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Stress-induced glucocorticoid response modulates mononuclear cell trafficking during an experimental influenza viral infection
Journal of Neuroimmunology
ELSEVIER
Journal
of Neuroimmunology
56 (1995) 179-186
Stress-induced glucocorticoid response modulates mononuclear cell trafficking during an experimental influenza viral infection Gerlinda Hermann a,b,*, F. Michael Beck ‘, John F. Sheridan a,b a Department of Medical Microbiology and Immunology, College of Medicine, The Ohio State University, Columbus, OH 43210, USA ’ Department of Oral Biology, College of Dentistry, The Ohio State University, Columbus, OH 43210, USA ’ Department of Endodontics, College of Dentistry, The Ohio State University, Columbus, OH 43210, USA Received
8 March
1994; revised
1 September
1994; accepted
14 September
1994
Abstract The migration, distribution, and localization of lymphoid cells throughout the body is critical to the efficiency and development of the immune response. This study examined the role of endogenous glucocorticoids in mononuclear cell (MNC) trafficking during the development of an immune response to infection by influenza A/PR8 virus. Accumulation of MNC in the draining lymph nodes and at the site of virus replication (lungs) was studied in infected mice, and infected mice subjected to a stressor (physical restraint). The glucocorticoid antagonist, RU486, was used to block the activity of endogenous corticosterone during development of the immune response. PR8-infected mice demonstrated an elevation in circulating corticosterone regardless of whether they were treated with RU486 or a placebo. Thus, some ‘afferent’ signal associated with the infection, and/or the immune response to infection, activated the hypothalamic-pituitary-adrenal axis (HPA) and was not subject to negative feedback regulation. The initial accumulation of MNC in the draining lymph nodes and lungs during infection, however, was independent of the glucocorticoid response. Our previous studies demonstrated that virally infected animals subjected to physical restraint had highly elevated plasma corticosterone levels, suppressed lymphadenopathy, and reduced accumulation of MNC in the lungs. In the present study, RU486 treatment restored cellularity to the draining lymph nodes and enhanced accumulation of MNC in lungs of stressed, A/PR8 virus-infected mice. Keywords:
Stress ; Influenza
virus; Glucocorticoids;
RU486; Inflammation
1. Introduction
A principal determinant of the effectiveness of the immune system is opportunity for antigen to meet antigen-specific lymphocytes. The migration, distribution, and localization of lymphoid cells throughout the body is critical to the efficiency and development of the immune response. Ottaway and Husband (1992) have recently reviewed the multiple means by which the central nervous system (CNS) influences systemic as well as local lymphocyte migration. Two of these neurally initiated processes include: (i> sympathetic inner-
* Corresponding author. Present address: Physiology Department, College of Medicine, 1645 Neil Avenue, Ohio State University, Columbus, OH 43210, USA. Phone (614) 292 4305; Fax (614) 292 4888. 01655728/95/$09.50 0 1995 Elsevier SSDI 0165-5728(94)00145-6
Science
B.V. All rights reserved
vation of the lymphoid tissue and altered hemodynamits; and (ii) activation of the hypothalamic-pituitaryadrenal axis. Given that responses to stressors are initiated by the hypothalamus and translated into action by the hypothalamic-pituitary-adrenal axis and the autonomic nervous system, these two pathways are available for modulating neurophysiological, neuroendocrine, and immune responses. Previous studies from our laboratory demonstrated that restraint stress, which elevated plasma corticosterone and tissue catecholamine levels, also reduced cellular accumulation and inflammation in target organs associated with pathogenic viral infections (Bonneau et al., 1991; Feng et al., 1991, Feng, 1992; Sheridan et al., 1991; Hermann et al., 1993). Restraint stress was found to suppress normal lymphadenopathy in lymph nodes draining the site of infection (Feng et al., 1991; Dobbs et al., 1993) and reduce mononuclear
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of Neuroimmunology 56 (1995) 179-186
cellular accumulation in the infected tissue (Hermann et al., 1993). In a murine HSV-1 viral infection, we demonstrated that blocking corticosterone activity (by treatment with the drug RU486) restored the cellularity of lymph nodes draining the site of viral replication in restraint-stressed animals (Dobbs et al., 1993). While RU486 has been shown to have a number of pharmacological activities, it is a potent glucocorticoid receptor antagonist (Moguilewsky and Philibert, 1984) that blocks HPA axis activity in stressed animals without interfering with basal HPA activity under nonstressed conditions (Ratka et al., 1989). Thus, the observation that RU486 restored the cellularity to draining lymph nodes in stressed mice suggested that a corticosterone response was responsible for suppression of lymphocyte accumulation. However, we did not examine the effect of RU486 treatment on the inflammatory response at the site of viral replication in previous studies (Dobbs et al., 1993). The ability of an experimental stressor to modulate immunity, viral pathogenesis and health outcomes may be related, in part, to changes in the migration, distribution, and localization of lymphocytes mediated by CNS pathways (see review by Ottaway and Husband, 1992). Lymphadenopathy, or its suppression, may be influenced by at least two different mechanisms. For example, glucocorticoids are known to modulate the expression of adhesion molecules, thus playing a critical role in changing trafficking patterns of lymphocytes as they are activated and become ‘memory’ cells (Cox and Ford, 1982; Chung et al., 1986; for reviews see Munck et al., 1984; Munck and Guyre, 1991). However, given that blood vessels and lymphatic ducts are innervated by adrenergic fibers, activation of the sympathetic nervous system might have an indirect, but significant, effect on blood flow dynamics and localization of lymphocytes. The specific aim of this study was to determine the role of stress-induced glucocorticoids in the accumulation of leukocytes at sites of inflammation and in draining lymph nodes. Therefore, the glucocorticoid antagonist, RU486, was used to block the activity of endogenous corticosteroids during an experimental influenza infection in the murine model. Subsequent changes in lymphadenopathy, corticosterone levels, catecholamine levels, and lung pathology were monitored.
