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or experimental influenza viral infection in two inbred strains of mice
J o u r n a l of Neuroimmunology ELSEVIER
Journal of Neuroimmunology 49 (1994) 25-33
Kinetics of glucocorticoid response to restraint stress a n d / o r experimental influenza viral infection in two inbred strains of mice Gerlinda Hermann .,a,b, C. Amy Tovar b, F. Michael Beck c, John F. Sheridan a,b a Department of Medical Microbiology and Immunology, Colleges of Medicine and Dentistry, The Ohio State University, Columbus, OH 43210, USA b Department of Oral Biology, Colleges of Medicine and Dentistry, The Ohio State University, Columbus, OH 43210, USA c Department of Endodontics, Colleges of Medicine and Dentistry, The Ohio State University, Columbus, OH 43210, USA (Received 28 May 1993) (Revision received 23 July 1993) (Accepted 23 July 1993)
Abstract
The murine model of influenza viral infection was used to evaluate the effects of restraint stress on pathogenesis and survival in inbred strains of mice. We recently reported that restraint stress was associated with an enhanced probability of survival in one strain of inbred mouse, DBA/2, and not in another, C57BL/6. Those studies suggested that the protective mechanism(s) of stress on mortality in the DBA/2 mice might be attributable to elevated levels of circulating glucocorticoids. Therefore, daily levels of plasma glucocorticoids were measured during influenza viral infection in both these strains. The present studies demonstrated that influenza infection itself is perceived as a stressor in both C57BL/6 and DBA/2 mice as evidenced by elevated plasma glucocorticoid levels within 48 h of infection. However, augmentation of glucocorticoid levels was not seen in the DBA/2 mice that were also subjected to restraint stress during the course of infection. Thus, corticosterone levels alone did not account for the enhanced survival seen in this group of animals.
Key words: Glucocorticoids; Restraint stress; Viral pathogenesis; Influenza virus; Strain differences
1. Introduction
Physiological responses to behavioral and psychological stressors are initiated by the hypothalamus and translated into action by the hypothalamic-pituitaryadrenal ( H P A ) axis and the sympathetic nervous system. Activation of H P A pathways results in secretion of hypothalamic corticotrophin-releasing factor (CRF), pituitary adrenocorticotrophic hormone ( A C T H ) and, ultimately, the release of glucocorticoid hormones from the adrenal cortex. Activation of the sympathetic nervous system provokes the release of catecholamines from the nerve terminal endings as well as the adrenal medulla. Co-activation of the cerebral noradrenergic and H P A systems has been observed with a variety of stressors such as restraint and electric footshock (Stone, 1975; D u n n et al., 1984, Anisman, 1987; Dunn, 1988).
* Corresponding author. Phone (614) 292 4305, Fax (614) 292 4888. 0165-5728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-5728(93)E0122-P
If such physiological responses are taken as a definition of stress, then infection with a viral pathogen is also considered a stressor. D u n n et al. (1988) demonstrated that infection of female B a l b / c mice with influenza virus PR8 resulted in an elevation of plasma corticosterone levels and co-activation of cerebral noradrenergic systems within 64 h following intranasal instillation of virus. Similar elevations in plasma corticosterone and central nervous system adrenergic systems have been elicited following administration of Newcastle disease virus to mice (Smith et al., 1982; D u n n et al., 1987). Natural and synthetic glucocorticoids inhibit a variety of lymphocyte functions and are successfully used clinically as anti-inflammatory and immunosuppressive agents (Claman, 1975; Fauci, 1979). Glucocorticoid actions on cytokines have been reviewed in detail (Munck et al., 1984; Munck and Guyre, 1991). Briefly, the propagation of both cellular and humoral immune responses is maintained by the cascading cytokine net-
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G. Hermann et al. / Journal of Neurotmmunology 49 (1994) 25-33
work which is subject to regulation (i.e. suppression) by glucocorticoids at multiple sites. For example, glucocorticoid inhibition of cytokine synthesis by macrophages and T cells, ultimately, inhibits or retards T cell activation, clonal expansion, and antibody production (Munck and Guyre, 1991). Additionally, glucocorticoids can affect the recirculation or trafficking of lymphocytes. Corticosteroids provoke redistribution of circulating lymphocytes back to the bone marrow (Fauci, 1975), impair the influx of mononuclear cells from the blood into lymph nodes (Cox and Ford, 1982), and prevent the localization of sensitized effector lymphocytes into sites of active inflammation (Chung et al., 1986). Our previous study (Hermann et al., 1993) demonstrated that restraint stress reduced influenza viral-induced mortality in one inbred strain of mouse, the DBA/2. This difference in mortality associated with stress was not seen in either the C57BL/6 or C 3 H / HeN strains despite the fact that all three strains showed similar effects of restraint on the immunological and pathological responses examined during influenza infection. The only parameter measured that distinguished the restrained D B A / 2 group from the control groups was the elevated corticosterone levels at day 10 post-infection. Given that glucocorticoid effects on inflammatory reactions, cellular functions, and immune effector cell trafficking have been well-documented (e.g. Parrillo and Fauci, 1979; Cohen and Crnic, 1984; Munck et al., 1984; Munck and Guyre, 1991), it is possible that elevated levels of corticosterone during the development of the immune response to influenza viral infection modulated the inflammation and cellular infiltration of the lung so as to reduce the mortality in the restraint stressed D B A / 2 group. Genetic factors contribute to the expression of behavioral and neurochemical responses elicited by stressors. Royce and colleagues (1970) described differences in 'emotionality' or coping behaviors of ten inbred strains of mice in response to various situations. Differences in behavioral responses were reflected in different biochemical profiles including endogenous opioids and plasma corticosterones (for review see Oliverio et al., 1979; Ingram and Corfman, 1980; Siegfried et al., 1984; Shanks et al., 1990). For example, Shanks et al. (1990) demonstrated that although the basal levels of plasma corticosterone did not differ across six inbred strains of mice, the dynamics of plasma corticosterone levels in response to unavoidable footshock stress showed differences in magnitude as well as clearance rates among the different strains of mice. Our previous study (Hermann et al., 1993) revealed that two inbred strains of mice, C57BL/6 and DBA/2, showed differences in mortality and terminal (i.e. 10 days post-infection) plasma corticosterone levels associated with influenza viral infection in association with
restraint stress. However, in that study the daily profile of the glucocorticoid response to stress a n d / o r infection was not characterized. Therefore, the specific aims of this study were to examine the dynamics of the glucocorticoid response to behavioral regimens (i.e. repetitive cycles of 12 h food and water deprivation or 12 h restraint stress) a n d / o r influenza viral infection during the 10 days post-infection period and evaluate the development of the immune response in these two inbred strains of mice.
2. Materials and methods 2.1. Animals
Virus-antibody-free C57BL/6 and D B A / 2 male mice 4-8 weeks old were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Mice were housed five per cage in laminar flow cabinets and provided food and water ad libitum. All mice were maintained on a 12-h light/dark cycle (lights ON at 06:00) and were allowed to acclimate to these conditions for 1 week prior to experimentation. 2.2. Virus
Influenza A / P u e r t o R i c o / 8 / 3 4 (PR8) virus was obtained from the American Type Culture Collection (Rockville, MD) and propagated in the allantoic cavity of 10-day-old embryonated chicken eggs. Infectious allantoic fluid was collected, clarified by low-speed centrifugation, and stored at -70°C. The virus titer was determined to be 3200 hemagglutinating units (HAU) per ml, using human type 'O' erythrocytes. 2.3. Infection o f mice
The lethal dose 50 (LDs0) of influenza PR8 was estimated for each strain of mouse (C57BL/6 = 32HAU; D B A / 2 = 8HAU). Mice were lightly anesthetized with an intraperitoneal injection (40 ~1/10 g body weight) of a mixture of 0.4 m g / m l xylazine (Rompun@; Haver-Lockhart, Shawnee, KS) and 7.8 m g / m l ketamine (Ketaset@; Bristol Laboratories, Syracruse, NY). All animals were intranasally infected (50 tzl) with an appropriate LDs0 dose of PR8 virus using an Eppendorf pipette. Pre-infection serum samples were routinely screened for antibody to influenza virus to assure that mice were seronegative prior to experimentation. 2.4. Restraint protocol
Mice were restrained/isolated (RST) in well-ventilated 50 ml conical polypropylene tubes for 12 h each day (from 21:00 to 09:00) within their home cages. While in the tubes, the animals could move back and forth freely but could not turn around nor did they have access to food or water during this time period.
G. Hermann et aL /Journal of Neuroimmunology 49 (1994) 25-33
Therefore, similarly food- and water-deprived (FWD), non-restrained groups were used as a control group. A third group of mice (non-stressed, NS) was included to control for the effects that 12 h FWD had on circadian rhythm of circulating corticosterone levels, alone, as well as during the development of an immune response to viral infection. The restraint (RST) or food-water deprivation (FWD) regimen was initiated one day prior (day - 1 ) to virus infection and continued for 10 days post-infection. At the end of each RST or FWD cycle, mice were released from their restraint tubes and given free access to food and water for the remainder of the day; food and water was returned to the FWD groups. The NS groups had access to food and water 24 h per day.
