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The Journal of Immunology, 1998, 160: 5441-5447.
Copyright © 1998 by The American Association of Immunologists

Role of the Hypothalamic Pituitary Adrenal Axis and IL-6 in Stress-Induced Reactivation of Latent Herpes Simplex Virus Type 11

Sansanee Noisakran, William P. Halford2, Livia Veress and Daniel J. J. Carr3

Department of Microbiology, Immunology, and Parasitology, Louisiana State University Medical Center, New Orleans, LA 70112


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hyperthermic stress induces reactivation of herpes simplex virus type 1 (HSV-1) in latently infected mice and also stimulates corticosterone release from the adrenals via activation of the hypothalamic pituitary adrenal axis. In the present study, we tested the hypothesis that stress-induced elevation of corticosterone potentiates HSV-1 reactivation in latently infected mice. Because of the putative role of IL-6 in facilitating HSV-1 reactivation in mice, the effect of hyperthermic stress and cyanoketone treatment on IL-6 expression in the trigeminal ganglion was also measured. Preadministration of cyanoketone, a glucocorticoid synthesis inhibitor, blocked the stress-induced elevation of corticosterone in a dose-dependent manner. Furthermore, inhibition of corticosterone synthesis was correlated with reduced levels of HSV-1 reactivation in latently infected mice. Hyperthermic stress elicited a transient rise in IL-6 mRNA levels in the trigeminal ganglion, but not other cytokine transcripts investigated. In addition, there was a significant reduction in MAC-3+, CD8+, and DX5+ (NK cell marker) cells in the trigeminal ganglion of latent HSV-1-infected mice 24 h after stress. Cyanoketone blocked the stress-induced rise in IL-6 mRNA and protein expression in the trigeminal ganglion latently infected with HSV-1. Collectively, the results indicate that the activation of the hypothalamic pituitary adrenal axis plays an important role in stimulating IL-6 expression and HSV-1 reactivation in the trigeminal ganglion following hyperthermic stress of mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The activation of the hypothalamic pituitary adrenal (HPA)4 axis and the sympathetic nervous system (SNS) following stress are two major pathways with a significant impact on the immune system. Catecholamines as a major product of SNS activation have been shown to affect the circulation of leukocytes and Ab synthesis (1, 2, 3, 4) through G-protein linked membrane bound receptors on the target lymphocytes (5). Glucocorticoids as the major product of HPA axis activation are potent immunosuppressive hormones (6, 7, 8, 9) that target the transcription of I{kappa}B{alpha} and thus block NF{kappa}B translocation to the nucleus and markedly reduce cytokine gene expression (10, 11). The result of the suppressive effects of stress on the immune system has been illustrated through experimental studies. Recent studies have shown that psychologic and physical stress can contribute to changes in susceptibility to viral pathogenesis and invasiveness (12, 13, 14, 15), metastatic spread (16), and active immunization (17).

One viral pathogen that has achieved remarkable success in the human population, herpes simplex virus type 1 (HSV-1), is sensitive to stressors and can reactivate following the establishment of latency in response to UV irradiation (18), epinephrine iontophoresis (19), and transient hyperthermia (20) in animal models. Moreover, the administration of dexamethasone alone has been shown to induce modest reactivation of cell cultures from the trigeminal ganglion (TG) latently infected with HSV-1 and facilitate reactivation following hyperthermic stress (21). Taken together, these results suggest that the activation of the HPA and SNS pathways either independently or in concert induces reactivation of latent HSV-1 by an undefined mechanism.

While HSV-1 latency in mice is defined through the sole expression of latency-associated transcript (LAT) RNAs in the infected tissue and the lack of a detectable HSV-1-encoded protein, there is a persistent immune response during the latent period including infiltrating inflammatory cells (22), cytokine gene and protein expression (23, 24, 25), and a continuous increase in Ab titer to viral glycoproteins (26). Since spontaneous reactivation of HSV-1 rarely occurs in mice, the presence of the immune effector cells or their cytokines during latency may, in part, block viral replication. To this end, cytokines present during latency (23, 24, 25) (our unpublished observation) and implicated in antagonizing HSV-1 replication include IFN-{alpha} (27, 28), IFN-ß (29), and TNF-{alpha} along with IFN-{gamma} (30, 31). Accordingly, stress may suppress the levels of one or more of these cytokines or effector T cells eliciting these cytokines which, in turn, allows the virus to replicate yielding infectious virions. Restraint stress has previously been shown to reduce HSV-specific memory CTL activation in the spleen by mechanisms that suppress selective cytokines including IL-2, IL-4, IL-6, and IFN-{gamma} (32, 33). These findings are consistent with the central role of CD8+ lymphocytes in the control of viral replication (34, 35). However, these studies have not addressed the immune events during the reactivation of HSV-1 in the sensory ganglia. Conversely, rather than reducing the presence of effector cells or cytokines, stress may elicit or increase the synthesis of other factors that promote viral reactivation locally within the sensory ganglia.

