01 ( N = 48).
to the PVN-lesioned animals (P < 0.01) which is reflected in the 2-way interaction between stress and lesion. These results clearly indicate that PVN lesions had no effect on baseline corticosterone secretion; however, they did significantly attenuate the stress-induced elevation of plasma corticosterone. Variations in baseline corticosterone secretion, levels of ambient environmental stress in the animal facility, and surgical expertise across the different experimental days most likely account for the significant repetition effect and the lesion by repetition interaction. All trends, however, were consistent across all three repetitions.
-/LESION
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Fig. 2. PBL mitogenic responses to PHA following footshock stress or home cage conditions in PVN- or sham-lesioned rats. The data in the figure represent mean cpm ( + S E ) for the PBL mitogenic responses to 5 p.g/ml of PHA. Note that the proliferative response to PHA was markedly suppressed in animals with sham PVN lesions exposed to electric footshock stress ( + / S H A M ) . Lesions of the PVN completely prevented the shock-induced suppression of PHA responsiveness ( + / L E S I O N ) . ** P < 0.01.
home cage/sham, and the shock/lesion groups were statistically equivalent ( P > 0.05). The variation in baseline lymphocyte mitogenic activity across experi160000 140000
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Figs. 2 and 3 display the peripheral blood lymphocyte (PBL) mitogenic responses to optimum concentrations of PHA (10 /.~g/ml) and ConA (5 /xg/ml), respectively. ANOVA of the PBL PHA data (Fig. 2) revealed significant main effects of stress (F{1,35} = 29.11, P < 0.0001), s u r g e r y (F{1,35} = 24.72, P < 0 . 0 0 0 1 ) , a n d r e p e t i t i o n ( F { 2 , 3 5 } = 11.74, P < 0 . 0 0 1 ) . There was also a significant 2-way interaction between s t r e s s a n d l e s i o n (F{1,35} = 5.57, P < 0.05). P o s t h o c tests for the PBL PHA data revealed a significant suppression of lymphocyte mitogenic activity in the shock/sham t r e a t m e n t g r o u p c o m p a r e d t o all o t h e r t r e a t m e n t g r o u p s ( P < 0.01). M e a n w h i l e , l e v e l s o f l y m p h o c y t e m i t o g e n i c a c t i v i t y in t h e h o m e c a g e / l e s i o n ,
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Fig. 3. PBL mitogenic responses to ConA following footshock stress and home cage conditions in PVN- and sham-lesioned rats. The data in the figure represent the mean cpm ( + SE) for the PBL mitogenic responses to 5 / x g / m l of ConA. Again, note the marked suppressive effect of electric footshock stress on the proliferative response of PBLs from animals with sham PVN lesions ( + / S H A M ) . Although lesions in the PVN significantly ameliorated the suppression of ConA responsiveness in shocked animals ( + / L E S I O N v s . + / SHAM), the mitogenic function of ConA-reactive lymphocytes was still significantly suppressed in comparison to non-shocked, sham( - / S H A M ) or PVN-lesioned ( - / L E S I O N ) controls. Thus, in contrast to the PHA-responsive PBLs, there was still a stress-induced suppression of the ConA-responsive PBLs in the lesioned animals. ** P < 0.01; * P < 0.05.
M.A. Pezzone et al. /Journal of Neuroimmunology 53 (1994) 39-46 mental days most likely accounts for the significant repetition effect. These results show that lesions of the PVN can completely prevent the shock-induced suppression of PBL mitogenic reactivity to P H A and slightly, although non-significantly, enhance the same response in home cage animals. A N O V A of the PBL ConA data (Fig. 3) revealed significant main effects of stress (F{1,35} = 42.00, P < 0.0001), surgery (F{1,35} = 4.44, P < 0.05), and repetition (F{2,35} = 11.32, P < 0 . 0 0 0 1 ) . Post hoc tests showed a significant suppression of PBL mitogenic proliferation in the s h o c k / s h a m treatment group compared to all other treatment groups ( P < 0.01) and in the shock/lesion treatment group compared to the home c a g e / s h a m ( P < 0.05) and the home cage/lesion treatment groups ( P < 0.01). These results suggest that PVN lesions only partially attenuate the shock-induced suppression of ConA-reactive PBLs, while baseline lymphocyte proliferation in home cage animals is not affected. Again, the variation in baseline lymphocyte mitogenic activity across experimental days most likely accounts for the significant main effect of repetition.
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Fig. 5. Splenic lymphocyte mitogenic responses to ConA following footshock stress and home cage conditions in PVN- and sham-lesioned rats. The data in the figure represent the mean cpm (+ SE) for the splenic lymphocyte mitogenic responses to 2.5 /zg/ml of ConA. The marked suppression of splenic lymphocyte mitogenic proliferation in home-caged, PVN-lesioned animals (-/LESION) is clearly demonstrated, while the suppressive effect of footshock was not evident in the sham-lesioned animals (+/SHAM). Combining PVN lesions with footshock(+/LESION), however, again elicited a further marked suppression of splenic lymphocytemitogenic activity. ** P < 0.01.
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All trends were consistent across experimental repetitions. 3.4. Splenic lymphocytes
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Fig. 4. Splenic lymphocyte mitogenic responses to PHA following footshock stress and home cage conditions in PVN- and sham-lesioned rats. The data in the figure represent the mean cpm (___SE) for the splenic lymphocytemitogenic responses to 5/zg/ml of PHA. The home-caged, PVN-lesioned animals (-/LESION) had a reduced (although not statistically significant) proliferative response to PHA in comparison to home-caged, sham-lesioned animals ( - / SHAM), suggesting that the PVN lesion itself may have lowered baseline splenic lymphocytemitogenic activity. Although the splenic lymphocyte mitogenic response to PHA was significantlysuppressed in sham-lesioned animals receiving electric footshock( +/SHAM) as compared to home-caged,sham-lesioned animals ( -/SHAM), there was no difference between this group (+/SHAM) and home-caged animals with PVN lesions (-/LESION), suggesting that the PVN lesion was comparable to footshock stress in lowering splenic lymphocyte mitogenic activity. Moreover, when the PVN-lesioned animals were exposed to footshock ( +/LESION), their splenic lymphocyte mitogenic response to PHA was markedly further suppressed. ** P < 0.0l.
