Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130
Received May 8, 2000; accepted July 27, 2000
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ABSTRACT |
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Key Words: stress; corticosterone; 3,4-dichloropropionanilide; predictive models.
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INTRODUCTION |
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In an effort to understand the quantitative relationships between stress responses and suppression of selected immunological parameters, this laboratory has obtained data from mice exposed to various dosages of exogenous corticosterone or to immobilization (restraint) stress for various periods of time. Previous work demonstrated that corticosterone accounts for some or all of the suppression of several immunological parameters in mice treated with the potent chemical stressor ethanol (Collier et al., 1998; Collier et al., 1999; Han and Pruett, 1995
; Han et al., 1993
; Weiss et al., 1996
; Wu and Pruett, 1997
). Therefore, corticosterone was selected as the neuroendocrine mediator to be used in these initial modeling efforts. The area under the corticosterone concentration vs. time curve was determined for mice treated with exogenous corticosterone or restraint. Linear models were then developed from these data, and these models indicate a clear relationship between cumulative corticosterone exposure and suppression of several immunological parameters.
The purpose of the present study was to examine a stress-inducing chemical, 3,4-dichloropropionanilide (propanil), to determine if the corticosterone AUC values induced by different dosages of that chemical could be used to predict the amount of suppression of several immunological parameters. Linear models were obtained that allow such predictions. In addition, the results for propanil were compared to results obtained in previous studies for exogenous corticosterone and restraint stress to determine if the immunological effects of propanil more closely matched the effects of exogenous corticosterone or restraint. Our hypothesis was that propanil would induce the whole range of neuroendocrine mediators induced by most stressors (including restraint), and that its effects would therefore more closely resemble the effects of restraint than the effects of exogenous corticosterone.
Propanil was selected as the chemical stressor for these studies because it is a widely used herbicide, and propanil-induced thymic atrophy seems to be mediated by corticosterone. Thymic atrophy occurred in normal mice but not in adrenalectomized mice that were treated with propanil (Cuff et al., 1996). Propanil suppresses a number of immunological parameters in mice, and T-dependent antibody responses and thymus cellularity seem to be among the more sensitive parameters (Barnett and Gandy, 1989
). Some direct effects of propanil have been identified using in vitro techniques, but it has not been determined if these effects are responsible for the alterations in immunological parameters noted in vivo (Xie et al., 1997
; Zhao et al., 1998
). Thus, it remains possible that suppression of a number of immunological parameters in vivo may be mediated by the neuroendocrine stress response to propanil.
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MATERIALS AND METHODS |
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Administration of propanil.
Propanil was obtained from Chem Service (West Chester, PA). It was administered intraperitoneally at dosages of 50, 75, or 100 mg/kg body weight in food-grade corn oil. In some experiments a dosage of 150 mg/kg was also used. Preliminary experiments demonstrated that the corticosterone response to vehicle administration subsided within 4 h. This yields a negligible corticosterone AUC value, which only serves to add an additional data point near the 0 value for corticosterone AUC to any models developed (Pruett et al., 1999). Therefore, naive control groups, not vehicle control groups, were used for these modeling studies.
Determination of corticosterone AUC values for propanil-treated mice.
Seven groups of mice (five mice per group) were treated with propanil at each of the four dosages used in this study (50, 75, 100, or 150 mg/kg body weight) at 9:00 P.M. One group at each dosage was bled by decapitation at 1, 2, 4, 6, 8, 12, and 18 h after propanil administration. Naive control groups were designated for each time point, and these mice remained undisturbed in a separate room from the treated mice until they were bled at the appropriate time point to match each treatment group. Mice were bled in an adjacent room, so neither naive nor treated mice were exposed to odors or noise associated with the bleeding process. Serum was separated from each blood sample and the corticosterone was analyzed using a radioimmunoassay kit (DPC, Los Angeles, CA) (Pruett et al., 1999). The data were graphed using DeltaGraph software, scanned to produce a digital image, and the area between the naive control values and the treated values was determined, as described previously, using NIH Image software (Pruett et al., 1999
).
Measurement of immunological parameters.
Immunological parameters were evaluated exactly as described in our previous studies at the time after administration of the stressor previously shown to produce maximum suppression (Han and Pruett, 1995; Pruett et al., 1999
; Pruett et al., 2000
; Weiss et al., 1996
; Wu et al., 1994
). In all experiments, the group size was five mice per group. Cell suspensions were prepared by pressing the spleen or thymus between the frosted ends of glass microscope slides, and spleen and thymus cellularity were determined using an electronic cell counter (Coulter model Z1, Coulter Electronics, Hialeah, FL). A previous time course study indicated that the greatest suppression of cell number in the spleen and thymus occurred 24 h after the initial administration of corticosterone (Pruett et al., 2000
). Therefore, this time point was used for the present study both for assessing cell number and examining cellular subpopulations by flow cytometry.
