Toxicology and Molecular Biology Branch, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, 1095 Willowdale Road, MS 3014, Morgantown, West Virginia 26505
Received February 19, 2001; accepted May 3, 2001
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ABSTRACT |
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Key Words: stress; corticosterone; RU486; skin; sensitization.
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INTRODUCTION |
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Psychological stressors activate the hypothalamic pituitary adrenal axis (HPAA), which in turn, stimulates the release of glucocorticoids and catecholamines from the adrenal gland (Tomaszewska and Przekop, 1997). Glucocorticoids are major mediators of the stress response and modulate many signaling events in the immune response. Previous studies have shown that inflammatory products, such as the cytokines IL-1 and tumor necrosis factor-
(TNF-
), can activate the HPAA and stimulate the release of neurohormones. Glucocorticoids, such as corticosterone, modulate antigen presentation, cytokine production, T-cell expansion, and natural killer cell activity (Belsito et al., 1982
; Bonneau et al., 1997
; Chrousos and Gold, 1992
; Maes et al., 1998
; Snyder and Unanue, 1982
; Steer et al., 1998
; Wiegers and Reul, 1998
).
Acute restraint of mice, which has many physiological similarities to emotional stress in humans, is often used to examine the influence of stress on the murine immune system (Sheridan et al., 1994; Dhabhar et al., 1997
, 1999
; 2000 2001; Zhang et al., 2000). However, different strains of rodents respond differently to the effects of restraint (Dhabhar et al., 1995a
,b
; Sternberg et al., 1992
). BALB/c mice appear to be highly responsive to stressors. They produce high levels of plasma adrenocorticotropin releasing hormone (ACTH) and corticotropin releasing hormone (CRH), and they exhibit more anxiety, as determined by behavioral disturbances than do DBA, C3H, and C57 mice (Anisman et al., 1998
; Thompson, 1953
). Shanks et al. (1990) demonstrated similar basal corticosterone levels in 6 strains of mice; however, in response to a stressor, the magnitude and the rate of clearance of corticosterone differed significantly between the strains. C57BL/6 mice have been reported to be relatively stress resistant and although they have basal corticosterone levels similar to BALB/c mice, they produce lower concentrations of ACTH in response to acute stressors (Anisman et al., 1998
; Shanks et al., 1990
). We have previously shown that, contrary to anti-inflammatory effects of chronic stress, acute restraint prior to chemical challenge enhances the chemical-induced ear swelling response and pro-inflammatory cytokine production in the stress susceptible BALB/c mice, and that these changes are partially corticosterone dependent (Flint et al., 2001
). We hypothesize that in contrast to our observations in BALB/c mice, restraint stress would not alter the DNFB-induced ear swelling response in C57BL/6 mice because of their blunted HPAA response to an acute stressor.
In this study, we evaluated acute restraint modulation of 2,4-dinitrofluorobenzene (DNFB)-induced cutaneous hypersensitivity in stress-resistant C57BL/6 mice. We measured restraint-induced changes in serum corticosterone and ear swelling, a well-established measure of CHS. To determine if changes in the concentration of serum corticosterone influenced our outcome measures, C57BL/6 mice were treated with exogenous corticosterone or the glucocorticoid receptor antagonist, RU486, in the presence or absence of restraint.
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MATERIALS AND METHODS |
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Adrenalectomized (ADX) C57BL/6 mice received 30 µg/ml of corticosterone in their drinking water during transportation and were housed individually. Upon arrival, ADX mice were rested for 5 days following removal of corticosterone from the water and administered 0.9% saline and sucrose in their drinking water throughout the course of the experiment.
Induction of allergic contact dermatitis (ACD).
Prior to the experiment, animals were weighed, numbered, and shaved on the back. On days 1 and 2 of the experiment 100 µl of 0.5% 2, 4-dinitrofluorobenzene (DNFB; Sigma-Aldrich, St. Louis, MO), diluted in a vehicle of 4:1 acetone:olive oil (AOO) was applied slowly to the back skin with a micropipette. On day 5, baseline ear thickness was measured for the right and left pinnae. On day 6, the right pinnae were challenged with 50 µl of 0.25% DNFB and the left pinnae were treated with AOO. For application of restraint prior to challenge, mice were restrained for 2 h on day 6. The thickness of the right and left ear pinnae were measured 24, 48, and 72 h after challenge using a digital micrometer.