Figs. 4 and 5 illustrate the splenic lymphocyte mitogenic responses to optimum concentrations of P H A (10 /xg/ml) and ConA (10 /xg/ml), respectively. A N O V A of the splenic lymphocyte P H A data (Fig. 4) revealed significant main effects of stress (F{1,35} = 104.1, P < 0.0001), surgery (F{1,35} = 51.42, P < 0.0001), and repetition (F{2,35} = 104.1, P < 0.0001). Additionally, there were significant 2-way interactions between stress and surgery (F{1,35} = 5.98, P < 0.05) and between stress and repetition (F{2,35} = 55.83, P < 0.0001) and a significant 3-way interaction between stress, surgery, and repetition (F{2,35} = 8.44, P < 0.01). Post hoc tests for the splenic lymphocyte P H A data revealed that the suppression of mitogenic activity in the shock/lesion group compared to all other treatment groups is highly significant ( P < 0.01). Relative to the home c a g e / s h a m group, splenic lymphocyte mitogenic activity in the home cage/lesion group was slightly suppressed although not statistically significant ( P > 0.05), while lymphocyte proliferation in the s h o c k / s h a m group was significantly suppressed ( P < 0.01). There was no statistical difference between the home cage/lesion and the s h o c k / s h a m groups. These data suggest that the PVN regulates the lymphocyte mitogenic response to P H A in the spleen. Moreover, when PVN lesions and
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M.A. Pezzone et al. /Journal of Neuroimmunology 53 (1994) 39-46
stress occurred together, the suppressive effects were compounded. The significant 3-way interaction between stress, surgery, and repetition not only reflects the variability in the mitogenic assay over experimental days and ambient stress levels in the animal facility but also reflects the compounding suppressive effects of stress and PVN lesions on splenic lymphocyte mitogenic reactivity to PHA. ANOVA of the spleen ConA data (Fig. 5) revealed significant main effects of stress (F{1,32} = 6.45, P < 0.05), surgery (F{1,32} = 48.46, P < 0.0001), and repetition (F{2,32} =56.36, P<0.0001), and a significant 2-way interaction between stress and repetition (F{2,32} = 10.67, P < 0.001). Post hoc tests showed that the proliferative responses of ConA-reactive splenic lymphocytes were significantly suppressed in the shock/lesion treatment group relative to the home c a g e / s h a m and the shock/sham groups ( P < 0.01). Lymphocyte proliferation in the home cage/sham and shock/sham groups were statistically equivalent (P > 0.05). These data suggest that the ConA-reactive lymphocytes taken from sham-operated rats have habituated to the effects of footshock stress. The suppression of ConA-reactive splenic lymphocytes by stress, however, was greatly exacerbated in animals receiving PVN lesions.
4. Discussion
This study has shown that lesions of the hypothalamic PVN can markedly alter the mitogenic responsiveness of peripheral blood and splenic lymphocytes following acute footshock stress. The resulting effects upon lymphocyte blastogenesis, which varied, depended upon the lymphoid compartment sampled and the mitogen used for stimulation. Interestingly, these findings concur with our previous studies which have shown that stress differentially affects peripheral blood and splenic lymphocytes by independent mechanisms (Cunniek et al., 1990) and studies which have shown that stress may differentially affect lymphocyte subpopulations as evidenced by differential reactivity to non-specific mitogenic stimulation (Haynes and Fauci, 1978). Although explicit characterization of these pathways remains to be elucidated, it appears that the PVN is capable of simultaneously influencing both splenic and peripheral blood lymphocytes by their respective pathways of activation. The sympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis are likely involved in the modulation of splenic and peripheral blood lymphocyte function during stress (Rabin et al., 1989). Correspondingly, a central role of the hypothalamic PVN in stressor-induced immune alterations is not unjustifiable, considering its neuronal projections can
influence both pituitary hormone secretion (Wiegand and Price, 1980) and preganglionic neurons of the sympathetic and parasympathetic divisions of the autonomic nervous system (Saper et al., 1976). Demonstrating that direct activation of the PVN follows the administration of stressful stimuli, the authors and other investigators have shown that levels of c-los messenger RNA a n d / o r its protein product are rapidly induced in cells of the medial parvocellular division of the PVN following various stressors (Ceccatelli et al., 1989; Sharp et al., 1991; Imaki et al., 1992; Kononen et al., 1992; Pezzone et al., 1992). Additionally, further characterization of these stress-activated neurons in the PVN has revealed that they contained corticotropin-releasing hormone (CRH) (Imaki et al., 1992; Pezzone et al., 1992), a neuropeptide that can activate both the autonomic nervous system as a neurotransmitter and the HPA axis as a neurohormone. Thus, CRH neurons in the PVN may play an integral role in stressor-induced immune alterations by activating the HPA axis and the sympathetic nervous system following stress. Indirectly suggesting the presence of a functional physiological relationship between CRH-containing neurons of the PVN and cells of the immune system, it has been demonstrated that increases in plasma levels of corticosterone correlate temporally with the formation of an immune response (Besedovsky et al., 1975; Shek and Sabiston, 1983). More direct evidence, however, specifically implicating the involvement of the PVN in immune modulation is apparent in studies which have shown that norepinephrine is selectively decreased in the PVN at the peak of an immune response in mice (Carlson et al., 1987) and that neuronal firing is decreased in the PVN of rats during the first 3 days after immunization later followed by an increased rate of firing by day 6 (Saphier et al., 1987). These studies suggest the presence of a neuroendocrine-immune circuit involving bi-directional interactions between the immune system and the HPA axis as previously hypothesized (Besedovsky and Sorkin, 1977). Considering that such a proposed neuroendocrineimmune circuit (most likely involving the PVN) does exist, one might expect to see alterations in the neuroendocrine and the immune responses to stress following its disruption. Indeed, previous studies have shown that destruction of the PVN does significantly inhibit activation of the HPA axis induced by stressors such as ether inhalation (Makara et al., 1981; Bruhn et al., 1984; Dohanics et al., 1986), surgical trauma (Makara et al., 1981), hypothalamic electrical stimulation (Makara et al., 1981), intermittent footshock (Rivest and Rivier, 1991), and hemorrhage (Darlington et al., 1988). It is unfortunate that concurrent assessment of immune function in these studies utilizing PVN lesions was not conducted, as immunological changes following footshock, anesthesia, and surgical
M.A. Pezzone et al. /Journal of Neuroimmunology 53 (1994) 39-46
trauma have all been well documented (Rabin et al., 1989; Salo, 1992). Specific alterations of immune function have been recorded following lesions of other hypothalamic nuclei, supporting the existence of and implicating the disruption of this neuroendocrine-immune circuit. In these studies, lesions of the anterior hypothalamus led to protection against lethal anaphylaxis (Luparello et al., 1964), reduced the intensity of Arthus and delayed skin reactions (Jankovic and Isakovic, 1973), reduced the rate of circulating antibody formation (Tyrey and Nalbandov, 1972; Jankovic and Isakovic, 1973), inhibited the development of experimental allergic encephalomyelitis (Wertman et al., 1985), inhibited lymphocyte stimulation in whole blood and spleen (Cross et al., 1980; Roszman et al., 1982; Keller et al., 1988), and decreased the cellularity of the thymus and spleen (Cross et al., 1980). Although the anterior hypothalamic nuclei are distinct from the PVN, it would appear that based on the data presented in this manuscript and findings from the previously mentioned studies that these two regions of the hypothalamus play important roles in neuroimmunomodulation. Although the pattern of immune responsiveness in the peripheral blood appears to be corticosteroid-dependent, the mitogen response pattern in the splenic lymphoid compartment appears to be entirely different (as previously alluded to) and warrants further discussion. As the suppression of splenic lymphocyte mitogenic activity has been shown to be mediated by catecholamines via a /3-adrenergic receptor mechanism (Cunnick et al., 1990), the markedly enhanced suppression of splenic lymphocyte mitogenic activity in PVNlesioned rats following stress (Figs. 4 and 5) suggests augmentation of SNS activity. Indeed, stress-induced increases in plasma epinephrine were significantly greater in PVN-lesioned rats when compared to those with sham lesions (Darlington et al., 1988). Likewise, adrenalectomy augmented stress-induced increments in catecholamine release, re-uptake, metabolism, turnover, and biosynthesis (Kvetnansky et al., 1993), suggesting that glucocorticoids exert negative feedback upon the SNS during stress. As always, one must be cautious in the interpretation of ablation studies especially those involving the PVN. Although the role of parvocellular CRH neurons is likely responsible for the observed alterations in immune function presented in this study, other PVN neurons or peptides could not be excluded since the entire PVN was indiscriminately destroyed.