Labeling for flow cytometry was conducted using fluorescent-labeled antibodies specific for CD4 and CD8 from Pharmingen (San Diego, CA) and B220 from Gibco/BRL (Grand Island, NY). Cells were labeled in V-bottom microtiter plates at a concentration of 106 cells per well. An isotype control was used for each antibody. Gates were set using the isotype controls, and less than 2% of the cells labeled with the isotype control were outside the gate defined as negative in any experiment.
Splenic natural killer (NK) cell activity was measured 12 h after initiation of propanil treatment (the same time used previously in experiments with exogenous corticosterone or restraint stress) (Wu et al., 1994) using a standard 4-h 51Cr release assay with YAC-1 target cells. Effector-to-target cell ratios of 100:1, 50:1, and 25:1 were used. Values were expressed as lytic units per 107 splenocytes (Bryant et al., 1992
) to provide a single value for modeling. Expression of MHC class II protein on splenocytes was evaluated using samples of the same splenocyte preparations used for the NK cell lytic assay. Cells were labeled as described above using anti-MHC class II protein (I-Ap,b,k,j,q,r,s) (Pharmingen, San Diego, CA) (Weiss et al., 1996
).
The IgG1 and IgG2a response to keyhole limpet hemocyanin (KLH, Pierce Chemical Co.) was measured by immunizing mice with KLH (100 µg/mouse) 12 h after treatment with propanil (the same as the optimum time previously identified for treatment with exogenous corticosterone) (manuscript submitted). Mice were then bled (under methyoxyflurane anesthesia) 2 weeks later (the time at which near maximum antibody levels occur) (Kruszewska et al., 1995). Serum was diluted 1/25, 1/50, and 1/100, and IgG1 and IgG2a antibodies specific for KLH were measured by ELISA. Wells in 96-well microtiter plates were coated with 100 µl KLH at 10 µg/ml in phosphate-buffered saline (PBS). The antigen solution was removed, and a blocking solution (1% bovine serum albumin in PBS) was added for 2 h at room temperature. Serum (100 µl diluted in PBS with 0.05% Tween 20, referred to hereafter as wash buffer) was added to the wells, and incubated 1.5 h at room temperature; the wells were washed three times with wash buffer using an automated plate washer. Secondary antibody (anti-mouse IgG1 or IgG2a-alkaline phosphatase conjugate, Caltag, Burlingame, CA) diluted 1/1600 in wash buffer was added to each well, and the wells were washed three times after a 1.5-h incubation period at room temperature. One hundred microliters of PNPP alkaline phosphatase substrate solution (Sigma Chemical Co, St. Louis, MO) was added to each well. Absorbance at 405 nm was determined using a plate reader (UV-3550, BioRad, Hercules, CA). Because models developed using all three serum dilutions were very similar, the 1/25 dilution only was used to obtain a single value for each mouse for modeling purposes. Serum from nonimmunized mice was diluted to the same extent as the test serum and included as a control in each experiment. Absorbance values for these controls were always less than 0.05.
Statistical and modeling methods.
Linear regression analysis was performed using the Prism software package (GraphPad, San Diego, CA). Replicate values for immunological parameters within each group were analyzed as separate values, not means. The program was also used to calculate and graphically illustrate the 95% confidence intervals of each line. The runs test (Motulsky, 1994) indicated no significant nonlinearity in any of the immunological parameters vs. corticosterone AUC plots. Statistical comparison of pairs of lines was done as described (Zar, 1984
) and as implemented by Prism software. This approach determines if the slope or the elevation (or Y-intercept) differ significantly between any pair of lines. A variety of nonlinear models were also examined, and in one case (spleen cell number) a nonlinear model yielded a larger correlation coefficient and a plot that more closely represented the apparent threshold effect in this data set. Therefore, the nonlinear model is shown for this parameter.
Linear models of the effects of exogenous corticosterone and restraint stress on the same immunological parameters examined in the present study were developed in previous studies in this lab (Pruett et al., 1999; Pruett et al., 2000
). These models were used to predict the effects of propanil (100 mg/kg). The regression equations were used to calculate the value for the immunological parameter that corresponds with the corticosterone AUC measured in mice treated with propanil at 100 mg/kg (4383 ng/ml h). The 95% confidence intervals for these calculated values were determined using a table of calculated confidence intervals over the entire range of the regression line, which is calculated by the Prism software package. In some cases, the regression equations and confidence intervals were confirmed using StatView software (SPSS, Chicago, IL), and the results were the same in all cases examined.