Manipulation of the HPAA
Restraint.
Each mouse was placed in an adequately ventilated 50 ml conical plastic tube (Corning Inc., Corning, NY) for 15 min to 2 h as specified in each experiment. Mice were not physically squeezed and felt no pain. They could rotate from a supine to prone position, but not turn head to tail. Restraint was applied at 1000 h for all experimental manipulations. Nonrestrained mice were left undisturbed in their home cages.
Corticosterone administration.
A pilot study was performed to confirm the dose of corticosterone necessary to produce the required serum concentration. Corticosterone (Sigma-Aldrich, St. Louis, MO) was dissolved in polyethylene glycol 400 (PEG), and each mouse received one sc injection of corticosterone (6 mg/kg in 0.01 ml volume in PEG) or PEG only at 1000 h. Mice were evaluated 0, 15, 30, 60, and 120 min after injection. To determine the serum concentration of corticosterone following injection and restraint, mice received an injection of corticosterone at 0800 h and restraint was applied at 1000 h for 0, 15, 30, 60, and 120 min. The effect of exogenous corticosterone on restraint modulation of ACD was evaluated within 4 h following administration, well within the peak response of the drug (Peeters et al., 1992).
RU486 treatment.
RU486 is a potent glucocorticoid type II receptor antagonist that can also block the progesterone receptor. Under conditions of our studies and using male mice, we interpreted the effects of RU486 in terms of the type II glucocorticoid receptor antagonism. RU486 peaks in the serum 1 h after injection and has an elimination half life of 86 h (Foldesi et al., 1996). Based on a pilot study in our laboratory, RU486 (Sigma-Aldrich, St. Louis, MO) was dissolved in polyethylene glycol 400 (PEG; Sigma-Aldrich, St. Louis, MO), and each mouse received one sc injection of RU486 (25 mg/kg in PEG; 0.01 ml/g body weight) or PEG 1 h prior to restraint. The effect of RU486 administration on restraint modulation of ACD was evaluated 3 h following administration, well within the peak response of the drug.
Adrenalectomy.
Bilaterally ADX C57BL/6 mice were purchased from Taconic (Germantown, NY). This surgical procedure significantly decreases the endogenous source of corticosterone and epinephrine. Auxillary nodes can produce corticosterone 1421 days post surgery, therefore all experiments were conducted within 14 days after surgery. Selected mice received 6 mg/kg of corticosterone in PEG or PEG alone 2 h prior to restraint or 4 h prior to challenge.
Corticosterone analysis.
Mice were sacrificed immediately following the 2 h restraint period, and blood was obtained by cardiac puncture and collected into plasma separator tubes containing lithium heparin (Becton-Dickinson & Co., Franklin Lakes, NJ). Serum was separated by centrifugation. Fifty µl samples were assayed in duplicate for corticosterone content using an anti-rat corticosterone-coated tube and 125I-corticosterone tracer protocol (Coat-A-Count RIA kit, DPC Inc., Los Angeles, CA). The RIA was performed according to the manufacturer's protocol, and samples were analyzed by gamma scintillation and duplicates were averaged. Because serum corticosterone concentrations can vary in mice due to handling techniques, laboratory baseline values were determined. The baseline range at noon in C57BL/6 mice was between 1832 ng/ml.
Statistical analysis.
Data are presented as the mean ± SEM for each experimental group. For the corticosterone data analysis, statistically significant differences (p < 0.05) between restrained and nonrestrained groups were determined by the Student's t-test.