The present study was undertaken to study the effects of hyperthermic stress and the activation of the HPA axis on HSV-1 reactivation and cytokine gene expression. The glucocorticoid synthesis inhibitor 2{alpha}-cyano-4,4,17{alpha}-trimethylandrost-5-en-17ß-ol-3-one (cyanoketone (CK)), which blocks the production of active steroids (36, 37), was used to block stress-induced increases in corticosterone. We hypothesized that the HPA axis was the primary means by which latent HSV-1 is signaled (via corticosterone) to reactivate and consequently, CK would block HSV-1 reactivation in latent mice. Finally, we anticipated that hyperthermic stress would modify the expression of cytokine genes in the TG. Accordingly, we measured the expression of cytokine genes before and after hyperthermic stress in an attempt to correlate the levels of their expression with HSV-1 reactivation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Virus and cells

Vero and CV-1 African monkey kidney cell lines were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 (Mediatech, Washington, D.C.) containing 5% FBS (Life Technologies, Gaithersburg, MD) and an antibiotic/antimycotic solution (Sigma Chemical, St. Louis, MO). Cells were incubated at 37°C, 5% CO2, and 95% humidity. HSV-1 was grown up and harvested as previously described (25).

Reverse transcription-PCR

RT-PCR of TG was performed as described (25). Briefly, TG RNA was extracted in Ultraspec RNA isolation reagent (Biotecx, Houston, TX). First-strand cDNA was synthesized using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI). PCR was performed in a thermal cycler (MJ Research, Watertown, MA) with 35 cycles of 94°C (1 min, 15 s)->57–60°C (1 min, 15 s)->72°C (30 s). PCR primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TNF-{alpha}, LAT, and RANTES were as previously described (25). IFN-ß and CD8 primer sequences were obtained from Clontech Laboratories (Palo Alto, CA). Primers for IL-1{alpha} were 5'-ATGGCCAAAGTTCCTGACTTGTTT-3' (sense) and 5'-CCTTCAGCAACACGGGCTGGT-3' (antisense) yielding a 620 bp product. Primers for IL-6 were 5'-TTCCATCCAGTTGCCTTCTTGG-3' (sense) and 5'-CTTCATGTACTCCAGGTAG-3' (antisense) yielding a 359-bp product. Following electrophoresis of the amplified product, ethidium bromide-stained PCR products were visualized with an Bio-Rad 1000 gel documentation system (Bio-Rad, Hercules, CA). Densitometric analysis of gel images was performed using Molecular analysis software 3.3 software (Bio-Rad).

Competitive RT-PCR

Our method involved the use of an external standard curve generated for each set of samples. The standard curve consisted of four PCR reactions containing a known amount of cloned target IL-6 or GAPDH ranging from 960 copies down to 15 copies per reaction. Three microliters of each undiluted sample was aliquoted into separate reaction tubes. Primer competition was produced by the addition of 3 µl containing 15 copies of IL-6 or 50 copies of GAPDH to both standard and sample reactions. After completion of the PCR, the amplified products were electrophoresed and analyzed densitometrically. Copy equivalence was determined by constructing a standard curve plotting the mimetic-to-standard ratio of intensities against the number of copies in each reaction. In this way, a mimetic-to-sample ratio of intensities could be used to determine the number of copies in each sample.