Acknowledgements The authors thank Ms. Ada Armfield, Ms. Ellen Hamill, and Mr. Paul Wood for their superb technical
45
assistance, Dr. Sheldon Cohen and Janet Schlarb for their expert statistical assistance, and Dr. Daohong Zhou for his helpful suggestions. This work was supported by national research service award F30 MH10157 to M.A.P. from the National Institute of Mental Health (NIMH) and by NIMH research grant MH43411-04 and a grant from the Pathology Education and Research Foundation to B.S.R. Preliminary results from these studies were presented at the 22nd Annual Meeting of the Society for Neuroscience, Anaheim, CA, 1992, and the Research Perspectives in Psychoneuroimmunology IV Conference in Boulder, CO, 1993.
References Antoni, F.A., Palkovits, M., Makara, G.B., Kiss, J.Z., Linton, E.A., Lowry, P.J. and Leranth, Cs. (1984) Immunoreactive ovine corticotropin-releasing factor (oCRF-41-LI) in the hypothalamo-hypophyseal tract of the rat. In: E. Usdin, R. Kvetnansky and J. Axelrod (Eds.), Stress: The Role of Catecholamines and other Neurotransmitters, Gordon and Breach, New York, NY, pp. 233-241. Besedovsky, H. and Sorkin, E. (1977) Network of immune-neuroendocrine interactions. Clin. Exp. Immunol. 27, 1-12. Besedovsky, H., Sorkin, E., Keller, M. and Muller, J. (1975) Changes in blood hormone levels during the immune response. Proc. Soc. Exp. Biol. Med. 150, 466-470. Bruhn, T.O., Plotsky, P.M. and Vale, W.W. (1984) Effect of paraventricular lesions on corticotropin-releasing factor (CRF)-like immunoreactivity in the stalk-median eminence: Studies on the adrenocorticotropin response to ether stress and exogenous CRF. Endocrinology 114, 57-62. Carlson, S.L., Felten, D.L., Livnat, S. and Felten, S.Y. (1987) Alterations of monoamines in specific central autonomic nuclei following immunization in mice. Brain Behav. Immun. 1, 52-63. Ceccatelli, S., Villar, M.J., Goldstein, M. and Hokfelt, T. (1989) Expression of c-Fos immunoreactivity in transmitter-characterized neurons after stress. Proc. Natl. Acad. Sci. USA 86, 9569-9573. Cross, R.J., Markesbery, W.R., Brooks, W.H. and Roszman, T.L. (1980) Hypothalamic-immune interactions. I. The acute effect of anterior hypothalamic lesions on the immune response. Brain Res. 196, 79-87. Cunnick, J.E., Lysle, D.T., Kuscinski, B.J. and Rabin, B.S. (1990) Evidence that shock-induced immune suppression is mediated by adrenal hormones and peripheral /3-adrenergic receptors. Pharmacol. Biochem. Behav. 36, 645-651. Darlington, D.N., Shinsako, J. and DaUman, M.F. (1988) Paraventricular lesions: hormonal and cardiovascular responses to hemorrhage. Brain Res. 439, 289-301. Dohanics, J., Kapocs, G., Janaky, T., Kiss, J.Z., Rappay, G., Laszlo, F.A. and Makara, G.B. (1986) Mechanism of restoration of ACTH release in rats with long-term lesions of the paraventricular nuclei. J. Endocrinol. 111, 75-82. Felten, D.L., Felten, S.Y., Carlson, S.L., Olschowka, J.A. and Livnat, S. (1985) Noradrenergic and peptidergic innervation of lymphoid tissue. J. Immunol. 135, 755s-765s. Haynes, B.F. and Fauci, A.S. (1978) The differential effect of in vivo hydrocortisone on the kinetics of subpopulations of human peripheral blood thymus-derived lymphocytes. J. Clin. Invest. 61, 703-707.
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Imaki, T., Shibasaki, T., Hotta, M. and Demura, H. (1992) Early induction of c-fos precedes increased expression of corticotropinreleasing factor messenger ribonucleic acid in the paraventricular nucleus after immobilization stress. Endocrinology 131,240-246. Jankovic, B.D. and Isakovic, K. (1973) Neuro-endocrine correlates of immune response. I. Effects of brain lesions on antibody production, arthus reactivity, and delayed hypersensitivity in the rat. Int. Arch. Allergy 45, 360-372. Keller, S.E., Schleifer, S.J., Liotta, A.S., Bond, R.N., Farhoody, N. and Stein, M. (1988) Stress-induced alterations of immunity in hypophysectomized rats. Proc. Natl. Acad. Sci. USA 85, 92979301. Kiecolt-Glaser, J.K. and Glaser, R. (1991) Stress and immune function in humans. In: R. Ader, D.L. Felten and N. Cohen (Eds.), Psychoneuroimmunology, 2nd edn., Academic Press, San Diego, CA, pp. 849-867. Kononen, J., Honkaniemi, J., Alho, H., Koistinaho, J., Iadarola, M. and Pelto-Huikko, M. (1992) Fos-like immunoreactivity in the rat bypotbalamlic-pituitary axis after immobilization stress. Endocrinology 130, 3041-3047. Kvetnansky, R., Fukuhara, K., Pacak, K., Cizza, G., Goldstein, D.S. and Kopin, I.J. (1993) Endogenous glucocorticoids restrain catecholamine synthesis and release at rest and during immobilization stress in rats. Endocrinology 133, 1411-1419. Luparello, T.J., Stein, M. and Park, C.D. (1964) Effect of hypothalamic lesions on rat anaphylaxis. Am. J. Physiol. 207, 911-914. Lysle, D.T., Lyte, M., Fowler, H. and Rabin, B.S. (1987) Shock-induced modulation of lymphocyte reactivity: Suppression, habituation, and recovery. Life Sci. 41, 1805-1814. Makara, G.B., Stark, E., Karteszi, M., Palkovits, M. and Rappay, G. (1981) Effects of paraventricular lesions on stimulated ACTH release and CRF in stalk-median eminence of the rat. Am. J. Physiol. 240, E441-E446. Makara, G.B., Stark, E., Kapocs, G. and Antoni, F.A. (1986) Longterm effects of bypothalamic paraventricular lesion on CRF content and stimulated ACTH secretion. Am. J. Physiol. 250, E319E324. Murphy, B.E.P. (1967) Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay. J. Clin. Endocrinol. Metab. 27, 973-990. Pezzone, M.A., Lee, W.S., Hoffman, G.E. and Rabin, B.S. (1992) Induction of c-Fos immunoreactivity in the rat forebrain by conditioned and unconditioned aversive stimuli. Brain Res. 595, 25-31.