Ear swelling data analysis was conducted using the SAS software program. Statistical analysis was performed on the raw data. Descriptive statistics such as means and standard deviations were calculated and compared using PROC MIXED, and adjusted for the covariate, initial ear thickness. The analysis combined 2 experiments done in different weeks. Each experiment was assumed to be a random effect that resulted in the interaction term of experiment by treatment being the appropriate error term for testing treatment effects. When analyzing repeated measures data, the REPEATED option was used to model the within subjects covariance structure. The best covariance structure was chosen by using 2 model-fit criteria, Akaike's Information Criterion (AIC) and Schwarz' Bayesian Criterion (SBC). The covariance structure that produced the largest value for these criteria was considered the best model-fit. The LSMEANS option was used to calculate means adjusted for unequal sample sizes among treatment groups.
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RESULTS |
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To extend these observations, we next asked if administration of exogenous corticosterone to C57BL/6 mice would produce serum corticosterone levels similar to the BALB/c mice and promote an enhanced CHS response. We evaluated the administration of exogenous corticosterone at 1000 h in nonrestrained mice at 0, 15, 30, 60, and 120 min following injection in order to verify the time course for the increase of the drug in the serum. Mice that received 6 mg/kg of corticosterone demonstrated a significantly increased serum concentration of corticosterone at 15 min, 1285 ± 122 ng/ml that remained elevated at 956 ± 126 ng/ml at 120 min (Table 1). The combined effect of exogenous corticosterone and restraint produced a similar time course, with no further increase in serum corticosterone attributable to the addition of restraint (Table 2
). Mice in this study received the corticosterone injection at 0800 h and restraint was applied for 0, 15, 30, 60, and 120 min, beginning at 1000 h. Serum corticosterone levels in this experiment were not statistically different from the mice that received the injection only, and the t = 0 time point for the injected and restrained mice occurred 2 h after injection and was equivalent in time after injection to the 120 min time point in Table 1
.
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The Effect of Restraint on Ear Swelling in C57BL/6 Mice Treated with DNFB
Chemical-induced ear swelling is characterized by edema, erythema, and influx of inflammatory cells throughout the epidermis and dermis that can be measured 24, 48, and 72 h postchallenge. To determine if C57BL/6 mice demonstrate the restraint-enhanced ear swelling response observed in BALB/c mice, C57BL/6 mice were dosed on the back with 0.5% DNFB on days 1 and 2, and restrained for 2 h prior to challenge on the ear with 0.25% DNFB on day 6 (Fig. 1). Nonrestrained C57BL/6 mice showed a chemical-induced increase in ear swelling at 24 h, 30% higher than the baseline measurement, that reached a maximum of 40% at 72 h. Restraint applied prior to challenge in C57BL/6 mice did not change the response at any time point. Ear swelling at 48 and 72 h were 45 and 40% respectively. These data show that, in contrast to our observations with BALB/c mice, the cutaneous immune response to chemical in C57BL/6 mice is insensitive to modulation by acute restraint stress administered prior to chemical challenge.
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The Effect of Corticosterone Administration on Ear Swelling in C57BL/6 Mice Treated with DNFB
To determine the effect of exogenous corticosterone on DNFB-induced ear swelling, C57BL/6 wild-type mice received a single injection of corticosterone, or the vehicle, PEG, 2 h prior to restraint. Additional mice were treated with corticosterone or PEG and left in their home cages. Although neither restraint nor corticosterone injection alone increased the DNFB-induced ear swelling response, the combination of restraint and corticosterone significantly increased ear swelling at all time points (Fig. 2). Administration of PEG to either restrained or nonrestrained mice did not significantly alter DNFB-induced ear swelling, indicating that the injection-induced increase in serum corticosterone, 8090 ng/ml, as measured for both wild type and ADX mice was insufficient to effect change in the ear thickness measurements (data not shown).
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DISCUSSION |
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Following initial application of chemical to the skin, the release of proinflammatory cytokines triggers LC migration from the epidermis to the draining lymph nodes where they activate CD4+ and CD8+ lymphocytes (Kimber, 1994). In response to cutaneous challenge with the same chemical, these activated leukocytes return to the skin to mount an antigen specific cell-mediated immune response. Glucocorticoids modulate the contact hypersensitivity response at many levels. For example, corticosterone can affect trafficking of lymphocytes from the peripheral blood to the lymph nodes and to sites of active inflammation (Davenpeck et al., 1998
; Dhabhar et al., 1996
; Schleimer, 1993
), vascular permeability and edema, or directly influence proinflammatory cytokine production (Flint et al., 2000
). These modulatory events are frequently accomplished by changes in the concentration of intercellular signaling molecules or in the pattern of receptor expression.