2.5. Experimental design and blood sample schedule After the 1 week acclimation period, blood samples from all groups were drawn each morning at 10:00. To avoid excessively stressing the animals, each Infection/ Behavioral group had three subsets of mice which were bled (approximately 0.1 ml) once every 3 days via the retro-orbital plexus. The original distribution of animals per group was the same in both strains of mice: no infection/no stress (NI/NS); three subgroups n = 5 ( N = 15) infection/no stress (I/NS); three subgroups n = 8 ( N = 24) no infection/FWD ( N I / F W D ) ; three subgroups n = 5 ( N = 15) infection/FWD ( I / F W D ) ; three subgroups n = 8 ( N = 24) no in f ectio n /R S T ( N I / R S T ) ; three subgroups n = 5 ( N = 15) infection/RST ( I / R S T ) ; three subgroups n = 8 ( N = 24),
2.6. Detection of anti-influenza antibody by enzymelinked immunosorbent assay (ELISA) Anti-influenza IgG antibody was determined by an ELISA assay described in detail in our previous paper (Hermann et al., 1993). Briefly, influenza virus was bound to the wells of a polyvinyl microtiter plate. Aliquots of test serum were added to the plates and incubated overnight at 4°C. Peroxidase-conjugated goat anti-mouse IgG was added to the plates and influenza-specific antibody complexes were demonstrated by addition of substrate (0.1% H 2 0 2 plus 0.1% o-phenylene-diamine).
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cycle and lowest levels coinciding with the inactive phase. Therefore, it was essential that all blood samples be obtained at the same time of day each day. Blood samples were drawn at 10:00 each day. Sera were collected and stored at -70° C until assayed for corticosterone by radioimmunoassay. [~25I]Corticosterone kits for rats and mice (ICN Biomedical, Costa Mesa, CA) were used to determine serum corticosterone levels. Corticosterone levels from individual mice were determined from a standard curve and expressed in ng/ml.
2.8. Determination of cell yield from mediastinal lymph nodes Single-cell suspensions of mediastinal lymph nodes were prepared (described in detail in Hermann et al., 1993) by macerating the tissue in Hanks' balanced salt solution. Samples were counted on a Coulter counter; cell yield per animal was determined.
2.9. Statistical analyses Serum corticosterone levels from the two strains of mice were analyzed separately according to which of the three behavioral groups they had been assigned. Differences were analyzed using factorial analysis of variance (ANOVA) with infection status and time (i.e. day post-infection) as the factors. A log transformation of the glucocorticoid data was necessary in order to stabilize the variance. When indicated by a significant F-test, post-hoc testing was done using the modified Tukey method. Differences in cell yield from the mediastinal lymph nodes also were analyzed separately for each strain using factorial analysis of variance (ANOVA) with infection status and behavioral group as the factors. A log transformation was again performed to stabilize the variance. Post hoc testing was done using the modified Tukey method. All analyses were done using SAS version 6.07.
3. Results
3.1. Determination of influenza-specific IgG titers All infected animals demonstrated IgG sero-conversion to influenza A / P R 8 based on an ELISA assay described above. On the day of sacrifice (day 11 p.i.), influenza-specific IgG titers within either mouse strain were quite similar with the exception being that the R S T / i n f e c t e d groups in both strains of mice showed a significantly lower titer (P < 0.05; data not shown).
2. 7. Determination of serum corticosterone levels Plasma levels of glucocorticoid hormones follow a predictable circadian variation in most species (reviewed in Krieger, 1979; Spackman and Riley, 1978; Cohen and Crnic, 1984;) with peak levels occurring as the animals enter their active phase of the light/dark
3.2. Effects of infection on basal corticosterone levels Daily basal corticosterone levels were obtained each morning at 10:00 from mice maintained in their home cage and not infected. Under this non-stressed, uninfected condition, both the C57BL/6 and D B A / 2
G. Hermann et al. /Journal of Neuroimmunology 49 (1994) 25-33
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strains had stable average p l a s m a corticosterone levels (less t h a n 50 n g / m l ) across the 12 days of s a m p l i n g (Figs. 1A a n d 2A). A N O V A results i n d i c a t e d a significant effect ( P < 0.01) in b o t h strains for (a) infection, (b) day, a n d (c) the infection by day i n t e r a c t i o n (i.e. t h e r e was a n i n t e r a c t i o n b e t w e e n w h e t h e r or n o t the a n i m a l s were infected a n d o n which day p o s t - i n f e c t i o n the samples were o b t a i n e d ) . Daily c o r t i c o s t e r o n e levels of infected mice m a i n t a i n e d u n d e r identical h o u s i n g c o n d i t i o n s a n d b l e d at the same time of day as the above groups d e m o n strated that i n f e c t i o n a l o n e was sufficient to elevate p l a s m a c o r t i c o s t e r o n e levels. T w o distinct peaks of p l a s m a c o r t i c o s t e r o n e levels o c c u r r e d at day 2 a n d
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Fig. 1. Kinetics of corticosterone responses by C57BL/6 mice over
days post-infection with influenza A PR/8. Daily plasma corticosterone levels were determined from blood samples obtained at 10:00 each morning. Day ' - 1 ' represents the basal plasma corticosterone levels for each group (i.e. no behavioral treatment nor infection). Day '0' represents the morning after the first exposure to the assigned behavioral treatment (i.e. non-stressed, FWD, or RST). Mice were infected at 13:00 on Day '0'. Day '2' represents plasma corticosterone levels 45 h post-infection. Open symbols represent non-infected animals; closed symbols represent infected animals. Data presented as mean + SEM. (A) Kinetics of glucocorticoid response over days in mice maintained in home cages with free access to food and water 24 h per day, i.e. non-stressed. (B) Kinetics of GC response over days in mice maintained in home cages with restricted access to food and water from 21:00 to 09:00 each day, i.e. FWD. (C) Kinetics of GC response over days in mice maintained in home cages and placed in isolation/restraint tubes from 21:00 to 09:00 each day, i.e. RST.
days post-infection with influenza A PR/8. Daily plasma corticosterone levels were determined from blood samples obtained at 10:00 each morning. Day ' - 1' represents the basal plasma corticosterone levels for each group (i.e. no behavioral treatment nor infection). Day '0' represents the morning after the first exposure to the assigned behavioral treatment (i.e. non-stressed, FWD, or RST). Mice were infected at 13:00 on Day '0'. Day '2' represents plasma corticosterone levels 45 h post-infection. Open symbols represent non-infected; closed symbols represent infected animals. Data presented as mean_+SEM. (A) Kinetics of glucocorticoid response over days in mice maintained in home cages with free access to food and water 24 h per day, i.e. non-stressed. (B) Kinetics of GC response over days in mice maintained in home cages with restricted access to food and water from 21:00 to 09:00 each day, i.e. FWD. (C) Kinetics of GC response over days in mice maintained in home cages and placed in isolation/restraint tubes from 21:00 to 09:00 each day, i.e. RST. approximately day 7 - 8 post-infection. T h e C 5 7 B L / 6 strain showed the first p e a k of a n average 166 n g / m l at approximately 2 days post-infection. This elevated p l a s m a corticosterone level was m a i n t a i n e d u n t i l a second p e a k o c c u r r e d at day 7 post-infection (432 n g / m l average) (Fig. 1A). As the infection resolved, p l a s m a corticosterone levels again d e c l i n e d a n d leveled off to approximately 100 n g / m l o n the day the a n i m a l s were killed (i.e. day 11 post-infection). A similar profile of daily p l a s m a corticosterone levels was s e e n in the infected, n o n - s t r e s s e d D B A / 2 mice (Fig. 2A). W i t h i n 2 - 3 days post-infection, the first p e a k (303 n g / m l average) in p l a s m a corticosterone levels occurred. S u b s e q u e n t daily p l a s m a corticosterone levels d e c r e a s e d to approximately 200 n g / m l u n t i l the
G. Hermann et al. /Journal of Neuroimmunology 49 (1994) 25-33
second peak occurred at day 8 post-infection (343 n g / m l average). Again, as the infection resolved, plasma cortisterone levels declined and approached baseline (87 n g / m l average).
3.3. Effects of FWD on corticosterone levels associated with infection Daily basal corticosterone levels were obtained each morning at 10:00 from healthy and infected mice that were maintained in their home cage and subjected to repetitive 12-h periods of food and water deprivation (FWD). ANOVA results for the C57BL/6 strain revealed a significant effect for (a) day (P < 0.0001), (b) infection (P < 0.0001) and (c) the day by infection interaction (P = 0.0006). The D B A / 2 strain showed a significant effect for (a) day (P < 0.0001) and (b) infection (P < 0.0001) but not for (c) the day by infection interaction (P = 0.0694). Under this behavioral condition, both the C57BL/6 and D B A / 2 strains demonstrated elevated plasma corticosterone levels as early as the first morning (see day '0') following the FWD (Figs. 1B and 2B). The C57BL/6 strain demonstrated a peak at 363 n g / m l on day '0'. Over time, this FWD group appeared to habituate to this behavioral regimen such that by day 5, plasma corticosterone levels were down around 83 ng/ml. By the day of sacrifice, the C57BL/6 I / F W D group plasma corticosterone returned to an average 64 ng/ml, i.e. not sigificantly different from baseline levels (P > 0.05). The daily profile of plasma corticosterone in inf e c t e d / F W D C57BL/6 mice demonstrated an additional increase in plasma corticosterone levels on day 2 post-infection and, otherwise, resembled the profile seen in the infected/non-stressed groups described above. The first peak (329 n g / m l average) occurred on day 2 post-infection. Although there was a slight decline in plasma corticosterone levels, a second peak (386 n g / m l ) occurred around day 8. As the infection resolved, plasma corticosterone levels continued to drop such that, on the day of sacrifice, there was no significant difference (P > 0.05) between the infected or healthy FWD groups (Fig. 1B). The healthy (i.e. uninfected), FWD D B A / 2 group also showed a marked elevation in plasma corticosterone levels on day '0' (133 n g / m l average). By day '3', plasma corticosterone levels peaked at 226 ng/ml. Although the healthy, FWD D B A / 2 group showed some habituation to the FWD schedule, they remained somewhat stressed by this behavioral regimen as indicated by the relative stability of the elevated plasma corticosterone levels seen right up to the day of sacrifice (140 n g / m l average; Fig. 2b). Infected/FWD groups of D B A / 2 mice demonstrated an additional increase in plasma corticosterone
29
levels on day 2 post-infection. Indeed, a similar daily plasma corticosterone profile was observed in the inf e c t e d / F W D groups as that seen in the infected/ non-stressed groups described in the preceding section. The first peak (362 n g / m l ) occurred on day 2 post-infection. Over successive days, there was a slight decline in plasma corticosterone levels. However, a second peak (376 ng/ml) occurred around day 8 post-infection. Again, as the infection resolved, plasma corticosterone levels continued to drop such that, on the day of sacrifice, there was a minimal difference between the infected or healthy FWD groups of DBA/2 mice (Fig. 2B).