2. Methods and materials 2.1. Animals
Virus-antibody-free C57BL/6 male mice 4-8 weeks old (n = 68) were purchased from Harlan SpragueDawley (Indianapolis, IN). Mice were housed four or
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:OO) and were allowed to acclimate to these conditions for 1 week prior to experimentation. Mice were randomly assigned to one of two drug treatment groups, RU486 or placebo (see details below). Within each drug group, four mice were assigned to the control group (CON) which was neither infected nor stressed; ten mice were assigned to the infected, non-stressed condition (NS); ten mice were assigned to the infected, food-water deprivation condition (FWD); and ten were assigned to the infected, restraint-stressed condition (RST). 2.2. RU486 The glucocorticoid receptor antagonist, RU486 (17b-hydroxy-llb-(4-dimethylaminophenyl)-17a-(l-propynyl)-estra-4,9-dien-3-one; gift from Roussel-UCLAF, Romainville, France), was dissolved in polyethylene glycol 400 (PEG; M, 400) at a concentration of 5 mg/ml. Mice received daily subcutaneous injections of either RU486 (25 mg/kg; 0.1 ml volume (Ratka et al., 1989)) or an equivalent volume of PEG 400 (vehicle control groups) approximately 1 h prior to the start of each RST cycle. This dose of drug previously was shown to be effective in restoring lymphadenopathy in stress animals (Dobbs et al., 1993). Daily injections began 2 days prior to infection and continued throughout the development of the immune response. 2.3. Restraint protocol One group of infected mice (RST) was restrained during their active period each day (from 21:00 to 09:OO) in well-ventilated 50-ml conical polypropylene tubes within their home cages. While in the tubes, the animals could move back and forth freely, but could not turn around. They did not have access to food or water during this time period. Therefore, a second group of infected mice (FWD) was similarly food- and water-deprived, but not restrained, to control for the effects of restraint. A third group of infected mice (NS) served as a home cage control and was not subjected to either RST or FWD. In some experiments, an uninfected control group was added (CON) that was not subjected to RST or FWD. These groups were included to compare the effects of the drugs alone. RST or FWD regimens were initiated 1 day prior (day - 1) to infection and continued for 10 days postinfection (p.i.1. 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 and CON groups had access to food and water 24 h per day.
G. Hermann et al. /Journal
of Neuroimmunology 56 (I 995) 179-186 Effects of RU486 and infection CORT levels on day 7
2.4. Virus and infection of mice
Influenza A/Puerto Rico/g/34 (A/PR8) virus was obtained from the American Type Culture Collection (Rockville, MD); propagation and determination of virus titer have been described (Feng et al., 1991; Sheridan et al., 1991). The lethal dose 50 (LD,,) of influenza A/PRS for C57BL/6 mice was estimated to be 32HAU. Intranasal (i.n.> infection of mice occurred on day ‘0’ and proceeded as previously described (Hermann et al., 1993, Hermann et al., 1994a, Hermann et al., 1994b).
181
plasma
on p.i.
600 s 2 2 !z
400
a B ._ r s
200
n uninfected q infected
0
Blood samples were drawn at 10:00 on the day of sacrifice (day 7 p.i.1. Sera were collected and stored at -70°C until assayed for corticosterone by radioimmunoassay. [‘251]Corticosterone kits for rats and mice (ICN Biomedical, Costa Mesa, CA) were used to determine serum corticosterone (CORT) levels (as described in Hermann et al., 1993). CORT levels from individual mice were determined from a standard curve and expressed in ng/ml. 2.6. Determination of cell yield from mediastinal lymph nodes Single-cell suspensions of mediastinal lymph nodes were prepared by macerating the tissue in 500 ~1 volume of Hanks’ balanced salt solution. Samples were counted on a Coulter counter (Coulter Diagnostics); cell yield per animal was determined as previously described (Hermann et al., 1994a).
Fig. 1. Influenza A/PRS viral infection alone was sufficient to elevate plasma CORT levels on day 7 p.i., regardless of whether the mice had been treated with RU486 or placebo ( * P < 0.0001; * * P = 0.0097).
norepinephrine (NE) levels were assessed using independent r-tests (two-tailed). Log transformation of the data for each of these variables was necessary in order to stabilize the variance. All analyses were done using SAS version 6.07.
3. Results 3.1. Effects of RU486 on plasma corticosterone levels
Mice infected i.n. with 32 HAU of influenza A/PR8 virus developed a respiratory tract infection that was
Effects of RU486, infection, and/or stress on cell number of draining lymph nodes
2.7. Histology The upper portion of the left lobe of the lung was obtained from half of the surviving animals in each group. Lung samples were prepared for histological examination and stained with Hematoxylin and Eosin as previously described (Hermann et al., 1993).
not infected
2.8. Catecholamine determinations >-
2.9. Statistical analysis Between-group differences in plasma glucocorticoid level, cell numbers within draining lymph nodes, and
infected
* ll!kIi L
I-
Spleens were quickly removed at time of sacrifice and placed on dry ice to halt degradation of catecholamines. Tissue was stored at -70°C until time of extraction. Catecholamine extraction and quantitative analysis were as described previously (Hermann et al., 1994b).
RU486
placebo
2.5. Determination of serum corticosterone levels
ICON
NS
WD
0
PLACEBO
n
RU466
RST
Fig. 2. Similar to our previous experiments, influenza A/PR8 viral infection was sufficient to increase the cell density in mediastinal lymph nodes at day 7 pi. (regardless of whether the mice had been treated with placebo or RU486) and restraint stress (RST) reduced the cell number in the placebo-treated, infected mice (open bars). In contrast, infected mice treated with RU486 (closed bars) and subjected to restraint stress (RST) showed an abrogation of this suppression in cell number (’ P < 0.05). CON, uninfected; NS, infected, nonstressed; FWD, infected/food-water-deprived: RST, infected/restraint-stressed.
182
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of Neuroimmunology 56 (199.5) 179-186
Fig. 3. RU486 treatment restored cellular infiltration in the lungs of restrained, influenza A/PR8 virus infected mice. All samples were obtained 7 days pi. (A) Lung tissue from uninfected, normal C57B1/6 mouse; note the paucity of cells in the pulmonary tissue, relative thinness of the septal walls, and the regularity of the bronchial epithelial cells. (B) Lung tissue from A/PRB virus-infected mouse; note the intensity of the cellular infiltration, swelling of the septal walls, and disarray of the bronchial epithelial cells. (C) Lung tissue from FWD, A/PR8 virus-infected mouse. (D) Lung tissue from RST, A/PR8 virus-infected mouse; note the swelling of the septal walls, disarray of the bronchial epithelial cells, but relative lack of cellular infiltration. (E) Lung tissue from uninfected normal mouse that received daily subcutaneous injections of RU486; note the thickness of the septal walls and relative increase in cellularity. (F) Lung tissue from A/PRI virus-infected mouse treated with RU486. (G) Lung tissue from A/PRS virus-infected mouse, FWD, and treated with RU486. (HI Lung tissue from A/PR8 virus-infected mouse, RST, and treated with RU486; note the swelling of the septal walls, disarray of the bronchial epithelial cells, and the intensity of the cellular infiltration. Magnification 100 x for all photomicrographs.