We have previously shown that restraint before chemical challenge significantly enhanced serum corticosterone and DNFB-induced ear swelling in BALB/c mice and that corticosterone was partially responsible for the enhanced ear swelling (Flint et al., 2001). In addition, other studies have shown that restraint stress enhances these parameters in rats and B6,129 mice, and Dhabhar and colleagues confirmed the additive effects of glucocorticoids and epinephrine in the enhanced response in rats (Dhabhar et al., 1996
). In this study we have demonstrated that, in spite of similar basal levels of serum corticosterone, C57BL/6 mice produce significantly lower levels of corticosterone in response to 2 h restraint than do BALB/c mice. In our laboratory, BALB/c mice typically produce 800900 ng/ml corticosterone in response to restraint whereas C57BL/6 mice routinely show an elevation of 450500 ng/ml (Flint et al., 2001
).
We also demonstrated that, in restrained mice, administration of corticosterone enhances DNFB-induced ear swelling and that surgical interruption of the HPAA diminished ear swelling, an effect that cannot be overcome by administration of exogenous corticosterone. Responses to stressors are initiated by the hypothalamus and translated by the HPAA and autonomic nervous system. Products from both systems, corticosteroids and catecholamines respectively, are able to modulate the activity of immune effector cells to restore homeostasis in the mouse. Studies have shown that strains of mice differ in terms of adrenal response and that acute stress does not significantly alter levels of pituitary CRH and plasma ACTH in C57BL/6 mice, a possible explanation for the lower levels of serum corticosterone we observed (Anisman et al., 1998). Interestingly, administration of either restraint or exogenous corticosterone to ADX mice depressed the mean ear swelling response. The adrenalectomy was performed prior to chemical sensitization, and we have shown previously that restraint applied to a naive mouse immediately before chemical sensitization suppresses development of contact hypersensitivity (Flint et al., 2001
). It is likely that immune suppression following a major surgical procedure such as adrenalectomy will have an extended time course. In addition to severely limiting corticosterone production, adrenalectomy also interrupts epinephrine production. Others have previously shown that epinephrine contributes the enhanced ear swelling in ADX mice (Dhabhar and McEwen, 1999). These findings highlight the importance of catecholamines in the stress response and the multifactorial link between the stress response and the cutaneous immune response.
Many studies have documented that corticosterone binds first to the high affinity type I receptor and only after saturation of the type I receptor does corticosterone bind the lower affinity type II receptor. Because RU486, a type II receptor antagonist, had no significant effects on ear swelling and increased corticosterone did not modulate cutaneous hypersensitivity in C57BL/6 mice, it is possible that there are strain dependent limitations in surface expression of the type II corticosterone receptor or changes in the receptor affinity. Consistent with this theory, earlier work suggested that corticosterone binding capacity in the hippocampus is lower in C57BL/6 mice than BALB/c mice, a condition that may extend to other organs in this mouse (Patacchioli et al., 1990). These data suggest that strain dependent differences in the HPAA underlie acute stress modulation of cutaneous hypersensitivity.
Taken together, our data demonstrate that C57BL/6 mice produce moderate levels of corticosterone in response to 2 h restraint and that increases in the concentration of serum corticosterone alone do not produce the enhanced ear swelling response observed in BALB/c mice. These studies support our hypothesis that acute restraint stress applied before chemical challenge has no effect on ear swelling in stress resistant C57BL/6 mice and suggest that strain-specific differences in the stress response impact the magnitude and direction of the cutaneous immune response to chemical. Additional studies are required to determine if, in humans, the combination of workplace stress and chemical exposure may alter the exposure-disease paradigm and indicate the need for additional considerations when establishing protective measures.
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ACKNOWLEDGMENTS |
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NOTES |
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