Infection and treatment of mice

All mice (female CD1 mice, 25–34 g, Harlan Sprague Dawley, Indianapolis, IN) were infected and monitored for the success of the infection as previously described (25). Mice were hyperthermically stressed by a published protocol (20) with minor modifications. Specifically, a Brinkman RC3 circulating water bath with feedthrough copper tubing placed in an acrylic bath designed to hold up to six 50-ml tubes at any one time was used to regulate the temperature of the water bath to within 0.1°C. Mice were placed in 50-ml restraining tubes with 5-mm-diameter holes drilled throughout each tube. The mice were gently placed into the bath of water at 43°C. The mice were situated such that the water level did not exceed the neck region such that no physical effort was required by the animal to remain above the water level. However, the mice were constrained in the tube, which limited their movement suggesting some degree of restraint. Following a 10-min bath, the mice were removed, gently blotted with paper towels, and placed in a warm room (34°C) for 30 min to prevent hypothermia. All animals were housed and cared for in accordance with National Institute of Health Guidelines on the Care and Use of Laboratory Animals (38). All procedures were approved by the Louisiana State University Medical Center Institutional Animal Care and Use Committee.

In those experiments in which CK was used, mice received 50 to 100 mg/kg CK i.p. 24 and 2 h before the stress episode. CK (gift from Sanofi-Winthrop Research Division, Malvern, PA) was reconstituted in DMSO, served as the vehicle in control animals. This regimen has previously been shown to block LPS-induced elevations in corticosterone (39).

Dexamethasone (ICN, Aurora, OH) was reconstituted in ethanol and added to the drinking water in a 0.5% ethanol solution for a final concentration of 1 mg/ml. Mice were exposed to the glucocorticoid for 24 h before the stress event. Blood was obtained from the retroorbital plexus at the indicated time for analysis of circulating corticosterone levels.

Detection of virus reactivated in vivo

Ganglia from hyperthermically stressed mice were removed aseptically and homogenized with the Pro-200 tissue homogenizer (ProScientific, Monroe, CT) in 1.0 ml of RPMI 1640 containing 5% FBS. Homogenates were centrifuged (1 min, 10,000 x g) to remove cellular debris and the supernatant was layered onto CV-1 monolayers. The supernatants were incubated with the indicator cells for 45 min at 37°C in a 5% CO2 incubator. Monolayers were then rinsed with PBS (pH 7.2), fresh RPMI 1640 containing 5% FBS and antibiotic/antimycotic solution was added, and the cultures were then placed in a 5% CO2 incubator and monitored daily for cytopathic effect for 7 days. Plaques generally appeared 72 to 96 h following supernatant addition ranging from 2 to 5 plaques/well.

Corticosterone determination

Sera from killed animals were assayed for corticosterone levels by RIA (ICN Biomedicals, Costa Mesa, CA). All samples were assayed in duplicate. The corticosterone levels were extrapolated from the standard curve (Rf >= 0.9900).

TG cell dissociation and flow cytometric analysis

Single-cell suspensions of TG cells were obtained by placing isolated TG in 0.5 ml of calcium- and magnesium-free HBSS (pH 7.0) containing collagenase type XI (1 mg/ml; Sigma) and collagenase type IV (1 mg/ml; Sigma). Tissue was triturated every 20 min for 1 h at 37°C with a 1-ml serologic pipette. Dissociated cells were washed twice in PBS (pH 7.4) containing 0.5% BSA (PBS-BSA) by centrifugation (300 x g, 5 min). Following the second wash, the cells were resuspended in 1.0 ml of PBS-BSA. Dissociated cells (in 0.1-ml aliquots) were subsequently labeled with FITC- or phycoerythrin-conjugated Ab to dendritic cells (40) (Leinco Technologies, Ballwin, MO), macrophages (41) (MAC-3, PharMingen, San Diego, CA), NK cells (42) (DX-5, PharMingen), or CD8+ cells (PharMingen). Cells were incubated for 25 min on ice in the dark and subsequently washed twice with PBS-BSA by centrifugation (300 x g, 5 min). Cells were resuspended in PBS containing 1% paraformaldehyde and analyzed on a Coulter Elite FACS (Coulter, Hialeah, FL). Log forward scatter vs log side scatter plot was assembled to gate viable cells for analysis to separate cells from debris. Light scatter was collected at 488 nm, and emitted light was passed through a long pass filter followed by narrow band filter and analyzed at 525 nm (FITC) and 575 nm (phycoerythrin). Five thousand gated events were collected and analyzed per sample. Isotypic controls (PharMingen) were used to subtract nonspecific labeling of cells. Compensation of signal noise was 38%.