G. Hermann et al. /Journal
of Neuroimmunology 56 (1995) 179-186
183
Fig. 3 (continued).
sufficient to elevate plasma CORT levels (Fig. 1) regardless of whether the mice were treated with RU486 or placebo (placebo, P < 0.0001; RU486, P = 0.0097). Similar to our previous study (Hermann et al., 1994a), on day 7 pi. RST did not further augment plasma CORT levels in either the placebo- or RU486-treated animals (placebo, P = 0.2795; RU486, P = 0.6113; data not shown).
3.2. Effects of RlJ486 lymph nodes
on cell number
of mediastinal
In response to an influenza A/PR8 viral infection in the respiratory tract regional draining mediastinal lymph nodes increased their cell density (Fig. 21, regardless of whether the mice were treated with RU486 or placebo. Our previous studies (Hermann et al., 1993, 1994a,b; Feng et al., 1991; Sheridan et al., 19911, which examined lymph nodes at day 10 post-infection, not only showed the same lymphadenopathy with infection, but also demonstrated that RST significantly suppressed this increase in cell number. In the present study, cell density was examined 7 days p.i. Lymph nodes from uninfected control mice (CON) averaged 1.4 x 106, while lymph nodes from A/PR8 virus-infected mice (NS) averaged 4.4 X 106. RST reduced the average cell density to 2.1 X lo6 ceils/node in A/PR8 virus-infected, placebo-treated mice (placebo-treated, infected/RST vs. RU486-treated, infected/RST; P < 0.05). In contrast, A/PR8 virus-infected mice treated with RU486 had very similar mediastinal lymph node cell densities regardless of the behavioral treatment, i.e. NS, FWD, or RST. Thus, drug treatment with a glucocorticoid receptor antagonist restored lymphadenopathy in the draining lymph nodes of restrained, A/PR8 virus-infected mice.
3.3. Effects of RU486 on pulmonary inflammation and pathosis In addition to suppressing mononuclear cellular accumulation in the draining lymph nodes, RST has been associated with diminished cellular infiltration in the lungs of A/PR8 virus-infected mice (Hermann et al., 1993, 1994a,b; Feng et al., 1991). To determine the role of CORT in reducing inflammatory responses in the lung, mice were treated with RU486 during infection. In the present study, infection with A/PR8 virus resulted in histopathological changes in the lungs which included swelling of the septal walls, disorganization of the bronchial epithelial cells, and mononuclear cellular infiltration (Fig. 3A, B). RST diminished cellular infiltration in the lungs of A/PR8 virus-infected mice (Fig. 3D1, while FWD had no effect (Fig. 30 Cellular
Effects of RU466, infection, and/or stress on splenic NE content in C57BU6 mice (day7 p.i.)
6
F 2
1.o
.-t i 8 .c it z z
0
placebo
n
RU486
0.5
0.0 CON
NS
f=WD
RST
Fig. 4. Treatment with RU486 was associated with a decrease in tissue catecholamine. Splenic norepinephrine levels in RU486-treated mice were diminished compared to placebo-treated mice across all behavioral treatments. These differences were statistically significant for the CON groups (* P = 0.0033) and the RST groups (* * P = 0.0015).
184
G. Hertnann et al. /Journal
of Neuroimmunology 56 (1995) 179-186
infiltration was restored in the lungs of restrained, A/PRS virus-infected mice by treatment with RU486 (Fig. 3H). In fact, all infected mice treated with RU486 demonstrated a high density of mononuclear cells in the lung tissue (Fig. 3F, G, H). 3.4. Effects of RU486 on splenic catecholamine content In addition to elevating plasma CORT in A/PR8 virus-infected mice, multiple cycles of RST have been shown to raise tissue catecholamine levels (Hermann et al., 1994b). However, in this study, treatment with RU486 was associated with a decrease in splenic norepinephrine (Fig. 4) compared to splenic norepinephrine content in placebo-treated mice across all behavioral groups. These differences were statistically significant for the uninfected (CON) groups (P = 0.0033) and the RST groups (P = 0.0015).
4. Discussion RU486 is a synthetic steroid analog which acts as a potent glucocorticoid receptor antagonist in vitro and in vivo (Gagne et al., 1985). The results presented here indicate that basal levels of plasma corticosterone were not affected by treatment with RU486. Ratka et al. (1989) have also reported that RU486 did not influence basal, non-stressed levels of CORT in the rat, although pretreatment with RU486 slightly reduced the elevation of plasma CORT normally seen in rats exposed to a novel environment. These different effects were attributed to two receptor types (type I, or mineralocorticoid receptors, and type II, or glucocorticoid receptors) within the brain with markedly different affinities for CORT (Rem and de Kloet, 1985; Reul et al., 1988; Spencer et al., 1990). In the studies presented here, influenza A/PR8 virus-infected mice had elevated circulating CORT levels regardless of whether they were treated with RU486 or placebo. Thus, some ‘afferent’ signal associated with viral replication, or the host’s response to infection, activated the hypothalamic-pituitary-adrenal axis (HPA). In studies by Hennet and colleagues (Hennet et al., 1991, 19921, proinflammatory cytokine responses were detected in the lung lavage samples of A/PR8 virus-infected mice within a few hours of infection. These cytokines, which included IL-l, IL-6, and TNF-(u, are known to stimulate the HPA axis (Sweep et al., 1991) and might be responsible for elevated CORT levels in an influenza viral infection. Furthermore, CORT secretion in response to influenza infection was not subject to negative feedback regulation by either of the above-mentioned receptor types as evidenced by the elevated CORT levels found in all the infected, RU486-treated mice.