3.4. Effects of R S T on corticosterone levels associated with infection Figures 1C and 2C illustrate that repetitive exposure to 12 h of restraint/isolation was sufficient to elicit an elevated glucocorticoid response in both C57BL/6 and D B A / 2 healthy strains of mice. ANOVA results for the C57BL/6 strain demonstrated a significant effect for (a) day (P < 0.0001) and (b) the day by infection interaction (P = 0.0006) but not (c) infection (P = 0.2702). The D B A / 2 strain revealed a significant effect for (a) day (P < 0.0001) and (b) infection (P = 0.0005) but not for (c) the day by infection interaction (P = 0.1256). In both strains, elevation of plasma corticosterone levels was quite pronounced following the first exposure to this behavioral regimen (i.e. 327 n g / m l average in C57BL/6 and 420 n g / m l average in DBA/2). The healthy C57BL/6 groups showed eventual habituation to repetitive cycles of restraint; plasma corticosterone level on the day of sacrifice averaged 125 ng/ml. Although the healthy D B A / 2 groups demonstrated maximal corticosterone elevation on day '0' and plasma corticosterone levels declined quickly, they remained relatively stable around 200 n g / m l until the day of sacrifice. Infection of C57BL/6 mice resulted in an augmentation of the increase in plasma glucocorticoids associated with restraint/isolation treatment. As seen in Fig. 1C, the basic corticosterone response profile associated with infection was still present. However, the first peak at days 2-3 was amplified to 700 ng/ml; the second peak at clay 8 post-infection was 491 ng/ml. In contrast to the infected/RST, C57BL/6 mice, infection of D B A / 2 mice resulted in similar plasma corticosterone profiles seen in either healthy D B A / 2 mice subjected to the restraint regimen (Fig. 2C) or infected D B A / 2 maintained in their home cage environment (Fig. 2A). Specifically, similar levels of plasma corticosterone were seen on day '0' in the healthy versus infected RST D B A / 2 groups (420 n g / m l vs. 345 ng/ml, respectively); on day '2' i n f e c t e d / N S and infected/RST groups were similar (235 n g / m l vs. 233
G. Hermann et al. / Journal of Neuroirnmunology 49 (1994) 25-33
30
Average cell yield from mediastinal
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Fig. 3. Effects of behavioral treatment a n d / o r infection on average cell yield from mediastinal lymph nodes. Nodes were taken from all groups on 'day 10 post-infection'. In the healthy state, behavioral treatments alone did not alter cell number within these nodes in either strain of mouse. Both C57BL/6 and D B A / 2 mice responded with a significant increase in cell number in association with influenza infection (non-infected/NS vs. infected/NS conditions; P < 0.05 ,a). Infected mice subjected to repetitive cycles of restraint (RST) showed a suppression of this lymphadenopathy associated with influenza infection. This suppression was statistically significant for the C57BL/6 group (infected/NS vs. infected/RST) and the D B A / 2 group (infected/FWD vs. infected/RST; P < 0.05 ,b).
n g / m l , respectively); day '3', all infected groups were the same ( I / N S = 303 ng/ m l ; I / F W D = 265 ng/ m l ; I / R S T = 359 ng/ml). Likewise, the plasma corticosterone levels of all infected groups were, essentially, the same on day 9 ( I / N S = 343 ng/ m l ; I / F W D = 376 n g /ml; I / RST --- 301 ng/ml). In other words, there was no significant interaction between the infection and behavioral treatment in terms of plasma corticosterone levels in the D B A / 2 mice.
3.5. Effects of RST on cell yield from medistinal lymph nodes (MLN) The mediastinal lymph nodes drain the respiratory tract. Changes in cell number of these nodes can be used as an index of the lymphadenopathy that is normally associated with infection of the respiratory tract. It is important to note that in the healthy state, both strains average approximately 1.5 x 106 cells per mediastinal lymph node. Behavioral treatments alone did not alter cell number within these nodes in either strain of mouse (Fig. 3A, B). However, both C57BL/6 and D B A / 2 strains responded with a significant (P < 0.05) increase in cell number in association with influenza infection in the infected/NS condition versus the non-infected/NS condition. Indeed, despite the different approximate LD50 doses of influenza A / P R 8 required to infect the two different strains, similar magnitudes in average cell yield from mediastinal lymph nodes were observed (C57BL/6 = 6.4 × 106; D B A / 2
= 6.6 X 106). Although the average cell yield in the i nfect ed/ FWD groups (both C57BL/6 and D B A / 2 ) appeared to be different from the infected/NS groups of the same strain (C57BL/6 = 4.0 X 106; D B A / 2 = 9.8 >(106), these were not statistically significant differences (P > 0.05). Infected mice subjected to repetitive cycles of restraint (RST) showed a suppression of this lymphadenopathy associated with influenza infection (Fig. 3). That is, cell density of the draining nodes was suppressed in the i n f e c t e d / R S T groups of both strains when compared with the infected/NS group of the C57BL/6 and infected/FWD group of the D B A / 2 strain (P < 0.05). These results are similar to our previous study (Hermann et al., 1993), which demonstrated that RST also reduced the average cell yield from the superficial cervical lymph nodes of infected mice by approximately 25% in both strains of mice.
4. D i s c u s s i o n
These studies demonstrated that the pathogenic process of influenza viral infection a n d / o r development of the immune response was sufficient to cause an elevation in plasma corticosterone levels in both strains of mice. Within 48 h of infection, even in the absence of any other imposed behavioral treatment, plasma corticosterone levels were elevated. Thus, some 'afferent' signal associated with the infection a n d / o r
G. Herrnann et al. /Journal of Neuroimmunology 49 (1994) 25-33
the immune response activated the hypothalamic-pituitary-adrenal axis (HPA). In the murine experimental influenza viral infection model, virus titer peaks occur between 48 and 72 h post-infection (Hennet et al., 1991). Within a few hours post-infection, products of activated macrophages, i.e. IL-6, IL-1 and TNFa are detectable in bronchoalveolar lavage fluid (Hennet et al., 1992). Thus, any of these soluble cytokines could act as potential signals to activate the HPA axis. A secondary peak in plasma corticosterone levels was seen around day 7-8 p.i., in both strains of mice. Peak mononuclear infiltration of lung tissue occurs around this time and contributes to the immunopathology (Beck and Sheridan, 1989; Sheridan et al., 1991; Hennet et al., 1992). At this point in the infection, respiration appeared labored and stressful, and may have been sufficient to account for the second peak in circulating corticosterone levels. As resolution of the infection continued, plasma levels of corticosterone declined (Figs. 1A and 2A). Clearly, behavioral treatments alone (either FWD or RST) provoked elevations in corticosterone levels in healthy mice of both strains (Figs. 1B,C and 2B,C). Over time, the animals seemed to habituate to the regimen, as expressed in plasma corticosterone levels, but in the D B A / 2 strain, circulating corticosterone amounts did not return to basal levels even after exposure to 12 cycles of the behavioral regimen; mean plasma corticosterone levels were well in excess of 100 n g / m l even on the final day sampled (P < 0.05). In the C57BL/6 mouse strain, the two peaks of corticosterone levels associated with the viral influenza infection were differentially affected by restraint stress (RST; Fig. 1C). The first peak, occurring at day 2 p.i., was augmented by the imposed RST; while the second peak, at day7-8 p.i., was not. It is possible that there is a synergism of activation of the HPA axis during the early phases of infection/immune response (i.e. soluble cytokine signals) and the stress of restraint. According to work by Hennet et al. (1992), levels of the pro-inflammatory cytokines (i.e. IL-1, IL-6, TNFa) have greatly declined by this late period of the infection. This second peak in plasma corticosterone levels was of comparable magnitude as that seen with infection alone (infection/NS), or infection/FWD groups (Fig. 1A, B). Thus, it appears to be a reflection of the immunopathology that was occurring, with the behavioral treatment having little or no impact on the activation of the HPA axis at this time. In contrast, this differential effect of RST on the two peaks of plasma corticosterone levels was NOT seen in the D B A / 2 mouse strain. Instead, development of the infection and immune response appeared to be the primary determining factors of corticosterone levels at either peak in the D B A / 2 strain, regardless of the behavioral treatment imposed. That is, there was
31