Rabin, B.S., Cohen, S., Ganguli, R., Lysle, D.T. and Cunnick, J.E. (1989) Bidirectional interaction between the central nervous system and the immune system. CRC Crit. Rev. Immunol. 9, 279312. Rabin, B.S., Cunnick, J.E. and Lysle, D.T. (1990) Stress-induced alteration of immune function. Prog. Neuroendocrinimmunol. 3, 116-124. Rivest, S. and Rivier, C. (1991) Influence of the paraventricular nucleus of the hypothalamus in the alteration of neuroendocrine functions induced by intermittent footshock or interleukin. Endocrinology 129, 2049-2057. Roszman, T.L., Cross, R.J., Brooks, W.H. and Markesbery, W.R. (1982) Hypothalamic-immune interactions II. The effect of hypothalamic lesions on the ability of adherent spleen cells to limit lymphocyte blastogenesis. Immunology 45, 737-742. Salo, M. (1992) Effects of anaesthesia and surgery on the immune response. Acta Anaesthesiol. Scand. 36, 201-220. Saper, C.B., Loewy, A.D., Swanson, L.W. and Cowan, W.M. (1976) Direct hypothalamo-autonomic connections. Brain Res. 117, 305-312. Saphier, D., Abramsky, O., Mor, G. and Ovadia, H. (1987) Multiunit electrical activity in conscious rats during and immune response. Brain Behav. Immun. 1, 40-51. Sharp, F.R., Sagar, S.M., Hicks, K., Lowenstein, D. and Hisanaga, K. (1991) c-los mRNA, Fos and Fos-related antigen induction by hypertonic saline and stress. J. Neurosci. 11, 2321-2331. Shek, P.N. and Sabiston, B.H. (1983) Neuroendocrine regulation of immune processes: Changes in circulating corticosterone levels induced by the primary antibody response in mice. Int. J. Immunopharmacol. 5, 23-33. Tilders, F.J.H., Schipper, J., Lowry, P.J. and Vermes, I. (1982) Effect of hypothalamus lesions on the presence of CRF-immunoreactive nerve terminals in the median eminence and on the pituitaryadrenal response to stress. Regul. Pept. 5, 77-84. Tyrey, L. and Nalbandov, A.V. (1972) Influence of anterior hypotbalamic lesions on circulating antibody titers in the rat. Am. J. Physiol. 222, 179-185. Wertman, E., Feldman, S., Ovadia, H. and Abramsky, O. (1985) Experimental allergic encephalomyelitis: prevention by electrical lesion in the anterior hypothalamus in rats. Isr. J. Med. Sci. 21, 72-73. Wiegand, S.J. and Price, J.L. (1980) The cells of origin of afferent fibers to the median eminence in the rat. J. Comp. Neurol. 192, 1-19.
ELSEVIER
Journal of Neuroimmunology 53 (1994) 39-46
Effects of footshock stress upon spleen and peripheral blood lymphocyte mitogenic responses in rats with lesions of the paraventricular nuclei Michael A. Pezzone a,., Janos Dohanics b, Bruce S. Rabin
a
a Department of Pathology, Brain, Behavior and Immunity Center, 200 Lothrop Street, Pittsburgh, PA 15213-2582, USA b Department of Medicine, University of Pittsburgh School of Medicine Pittsburgh, PA 15213, USA Received 26 October 1993; revised received 25 February 1994; accepted 18 April 1994
Abstract
To assess the role of the hypothalamic paraventricular nucleus (PVN) in mediating stressor-induced immune alterations, male Lewis rats were subjected to a 1-h session of intermittent footshock stress or home cage conditions 6 days after receiving bilateral or sham PVN lesions. Splenic and peripheral blood lymphocyte proliferative responses to the non-specific mitogens, concanavalin A (ConA) and phytohemagglutinin (PHA), were subsequently measured as were plasma corticosterone levels. In sham-operated rats, footshock markedly elevated plasma corticosterone levels and concurrently suppressed the proliferative responses of peripheral blood and splenic lymphocytes. In PVN-lesioned rats, however, the shock-induced suppression of lymphocyte proliferation in the peripheral blood and the elevation of plasma corticosterone were significantly attenuated, while lymphocyte proliferation in the spleen was suppressed below the level of the sham-treated animals. Thus, by utilizing ablation studies, we have determined that the PVN may play a direct role in the alteration of lymphocyte function during stress, and an intact PVN buffers the effect of stress on the responsiveness of spleen lymphocytes to non-specific mitogens.
Key words: Stress; Glucocorticoid; Lymphocyte proliferation; Hypothalamic-pituitary-adrenal axis; Paraventricular nucleus; Neuroimmunomodulation
I. Introduction
Stress is capable of altering the function of the immune system (Rabin et al., 1989, 1990; Kiecolt-Glaser and Glaser, 1991). Although the definitive pathways mediating these immune alterations have not been fully characterized, involvement of the sympathetic nervous system (SNS) and the hypothalamic-pituitaryadrenal (HPA) axis has been strongly suggested (Rabin et al., 1989). Through direct innervation of lymphoid tissue, as well as through endocrine mechanisms, the central nervous system (CNS) may exert its influences upon cells of the immune system (Felten et al., 1985). By acting as a central coordinator of an integrated stress response, the paraventricular nucleus (PVN) of the hypothalamus may be responsible for mediating many of the observed physiological effects of stress.