Increases in size, weight, and cell density of draining lymph nodes, i.e. lymphadenopathy, is normally associated with infection. The present studies demonstrated that the initial accumulation of cells in the draining lymph nodes is independent of corticosterone in that all A/PR8 virus-infected, non-stressed animals showed a significant increase in cell number regardless of whether they had been treated with RU486 or placebo. Our previous studies (Feng et al., 1991, Feng, 1992; Hermann et al., 1993, 1994a,b) demonstrated reduced cell densities in A/PR8 virus-infected mice exposed to RST. This phenomenon has been attributed to an elevation of CORT associated with the stress and its effects on lymphoid cell trafficking (see reviews by Munck et al., 1984; Munck and Guyre, 1991). The results presented in this study suggest that suppression of lymphadenopathy associated with RST was attributable to glucocorticoid activity since all infected mice treated with the anti-glucocorticoid RU486 failed to suppress lymphadenopathy regardless of the behavioral treatment they received. Thus, initial cell trafficking to lymph nodes was glucocorticoid-independent, while suppression of lymphadenopathy was attributable to CORT. Modulation of cell trafficking was also reflected in the lungs. Influenza viral infection of the respiratory tract induces an inflammatory response in the lung characterized by severe mononuclear cell infiltration, edema, and tissue consolidation. As reported in our previous studies (Sheridan et al., 1991; Hermann et al., 1993), RST was associated with a lesser extent of infiltration and inflammation in the placebo-treated, infected mice when compared to the FWD or NS cohorts. In contrast, all infected mice that had been treated with RU486 demonstrated marked cellular infiltration and tissue consolidation regardless of the behavioral treatment, and high levels of mortality in these groups required termination of all studies on day 7 p.i. Activated macrophages and polymorphonuclear leukocytes produce large quantities of nitric oxide (NO). NO and its metabolites display vasodilator properties (Hughes et al., 1990; Ialenti et al., 1992) and cytotoxic effects at moderate levels and may play a role in inflammation (for review, see Moncada, 1992). However, when produced in greater quantities, they can induce tissue damage (Kroencke et al., 1991). Indeed, NO has been reported as a major mediator of lung damage (Pendino, et al., 1992). NO synthase, the enzyme responsible for conversion of L-arginine into NO, is induced by immunological stimuli. Its induction is inhibited by glucocorticoids (for review, see Moncada, 1992). Thus, during the course of an infection, as glucocorticoid production is elevated (Hermann et al., 1994a), the extent of edema may be self-limited by the effect of glucocorticoid on cell trafficking and adhesion
G. Hermann et al. /Journal
of Neuroimmunology 56 (1995) 179-186
molecule expression as well as inhibition of NO synthase. Use of the anti-glucocorticoid, RU486 in our present studies, may have blocked glucocorticoid ability to block NO synthase activity (currently under investigation). This may have contributed to the pronounced tissue damage that was observed in the infected lungs. Although neither infection alone, nor restraint alone, affected tissue catecholamine levels (Hermann et al., 1994b), the combination of infection and restraint increased splenic norepinephrine. The present studies demonstrated that RU486 treatment alone reduced splenic NE content (even in uninfected mice; Fig. 4). This effect is a reflection of the complex relationship between the HPA axis and the sympathetic nervous system. For example, steroids have been shown to induce up-regulation of P-receptors (Cheng et al., 19801, as well as participate in the regulation of tyrosine hydroxylase (i.e. a rate-limiting enzyme in catecholamine biosynthesis) (Plotsky et al., 1989). Conversely, catecholamines have been demonstrated to stimulate secretion of adrenocorticotropin and, ultimately, glucocorticoids (Mezey et al., 1983). Thus, it is clear that the HPA axis and the autonomic nervous system are coordinated and dedicated to minimize deviations from the homeostatic state, even during the development of an immune response to a pathogenic challenge (Axelrod and Reisine, 1984). Activation of these neuroendocrine pathways by a stressor tend to reduce inflammatory responses during viral infection.
Acknowledgements
The authors gratefully acknowledge the invaluable technical skills of Sue Moseley and Mark Mengerink. We, also, thank Roussel-UCLAF (France) for their gift of RU486. This work was supported, in part, by Grants MH46801 and HL38485 from the NIH (to J.F.S.) and a postdoctoral fellowship (to G.H.) on Training Grant MH18831 from the NIMH.
References Axelrod, .I. and Reisine, T.D. (1984) Stress hormones: their interaction and regulation. Science 224, 452-459. Bonneau, R.H., Sheridan, J.F., Feng, N. and Glaser, R. (1991) Stress-induced suppression of herpes simplex virus (HSVI-specific cytotoxic T lymphocytes and natural killer cell activity and enhancement of acute pathogenesis following local HSV infection. Brain Behav. Immun. 5, 170-192. Cheng, J.B., Goldfien, A., Ballard, P.L. and Roberts, J.M. (1980) Glucocorticoids increase pulmonary P-adrenergic receptors in fetal rabbits. Endocrinology 107, 1646-1648. Chung, H.-T., Samlowski, W.E. and Daynes, R.A. (1986) Modifications of the murine immune system by glucocorticoids: alterations
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of the tissue lo&lization properties of circulating lymphocytes. Cell. Immunol. 101, 571-585. Cox, J.H. and Ford, W.L. (19821 The migration of lymphocytes across specialized vascular endothelium. Cell. Immunol. 66, 407-422. Dobbs, C.M., Vasquez, M., Glaser, R. and Sheridan, J.F. (1993) Mechanisms of stress-induced modulation of viral pathogenesis and immunity. J. Neuroimmunol. 48, 151-160. Feng, N. (19921 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, CA., 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. Gagne, D., Pons, M. and Philibert, D. (19851 RU38486: a potent antiglucocorticoid in vitro and in vivo. J. Steroid Biochem. 23. 247-251. Hennet, T., Peterhans, E. and Stocker, R. (1991) Alterations in 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, 9322939. 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. Hermann, G., Tovar, CA., Beck, F.M. and Sheridan, J.F. (1994al Kinetics of glucocorticoid response to restraint stress and/or experimental influenza viral infection in two inbred strains of mice. J. Neuroimmunol. 