no significant interaction of infection and behavior (Fig. 2C). Indeed, the kinetics of the plasma glucocorticoid levels of the infected D B A / 2 mice were quite similar across all three behavioral treatments. Despite the lack of difference in plasma corticosterone response profiles of the NS, FWD, or RST/infected D B A / 2 groups, we have seen differences in mortality following influenza infection associated with RST in the D B A / 2 strain (Hermann et al., 1993; and in this study, data not shown). Therefore, some physiological parameter associated with restraint stress, other than glucocorticoids, may be responsible for this difference in survival of the infected D B A / 2 mice. Similar to our previous studies (Feng et al., 1991, 1992; Hermann et al., 1993), changes in cell number within the draining lymph nodes (e.g. mediastinal lymph nodes) normally associated with infection of the respiratory tract were seen when the animals from either strain were exposed to RST. It is important to note that behavioral treatments alone did not alter cell number within these nodes in either strain of mouse (Fig. 3). Both C57BL/6 and D B A / 2 strains responded with a significant increase in cell number in association with influenza infection in the infected/NS condition despite the different approximate LDs0 doses of influenza A / P R 8 required to infect the two different strains. Suppression of the increased cell number normally associated with influenza infection was observed when the mice of either strain were subjected to repetitive cycles of restraint/isolation (RST). Glucocorticoids have been credited with a role in trafficking or the redistribution of lymphocytes as well as controlling edema and inflammation (reviewed in Munck and Guyre, 1991; Claman, 1975; Fauci, 1975, 1979; Cox and Ford, 1982; Chung et al., 1986). The studies presented here support these concepts by showing an association between plasma corticosterone levels and distribution of lymphocytes in infected/RST C57BL/6 mice as reflected in the cell density of the draining lymph nodes. However, in the D B A / 2 inbred mouse strain, the diminished cell density, reduction in mortality, and reduction in pathosis (as seen in this study and our previous study, Hermann et al., 1993) cannot be attributed solely to plasma corticosterone levels. Responses to stressors are initiated by the hypothalamus and translated into action by the hypothalamicpituitary-adrenal axis and the autonomic nervous system. Products from both of these systems (e.g. corticosteroid hormones and catecholamines) are able to modulate the activity of various immune effector cells directly (see reviews by Livnat et al., 1985; Ader et al., 1990; Besedovsky and del Rey, 1991; Madden and Livnat, 1991; Munck and Guyre, 1991) as well as alter the general physiology of the animal. There is extensive documentation of functional adrenergic receptors on
32
G. Hermann et al. /Journal of Neuroimmunology 49 (1994) 25-33
m a c r o p h a g e s which c a n modify the p r o d u c t i o n a n d / o r secretion of t u m o r necrosis factor ( T N F ) a n d lymphocyte activating factors ( L A F ) (e.g. S p e n g l e r et al., 1990; H u et al., 1991). A n a p p r e c i a t i o n for the roles that these cytokines play in d e v e l o p m e n t of the cascading i m m u n e r e s p o n s e d e m o n s t r a t e s how c a t e c h o l a m i n e s m a y i n d u c e p o t e n t i m m u n o r e g u l a t o r y a n d imm u n o p a t h o l o g i c a l effects. A d d i t i o n a l l y , a d r e n e r g i c inn e r v a t i o n of the v a s c u l a t u r e a n d lymphatic ducts ( T o d d a n d B e r n a r d , 1973; W a n g a n d Z h o n g , 1985; M c H a l e , 1990) may play a critical role in r e c i r c u l a t i o n or distrib u t i o n of lymphocytes. T h u s , the a n i m a l has at least two pathways (i.e. the h y p o t h a l a m i c - p i t u i t a r y - a d r e n a l axis a n d the sympathetic n e r v o u s system) which, ultimately, may affect the n u m b e r , distribution, or state of activation of i m m u n e effector cells d u r i n g stress. T h e s e two pathways p r o b a b l y work cooperatively to try to m a i n t a i n , or regain, homeostasis. T h e results of the p r e s e n t studies suggest that co-activation of the sympathetic n e r v o u s system a n d the H P A axis is responsible for the c h a n g e s in mortality, pathology, s u p p r e s s e d lymphadenopathy, and diminished immune response s e e n in the D B A / 2 i n f e c t e d / R S T mice. T h e r e f o r e , f u t u r e studies will investigate the possible role of the s y m p a t h e t i c n e r v o u s system d u r i n g the d e v e l o p m e n t of the i m m u n e r e s p o n s e to i n f l u e n z a viral infection in the D B A / 2 i n b r e d m o u s e strain.
Acknowledgements W e t h a n k Dr. A.C. G r i f f i n for t h o u g h t f u l discussion a n d critical review of this m a n u s c r i p t . This work was s u p p o r t e d in part by grants to J.F.S. from N I H (HL38485) a n d N I M H (MH46801) a n d a n N I M H Postdoctoral T r a i n i n g G r a n t fellowship to G.H. (MH18831).
References Ader, R., Felten, D. and Cohen, N. (1990) Interaction between brain and the immune system. Ann. Rev. Pharmacol. Toxicol. 30, 561-602. Anisman, H. (1987) Neurochemical changes elicited by stress. In: Anisman and Bignami (Eds.), Psychopharmacology of Aversively Motivated Behavior. Plenum Press, New York, NY, pp. 119-172. Beck, M.A. and Sheridan, J.F. (1989) Regulation of lymphokine response during reinfection by influenza virus. J. Immunol. 142, 3560-3567. Besedovsky, H.O. and del Rey, A. (1991) Physiological implications of the immune-neuro-endocrine network. In: Ader, Felten and Cohen (Eds.), Psychoneuroimmunology,2nd edn. Academic Press, San Diego, CA, pp. 589-608. Chung, H.-T., Samlowski, W.E. and Daynes, R.A. (1986) Modifications of the murine immune system by glucocorticoids: alterations of the tissue localization properties of circulating lymphocytes. Cell. Immunol. 101, 571-585. Claman, H.N. (1975) How corticosteroids work. J. Allergy Clin. Immunol. 55, 145-151.
Cohen, J.J. and Crnic, L.A. (1984) Glucocorticoids, stress and the immune response. In: D.R. Webb (Ed.), Immunopharmacology and the Regulation of Leukocyte Function. Marcel Dekker, New York, NY, pp. 61-91. Cox, J.H. and Ford, W.L. (1982) The migration of lymphocytesacross specialized vascular endothelium. Cell. Immunol. 66, 407-422. Dunn, A.J. (1988) Stress-related activation of cerebral dopaminergic systems. Ann. N.Y. Acad. Sci. 537, 188-205. Dunn, A.J. and Kramarcy, N.R. (1984) Neurochemical responses m stress: relationships between the hypothalamic-pituitary-adrenal and catecholamine systems. In: Iversen, Iversen, Snyder (Eds.), Handbook of Psychopharmacology, Vol. 18. Plenum Press, New York, NY, pp. 455-515. Dunn, A.J., Powell, M.L., Moreshead, W.V., Gaskin, J.M. and Hall, N.R. (1987) Effects of Newcastle disease virus administration to mice on the metabolism of cerebral biogenic amines, plasma corticosterone, and lymphocyte proliferation. Brain Behav. Immunol. 1,216-230. Dunn, A.J., Powell, M.L., Meitin, C. and Small, P.A. Jr. (1988) Virus infection as a stressor: influenza virus elevates plasma concentrations of corticosterone, and brain concentrations of MHPG and tryptophan. Physiol. Behav. 45, 591-594. Fauci, A.S. (1975) Mechanisms of corticosteroid action on lymphocyte subpopulations. Immunology 28, 669-680. Fauci, A.S. (1979) Mechanisms of immunosuppressive and anti-inflammatory effects of glucocorticoids. J. lmmunopharmacol. 1,
1-25. Feng, N. (1992) Restraint Stress-induced Alteration of the Pathogenesis and Immune Response to Influenza Virus Infection in Mice. Ph.D. Thesis, Ohio State University Health Sciences Library. Feng, N., Pagniano, R., Tovar, C.A., Bonneau, R.H., Glaser, R. and Sheridan, J.F. (1991) The effect of restraint stress on the kinetics, magnitude, and isotype of the humoral immune response to influenza virus infection. Brain Behav. Immun. 5, 370-382. Hennet, T., Peterhans, E. and Stocker, R. (1991) Alterations m antioxidant defences in lung and liver of mice infected with influenza A virus. J. Gen. Virol. 73, 39-46. Hennet, T., Ziltener, H.J., Frei, K. and Peterhans, P. (1992) A kinetic study of immune mediators in the lungs of mice infected with influenza A virus. J. Immunol. 149, 932-939. Hermann, G., Tovar, C.A., Beck, F.M., Allen, C. and Sheridan, J.F. (1993) Restraint stress differentially affects the pathogenesis of an experimental influenza viral infection in three inbred strains of mice. J. Neuroimmunol. 47, 83-94. Hu, X., Goldmuntz, E.A. and Brosnan, C.F. (1991) The effect of norepinephrine on endotoxin-mediated macrophage activation. J. Neuroimmunol. 31, 35-42. Ingram, D.K. and Corfman, T.P. (1980) An overview of neurobiological comparisons in mouse strains. Neurosci. Biobehav. Rev. 4, 421-435. Kreiger, D. (1979) Rhythms in CRF, ACTH, and corticosteroids. In: L. Martini (Ed.), Comprehensive Endocrinology Series, Endocrine Rhythms. Raven Press, New York, NY, pp. 123-142. Livnat, S., Felten, S.Y., Carlson, S.L., Bellinger, D.L. and Felten, D.L. (1985) Involvement of peripheral and central catecholamine systems in neural-immune interactions. J. Neuroimmunol. 10, 5-30. Madden, K.S. and Livnat, S. (1991) Catecholamine action and immunologic reactivity. In: Ader, Felten and Cohen (Eds.), Psychoneuroimmunology, 2nd edn. Academic Press, San Diego, CA, pp. 283-310. McHale, N.G. (1990) Lymphatic innervation. Blood Vessels 27, 127136. Munck, A. and Guyre, P.M. (1991) Glucocorticoids and immune function. In: Ader, Felton and Cohen (Eds.), Psychoneuroimmunology, Academic Press, San Diego, CA, pp. 447-474. Munck, A., Guyre, P.M. and Holbrook, N.J. (1984) Physiological
G. Hermann et al. /Journal of Neuroimmunology 49 (1994) 25-33 functions of glucocorticoids in stress and their relation to pharmacological actions. Endocrin. Rev. 5, 25-44. Oliverio, A., Castellano, C. and Puglisi-Allegra, S. (1979) A genetic approach to behavioral plasticity and rigidity. In: J.R. Royce and L.P. Mos (Eds.), Theoretical Advances in Behavioral Genetics. Sijthoff and Noordhoff, Alphen aan de Rijn, pp. 139-165. Parrillo, J.E. and Fauci, A.S. (1979) Mechanisms of glucocorticoid action on immune processes. Ann. Rev. Pharmacol. Toxicol. 19, 179-201. Royce, J.R., Carran, A. and Howarth, E. (1970) Factor analysis of cmotionality in ten inbred strains of mice. Multivar. Behav. Res. 5, 19-48. Shanks, N., Griffiths, J., Zalcman, S., Zacharko, R.M. and Anisman, H. (1990) Mouse strain differences in plasma corticosterone following uncontrollable footshock. Pharmacol. Biochem. Behav. 36, 515-519. Sheridan, J.F., Feng, N., Bonneau, R.H., Allen, C.M., Huneycutt, B.S. and Glaser, R. (1991) Restraint stress differentially affects anti-viral cellular and humoral immune responses in mice. J. Neuroimmunol. 31,245-255.