Statistics

One-way analysis of variance and the Scheffé multiple comparison test were used to determine significant (p < 0.05) differences between the indicated groups using the GBSTAT program (Dynamic Microsystems, Silver Spring, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hyperthermic stress increases IL-6 but reduces CD8 transcript expression in the trigeminal ganglion of latent HSV-1-infected mice

A study was conducted to determine the effects of hyperthermic stress on cytokine gene expression and the reactivation of virus from the TG in mice latently infected with HSV-1. The results show that hyperthermic stress significantly increased the expression of IL-6 mRNA in the TG of latently infected mice in a time-dependent fashion, peaking 2 h poststress (Fig. 1Go, Table IGo). Although there was an indication that IL-6 levels were elevated in the uninfected, stressed animals, the levels did not reach significance. The stress episode was found to decrease CD8 transcript expression in the TG becoming significant 24 h poststress. No other cytokine mRNA was found to be modified in the TG, suggesting that the outcome of the stress response was specific for IL-6 and CD8. While there were significant differences comparing the levels of LAT, CD8, RANTES, and IL-1{alpha} transcripts in the TG between infected and uninfected mice, there were no differences in IL-6, TNF-{alpha}, and IFN-ß.



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  		FIGURE 1. Hyperthermic stress transiently increases IL-6 mRNA expression in mice latently infected with HSV-1. Latently infected (I) (>=30 days postinfection) and uninfected (UI) mice were subjected to hyperthermic stress and killed 2 to 24 h later. Nonstressed animals (0 h) served as controls for basal expression. TGs were removed, and processed RNA was subjected to RT-PCR for the genes of interest. Results are representative of three experiments (n = 2–3/group/experiment). A summary is found in Table 1.

 

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  		Table I. Summary of cytokine and CD8 transcript expression in the TG of mice following hyperthermic stressa

 
Since qualitative changes were observed in the RT-PCR for IL-6, we assessed IL-6 mRNA using a quantitative RT-PCR method. Using competitive RT-PCR, we could confirm the RT-PCR results by determining the copy numbers of IL-6 mRNA as well as the constitutively expressed gene, GAPDH, from TG of latently infected mice following stress. Consistent with the previous observations, IL-6 mRNA copy numbers were significantly elevated in the TG of infected mice 2 h poststress but returned to prestress levels by 6 h poststress (Fig. 2Go). Uninfected mice had a delayed elevation in IL-6 mRNA in the TG peaking 6 h poststress but never attaining the significant level observed in the infected animals (Fig. 2Go). Competitive RT-PCR for GAPDH using the identical templates as the IL-6 competitive RT-PCR showed similar levels of this transcript in both infected and uninfected mice at all time points tested, suggesting that the elevation observed in IL-6 mRNA 2 h poststress was due to a specific increase in the target and not discrepancies in the quantity or quality of total template (Fig. 3Go).



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  		FIGURE 2. Quantitative competitive PCR reveals a fivefold increase in IL-6 mRNA in mice latently infected with HSV-1 2 h poststress. Infected (n = 3/group) and uninfected (n = 2/group) mice were subjected to hyperthermic stress and killed 2 to 24 h poststress. Nonstressed groups (0 h) served as controls for basal expression. RNA was obtained from the TG of the killed mice and subjected to quantitative RT-PCR for IL-6. A summary of the results of two experiments for the quantitative RT-PCR for IL-6 is shown.

 


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  		FIGURE 3. Quantitative competitive PCR reveals no difference in GAPDH mRNA expression in mice latently infected with HSV-1 2 h poststress. Infected (n = 3/group) and uninfected (UI) (n = 2/group) mice were subjected to hyperthermic stress and killed 2 to 24 h poststress. Nonstressed groups (0 h) served as controls for basal expression. RNA was obtained from the TG of the killed mice and subjected to quantitative RT-PCR for GAPDH. A summary of the results of two experiments for the quantitative RT-PCR for GAPDH is shown.