* Corresponding author. Phone (412) 647 3787; Fax (412) 647 7741. 0165-5728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 5 7 2 8 ( 9 4 ) 0 0 0 6 2 - S
Possessing neuronal projections that can influence both pituitary hormone secretion (Wiegand and Price, 1980) and preganglionic neurons of the sympathetic and parasympathetic divisions of the autonomic nervous system (Saper et al., 1976), the PVN is capable of initiating a stress response characterized by concomitant activation of the H P A axis and the SNS. Attempting to characterize the role of the PVN during stress, we have previously used c-Fos induction as a marker of neuronal activity in rats following exposure to footshock and conditioned, immunomodulating stimuli (Pezzone et al., 1992). Our studies showed that c-Fos immunoreactivity was strongly expressed in cells of the medial parvocellular division of the PVN and in cells bordering the posterior magnocellular division of the PVN following administration of the immune-modulating stimuli. Moreover, because many of the activated PVN neurons also exhibited corticotropin-releasing hormone (CRH) immunoreactivity, a role of CRH in stressor-induced immune alterations was further substantiated (Pezzone et al., 1992).
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M.A. Pezzone et al. /Journal of Neuroimmunology 53 (1994) 39-46
To further investigate the immunomodulatory role of the PVN, splenic and peripheral blood lymphocyte mitogenic responses and plasma corticosterone levels were measured following acute footshock stress in male Lewis rats subjected to bilateral or sham PVN lesions. Because PVN lesions cause complete disappearance of CRH-immunoreactive fibers from the median eminence (Tilders et al., 1982; Antoni et al., 1984; Bruhn et al., 1984) and block the adrenocorticotropic hormone (ACTH) (Makara et al., 1981; Bruhn et al., 1984; Darlington et al., 1988) and corticosterone responses to stress (Makara et al., 1981; Tilders et al., 1982), the immunological effects of footshock stress were consequently studied without activation of the H P A axis by the PVN.
2. Materials and methods
2.1. Experimental animals Male rats of the Lewis strain, 65 days old and 250-300 g in weight were purchased from Harlan Sprague Dawley (Indianapolis, IN). The animals were individually housed in hanging wire cages under a 12-h day-night cycle (lights on at 0700) of artificial illumination and ad lib access to food and water. Upon arrival in the animal housing facility, rats were given a 2-week acclimatization to the new surroundings. Furthermore, the animals were gently handled approximately 2 m i n / d a y for 5 consecutive days during their first week in the new colony room.
2.3. Shock apparatus Eight identical rodent chambers ( B R S / L V E ) measuring 27 x 30 X 24 cm, served as the shock apparatuses. The chambers had clear Plexiglas front, top, and back panels, stainless steel side panels, and a grid floor consisting of stainless steel bars 0.48 cm in diameter, spaced 1.9 cm apart. Chamber illumination was provided by a houselight externally mounted on the wall of each chamber and operated at 28 V, dc. The grid floor of each chamber was connected through timer circuitry to the output of a shock generator and scrambler ( B R S / L V E Models SG 903 and SC 922) to provide a 5.0-s, 1.6-mA, footshock. The chambers were individually enclosed in identical sound-attenuating cubicles, 42 x 56 x 41 cm, with an ambient sound level of 72 dB provided by operating the cubicle's ventilating fan at 110 V, ac.
2.4. Parameters of shock Six days following surgery (an adequate period of time to permit physiological recovery from surgery and to minimize neuronal regeneration), PVN- and shamlesioned rats were exposed to either one session of footshock or home cage conditions. Shocked animals received 16 presentations of a 5-s, 1.6-mA footshock that was delivered once every 4 min; hence, one daily shock session consisted of 16 shocks and lasted 64 rain. Immediately following the last footshock, the shocked animals were sacrificed and prepared for specimen collection. Likewise, the control animals were transported from their home cages to the procedure room and sacrificed at the same time as the shocked animals.
2.2. Paraventricular nucleus lesions 2.5. Specimen collection Rats were anesthetized with methoxyflurane (Metafane) (Pitman-Moore) and mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) with toothbar 6.5 mm below the interaural line. Lesions of the paraventricular nucleus of the hypothalamus (PVN) were performed as previously described (Makara et al., 1986) using a rotating triangular knife with a shape similar to the coronal cross section of the PVN. The knife was lowered into the brain 2.0 mm behind the bregma in the midline with the blade pointing caudally until the tip of the wire touched the floor of the skull; the knife was then rotated 360 ° in both directions and removed, producing a lesion in the shape of an inverted cone. In sham-operated rats, the knife was lowered but not rotated. Upon sacrificing the rats at the conclusion of the study, histological verification of the lesions was determined. Brains were fixed in 10% paraformaldehyde, cut into 40-/zm serial sections using a Reichert freezing microtome, and were stained with Cresyl violet.
Each rat was rapidly sacrificed by cervical dislocation using a clamp. The animal was placed on its back and sprayed with alcohol, and a mid-abdominal incision was made to expose the abdominal aorta. Within 1 min, blood was collected into a 5-ml heparinized syringe fitted with a 21-gauge needle. Approximately 1.5 ml of this blood was placed into an Eppendorf microcentrifuge tube and kept on ice for later corticosterone measurement. From the same animal, the spleen was then extracted and placed into a 15-ml polypropylene centrifuge tube, containing 7 ml of RPMI 1640 tissue culture medium supplemented with 10 mM Hepes, 2 mM glutamine, and 50 ~g gentamicin/ml. After all specimens were collected, the samples of heparinized blood collected for corticosterone measurement were centrifuged at 12000 rpm for 3 min in a microcentrifuge. The plasma layer was removed and frozen at -70°C. The remainder of the blood and spleen were later processed for lymphocyte mitogenic proliferation.