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Siegfried, B., Frischknecht, H.-R. and Waser, P.G. (1984) Defeat, learned submissiveness, and analgesia in mice: effect of genotype, Behav. Neural Biol. 42, 91-97. Smith, E.M., Meyer, W.J. and Blalock, J.E. (1982) Virus-induced corticosterone in hypophysectomized mice: a possible lymphoid adrenal axis. Science 218, 1311-2. Spackman, D.H. and Riley, V. (1978) Corticosterone concentrations in the mouse. Science 200, 87. Spengler, R.N., Allen, R.M., Remick, D.G., Strieter, R.M. and Kunkel, S.L. (1990) Stimulation of alpha-adrenergic receptor augments the production of macrophage-derived tumor necrosis factor. J. lmmunol. 145 (5), 1430-1434. Stone, E.A. (1975) Stress and catecholamines. In: Friedhof (Ed.), Catecholamines and Behavior, Vol. 2, Neuropsychopharmacology. Plenum Press, New York, NX/, pp. 31-72. Todd, G . L and Bernard, G.R. (1973) The sympathetic innervation of the cervical lymphatic duct of the dog. Ana. Rec. 177, 303-316. Wang, G.¥. and Zhong, S.Z. (1985) Experimental study of lymphatic contractility and its clinical importance. Ann. Plast. Surg. 15, 278-284.
Journal of Neuroimmunology 49 (1994) 25-33
Kinetics of glucocorticoid response to restraint stress a n d / o r experimental influenza viral infection in two inbred strains of mice Gerlinda Hermann .,a,b, C. Amy Tovar b, F. Michael Beck c, John F. Sheridan a,b a Department of Medical Microbiology and Immunology, Colleges of Medicine and Dentistry, The Ohio State University, Columbus, OH 43210, USA b Department of Oral Biology, Colleges of Medicine and Dentistry, The Ohio State University, Columbus, OH 43210, USA c Department of Endodontics, Colleges of Medicine and Dentistry, The Ohio State University, Columbus, OH 43210, USA (Received 28 May 1993) (Revision received 23 July 1993) (Accepted 23 July 1993)
Abstract
The murine model of influenza viral infection was used to evaluate the effects of restraint stress on pathogenesis and survival in inbred strains of mice. We recently reported that restraint stress was associated with an enhanced probability of survival in one strain of inbred mouse, DBA/2, and not in another, C57BL/6. Those studies suggested that the protective mechanism(s) of stress on mortality in the DBA/2 mice might be attributable to elevated levels of circulating glucocorticoids. Therefore, daily levels of plasma glucocorticoids were measured during influenza viral infection in both these strains. The present studies demonstrated that influenza infection itself is perceived as a stressor in both C57BL/6 and DBA/2 mice as evidenced by elevated plasma glucocorticoid levels within 48 h of infection. However, augmentation of glucocorticoid levels was not seen in the DBA/2 mice that were also subjected to restraint stress during the course of infection. Thus, corticosterone levels alone did not account for the enhanced survival seen in this group of animals.
Key words: Glucocorticoids; Restraint stress; Viral pathogenesis; Influenza virus; Strain differences
1. Introduction
Physiological responses to behavioral and psychological stressors are initiated by the hypothalamus and translated into action by the hypothalamic-pituitaryadrenal ( H P A ) axis and the sympathetic nervous system. Activation of H P A pathways results in secretion of hypothalamic corticotrophin-releasing factor (CRF), pituitary adrenocorticotrophic hormone ( A C T H ) and, ultimately, the release of glucocorticoid hormones from the adrenal cortex. Activation of the sympathetic nervous system provokes the release of catecholamines from the nerve terminal endings as well as the adrenal medulla. Co-activation of the cerebral noradrenergic and H P A systems has been observed with a variety of stressors such as restraint and electric footshock (Stone, 1975; D u n n et al., 1984, Anisman, 1987; Dunn, 1988).
* Corresponding author. Phone (614) 292 4305, Fax (614) 292 4888. 0165-5728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-5728(93)E0122-P
If such physiological responses are taken as a definition of stress, then infection with a viral pathogen is also considered a stressor. D u n n et al. (1988) demonstrated that infection of female B a l b / c mice with influenza virus PR8 resulted in an elevation of plasma corticosterone levels and co-activation of cerebral noradrenergic systems within 64 h following intranasal instillation of virus. Similar elevations in plasma corticosterone and central nervous system adrenergic systems have been elicited following administration of Newcastle disease virus to mice (Smith et al., 1982; D u n n et al., 1987). Natural and synthetic glucocorticoids inhibit a variety of lymphocyte functions and are successfully used clinically as anti-inflammatory and immunosuppressive agents (Claman, 1975; Fauci, 1979). Glucocorticoid actions on cytokines have been reviewed in detail (Munck et al., 1984; Munck and Guyre, 1991). Briefly, the propagation of both cellular and humoral immune responses is maintained by the cascading cytokine net-
26
G. Hermann et al. / Journal of Neurotmmunology 49 (1994) 25-33
work which is subject to regulation (i.e. suppression) by glucocorticoids at multiple sites. For example, glucocorticoid inhibition of cytokine synthesis by macrophages and T cells, ultimately, inhibits or retards T cell activation, clonal expansion, and antibody production (Munck and Guyre, 1991). Additionally, glucocorticoids can affect the recirculation or trafficking of lymphocytes. Corticosteroids provoke redistribution of circulating lymphocytes back to the bone marrow (Fauci, 1975), impair the influx of mononuclear cells from the blood into lymph nodes (Cox and Ford, 1982), and prevent the localization of sensitized effector lymphocytes into sites of active inflammation (Chung et al., 1986). Our previous study (Hermann et al., 1993) demonstrated that restraint stress reduced influenza viral-induced mortality in one inbred strain of mouse, the DBA/2. This difference in mortality associated with stress was not seen in either the C57BL/6 or C 3 H / HeN strains despite the fact that all three strains showed similar effects of restraint on the immunological and pathological responses examined during influenza infection. The only parameter measured that distinguished the restrained D B A / 2 group from the control groups was the elevated corticosterone levels at day 10 post-infection. Given that glucocorticoid effects on inflammatory reactions, cellular functions, and immune effector cell trafficking have been well-documented (e.g. Parrillo and Fauci, 1979; Cohen and Crnic, 1984; Munck et al., 1984; Munck and Guyre, 1991), it is possible that elevated levels of corticosterone during the development of the immune response to influenza viral infection modulated the inflammation and cellular infiltration of the lung so as to reduce the mortality in the restraint stressed D B A / 2 group. Genetic factors contribute to the expression of behavioral and neurochemical responses elicited by stressors. Royce and colleagues (1970) described differences in 'emotionality' or coping behaviors of ten inbred strains of mice in response to various situations. Differences in behavioral responses were reflected in different biochemical profiles including endogenous opioids and plasma corticosterones (for review see Oliverio et al., 1979; Ingram and Corfman, 1980; Siegfried et al., 1984; Shanks et al., 1990). For example, Shanks et al. (1990) demonstrated that although the basal levels of plasma corticosterone did not differ across six inbred strains of mice, the dynamics of plasma corticosterone levels in response to unavoidable footshock stress showed differences in magnitude as well as clearance rates among the different strains of mice. Our previous study (Hermann et al., 1993) revealed that two inbred strains of mice, C57BL/6 and DBA/2, showed differences in mortality and terminal (i.e. 10 days post-infection) plasma corticosterone levels associated with influenza viral infection in association with
restraint stress. However, in that study the daily profile of the glucocorticoid response to stress a n d / o r infection was not characterized. Therefore, the specific aims of this study were to examine the dynamics of the glucocorticoid response to behavioral regimens (i.e. repetitive cycles of 12 h food and water deprivation or 12 h restraint stress) a n d / o r influenza viral infection during the 10 days post-infection period and evaluate the development of the immune response in these two inbred strains of mice.