 
Hyperthermic stress reduces CD8+, MAC-3+ (macrophages), and DX5+ (NK cells) cells in the TG

Since the RT-PCR analysis showed a decrease in the expression of CD8 transcript 24 h poststress, immune cell composition before and after stress in latently infected and uninfected mice was investigated. There was a significant increase in the percentage of MAC-3+ and DX5+ cells but not dendritic cells or CD8+ cells in the TG of latently infected mice compared with the uninfected controls (Table IIGo). Consistent with the RT-PCR mRNA levels measured 24 h poststress, there was a significant decrease in the percentage of CD8+ cells in the TG of latently infected mice 24 h poststress. Likewise, there was a significant decrease in MAC-3+ and DX5+ cells in the TG of latently infected animals following stress. The dendritic cell population (as measured by a percentage of the total population) was not affected by hyperthermic stress in the latent HSV-1-infected mice. Likewise, none of the immune cell populations in the uninfected mouse TG was modified following stress (Table IIGo).


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  		Table II. Hyperthermic stress reduces the percentage of CD8+, MAC-3+, and DX5+ cells in the TG 24 h poststress1

 
CK blocks stress-induced increases in corticosterone and HSV-1 reactivation

An association between stress and HSV-1 reactivation suggested that the activation of the HPA axis might be directly involved in this process. To investigate this possibility, the corticosterone synthesis inhibitor CK was administered to mice before hyperthermic stress. Consistent with previous observations, CK blocked stress-induced increases in circulating corticosterone levels in a dose-dependent fashion (Table IIIGo). Likewise, CK blocked HSV-1 reactivation in latent mice in a dose-dependent fashion (Table IIIGo). Since the results showed that increases in corticosterone coincided with viral reactivation, we next asked whether the exogenous administration of dexamethasone (a glucocorticoid analogue) alone could induce reactivation of latent HSV-1. The results show that unlike stress, dexamethasone alone did not induce reactivation nor did it augment hyperthermic stress-induced reactivation (Table IIIGo). However, dexamethasone did block stress-induced increases in corticosterone levels, suggesting that it was at a concentration that induced a negative feedback loop on the HPA axis (Table IIIGo).


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  		Table III. CK blocks stress-induced reactivation of HSV-1a

 
CK treatment antagonizes the stress-induced increase in IL-6 mRNA and protein in the TG of mice latently infected with HSV-1

Since hyperthermic stress induced reactivation of latent HSV-1 and transiently increased the expression of IL-6 in the TG and CK blocked HSV-1 reactivation, a study was conducted to determine whether CK could block the stress-induced elevation in IL-6 mRNA expression. CK treatment (100 mg/kg) of mice was found to partially block the increase in IL-6 gene expression 2 h poststress compared with the vehicle-treated group (Fig. 4Go). Consistent with the mRNA expression, there was a reduction in IL-6 protein measured in the TG of the 100 mg/kg CK-treated mice compared with the vehicle-treated controls 12 h poststress (Fig. 5Go). The effect mediated by CK treatment on IL-6 mRNA expression was specific, since there were no changes in the expression in the housekeeping gene, GAPDH (Fig. 4Go). However, a lower concentration of CK (50 mg/kg) had no effect on IL-6 mRNA expression 2 h poststress. Specifically, prestress IL-6:GAPDH image pixel ratios were 0.31 ± 0.02. Two hours after hyperthermic stress, IL-6:GAPDH image pixel ratio of the vehicle-treated group was 0.48 ± 0.05, while the IL-6:GAPDH ratio of the 50.0 mg/kg CK-treated group was 0.52 ± 0.07.



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  		FIGURE 4. CK (100 mg/kg) blocks the hyperthermic stress-induced increase in TG IL-6 mRNA. Mice were pretreated with vehicle (VEH) or CK and subsequently hyperthermically stressed. Two hours poststress, the mice were killed and RNA extracted from the TG was used in RT-PCR assays measuring IL-6 and GAPDH mRNA. Nonstressed mice served to establish baseline levels of cytokine gene expression. No template (-) and IL-6 DNA (+) served as negative and positive controls, respectively, in the PCR assay. Results are representative of three experiments (n = 2 mice/group/experiment), the summary of which is shown under the representative gel. aNumbers represent the summary of the ratio of IL-6:GAPDH image pixel values ± SEM. bThe number is the percent increase in the expression of the IL-6:GAPDH ratio following hyperthermic stress.