M.4. Pezzone et al. / Journal of Neuroimmunology 53 (1994) 39-46 2.6. Lymphocyte mitogenic assay
Mitogen stimulation assays were performed as previously described (Lysle et al., 1987) on whole blood and splenic lymphocytes by using the non-specific T-cell mitogens, concanavalin A (ConA) (Difco) and phytohemagglutinin (PHA) type HA-16 (Wellcome). Spleens were dissociated into a single-cell suspension using sterile frosted glass slides. Splenic leukocytes were counted using a Coulter counter (Model ZBI) and diluted to 5 x 106 leukocytes/ml in supplemented RPMI 1640 with 10% fetal calf serum (FCS) (Difco) without further washing. Whole blood leukocytes were counted using a Unopette and hemocytometer and diluted 1 : 10 with supplemented media containing heparin, 5 units/ml. ConA was prepared in supplemented RPMI 1640 at concentrations of 1.0, 5.0, and 10/.~g/ml, and 100 /xl was added in triplicate to the wells of a 96-well, fiat-bottom, microtiter plate (Costar No. 3596). PHA was prepared in supplemented RPMI-1640 at concentrations of 1.0 and 10 p.g/ml, and 100 /~1 was added in triplicate to the wells of a 96-well, fiat-bottom, microtiter plate. To background culture wells, 100 /zl of supplemented RPMI 1640 was added. Then, 100 /zl of the adjusted cell suspension was added to each well, and the plates were incubated at 37°C in a humidified incubator with 5% CO 2. Preliminary investigations showed that ConA at a final concentration of 2.5-5.0 /xg/ml provided optimal lymphocyte stimulation, while PHA provided optimal stimulation at a final concentration of 5 /zg/ml. The spleen cultures were pulsed with 1 /xCi [3H]thymidine (sp. act. 6.7 Ci/mM; Dupont-New England Nuclear) in 50 /~1 of supplemented RPMI 1640 during the last 5 h of a 48-h incubation. The cultures were harvested onto glass filter paper using a Skatron Cell Harvester (Skatron Inc.), and the incorporation of [3H]thymidine was determined with a liquid scintillation counter (Packard Model 1500) and was expressed as counts per min (cpm). The blood leukocyte cultures were incubated for a total of 96 h and were pulsed with [3H]thymidine (1 /~Ci/well) during the last 18 h of the incubation. The blood cultures were harvested and counted in the same manner as the spleen cultures. The average cpm/well for the blood cultures were normalized to 105 leukocytes/well. 2. 7. Plasma corticosterone assay
Plasma corticosterone concentrations were determined by a previously described competitive proteinbinding radioassay, sensitive to 0.2 /zg/dl (Murphy, 1967). 2.8. Data analysis
The data presented in this study were obtained from three identical experiments, each with 3-6 rats per
41
group. The four groups studied included: (i) nonshocked rats with sham lesions ( - / S H A M ) (n = 13); (ii) non-shocked rats with PVN lesions ( - / L E S I O N ) (n = 13); (iii) shocked rats with sham lesions ( + / S H A M ) (n = 12); and (iv) shocked rats with PVN lesions ( + / L E S I O N ) (n = 10). The total subject population (N) was 48. Plasma corticosterone levels and the means of the triplicate counts per min for the peripheral blood and splenic mitogenic assays were separately analyzed using a standard two-two-three analysis of variance (ANOVA). One factor consisted of stress, a session of footshock or home cage conditions. The second factor was surgery, PVN lesion or sham lesion, and the third factor was repetition, one, two or three. In the event of a significant main effect, the group means were subjected to multiple post-hoc comparisons using the Neuman-Keuls test. Because the mitogen concentrations used in this study produced the same overall effects in the lymphocyte stimulation assays, only data from mitogen concentrations producing optimal lymphocyte stimulation are presented.
3. Results
3.1. P V N histology
Histological assessment of the sectioned brains was utilized to ensure completeness of the PVN lesions. The very discrete, rotating knife used to destroy the PVN, however, occasionally damaged surrounding structures, such as the anterior commissure, the anterior commissural nucleus, and the periventricular hypothalamic nucleus. In rats receiving PVN lesions, additional damage to the above structures caused no apparent difference in their corticosterone and immune responses when compared to rats of the same treatment group with lesions limited solely to the PVN (data not shown). 3.2. Plasma corticosterone
The results of the plasma corticosterone analysis are depicted in Fig. 1. ANOVA of the corticosterone data revealed significant main effects of stress (F{1,36} = 260.4, P < 0.0001), surgery (F{1,36} = 108.2, P < 0.0001), and repetition (F{2,36} = 4.54, P < 0.05). In addition, there were significant 2-way interactions between stress and lesion (F{1,36} = 115.4, P < 0.0001) and between lesion and repetition (F{2,36} = 5.37, P < 0.01). Post hoc multiple comparisons of the group means revealed highly significant elevations of plasma corticosterone in the shock/sham and shock/lesion treatment groups relative to the home cage/sham and home cage/lesion groups (P < 0.01). Furthermore, the shock-induced elevation of plasma corticosterone was much greater in the sham-operated animals compared
M.A. Pezzone et al. /Journal of Neuroimmunology 53 (1994) 39-46
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-/SHAM -/LESION +/SHAM +/LESION Fig. 1. Corticosterone responses to footshock stress and home cage conditions in PVN- and sham-lesioned rats. The data in the figure represent the plasma levels of corticosterone (p.g/dl) ( + S E ) for 10-13 rats per group. Non-shocked ( - ) animals with sham ( - / S H A M ) or PVN lesions ( - / L E S I O N ) had plasma corticosterone concentrations that were significantly lower than the shocked ( + ) animals. Lesions of the PVN significantly reduced the levels of plasma corticosterone in shocked animals ( + / L E S I O N ) in comparison to shocked animals receiving only sham PVN lesions ( + / S H A M ) . ** P < 0.01 ( N = 48).
to the PVN-lesioned animals (P < 0.01) which is reflected in the 2-way interaction between stress and lesion. These results clearly indicate that PVN lesions had no effect on baseline corticosterone secretion; however, they did significantly attenuate the stress-induced elevation of plasma corticosterone. Variations in baseline corticosterone secretion, levels of ambient environmental stress in the animal facility, and surgical expertise across the different experimental days most likely account for the significant repetition effect and the lesion by repetition interaction. All trends, however, were consistent across all three repetitions.
-/LESION
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Fig. 2. PBL mitogenic responses to PHA following footshock stress or home cage conditions in PVN- or sham-lesioned rats. The data in the figure represent mean cpm ( + S E ) for the PBL mitogenic responses to 5 p.g/ml of PHA. Note that the proliferative response to PHA was markedly suppressed in animals with sham PVN lesions exposed to electric footshock stress ( + / S H A M ) . Lesions of the PVN completely prevented the shock-induced suppression of PHA responsiveness ( + / L E S I O N ) . ** P < 0.01.