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RESULTS |
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Linear Models of Corticosterone AUC versus NK Cell Activity in Mice Treated with Propanil
Data shown in Figure 5 indicate that propanil dose-responsively decreases splenic NK cell activity. For modeling purposes, the highest dosage of propanil is not shown. It induced only a slight decrease in NK cell lytic function (26 ± 4 LU/107 splenocytes compared to 30 ± 3 for the control group) that was not consistent with the dose-responsive decreases produced by the lower dosages. This may represent NK cell activation associated with increased spleen weight and cellularity reported after high dosages of propanil (Barnett and Gandy, 1989
), or it may represent direct activation of NK cells by propanil at high dosages, which overcomes the suppression induced by stress-related mediators. However, the effect of propanil on NK cell function at dosages of 0, 50, 75, and 100 mg/kg yielded a linear model that was used to describe suppression of NK activity within this dosage range. Linear models obtained in a previous study (Pruett et al., 1999
) using mice treated with exogenous corticosterone or restraint were consistent with data from mice treated with propanil at 50, 75, and 100 mg/kg. The line obtained using data from propanil-treated mice was not significantly different from the line obtained using data from restrained mice, but it was different from the line obtained from corticosterone treated mice (the elevations of these lines differ significantly; p = 0.0015). The elevation of the propanil line was lower than the exogenous corticosterone line, suggesting greater suppression of NK cell function by propanil than by an equivalent exposure to corticosterone alone.
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DISCUSSION |
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Although glucocorticoid-induced changes in lymphocyte trafficking could be involved in the changes in cell types both in the spleen and in the blood, this seems unlikely. Glucocorticoid-induced changes in lymphocyte trafficking typically occur very quickly, and values return to normal within 4 h (Dhabhar et al., 1995). In addition, the finding that MHC class II protein expression is decreased similarly at 12 h in the spleen and in peripheral blood (Fig. 4
) is more consistent with decreased MHC II expression than with altered trafficking. Altered trafficking would be a more reasonable explanation if an increase in MHC II expression was noted in the blood, suggesting that the cells lost from the spleen were mobilized to the blood.
Only one of the immunological parameters (the IgG2a response to KLH) evaluated in the present study was suppressed by propanil to a significantly greater extent than predicted from corticosterone AUC values using either restraint or exogenous corticosterone models. This suggests that most of the effects of propanil noted here could have been mediated by the neuroendocrine stress response to this compound. The additional suppression of the IgG2a response to KLH may be a consequence of the ability of propanil to directly suppress the production of some cytokines (Zhao et al., 1999). In addition, it should be noted that propanil exerts immunosuppressive effects that were not evaluated in the present study, and it remains possible that some of these effects are mediated directly by propanil or indirectly by mechanisms that do not involve the neuroendocrine stress response. The failure of the highest dosage of propanil to suppress NK cell activity in a manner consistent with the suppression expected from corticosterone exposure suggests that some dosages of some immunotoxicants may counteract the effects of stressors, leading to nonlinear (biphasic) relationships. Additional data are needed to determine how common such occurrences will be, but such departures from linearity can be easily identified by visual inspection or by mathematical methods (Luster et al., 1993
). In any case, the effectiveness of the restraint models in predicting important immunological effects of a chemical immunotoxicant suggests that further development of this approach is warranted.
The basis for the nonlinear effect of propanil on spleen cell number is not clear. A number of immunotoxic chemicals can induce hematopoiesis, and it is possible that increased hematopoiesis in the spleen overcomes the effect of corticosterone at low propanil dosages. In any case, the departure from a linear model is informative. It demonstrates that the effects of corticosterone alone or in combination with the other neuroendocrine mediators induced by restraint are not the only factors involved in the effects of propanil on spleen cellularity.
It is not surprising that models developed using data from mice subjected to restraint stress provide better predictions of the effect of propanil on a number of immunological parameters than models developed using data from mice treated with exogenous corticosterone. We had hypothesized that this would be the case, because a wide variety of stressors induce similar (though not identical) patterns of increased concentrations of several neuroendocrine mediators (Pacák et al., 1998). In contrast, mice treated with exogenous corticosterone presumably only exhibit changes in these neuroendocrine mediators for a few minutes as a result of the brief period of handling and restraint involved in dosing. Our previous studies and studies from other labs demonstrate that some immunological parameters can be affected by corticosterone and other neuroendocrine mediators (Ader et al., 1990
; Wu and Pruett, 1997
), and that interactions between mediators can occur. For example, the presence of restraint stress-induced mediators apparently modifies the effect of corticosterone on the thymus so that all subpopulations are depleted equally, without preferential loss of CD4+CD8+ cells noted in mice treated with exogenous corticosterone (Pruett et al., 1999
). Fortunately, these mechanistic considerations do not preclude the use of modeling on the basis of corticosterone AUC alone. Data presented here demonstrate that corticosterone AUC measured in restrained mice effectively predicts suppression of several important immunological parameters. Presumably, the other neuroendocrine mediators that contribute to these effects exhibit covariance with corticosterone AUC such that corticosterone AUC acts as a suitable surrogate for these mediators for modeling purposes. Considering that the data for modeling the effects of restraint and data for modeling the effects of propanil were obtained during a 3-year time period and that different personnel were involved, the results seem quite robust.
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ACKNOWLEDGMENTS |
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NOTES |
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