49, 25-33. Hermann, G., Beck, F.M., Tovar, C.A., Malarkey, W.B., Allen, C. and Sheridan, J.F. (1994bl Stress-induced changes attributed to the sympathetic nervous system during experimental influenza viral infection in DBA/2 inbred mouse strain. J. Neuroimmunol. 53, 173-180. Hughes, S.R., Williams, T.J. and Brain, S.D. (19901 Evidence that endogenous nitric oxide modulates edema formation induced by substance P. Eur. J. Pharmacol. 191, 481-484. Ialenti, A., Ianaro, A., Moncada, S. and DiRosa, M. (19921 Modulation of acute inflammation by endogenous nitric oxide. Eur. J. Pharmacol. 211, 177-182. Kroencke, K.D., Kolb-Bachofen, V., Berschick, B., Burkart, V. and Kolb, H. (1991) Activated macrophages kill pancreatic syngeneic islet cells via arginine-dependent nitric oxide generation. Biochem. Biophys. Res. Commun. 175, 752-758. Mezey, E., Reisine, T.D., Palkovits, M., Brownstein, M.J. and Axelrod, J. (1983) Direct stimulation of p2-adrenergic receptors in the rat anterior pituitary induced the release of adrenocorticotropin in vivo. Proc. Natl. Acad. Sci. USA 80, 6728-6731. Moguilewsky, M. and Philibert, D. (1984) RU486: potent antiglucocorticoid activity correlated with strong binding to the cytosolic glucocorticoid receptor followed by an impaired activation. J. Steroid Biochem. 20, 271-276. Moncada, S. (1992) The L-arginine:nitric oxide pathway. Acta Physiol. Stand. 145, 201-227. Munck, A. and Guyre, P.M. (19911 Glucocorticoids and immune function. In: Ader, Felton and Cohen (Eds.1, Psychoneuroimmunology, 2nd edn. Academic Press, San Diego, CA, pp. 447-474. Munck, A., Guyre, P.M. and Holbrook, N.J. (1984) Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocr. Rev. 5, 25-44. Ottaway, CA. and Husband, A.J. (1992) Central nervous system influences on lymphocyte migration. Brain Behav. Immun. 6, 97-116. Pendino, K., Punjabi, C., Gardner, C., Laskin, J. and Laskin, D.
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Relative occupation of type I and type II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications. J. Endocrinol. 115, 459-467. 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. Spencer, R.L., Young, E.A., Choo, P.H. and McEwen, B.S. (1990) Adrenal steroid type I and type II receptor binding: estimates of in vivo receptor number, occupancy, and activation with varying level of steroid. Brain Res. 514, 37-48. Sweep, F., Rijinkels, C. and Hermus, A. (1991) Activation of the hypothalamus-pituitary-adrenal axis by cytokines. Acta Endocrinol. (Copenh.) 125, 84-91.
ELSEVIER
Journal
of Neuroimmunology
56 (1995) 179-186
Stress-induced glucocorticoid response modulates mononuclear cell trafficking during an experimental influenza viral infection Gerlinda Hermann a,b,*, F. Michael Beck ‘, John F. Sheridan a,b a Department of Medical Microbiology and Immunology, College of Medicine, The Ohio State University, Columbus, OH 43210, USA ’ Department of Oral Biology, College of Dentistry, The Ohio State University, Columbus, OH 43210, USA ’ Department of Endodontics, College of Dentistry, The Ohio State University, Columbus, OH 43210, USA Received
8 March
1994; revised
1 September
1994; accepted
14 September
1994
Abstract The migration, distribution, and localization of lymphoid cells throughout the body is critical to the efficiency and development of the immune response. This study examined the role of endogenous glucocorticoids in mononuclear cell (MNC) trafficking during the development of an immune response to infection by influenza A/PR8 virus. Accumulation of MNC in the draining lymph nodes and at the site of virus replication (lungs) was studied in infected mice, and infected mice subjected to a stressor (physical restraint). The glucocorticoid antagonist, RU486, was used to block the activity of endogenous corticosterone during development of the immune response. PR8-infected mice demonstrated an elevation in circulating corticosterone regardless of whether they were treated with RU486 or a placebo. Thus, some ‘afferent’ signal associated with the infection, and/or the immune response to infection, activated the hypothalamic-pituitary-adrenal axis (HPA) and was not subject to negative feedback regulation. The initial accumulation of MNC in the draining lymph nodes and lungs during infection, however, was independent of the glucocorticoid response. Our previous studies demonstrated that virally infected animals subjected to physical restraint had highly elevated plasma corticosterone levels, suppressed lymphadenopathy, and reduced accumulation of MNC in the lungs. In the present study, RU486 treatment restored cellularity to the draining lymph nodes and enhanced accumulation of MNC in lungs of stressed, A/PR8 virus-infected mice. Keywords:
Stress ; Influenza
virus; Glucocorticoids;
RU486; Inflammation
1. Introduction
A principal determinant of the effectiveness of the immune system is opportunity for antigen to meet antigen-specific lymphocytes. The migration, distribution, and localization of lymphoid cells throughout the body is critical to the efficiency and development of the immune response. Ottaway and Husband (1992) have recently reviewed the multiple means by which the central nervous system (CNS) influences systemic as well as local lymphocyte migration. Two of these neurally initiated processes include: (i> sympathetic inner-
* Corresponding author. Present address: Physiology Department, College of Medicine, 1645 Neil Avenue, Ohio State University, Columbus, OH 43210, USA. Phone (614) 292 4305; Fax (614) 292 4888. 01655728/95/$09.50 0 1995 Elsevier SSDI 0165-5728(94)00145-6
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vation of the lymphoid tissue and altered hemodynamits; and (ii) activation of the hypothalamic-pituitaryadrenal axis. Given that responses to stressors are initiated by the hypothalamus and translated into action by the hypothalamic-pituitary-adrenal axis and the autonomic nervous system, these two pathways are available for modulating neurophysiological, neuroendocrine, and immune responses. Previous studies from our laboratory demonstrated that restraint stress, which elevated plasma corticosterone and tissue catecholamine levels, also reduced cellular accumulation and inflammation in target organs associated with pathogenic viral infections (Bonneau et al., 1991; Feng et al., 1991, Feng, 1992; Sheridan et al., 1991; Hermann et al., 1993). Restraint stress was found to suppress normal lymphadenopathy in lymph nodes draining the site of infection (Feng et al., 1991; Dobbs et al., 1993) and reduce mononuclear