home cage/sham, and the shock/lesion groups were statistically equivalent ( P > 0.05). The variation in baseline lymphocyte mitogenic activity across experi160000 140000
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Figs. 2 and 3 display the peripheral blood lymphocyte (PBL) mitogenic responses to optimum concentrations of PHA (10 /.~g/ml) and ConA (5 /xg/ml), respectively. ANOVA of the PBL PHA data (Fig. 2) revealed significant main effects of stress (F{1,35} = 29.11, P < 0.0001), s u r g e r y (F{1,35} = 24.72, P < 0 . 0 0 0 1 ) , a n d r e p e t i t i o n ( F { 2 , 3 5 } = 11.74, P < 0 . 0 0 1 ) . There was also a significant 2-way interaction between s t r e s s a n d l e s i o n (F{1,35} = 5.57, P < 0.05). P o s t h o c tests for the PBL PHA data revealed a significant suppression of lymphocyte mitogenic activity in the shock/sham t r e a t m e n t g r o u p c o m p a r e d t o all o t h e r t r e a t m e n t g r o u p s ( P < 0.01). M e a n w h i l e , l e v e l s o f l y m p h o c y t e m i t o g e n i c a c t i v i t y in t h e h o m e c a g e / l e s i o n ,
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Fig. 3. PBL mitogenic responses to ConA following footshock stress and home cage conditions in PVN- and sham-lesioned rats. The data in the figure represent the mean cpm ( + SE) for the PBL mitogenic responses to 5 / x g / m l of ConA. Again, note the marked suppressive effect of electric footshock stress on the proliferative response of PBLs from animals with sham PVN lesions ( + / S H A M ) . Although lesions in the PVN significantly ameliorated the suppression of ConA responsiveness in shocked animals ( + / L E S I O N v s . + / SHAM), the mitogenic function of ConA-reactive lymphocytes was still significantly suppressed in comparison to non-shocked, sham( - / S H A M ) or PVN-lesioned ( - / L E S I O N ) controls. Thus, in contrast to the PHA-responsive PBLs, there was still a stress-induced suppression of the ConA-responsive PBLs in the lesioned animals. ** P < 0.01; * P < 0.05.
M.A. Pezzone et al. /Journal of Neuroimmunology 53 (1994) 39-46 mental days most likely accounts for the significant repetition effect. These results show that lesions of the PVN can completely prevent the shock-induced suppression of PBL mitogenic reactivity to P H A and slightly, although non-significantly, enhance the same response in home cage animals. A N O V A of the PBL ConA data (Fig. 3) revealed significant main effects of stress (F{1,35} = 42.00, P < 0.0001), surgery (F{1,35} = 4.44, P < 0.05), and repetition (F{2,35} = 11.32, P < 0 . 0 0 0 1 ) . Post hoc tests showed a significant suppression of PBL mitogenic proliferation in the s h o c k / s h a m treatment group compared to all other treatment groups ( P < 0.01) and in the shock/lesion treatment group compared to the home c a g e / s h a m ( P < 0.05) and the home cage/lesion treatment groups ( P < 0.01). These results suggest that PVN lesions only partially attenuate the shock-induced suppression of ConA-reactive PBLs, while baseline lymphocyte proliferation in home cage animals is not affected. Again, the variation in baseline lymphocyte mitogenic activity across experimental days most likely accounts for the significant main effect of repetition.
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Fig. 5. Splenic lymphocyte mitogenic responses to ConA following footshock stress and home cage conditions in PVN- and sham-lesioned rats. The data in the figure represent the mean cpm (+ SE) for the splenic lymphocyte mitogenic responses to 2.5 /zg/ml of ConA. The marked suppression of splenic lymphocyte mitogenic proliferation in home-caged, PVN-lesioned animals (-/LESION) is clearly demonstrated, while the suppressive effect of footshock was not evident in the sham-lesioned animals (+/SHAM). Combining PVN lesions with footshock(+/LESION), however, again elicited a further marked suppression of splenic lymphocytemitogenic activity. ** P < 0.01.
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+/SHAM +/LESION
Fig. 4. Splenic lymphocyte mitogenic responses to PHA following footshock stress and home cage conditions in PVN- and sham-lesioned rats. The data in the figure represent the mean cpm (___SE) for the splenic lymphocytemitogenic responses to 5/zg/ml of PHA. The home-caged, PVN-lesioned animals (-/LESION) had a reduced (although not statistically significant) proliferative response to PHA in comparison to home-caged, sham-lesioned animals ( - / SHAM), suggesting that the PVN lesion itself may have lowered baseline splenic lymphocytemitogenic activity. Although the splenic lymphocyte mitogenic response to PHA was significantlysuppressed in sham-lesioned animals receiving electric footshock( +/SHAM) as compared to home-caged,sham-lesioned animals ( -/SHAM), there was no difference between this group (+/SHAM) and home-caged animals with PVN lesions (-/LESION), suggesting that the PVN lesion was comparable to footshock stress in lowering splenic lymphocyte mitogenic activity. Moreover, when the PVN-lesioned animals were exposed to footshock ( +/LESION), their splenic lymphocyte mitogenic response to PHA was markedly further suppressed. ** P < 0.0l.
Figs. 4 and 5 illustrate the splenic lymphocyte mitogenic responses to optimum concentrations of P H A (10 /xg/ml) and ConA (10 /xg/ml), respectively. A N O V A of the splenic lymphocyte P H A data (Fig. 4) revealed significant main effects of stress (F{1,35} = 104.1, P < 0.0001), surgery (F{1,35} = 51.42, P < 0.0001), and repetition (F{2,35} = 104.1, P < 0.0001). Additionally, there were significant 2-way interactions between stress and surgery (F{1,35} = 5.98, P < 0.05) and between stress and repetition (F{2,35} = 55.83, P < 0.0001) and a significant 3-way interaction between stress, surgery, and repetition (F{2,35} = 8.44, P < 0.01). Post hoc tests for the splenic lymphocyte P H A data revealed that the suppression of mitogenic activity in the shock/lesion group compared to all other treatment groups is highly significant ( P < 0.01). Relative to the home c a g e / s h a m group, splenic lymphocyte mitogenic activity in the home cage/lesion group was slightly suppressed although not statistically significant ( P > 0.05), while lymphocyte proliferation in the s h o c k / s h a m group was significantly suppressed ( P < 0.01). There was no statistical difference between the home cage/lesion and the s h o c k / s h a m groups. These data suggest that the PVN regulates the lymphocyte mitogenic response to P H A in the spleen. Moreover, when PVN lesions and
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M.A. Pezzone et al. /Journal of Neuroimmunology 53 (1994) 39-46
stress occurred together, the suppressive effects were compounded. The significant 3-way interaction between stress, surgery, and repetition not only reflects the variability in the mitogenic assay over experimental days and ambient stress levels in the animal facility but also reflects the compounding suppressive effects of stress and PVN lesions on splenic lymphocyte mitogenic reactivity to PHA. ANOVA of the spleen ConA data (Fig. 5) revealed significant main effects of stress (F{1,32} = 6.45, P < 0.05), surgery (F{1,32} = 48.46, P < 0.0001), and repetition (F{2,32} =56.36, P<0.0001), and a significant 2-way interaction between stress and repetition (F{2,32} = 10.67, P < 0.001). Post hoc tests showed that the proliferative responses of ConA-reactive splenic lymphocytes were significantly suppressed in the shock/lesion treatment group relative to the home c a g e / s h a m and the shock/sham groups ( P < 0.01). Lymphocyte proliferation in the home cage/sham and shock/sham groups were statistically equivalent (P > 0.05). These data suggest that the ConA-reactive lymphocytes taken from sham-operated rats have habituated to the effects of footshock stress. The suppression of ConA-reactive splenic lymphocytes by stress, however, was greatly exacerbated in animals receiving PVN lesions.