 


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  		FIGURE 5. CK partially blocks IL-6 protein in the TG of hyperthermically stressed, mice latently infected with HSV-1. CK (100 mg/kg) or vehicle was administered to mice latently infected with HSV-1 24 and 2 h before hyperthermic stress. At 7 and 12 h poststress, the mice (n = 4/time point/group) were killed, and the TG was removed and processed as previously described (25). Supernatants from homogenized TGs were assayed for IL-6 by ELISA as described (25). *p < 0.05 comparing the stressed, vehicle-treated group with the nonstressed control. Bars represent SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Glucocorticoids have previously been shown to modestly induce reactivation of latent HSV-1 both in vitro (21) and in vivo (43). Moreover, there is clinical precedence for herpes virus reactivation following systemic glucocorticoid administration (44, 45). Previous studies have shown the addition of dexamethasone (10-7 M) added to in vitro TG cell cultures enhanced the kinetics of reactivation of HSV-1 (25). However, data in the present study showed the lack of an effect in viral reactivation following dexamethasone treatment in vivo. The concentration administered to mice (1 mg/ml) significantly reduced circulating levels of corticosterone and may have interfered with other endogenous hormonal pathways that influence HSV-1 reactivation including ß-adrenoceptor-sensitive pathways (46). A previous study has shown that 10 µg/ml dexamethasone in the drinking water of rats occupies 80% of the type II glucocorticoid receptor, reducing the affinity by 10-fold following an overnight exposure to the drug (47). Alternatively, the glucocorticoid may have suppressed the production of factors/cytokines that promote HSV-1 reactivation. Cytokines that are sensitive to the effects of glucocorticoids include IL-6 (48) which is known to have glucocorticoid-responsive elements in the 5'-flanking region of the IL-6 gene (49) as well as TNF-{alpha} (50, 51). TNF-{alpha} also augments HSV-1 reactivation and replication in TG explants (52). While TNF-{alpha} has previously been shown to elicit IL-6 production by astrocytes (53, 54), the relationship between IL-6 and TNF-{alpha} has not been established in the peripheral nervous system including the TG.

The coincidental occurrence of IL-6 and HSV-1 has previously been observed. For example, UV light which reactivates HSV-1 has been shown to induce IL-6 (55). Likewise, high levels of IL-6 in the cerebrospinal fluid of patients with HSV-1 encephalitis during the acute stage of the infection have been reported (56). Furthermore, the selective expression of the IL-6 gene in an HSV-1 permissive cell line following infection has been described (57). While the appearance of IL-6 may simply be due to its proinflammatory nature, a recent study suggests that IL-6 may, in part, facilitate HSV-1 replication or reactivation. Specifically, pretreatment of mice latently infected with HSV-1 with a mAb to IL-6 significantly blocked recoverable virus from the eye following hyperthermic stress (58). These authors suggest that IL-6 induces the activation of the transcription factors STAT3/APRF and nuclear factor-IL-6, which in turn promotes viral DNA replication. Neutralizing Ab to IL-6 has been found to partially block (50%) HSV-1 reactivation in TG explant cultures again supporting a role for IL-6 in viral reactivation.5

In the present study, hyperthermic stress induced HSV-1 reactivation and elicitation of a transient increase in IL-6 mRNA in the TG of latent mice. In addition, CK, which blocked stress-induced HSV-1 reactivation in a dose-dependent manner, also partially blocked the expression of the IL-6 transcript and protein. Taken together, these observations seem to support Kriesel’s work suggesting a role for IL-6 and HSV-1 reactivation (58). Hyperthermic stress was found to specifically elevate IL-6 mRNA but not other proinflammatory cytokines including IL-1{alpha} or TNF-{alpha} assessed during latency. However, CD8 transcript levels were significantly decreased 24 h poststress in the TG compared with the 0- to 12-h poststress time points. Consistent with these observations, hyperthermic stress of in vitro latently infected TG cell cultures results in the disappearance of CD8 transcripts 24 h poststress (59). The reduction of CD8 mRNA following stress and reduction of CD8+ lymphocytes, NK cells, and macrophages in the TG may allow viral replication and assembly to occur without interference by the would-be resident effector cells. However, the time frame of the absence of these immune cells is short lived (24–48 h) with the recruitment of infiltrating cells following viral reactivation as has been reported (60). The infiltrating CD8+ effector cells would limit the spread of the virus from the TG to the eye as suggested by the difference in frequency of detection of HSV-1 comparing the TG and eye following hyperthermic stress (20).