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cellular accumulation in the infected tissue (Hermann et al., 1993). In a murine HSV-1 viral infection, we demonstrated that blocking corticosterone activity (by treatment with the drug RU486) restored the cellularity of lymph nodes draining the site of viral replication in restraint-stressed animals (Dobbs et al., 1993). While RU486 has been shown to have a number of pharmacological activities, it is a potent glucocorticoid receptor antagonist (Moguilewsky and Philibert, 1984) that blocks HPA axis activity in stressed animals without interfering with basal HPA activity under nonstressed conditions (Ratka et al., 1989). Thus, the observation that RU486 restored the cellularity to draining lymph nodes in stressed mice suggested that a corticosterone response was responsible for suppression of lymphocyte accumulation. However, we did not examine the effect of RU486 treatment on the inflammatory response at the site of viral replication in previous studies (Dobbs et al., 1993). The ability of an experimental stressor to modulate immunity, viral pathogenesis and health outcomes may be related, in part, to changes in the migration, distribution, and localization of lymphocytes mediated by CNS pathways (see review by Ottaway and Husband, 1992). Lymphadenopathy, or its suppression, may be influenced by at least two different mechanisms. For example, glucocorticoids are known to modulate the expression of adhesion molecules, thus playing a critical role in changing trafficking patterns of lymphocytes as they are activated and become ‘memory’ cells (Cox and Ford, 1982; Chung et al., 1986; for reviews see Munck et al., 1984; Munck and Guyre, 1991). However, given that blood vessels and lymphatic ducts are innervated by adrenergic fibers, activation of the sympathetic nervous system might have an indirect, but significant, effect on blood flow dynamics and localization of lymphocytes. The specific aim of this study was to determine the role of stress-induced glucocorticoids in the accumulation of leukocytes at sites of inflammation and in draining lymph nodes. Therefore, the glucocorticoid antagonist, RU486, was used to block the activity of endogenous corticosteroids during an experimental influenza infection in the murine model. Subsequent changes in lymphadenopathy, corticosterone levels, catecholamine levels, and lung pathology were monitored.
2. Methods and materials 2.1. Animals
Virus-antibody-free C57BL/6 male mice 4-8 weeks old (n = 68) were purchased from Harlan SpragueDawley (Indianapolis, IN). Mice were housed four or
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:OO) and were allowed to acclimate to these conditions for 1 week prior to experimentation. Mice were randomly assigned to one of two drug treatment groups, RU486 or placebo (see details below). Within each drug group, four mice were assigned to the control group (CON) which was neither infected nor stressed; ten mice were assigned to the infected, non-stressed condition (NS); ten mice were assigned to the infected, food-water deprivation condition (FWD); and ten were assigned to the infected, restraint-stressed condition (RST). 2.2. RU486 The glucocorticoid receptor antagonist, RU486 (17b-hydroxy-llb-(4-dimethylaminophenyl)-17a-(l-propynyl)-estra-4,9-dien-3-one; gift from Roussel-UCLAF, Romainville, France), was dissolved in polyethylene glycol 400 (PEG; M, 400) at a concentration of 5 mg/ml. Mice received daily subcutaneous injections of either RU486 (25 mg/kg; 0.1 ml volume (Ratka et al., 1989)) or an equivalent volume of PEG 400 (vehicle control groups) approximately 1 h prior to the start of each RST cycle. This dose of drug previously was shown to be effective in restoring lymphadenopathy in stress animals (Dobbs et al., 1993). Daily injections began 2 days prior to infection and continued throughout the development of the immune response. 2.3. Restraint protocol One group of infected mice (RST) was restrained during their active period each day (from 21:00 to 09:OO) in well-ventilated 50-ml conical polypropylene tubes within their home cages. While in the tubes, the animals could move back and forth freely, but could not turn around. They did not have access to food or water during this time period. Therefore, a second group of infected mice (FWD) was similarly food- and water-deprived, but not restrained, to control for the effects of restraint. A third group of infected mice (NS) served as a home cage control and was not subjected to either RST or FWD. In some experiments, an uninfected control group was added (CON) that was not subjected to RST or FWD. These groups were included to compare the effects of the drugs alone. RST or FWD regimens were initiated 1 day prior (day - 1) to infection and continued for 10 days postinfection (p.i.1. 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 and CON groups had access to food and water 24 h per day.
G. Hermann et al. /Journal
of Neuroimmunology 56 (I 995) 179-186 Effects of RU486 and infection CORT levels on day 7
2.4. Virus and infection of mice
Influenza A/Puerto Rico/g/34 (A/PR8) virus was obtained from the American Type Culture Collection (Rockville, MD); propagation and determination of virus titer have been described (Feng et al., 1991; Sheridan et al., 1991). The lethal dose 50 (LD,,) of influenza A/PRS for C57BL/6 mice was estimated to be 32HAU. Intranasal (i.n.> infection of mice occurred on day ‘0’ and proceeded as previously described (Hermann et al., 1993, Hermann et al., 1994a, Hermann et al., 1994b).
181
plasma
on p.i.
600 s 2 2 !z
400
a B ._ r s
200
n uninfected q infected
0
Blood samples were drawn at 10:00 on the day of sacrifice (day 7 p.i.1. Sera were collected and stored at -70°C until assayed for corticosterone by radioimmunoassay. [‘251]Corticosterone kits for rats and mice (ICN Biomedical, Costa Mesa, CA) were used to determine serum corticosterone (CORT) levels (as described in Hermann et al., 1993). CORT levels from individual mice were determined from a standard curve and expressed in ng/ml. 2.6. Determination of cell yield from mediastinal lymph nodes Single-cell suspensions of mediastinal lymph nodes were prepared by macerating the tissue in 500 ~1 volume of Hanks’ balanced salt solution. Samples were counted on a Coulter counter (Coulter Diagnostics); cell yield per animal was determined as previously described (Hermann et al., 1994a).
Fig. 1. Influenza A/PRS viral infection alone was sufficient to elevate plasma CORT levels on day 7 p.i., regardless of whether the mice had been treated with RU486 or placebo ( * P < 0.0001; * * P = 0.0097).
norepinephrine (NE) levels were assessed using independent r-tests (two-tailed). Log transformation of the data for each of these variables was necessary in order to stabilize the variance. All analyses were done using SAS version 6.07.