2. Materials and methods 2.1. Animals
Virus-antibody-free C57BL/6 and D B A / 2 male mice 4-8 weeks old were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Mice were housed five per cage in laminar flow cabinets and provided food and water ad libitum. All mice were maintained on a 12-h light/dark cycle (lights ON at 06:00) and were allowed to acclimate to these conditions for 1 week prior to experimentation. 2.2. Virus
Influenza A / P u e r t o R i c o / 8 / 3 4 (PR8) virus was obtained from the American Type Culture Collection (Rockville, MD) and propagated in the allantoic cavity of 10-day-old embryonated chicken eggs. Infectious allantoic fluid was collected, clarified by low-speed centrifugation, and stored at -70°C. The virus titer was determined to be 3200 hemagglutinating units (HAU) per ml, using human type 'O' erythrocytes. 2.3. Infection o f mice
The lethal dose 50 (LDs0) of influenza PR8 was estimated for each strain of mouse (C57BL/6 = 32HAU; D B A / 2 = 8HAU). Mice were lightly anesthetized with an intraperitoneal injection (40 ~1/10 g body weight) of a mixture of 0.4 m g / m l xylazine (Rompun@; Haver-Lockhart, Shawnee, KS) and 7.8 m g / m l ketamine (Ketaset@; Bristol Laboratories, Syracruse, NY). All animals were intranasally infected (50 tzl) with an appropriate LDs0 dose of PR8 virus using an Eppendorf pipette. Pre-infection serum samples were routinely screened for antibody to influenza virus to assure that mice were seronegative prior to experimentation. 2.4. Restraint protocol
Mice were restrained/isolated (RST) in well-ventilated 50 ml conical polypropylene tubes for 12 h each day (from 21:00 to 09:00) within their home cages. While in the tubes, the animals could move back and forth freely but could not turn around nor did they have access to food or water during this time period.
G. Hermann et aL /Journal of Neuroimmunology 49 (1994) 25-33
Therefore, similarly food- and water-deprived (FWD), non-restrained groups were used as a control group. A third group of mice (non-stressed, NS) was included to control for the effects that 12 h FWD had on circadian rhythm of circulating corticosterone levels, alone, as well as during the development of an immune response to viral infection. The restraint (RST) or food-water deprivation (FWD) regimen was initiated one day prior (day - 1 ) to virus infection and continued for 10 days post-infection. At the end of each RST or FWD cycle, mice were released from their restraint tubes and given free access to food and water for the remainder of the day; food and water was returned to the FWD groups. The NS groups had access to food and water 24 h per day.
2.5. Experimental design and blood sample schedule After the 1 week acclimation period, blood samples from all groups were drawn each morning at 10:00. To avoid excessively stressing the animals, each Infection/ Behavioral group had three subsets of mice which were bled (approximately 0.1 ml) once every 3 days via the retro-orbital plexus. The original distribution of animals per group was the same in both strains of mice: no infection/no stress (NI/NS); three subgroups n = 5 ( N = 15) infection/no stress (I/NS); three subgroups n = 8 ( N = 24) no infection/FWD ( N I / F W D ) ; three subgroups n = 5 ( N = 15) infection/FWD ( I / F W D ) ; three subgroups n = 8 ( N = 24) no in f ectio n /R S T ( N I / R S T ) ; three subgroups n = 5 ( N = 15) infection/RST ( I / R S T ) ; three subgroups n = 8 ( N = 24),
2.6. Detection of anti-influenza antibody by enzymelinked immunosorbent assay (ELISA) Anti-influenza IgG antibody was determined by an ELISA assay described in detail in our previous paper (Hermann et al., 1993). Briefly, influenza virus was bound to the wells of a polyvinyl microtiter plate. Aliquots of test serum were added to the plates and incubated overnight at 4°C. Peroxidase-conjugated goat anti-mouse IgG was added to the plates and influenza-specific antibody complexes were demonstrated by addition of substrate (0.1% H 2 0 2 plus 0.1% o-phenylene-diamine).
27
cycle and lowest levels coinciding with the inactive phase. Therefore, it was essential that all blood samples be obtained at the same time of day each day. Blood samples were drawn at 10:00 each day. Sera were collected and stored at -70° C until assayed for corticosterone by radioimmunoassay. [~25I]Corticosterone kits for rats and mice (ICN Biomedical, Costa Mesa, CA) were used to determine serum corticosterone levels. Corticosterone levels from individual mice were determined from a standard curve and expressed in ng/ml.
2.8. Determination of cell yield from mediastinal lymph nodes Single-cell suspensions of mediastinal lymph nodes were prepared (described in detail in Hermann et al., 1993) by macerating the tissue in Hanks' balanced salt solution. Samples were counted on a Coulter counter; cell yield per animal was determined.
2.9. Statistical analyses Serum corticosterone levels from the two strains of mice were analyzed separately according to which of the three behavioral groups they had been assigned. Differences were analyzed using factorial analysis of variance (ANOVA) with infection status and time (i.e. day post-infection) as the factors. A log transformation of the glucocorticoid data was necessary in order to stabilize the variance. When indicated by a significant F-test, post-hoc testing was done using the modified Tukey method. Differences in cell yield from the mediastinal lymph nodes also were analyzed separately for each strain using factorial analysis of variance (ANOVA) with infection status and behavioral group as the factors. A log transformation was again performed to stabilize the variance. Post hoc testing was done using the modified Tukey method. All analyses were done using SAS version 6.07.
3. Results
3.1. Determination of influenza-specific IgG titers All infected animals demonstrated IgG sero-conversion to influenza A / P R 8 based on an ELISA assay described above. On the day of sacrifice (day 11 p.i.), influenza-specific IgG titers within either mouse strain were quite similar with the exception being that the R S T / i n f e c t e d groups in both strains of mice showed a significantly lower titer (P < 0.05; data not shown).
2. 7. Determination of serum corticosterone levels Plasma levels of glucocorticoid hormones follow a predictable circadian variation in most species (reviewed in Krieger, 1979; Spackman and Riley, 1978; Cohen and Crnic, 1984;) with peak levels occurring as the animals enter their active phase of the light/dark
3.2. Effects of infection on basal corticosterone levels Daily basal corticosterone levels were obtained each morning at 10:00 from mice maintained in their home cage and not infected. Under this non-stressed, uninfected condition, both the C57BL/6 and D B A / 2
G. Hermann et al. /Journal of Neuroimmunology 49 (1994) 25-33
28
strains had stable average p l a s m a corticosterone levels (less t h a n 50 n g / m l ) across the 12 days of s a m p l i n g (Figs. 1A a n d 2A). A N O V A results i n d i c a t e d a significant effect ( P < 0.01) in b o t h strains for (a) infection, (b) day, a n d (c) the infection by day i n t e r a c t i o n (i.e. t h e r e was a n i n t e r a c t i o n b e t w e e n w h e t h e r or n o t the a n i m a l s were infected a n d o n which day p o s t - i n f e c t i o n the samples were o b t a i n e d ) . Daily c o r t i c o s t e r o n e levels of infected mice m a i n t a i n e d u n d e r identical h o u s i n g c o n d i t i o n s a n d b l e d at the same time of day as the above groups d e m o n strated that i n f e c t i o n a l o n e was sufficient to elevate p l a s m a c o r t i c o s t e r o n e levels. T w o distinct peaks of p l a s m a c o r t i c o s t e r o n e levels o c c u r r e d at day 2 a n d
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Fig. 1. Kinetics of corticosterone responses by C57BL/6 mice over
days post-infection with influenza A PR/8. Daily plasma corticosterone levels were determined from blood samples obtained at 10:00 each morning. Day ' - 1 ' represents the basal plasma corticosterone levels for each group (i.e. no behavioral treatment nor infection). Day '0' represents the morning after the first exposure to the assigned behavioral treatment (i.e. non-stressed, FWD, or RST). Mice were infected at 13:00 on Day '0'. Day '2' represents plasma corticosterone levels 45 h post-infection. Open symbols represent non-infected animals; closed symbols represent infected animals. Data presented as mean + SEM. (A) Kinetics of glucocorticoid response over days in mice maintained in home cages with free access to food and water 24 h per day, i.e. non-stressed. (B) Kinetics of GC response over days in mice maintained in home cages with restricted access to food and water from 21:00 to 09:00 each day, i.e. FWD. (C) Kinetics of GC response over days in mice maintained in home cages and placed in isolation/restraint tubes from 21:00 to 09:00 each day, i.e. RST.