4. Discussion
This study has shown that lesions of the hypothalamic PVN can markedly alter the mitogenic responsiveness of peripheral blood and splenic lymphocytes following acute footshock stress. The resulting effects upon lymphocyte blastogenesis, which varied, depended upon the lymphoid compartment sampled and the mitogen used for stimulation. Interestingly, these findings concur with our previous studies which have shown that stress differentially affects peripheral blood and splenic lymphocytes by independent mechanisms (Cunniek et al., 1990) and studies which have shown that stress may differentially affect lymphocyte subpopulations as evidenced by differential reactivity to non-specific mitogenic stimulation (Haynes and Fauci, 1978). Although explicit characterization of these pathways remains to be elucidated, it appears that the PVN is capable of simultaneously influencing both splenic and peripheral blood lymphocytes by their respective pathways of activation. The sympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis are likely involved in the modulation of splenic and peripheral blood lymphocyte function during stress (Rabin et al., 1989). Correspondingly, a central role of the hypothalamic PVN in stressor-induced immune alterations is not unjustifiable, considering its neuronal projections can
influence both pituitary hormone secretion (Wiegand and Price, 1980) and preganglionic neurons of the sympathetic and parasympathetic divisions of the autonomic nervous system (Saper et al., 1976). Demonstrating that direct activation of the PVN follows the administration of stressful stimuli, the authors and other investigators have shown that levels of c-los messenger RNA a n d / o r its protein product are rapidly induced in cells of the medial parvocellular division of the PVN following various stressors (Ceccatelli et al., 1989; Sharp et al., 1991; Imaki et al., 1992; Kononen et al., 1992; Pezzone et al., 1992). Additionally, further characterization of these stress-activated neurons in the PVN has revealed that they contained corticotropin-releasing hormone (CRH) (Imaki et al., 1992; Pezzone et al., 1992), a neuropeptide that can activate both the autonomic nervous system as a neurotransmitter and the HPA axis as a neurohormone. Thus, CRH neurons in the PVN may play an integral role in stressor-induced immune alterations by activating the HPA axis and the sympathetic nervous system following stress. Indirectly suggesting the presence of a functional physiological relationship between CRH-containing neurons of the PVN and cells of the immune system, it has been demonstrated that increases in plasma levels of corticosterone correlate temporally with the formation of an immune response (Besedovsky et al., 1975; Shek and Sabiston, 1983). More direct evidence, however, specifically implicating the involvement of the PVN in immune modulation is apparent in studies which have shown that norepinephrine is selectively decreased in the PVN at the peak of an immune response in mice (Carlson et al., 1987) and that neuronal firing is decreased in the PVN of rats during the first 3 days after immunization later followed by an increased rate of firing by day 6 (Saphier et al., 1987). These studies suggest the presence of a neuroendocrine-immune circuit involving bi-directional interactions between the immune system and the HPA axis as previously hypothesized (Besedovsky and Sorkin, 1977). Considering that such a proposed neuroendocrineimmune circuit (most likely involving the PVN) does exist, one might expect to see alterations in the neuroendocrine and the immune responses to stress following its disruption. Indeed, previous studies have shown that destruction of the PVN does significantly inhibit activation of the HPA axis induced by stressors such as ether inhalation (Makara et al., 1981; Bruhn et al., 1984; Dohanics et al., 1986), surgical trauma (Makara et al., 1981), hypothalamic electrical stimulation (Makara et al., 1981), intermittent footshock (Rivest and Rivier, 1991), and hemorrhage (Darlington et al., 1988). It is unfortunate that concurrent assessment of immune function in these studies utilizing PVN lesions was not conducted, as immunological changes following footshock, anesthesia, and surgical
M.A. Pezzone et al. /Journal of Neuroimmunology 53 (1994) 39-46
trauma have all been well documented (Rabin et al., 1989; Salo, 1992). Specific alterations of immune function have been recorded following lesions of other hypothalamic nuclei, supporting the existence of and implicating the disruption of this neuroendocrine-immune circuit. In these studies, lesions of the anterior hypothalamus led to protection against lethal anaphylaxis (Luparello et al., 1964), reduced the intensity of Arthus and delayed skin reactions (Jankovic and Isakovic, 1973), reduced the rate of circulating antibody formation (Tyrey and Nalbandov, 1972; Jankovic and Isakovic, 1973), inhibited the development of experimental allergic encephalomyelitis (Wertman et al., 1985), inhibited lymphocyte stimulation in whole blood and spleen (Cross et al., 1980; Roszman et al., 1982; Keller et al., 1988), and decreased the cellularity of the thymus and spleen (Cross et al., 1980). Although the anterior hypothalamic nuclei are distinct from the PVN, it would appear that based on the data presented in this manuscript and findings from the previously mentioned studies that these two regions of the hypothalamus play important roles in neuroimmunomodulation. Although the pattern of immune responsiveness in the peripheral blood appears to be corticosteroid-dependent, the mitogen response pattern in the splenic lymphoid compartment appears to be entirely different (as previously alluded to) and warrants further discussion. As the suppression of splenic lymphocyte mitogenic activity has been shown to be mediated by catecholamines via a /3-adrenergic receptor mechanism (Cunnick et al., 1990), the markedly enhanced suppression of splenic lymphocyte mitogenic activity in PVNlesioned rats following stress (Figs. 4 and 5) suggests augmentation of SNS activity. Indeed, stress-induced increases in plasma epinephrine were significantly greater in PVN-lesioned rats when compared to those with sham lesions (Darlington et al., 1988). Likewise, adrenalectomy augmented stress-induced increments in catecholamine release, re-uptake, metabolism, turnover, and biosynthesis (Kvetnansky et al., 1993), suggesting that glucocorticoids exert negative feedback upon the SNS during stress. As always, one must be cautious in the interpretation of ablation studies especially those involving the PVN. Although the role of parvocellular CRH neurons is likely responsible for the observed alterations in immune function presented in this study, other PVN neurons or peptides could not be excluded since the entire PVN was indiscriminately destroyed.
Acknowledgements The authors thank Ms. Ada Armfield, Ms. Ellen Hamill, and Mr. Paul Wood for their superb technical
45
assistance, Dr. Sheldon Cohen and Janet Schlarb for their expert statistical assistance, and Dr. Daohong Zhou for his helpful suggestions. This work was supported by national research service award F30 MH10157 to M.A.P. from the National Institute of Mental Health (NIMH) and by NIMH research grant MH43411-04 and a grant from the Pathology Education and Research Foundation to B.S.R. Preliminary results from these studies were presented at the 22nd Annual Meeting of the Society for Neuroscience, Anaheim, CA, 1992, and the Research Perspectives in Psychoneuroimmunology IV Conference in Boulder, CO, 1993.
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