3. Results 3.1. Effects of RU486 on plasma corticosterone levels
Mice infected i.n. with 32 HAU of influenza A/PR8 virus developed a respiratory tract infection that was
Effects of RU486, infection, and/or stress on cell number of draining lymph nodes
2.7. Histology The upper portion of the left lobe of the lung was obtained from half of the surviving animals in each group. Lung samples were prepared for histological examination and stained with Hematoxylin and Eosin as previously described (Hermann et al., 1993).
not infected
2.8. Catecholamine determinations >-
2.9. Statistical analysis Between-group differences in plasma glucocorticoid level, cell numbers within draining lymph nodes, and
infected
* ll!kIi L
I-
Spleens were quickly removed at time of sacrifice and placed on dry ice to halt degradation of catecholamines. Tissue was stored at -70°C until time of extraction. Catecholamine extraction and quantitative analysis were as described previously (Hermann et al., 1994b).
RU486
placebo
2.5. Determination of serum corticosterone levels
ICON
NS
WD
0
PLACEBO
n
RU466
RST
Fig. 2. Similar to our previous experiments, influenza A/PR8 viral infection was sufficient to increase the cell density in mediastinal lymph nodes at day 7 pi. (regardless of whether the mice had been treated with placebo or RU486) and restraint stress (RST) reduced the cell number in the placebo-treated, infected mice (open bars). In contrast, infected mice treated with RU486 (closed bars) and subjected to restraint stress (RST) showed an abrogation of this suppression in cell number (’ P < 0.05). CON, uninfected; NS, infected, nonstressed; FWD, infected/food-water-deprived: RST, infected/restraint-stressed.
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Fig. 3. RU486 treatment restored cellular infiltration in the lungs of restrained, influenza A/PR8 virus infected mice. All samples were obtained 7 days pi. (A) Lung tissue from uninfected, normal C57B1/6 mouse; note the paucity of cells in the pulmonary tissue, relative thinness of the septal walls, and the regularity of the bronchial epithelial cells. (B) Lung tissue from A/PRB virus-infected mouse; note the intensity of the cellular infiltration, swelling of the septal walls, and disarray of the bronchial epithelial cells. (C) Lung tissue from FWD, A/PR8 virus-infected mouse. (D) Lung tissue from RST, A/PR8 virus-infected mouse; note the swelling of the septal walls, disarray of the bronchial epithelial cells, but relative lack of cellular infiltration. (E) Lung tissue from uninfected normal mouse that received daily subcutaneous injections of RU486; note the thickness of the septal walls and relative increase in cellularity. (F) Lung tissue from A/PRI virus-infected mouse treated with RU486. (G) Lung tissue from A/PRS virus-infected mouse, FWD, and treated with RU486. (HI Lung tissue from A/PR8 virus-infected mouse, RST, and treated with RU486; note the swelling of the septal walls, disarray of the bronchial epithelial cells, and the intensity of the cellular infiltration. Magnification 100 x for all photomicrographs.
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of Neuroimmunology 56 (1995) 179-186
183
Fig. 3 (continued).
sufficient to elevate plasma CORT levels (Fig. 1) regardless of whether the mice were treated with RU486 or placebo (placebo, P < 0.0001; RU486, P = 0.0097). Similar to our previous study (Hermann et al., 1994a), on day 7 pi. RST did not further augment plasma CORT levels in either the placebo- or RU486-treated animals (placebo, P = 0.2795; RU486, P = 0.6113; data not shown).
3.2. Effects of RlJ486 lymph nodes
on cell number
of mediastinal
In response to an influenza A/PR8 viral infection in the respiratory tract regional draining mediastinal lymph nodes increased their cell density (Fig. 21, regardless of whether the mice were treated with RU486 or placebo. Our previous studies (Hermann et al., 1993, 1994a,b; Feng et al., 1991; Sheridan et al., 19911, which examined lymph nodes at day 10 post-infection, not only showed the same lymphadenopathy with infection, but also demonstrated that RST significantly suppressed this increase in cell number. In the present study, cell density was examined 7 days p.i. Lymph nodes from uninfected control mice (CON) averaged 1.4 x 106, while lymph nodes from A/PR8 virus-infected mice (NS) averaged 4.4 X 106. RST reduced the average cell density to 2.1 X lo6 ceils/node in A/PR8 virus-infected, placebo-treated mice (placebo-treated, infected/RST vs. RU486-treated, infected/RST; P < 0.05). In contrast, A/PR8 virus-infected mice treated with RU486 had very similar mediastinal lymph node cell densities regardless of the behavioral treatment, i.e. NS, FWD, or RST. Thus, drug treatment with a glucocorticoid receptor antagonist restored lymphadenopathy in the draining lymph nodes of restrained, A/PR8 virus-infected mice.
3.3. Effects of RU486 on pulmonary inflammation and pathosis In addition to suppressing mononuclear cellular accumulation in the draining lymph nodes, RST has been associated with diminished cellular infiltration in the lungs of A/PR8 virus-infected mice (Hermann et al., 1993, 1994a,b; Feng et al., 1991). To determine the role of CORT in reducing inflammatory responses in the lung, mice were treated with RU486 during infection. In the present study, infection with A/PR8 virus resulted in histopathological changes in the lungs which included swelling of the septal walls, disorganization of the bronchial epithelial cells, and mononuclear cellular infiltration (Fig. 3A, B). RST diminished cellular infiltration in the lungs of A/PR8 virus-infected mice (Fig. 3D1, while FWD had no effect (Fig. 30 Cellular
Effects of RU466, infection, and/or stress on splenic NE content in C57BU6 mice (day7 p.i.)
6
F 2
1.o
.-t i 8 .c it z z
0
placebo
n
RU486
0.5
0.0 CON
NS
f=WD
RST
Fig. 4. Treatment with RU486 was associated with a decrease in tissue catecholamine. Splenic norepinephrine levels in RU486-treated mice were diminished compared to placebo-treated mice across all behavioral treatments. These differences were statistically significant for the CON groups (* P = 0.0033) and the RST groups (* * P = 0.0015).