days post-infection with influenza A PR/8. Daily plasma corticosterone levels were determined from blood samples obtained at 10:00 each morning. Day ' - 1' represents the basal plasma corticosterone levels for each group (i.e. no behavioral treatment nor infection). Day '0' represents the morning after the first exposure to the assigned behavioral treatment (i.e. non-stressed, FWD, or RST). Mice were infected at 13:00 on Day '0'. Day '2' represents plasma corticosterone levels 45 h post-infection. Open symbols represent non-infected; closed symbols represent infected animals. Data presented as mean_+SEM. (A) Kinetics of glucocorticoid response over days in mice maintained in home cages with free access to food and water 24 h per day, i.e. non-stressed. (B) Kinetics of GC response over days in mice maintained in home cages with restricted access to food and water from 21:00 to 09:00 each day, i.e. FWD. (C) Kinetics of GC response over days in mice maintained in home cages and placed in isolation/restraint tubes from 21:00 to 09:00 each day, i.e. RST. approximately day 7 - 8 post-infection. T h e C 5 7 B L / 6 strain showed the first p e a k of a n average 166 n g / m l at approximately 2 days post-infection. This elevated p l a s m a corticosterone level was m a i n t a i n e d u n t i l a second p e a k o c c u r r e d at day 7 post-infection (432 n g / m l average) (Fig. 1A). As the infection resolved, p l a s m a corticosterone levels again d e c l i n e d a n d leveled off to approximately 100 n g / m l o n the day the a n i m a l s were killed (i.e. day 11 post-infection). A similar profile of daily p l a s m a corticosterone levels was s e e n in the infected, n o n - s t r e s s e d D B A / 2 mice (Fig. 2A). W i t h i n 2 - 3 days post-infection, the first p e a k (303 n g / m l average) in p l a s m a corticosterone levels occurred. S u b s e q u e n t daily p l a s m a corticosterone levels d e c r e a s e d to approximately 200 n g / m l u n t i l the
G. Hermann et al. /Journal of Neuroimmunology 49 (1994) 25-33
second peak occurred at day 8 post-infection (343 n g / m l average). Again, as the infection resolved, plasma cortisterone levels declined and approached baseline (87 n g / m l average).
3.3. Effects of FWD on corticosterone levels associated with infection Daily basal corticosterone levels were obtained each morning at 10:00 from healthy and infected mice that were maintained in their home cage and subjected to repetitive 12-h periods of food and water deprivation (FWD). ANOVA results for the C57BL/6 strain revealed a significant effect for (a) day (P < 0.0001), (b) infection (P < 0.0001) and (c) the day by infection interaction (P = 0.0006). The D B A / 2 strain showed a significant effect for (a) day (P < 0.0001) and (b) infection (P < 0.0001) but not for (c) the day by infection interaction (P = 0.0694). Under this behavioral condition, both the C57BL/6 and D B A / 2 strains demonstrated elevated plasma corticosterone levels as early as the first morning (see day '0') following the FWD (Figs. 1B and 2B). The C57BL/6 strain demonstrated a peak at 363 n g / m l on day '0'. Over time, this FWD group appeared to habituate to this behavioral regimen such that by day 5, plasma corticosterone levels were down around 83 ng/ml. By the day of sacrifice, the C57BL/6 I / F W D group plasma corticosterone returned to an average 64 ng/ml, i.e. not sigificantly different from baseline levels (P > 0.05). The daily profile of plasma corticosterone in inf e c t e d / F W D C57BL/6 mice demonstrated an additional increase in plasma corticosterone levels on day 2 post-infection and, otherwise, resembled the profile seen in the infected/non-stressed groups described above. The first peak (329 n g / m l average) occurred on day 2 post-infection. Although there was a slight decline in plasma corticosterone levels, a second peak (386 n g / m l ) occurred around day 8. As the infection resolved, plasma corticosterone levels continued to drop such that, on the day of sacrifice, there was no significant difference (P > 0.05) between the infected or healthy FWD groups (Fig. 1B). The healthy (i.e. uninfected), FWD D B A / 2 group also showed a marked elevation in plasma corticosterone levels on day '0' (133 n g / m l average). By day '3', plasma corticosterone levels peaked at 226 ng/ml. Although the healthy, FWD D B A / 2 group showed some habituation to the FWD schedule, they remained somewhat stressed by this behavioral regimen as indicated by the relative stability of the elevated plasma corticosterone levels seen right up to the day of sacrifice (140 n g / m l average; Fig. 2b). Infected/FWD groups of D B A / 2 mice demonstrated an additional increase in plasma corticosterone
29
levels on day 2 post-infection. Indeed, a similar daily plasma corticosterone profile was observed in the inf e c t e d / F W D groups as that seen in the infected/ non-stressed groups described in the preceding section. The first peak (362 n g / m l ) occurred on day 2 post-infection. Over successive days, there was a slight decline in plasma corticosterone levels. However, a second peak (376 ng/ml) occurred around day 8 post-infection. Again, as the infection resolved, plasma corticosterone levels continued to drop such that, on the day of sacrifice, there was a minimal difference between the infected or healthy FWD groups of DBA/2 mice (Fig. 2B).
3.4. Effects of R S T on corticosterone levels associated with infection Figures 1C and 2C illustrate that repetitive exposure to 12 h of restraint/isolation was sufficient to elicit an elevated glucocorticoid response in both C57BL/6 and D B A / 2 healthy strains of mice. ANOVA results for the C57BL/6 strain demonstrated a significant effect for (a) day (P < 0.0001) and (b) the day by infection interaction (P = 0.0006) but not (c) infection (P = 0.2702). The D B A / 2 strain revealed a significant effect for (a) day (P < 0.0001) and (b) infection (P = 0.0005) but not for (c) the day by infection interaction (P = 0.1256). In both strains, elevation of plasma corticosterone levels was quite pronounced following the first exposure to this behavioral regimen (i.e. 327 n g / m l average in C57BL/6 and 420 n g / m l average in DBA/2). The healthy C57BL/6 groups showed eventual habituation to repetitive cycles of restraint; plasma corticosterone level on the day of sacrifice averaged 125 ng/ml. Although the healthy D B A / 2 groups demonstrated maximal corticosterone elevation on day '0' and plasma corticosterone levels declined quickly, they remained relatively stable around 200 n g / m l until the day of sacrifice. Infection of C57BL/6 mice resulted in an augmentation of the increase in plasma glucocorticoids associated with restraint/isolation treatment. As seen in Fig. 1C, the basic corticosterone response profile associated with infection was still present. However, the first peak at days 2-3 was amplified to 700 ng/ml; the second peak at clay 8 post-infection was 491 ng/ml. In contrast to the infected/RST, C57BL/6 mice, infection of D B A / 2 mice resulted in similar plasma corticosterone profiles seen in either healthy D B A / 2 mice subjected to the restraint regimen (Fig. 2C) or infected D B A / 2 maintained in their home cage environment (Fig. 2A). Specifically, similar levels of plasma corticosterone were seen on day '0' in the healthy versus infected RST D B A / 2 groups (420 n g / m l vs. 345 ng/ml, respectively); on day '2' i n f e c t e d / N S and infected/RST groups were similar (235 n g / m l vs. 233
G. Hermann et al. / Journal of Neuroirnmunology 49 (1994) 25-33
30
Average cell yield from mediastinal
Average cell yield from mediastinal lymph nodes (C57BL/6; 10 days p.i.)
lymph nodes (DBM2; 10 days p.i.)
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Fig. 3. Effects of behavioral treatment a n d / o r infection on average cell yield from mediastinal lymph nodes. Nodes were taken from all groups on 'day 10 post-infection'. In the healthy state, behavioral treatments alone did not alter cell number within these nodes in either strain of mouse. Both C57BL/6 and D B A / 2 mice responded with a significant increase in cell number in association with influenza infection (non-infected/NS vs. infected/NS conditions; P < 0.05 ,a). Infected mice subjected to repetitive cycles of restraint (RST) showed a suppression of this lymphadenopathy associated with influenza infection. This suppression was statistically significant for the C57BL/6 group (infected/NS vs. infected/RST) and the D B A / 2 group (infected/FWD vs. infected/RST; P < 0.05 ,b).
n g / m l , respectively); day '3', all infected groups were the same ( I / N S = 303 ng/ m l ; I / F W D = 265 ng/ m l ; I / R S T = 359 ng/ml). Likewise, the plasma corticosterone levels of all infected groups were, essentially, the same on day 9 ( I / N S = 343 ng/ m l ; I / F W D = 376 n g /ml; I / RST --- 301 ng/ml). In other words, there was no significant interaction between the infection and behavioral treatment in terms of plasma corticosterone levels in the D B A / 2 mice.
3.5. Effects of RST on cell yield from medistinal lymph nodes (MLN) The mediastinal lymph nodes drain the respiratory tract. Changes in cell number of these nodes can be used as an index of the lymphadenopathy that is normally associated with infection of the respiratory tract. It is important to note that in the healthy state, both strains average approximately 1.5 x 106 cells per mediastinal lymph node. Behavioral treatments alone did not alter cell number within these nodes in either strain of mouse (Fig. 3A, B). However, both C57BL/6 and D B A / 2 strains responded with a significant (P < 0.05) increase in cell number in association with influenza infection in the infected/NS condition versus the non-infected/NS condition. Indeed, despite the different approximate LD50 doses of influenza A / P R 8 required to infect the two different strains, similar magnitudes in average cell yield from mediastinal lymph nodes were observed (C57BL/6 = 6.4 × 106; D B A / 2
= 6.6 X 106). Although the average cell yield in the i nfect ed/ FWD groups (both C57BL/6 and D B A / 2 ) appeared to be different from the infected/NS groups of the same strain (C57BL/6 = 4.0 X 106; D B A / 2 = 9.8 >(106), these were not statistically significant differences (P > 0.05). Infected mice subjected to repetitive cycles of restraint (RST) showed a suppression of this lymphadenopathy associated with influenza infection (Fig. 3). That is, cell density of the draining nodes was suppressed in the i n f e c t e d / R S T groups of both strains when compared with the infected/NS group of the C57BL/6 and infected/FWD group of the D B A / 2 strain (P < 0.05). These results are similar to our previous study (Hermann et al., 1993), which demonstrated that RST also reduced the average cell yield from the superficial cervical lymph nodes of infected mice by approximately 25% in both strains of mice.