Changes in the immune cell constituency within the TG of latently infected mice with HSV-1 following hyperthermic stress may be due to corticosteroids. Previous studies have shown that stress-induced increases in corticosterone was accompanied by decreases in the circulating population of immune cells including B lymphocytes, NK cells, and monocytes (61). The changes in the circulating population of cells following stress were negated in adrenalectomized animals but mimicked by type 1 but not type 2 glucocorticoid receptor agonists suggesting that adrenal hormones are the primary mediators altering lymphoid and myeloid cell trafficking (61, 62). Consequently, one possible scenario is that by blocking the stress-induced increase in corticosterone with CK the successful antagonism of corticosterone modulation of the immune cell profile within the TG is achieved and viral reactivation/replication is limited.

The proinflammatory cytokines including TNF-{alpha}, IL-1, and IL-6 are known to activate the HPA axis (63, 64). A recent study has found that IL-6 is required for glucocorticoid production during murine CMV infection through the induction of IL-1{alpha} (65). However, in the present study hyperthermic stress was found to specifically alter IL-6 mRNA expression but not other cytokines including IL-1{alpha} in the TG of latently infected mice, suggesting that the induction of IL-6 is IL-1{alpha} independent in this stress paradigm. Yet, in another study, changes in the immune profile of restrained mice infected with influenza A virus (strain PR834) included a selective rise in IL-6 by cells from the regional lymph node (66). The restraint stress-induced rise in IL-6 could be prevented by the administration of the type II glucocorticoid antagonist RU486, suggesting the involvement of corticosteroids. This notion was further supported by data showing that low concentrations (10-10 M) of corticosterone could augment IL-6 but not IL-2, IL-10, or IFN-{gamma} production by splenocytes (66). However, in the present study, it is difficult to determine the mechanism for IL-6 induction. Since there is no significant rise in IL-6 mRNA in uninfected TG following hyperthermic stress, the presence of latent HSV-1 would appear to be involved in the augmentation of this cytokine. Similar to the findings of Dobbs et al. (66), it is conceivable that modest concentrations of corticosterone reach the TG immediately following hyperthermic stress that through either an additive or a synergistic effect with HSV-1 reactivation augment IL-6 production. The transient rise and fall of IL-6 mRNA expression in the TG is consistent with the continued rise in circulating corticosterone that ultimately reaches a critical level and acts as a feedback inhibitor to IL-6 production (64).

The cells that express IL-6 in the TG are presently unknown but could include resident dendritic cells and macrophages (59), Schwann cells (67), or neurons (68). Since hyperthermic stress did not change the percentage of dendritic cells in the TG of latent HSV-1-infected mice, these cells are a likely candidate for IL-6 expression. Dendritic cells are potent producers of IL-6 following HSV-1 infection (69). Likewise, Schwann cells have been shown to express IL-6 and IL-6 receptors following trauma (67). It is tempting to speculate that hyperthermic stress can induce the expression of IL-6 and IL-6 receptors by resident cells within the TG that subsequently allow the peripheral nerves to respond to IL-6 as previously suggested (67) potentially thru gp130 dimerization and subsequently cytoplasmic tyrosine kinase activation (70). Future work is required to address this tempting association more closely.


   Footnotes
 
1 This work was supported by grants from the Department of the Army, Cooperative Agreement DAMD17-93-V-3013, and National Institute of Neurologic Disorders and Stroke Grant NS35470. This work does not necessarily reflect the position or policy of the United States government, and no official endorsement should be inferred. Back

2 Present address: Department of Microbiology, University of Pennsylvania, 221 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104-6076. Back

3 Address correspondence and reprint requests to Dr. Daniel J. J. Carr, Department of Microbiology and Immunology, Louisiana State University Medical Center Box P6-1, 1901 Perdido Street, New Orleans, LA 70112-1393. Back

4 Abbreviations used in this paper: HPA, hypothalamic pituitary adrenal; SNS, sympathetic nervous system; HSV-1, herpes simplex virus type 1; LAT, latency-associated transcript; CK, cyanoketone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TG, trigeminal ganglion. Back

5 S. Noisakran and D. J. J. Carr. Submitted for publication. Back

Received for publication September 26, 1997. Accepted for publication January 30, 1991.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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