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infiltration was restored in the lungs of restrained, A/PRS virus-infected mice by treatment with RU486 (Fig. 3H). In fact, all infected mice treated with RU486 demonstrated a high density of mononuclear cells in the lung tissue (Fig. 3F, G, H). 3.4. Effects of RU486 on splenic catecholamine content In addition to elevating plasma CORT in A/PR8 virus-infected mice, multiple cycles of RST have been shown to raise tissue catecholamine levels (Hermann et al., 1994b). However, in this study, treatment with RU486 was associated with a decrease in splenic norepinephrine (Fig. 4) compared to splenic norepinephrine content in placebo-treated mice across all behavioral groups. These differences were statistically significant for the uninfected (CON) groups (P = 0.0033) and the RST groups (P = 0.0015).
4. Discussion RU486 is a synthetic steroid analog which acts as a potent glucocorticoid receptor antagonist in vitro and in vivo (Gagne et al., 1985). The results presented here indicate that basal levels of plasma corticosterone were not affected by treatment with RU486. Ratka et al. (1989) have also reported that RU486 did not influence basal, non-stressed levels of CORT in the rat, although pretreatment with RU486 slightly reduced the elevation of plasma CORT normally seen in rats exposed to a novel environment. These different effects were attributed to two receptor types (type I, or mineralocorticoid receptors, and type II, or glucocorticoid receptors) within the brain with markedly different affinities for CORT (Rem and de Kloet, 1985; Reul et al., 1988; Spencer et al., 1990). In the studies presented here, influenza A/PR8 virus-infected mice had elevated circulating CORT levels regardless of whether they were treated with RU486 or placebo. Thus, some ‘afferent’ signal associated with viral replication, or the host’s response to infection, activated the hypothalamic-pituitary-adrenal axis (HPA). In studies by Hennet and colleagues (Hennet et al., 1991, 19921, proinflammatory cytokine responses were detected in the lung lavage samples of A/PR8 virus-infected mice within a few hours of infection. These cytokines, which included IL-l, IL-6, and TNF-(u, are known to stimulate the HPA axis (Sweep et al., 1991) and might be responsible for elevated CORT levels in an influenza viral infection. Furthermore, CORT secretion in response to influenza infection was not subject to negative feedback regulation by either of the above-mentioned receptor types as evidenced by the elevated CORT levels found in all the infected, RU486-treated mice.
Increases in size, weight, and cell density of draining lymph nodes, i.e. lymphadenopathy, is normally associated with infection. The present studies demonstrated that the initial accumulation of cells in the draining lymph nodes is independent of corticosterone in that all A/PR8 virus-infected, non-stressed animals showed a significant increase in cell number regardless of whether they had been treated with RU486 or placebo. Our previous studies (Feng et al., 1991, Feng, 1992; Hermann et al., 1993, 1994a,b) demonstrated reduced cell densities in A/PR8 virus-infected mice exposed to RST. This phenomenon has been attributed to an elevation of CORT associated with the stress and its effects on lymphoid cell trafficking (see reviews by Munck et al., 1984; Munck and Guyre, 1991). The results presented in this study suggest that suppression of lymphadenopathy associated with RST was attributable to glucocorticoid activity since all infected mice treated with the anti-glucocorticoid RU486 failed to suppress lymphadenopathy regardless of the behavioral treatment they received. Thus, initial cell trafficking to lymph nodes was glucocorticoid-independent, while suppression of lymphadenopathy was attributable to CORT. Modulation of cell trafficking was also reflected in the lungs. Influenza viral infection of the respiratory tract induces an inflammatory response in the lung characterized by severe mononuclear cell infiltration, edema, and tissue consolidation. As reported in our previous studies (Sheridan et al., 1991; Hermann et al., 1993), RST was associated with a lesser extent of infiltration and inflammation in the placebo-treated, infected mice when compared to the FWD or NS cohorts. In contrast, all infected mice that had been treated with RU486 demonstrated marked cellular infiltration and tissue consolidation regardless of the behavioral treatment, and high levels of mortality in these groups required termination of all studies on day 7 p.i. Activated macrophages and polymorphonuclear leukocytes produce large quantities of nitric oxide (NO). NO and its metabolites display vasodilator properties (Hughes et al., 1990; Ialenti et al., 1992) and cytotoxic effects at moderate levels and may play a role in inflammation (for review, see Moncada, 1992). However, when produced in greater quantities, they can induce tissue damage (Kroencke et al., 1991). Indeed, NO has been reported as a major mediator of lung damage (Pendino, et al., 1992). NO synthase, the enzyme responsible for conversion of L-arginine into NO, is induced by immunological stimuli. Its induction is inhibited by glucocorticoids (for review, see Moncada, 1992). Thus, during the course of an infection, as glucocorticoid production is elevated (Hermann et al., 1994a), the extent of edema may be self-limited by the effect of glucocorticoid on cell trafficking and adhesion
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molecule expression as well as inhibition of NO synthase. Use of the anti-glucocorticoid, RU486 in our present studies, may have blocked glucocorticoid ability to block NO synthase activity (currently under investigation). This may have contributed to the pronounced tissue damage that was observed in the infected lungs. Although neither infection alone, nor restraint alone, affected tissue catecholamine levels (Hermann et al., 1994b), the combination of infection and restraint increased splenic norepinephrine. The present studies demonstrated that RU486 treatment alone reduced splenic NE content (even in uninfected mice; Fig. 4). This effect is a reflection of the complex relationship between the HPA axis and the sympathetic nervous system. For example, steroids have been shown to induce up-regulation of P-receptors (Cheng et al., 19801, as well as participate in the regulation of tyrosine hydroxylase (i.e. a rate-limiting enzyme in catecholamine biosynthesis) (Plotsky et al., 1989). Conversely, catecholamines have been demonstrated to stimulate secretion of adrenocorticotropin and, ultimately, glucocorticoids (Mezey et al., 1983). Thus, it is clear that the HPA axis and the autonomic nervous system are coordinated and dedicated to minimize deviations from the homeostatic state, even during the development of an immune response to a pathogenic challenge (Axelrod and Reisine, 1984). Activation of these neuroendocrine pathways by a stressor tend to reduce inflammatory responses during viral infection.
Acknowledgements
The authors gratefully acknowledge the invaluable technical skills of Sue Moseley and Mark Mengerink. We, also, thank Roussel-UCLAF (France) for their gift of RU486. This work was supported, in part, by Grants MH46801 and HL38485 from the NIH (to J.F.S.) and a postdoctoral fellowship (to G.H.) on Training Grant MH18831 from the NIMH.
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