4. D i s c u s s i o n
These studies demonstrated that the pathogenic process of influenza viral infection a n d / o r development of the immune response was sufficient to cause an elevation in plasma corticosterone levels in both strains of mice. Within 48 h of infection, even in the absence of any other imposed behavioral treatment, plasma corticosterone levels were elevated. Thus, some 'afferent' signal associated with the infection a n d / o r
G. Herrnann et al. /Journal of Neuroimmunology 49 (1994) 25-33
the immune response activated the hypothalamic-pituitary-adrenal axis (HPA). In the murine experimental influenza viral infection model, virus titer peaks occur between 48 and 72 h post-infection (Hennet et al., 1991). Within a few hours post-infection, products of activated macrophages, i.e. IL-6, IL-1 and TNFa are detectable in bronchoalveolar lavage fluid (Hennet et al., 1992). Thus, any of these soluble cytokines could act as potential signals to activate the HPA axis. A secondary peak in plasma corticosterone levels was seen around day 7-8 p.i., in both strains of mice. Peak mononuclear infiltration of lung tissue occurs around this time and contributes to the immunopathology (Beck and Sheridan, 1989; Sheridan et al., 1991; Hennet et al., 1992). At this point in the infection, respiration appeared labored and stressful, and may have been sufficient to account for the second peak in circulating corticosterone levels. As resolution of the infection continued, plasma levels of corticosterone declined (Figs. 1A and 2A). Clearly, behavioral treatments alone (either FWD or RST) provoked elevations in corticosterone levels in healthy mice of both strains (Figs. 1B,C and 2B,C). Over time, the animals seemed to habituate to the regimen, as expressed in plasma corticosterone levels, but in the D B A / 2 strain, circulating corticosterone amounts did not return to basal levels even after exposure to 12 cycles of the behavioral regimen; mean plasma corticosterone levels were well in excess of 100 n g / m l even on the final day sampled (P < 0.05). In the C57BL/6 mouse strain, the two peaks of corticosterone levels associated with the viral influenza infection were differentially affected by restraint stress (RST; Fig. 1C). The first peak, occurring at day 2 p.i., was augmented by the imposed RST; while the second peak, at day7-8 p.i., was not. It is possible that there is a synergism of activation of the HPA axis during the early phases of infection/immune response (i.e. soluble cytokine signals) and the stress of restraint. According to work by Hennet et al. (1992), levels of the pro-inflammatory cytokines (i.e. IL-1, IL-6, TNFa) have greatly declined by this late period of the infection. This second peak in plasma corticosterone levels was of comparable magnitude as that seen with infection alone (infection/NS), or infection/FWD groups (Fig. 1A, B). Thus, it appears to be a reflection of the immunopathology that was occurring, with the behavioral treatment having little or no impact on the activation of the HPA axis at this time. In contrast, this differential effect of RST on the two peaks of plasma corticosterone levels was NOT seen in the D B A / 2 mouse strain. Instead, development of the infection and immune response appeared to be the primary determining factors of corticosterone levels at either peak in the D B A / 2 strain, regardless of the behavioral treatment imposed. That is, there was
31
no significant interaction of infection and behavior (Fig. 2C). Indeed, the kinetics of the plasma glucocorticoid levels of the infected D B A / 2 mice were quite similar across all three behavioral treatments. Despite the lack of difference in plasma corticosterone response profiles of the NS, FWD, or RST/infected D B A / 2 groups, we have seen differences in mortality following influenza infection associated with RST in the D B A / 2 strain (Hermann et al., 1993; and in this study, data not shown). Therefore, some physiological parameter associated with restraint stress, other than glucocorticoids, may be responsible for this difference in survival of the infected D B A / 2 mice. Similar to our previous studies (Feng et al., 1991, 1992; Hermann et al., 1993), changes in cell number within the draining lymph nodes (e.g. mediastinal lymph nodes) normally associated with infection of the respiratory tract were seen when the animals from either strain were exposed to RST. It is important to note that behavioral treatments alone did not alter cell number within these nodes in either strain of mouse (Fig. 3). Both C57BL/6 and D B A / 2 strains responded with a significant increase in cell number in association with influenza infection in the infected/NS condition despite the different approximate LDs0 doses of influenza A / P R 8 required to infect the two different strains. Suppression of the increased cell number normally associated with influenza infection was observed when the mice of either strain were subjected to repetitive cycles of restraint/isolation (RST). Glucocorticoids have been credited with a role in trafficking or the redistribution of lymphocytes as well as controlling edema and inflammation (reviewed in Munck and Guyre, 1991; Claman, 1975; Fauci, 1975, 1979; Cox and Ford, 1982; Chung et al., 1986). The studies presented here support these concepts by showing an association between plasma corticosterone levels and distribution of lymphocytes in infected/RST C57BL/6 mice as reflected in the cell density of the draining lymph nodes. However, in the D B A / 2 inbred mouse strain, the diminished cell density, reduction in mortality, and reduction in pathosis (as seen in this study and our previous study, Hermann et al., 1993) cannot be attributed solely to plasma corticosterone levels. Responses to stressors are initiated by the hypothalamus and translated into action by the hypothalamicpituitary-adrenal axis and the autonomic nervous system. Products from both of these systems (e.g. corticosteroid hormones and catecholamines) are able to modulate the activity of various immune effector cells directly (see reviews by Livnat et al., 1985; Ader et al., 1990; Besedovsky and del Rey, 1991; Madden and Livnat, 1991; Munck and Guyre, 1991) as well as alter the general physiology of the animal. There is extensive documentation of functional adrenergic receptors on
32
G. Hermann et al. /Journal of Neuroimmunology 49 (1994) 25-33
m a c r o p h a g e s which c a n modify the p r o d u c t i o n a n d / o r secretion of t u m o r necrosis factor ( T N F ) a n d lymphocyte activating factors ( L A F ) (e.g. S p e n g l e r et al., 1990; H u et al., 1991). A n a p p r e c i a t i o n for the roles that these cytokines play in d e v e l o p m e n t of the cascading i m m u n e r e s p o n s e d e m o n s t r a t e s how c a t e c h o l a m i n e s m a y i n d u c e p o t e n t i m m u n o r e g u l a t o r y a n d imm u n o p a t h o l o g i c a l effects. A d d i t i o n a l l y , a d r e n e r g i c inn e r v a t i o n of the v a s c u l a t u r e a n d lymphatic ducts ( T o d d a n d B e r n a r d , 1973; W a n g a n d Z h o n g , 1985; M c H a l e , 1990) may play a critical role in r e c i r c u l a t i o n or distrib u t i o n of lymphocytes. T h u s , the a n i m a l has at least two pathways (i.e. the h y p o t h a l a m i c - p i t u i t a r y - a d r e n a l axis a n d the sympathetic n e r v o u s system) which, ultimately, may affect the n u m b e r , distribution, or state of activation of i m m u n e effector cells d u r i n g stress. T h e s e two pathways p r o b a b l y work cooperatively to try to m a i n t a i n , or regain, homeostasis. T h e results of the p r e s e n t studies suggest that co-activation of the sympathetic n e r v o u s system a n d the H P A axis is responsible for the c h a n g e s in mortality, pathology, s u p p r e s s e d lymphadenopathy, and diminished immune response s e e n in the D B A / 2 i n f e c t e d / R S T mice. T h e r e f o r e , f u t u r e studies will investigate the possible role of the s y m p a t h e t i c n e r v o u s system d u r i n g the d e v e l o p m e n t of the i m m u n e r e s p o n s e to i n f l u e n z a viral infection in the D B A / 2 i n b r e d m o u s e strain.
Acknowledgements W e t h a n k Dr. A.C. G r i f f i n for t h o u g h t f u l discussion a n d critical review of this m a n u s c r i p t . This work was s u p p o r t e d in part by grants to J.F.S. from N I H (HL38485) a n d N I M H (MH46801) a n d a n N I M H Postdoctoral T r a i n i n g G r a n t fellowship to G.H. (MH18831).
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Siegfried, B., Frischknecht, H.-R. and Waser, P.G. (1984) Defeat, learned submissiveness, and analgesia in mice: effect of genotype, Behav. Neural Biol. 42, 91-97. Smith, E.M., Meyer, W.J. and Blalock, J.E. (1982) Virus-induced corticosterone in hypophysectomized mice: a possible lymphoid adrenal axis. Science 218, 1311-2. Spackman, D.H. and Riley, V. (1978) Corticosterone concentrations in the mouse. Science 200, 87. Spengler, R.N., Allen, R.M., Remick, D.G., Strieter, R.M. and Kunkel, S.L. (1990) Stimulation of alpha-adrenergic receptor augments the production of macrophage-derived tumor necrosis factor. J. lmmunol. 145 (5), 1430-1434. Stone, E.A. (1975) Stress and catecholamines. In: Friedhof (Ed.), Catecholamines and Behavior, Vol. 2, Neuropsychopharmacology. Plenum Press, New York, NX/, pp. 31-72. Todd, G . L and Bernard, G.R. (1973) The sympathetic innervation of the cervical lymphatic duct of the dog. Ana. Rec. 177, 303-316. Wang, G.¥. and Zhong, S.Z. (1985) Experimental study of lymphatic contractility and its clinical importance. Ann. Plast. Surg. 15, 278-284.