Immunotoxicology Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received July 26, 2001; accepted November 19, 2001
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ABSTRACT |
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Key Words: T-helper lymphocytes; cytokines; influenza virus; mice; ultraviolet radiation; inflammation.
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INTRODUCTION |
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Because of the public health importance of influenza, several investigators have used this disease in rodents as host resistance models to assess immunotoxicity following exposure to chemicals or other agents. Others and we have demonstrated that enhanced mortality caused by xenobiotic exposure in this model is not related to suppression of virus-specific immune responses. When mice were exposed to 0.01 µg/kg of dioxin one week prior to infection, mortality doubled without increasing virus titers (Burleson et al., 1996; House et al., 1990
; Warren et al., 2000
). In another study, mortality doubled without an increase in lung virus titers in mice that were exposed to 1 ppm ozone for 3 h on 5 consecutive days, and then were infected after the second day of exposure (Selgrade et al., 1988
). In contrast, work with more traditional immunosuppressive agents (X-irradiation and cyclophosphamide) demonstrated increased virus titers in association with increased mortality in influenza-infected rodents (Frankova, 1989
, Hurd and Heath, 1975
; Singer et al., 1972
).
Ultraviolet radiation (UVR) causes systemic immune suppression, decreasing the delayed type (DTH) and contact hypersensitivity (CHS) responses in animals and humans and enhancing mycobacterial, parasitic, and herpes viral infections in mice (reviewed in Norval and El-Ghorr, 1998; Norval et al., 1999
; Selgrade et al., 1997
; Ullrich, 1998
). We previously demonstrated that UVR exposure increased influenza virus-associated mortality in a dose-dependent manner (up to a 2-fold increase at 8.2 kJ/m2; Ryan et al., 2000
). The increased mortality was not associated with bacterial pneumonia. UVR also accelerated the body weight loss and increased the severity and incidence of thymic atrophy associated with influenza infection. However, UVR treatment had little effect on the increase in lung wet weight seen with viral infection, and did not cause an increase in virus titers in the lung or dissemination of virus to the brain. The mice died 56 days postinfection, too early for adaptive immune responses to have much impact against viral infection. Protective immunity to the virus (as measured by adoptive transfer of immune spleen cells from UVR-treated mice and resistance to reinfection of UVR-treated immunized mice with a lethal challenge of influenza) was unaffected.
We chose to use the UVR/influenza virus mouse model system to study the mechanism of enhanced mortality in the absence of effects on virus-specific immunity. The symptoms resembled those of mice succumbing to endotoxin. Because UVR induces some of the same proinflammatory cytokines (TNF-, IL-1, and IL-6) associated with endotoxin-induced septic shock syndrome (Akira et al., 1993
; Beutler and Cerami, 1987
; Dinarello, 1996
), we tested the hypothesis that modulation of these cytokine responses was associated with the increased deaths seen in this model. In addition, because UVR is known to suppress Th1 responses, and Th2 responses exacerbate influenza pathogenesis (Graham et al., 1994
), we also measured levels of Th1 and Th2 cytokines to test the hypothesis that the increased morbidity and mortality was due to changes in the balance of these 2 cell types.
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MATERIALS AND METHODS |
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Ultraviolet irradiation.
Prior to irradiation, mice were lightly anesthetized with metafane (Pitman-Moore, Chicago, IL) and an area of 8 cm2 was shaved on the dorsal side of each mouse. Mice were then irradiated 24 h later with either UVR or fluorescent room light that contained no UV-B (controls) for 30 min. All mice, including controls (room light exposed), were exposed in wire cages designed to hold individual mice, and their ears were protected with white labeling tape during exposure. Except where indicated, irradiation with 8.2 kJ/m2 UVR occurred 3 days prior to infection with influenza virus. Ultraviolet irradiation was accomplished using five 20-watt (W) FS20 fluorescent lamps (Westinghouse SunLamp, Pittsburgh, PA) that emit wavelengths between 270 and 390 nm with a peak emission at 313 nm. Approximately 55% of the UVR output of this system was in the UV-B range (280320 nm), 36% was in the UV-A range (320400 nm), and 3.7% was in the UV-C range (200290 nm). Light intensity was determined before and after every exposure, using an LMB-304 UVB detector (National Biological Corp., Twinsburg, OH). Intensity remained consistent during exposure, ranging from 0.38 to 0.54 mW/cm2 and averaging 0.48 ± 0.03 mW/cm2 for all experiments. Readings and exposure occurred 15 cm from the source. The total incident dose received during a 30-min exposure, including shielding from the exposure cage lids, was approximately 8.2 kJ/m2 (approximately 4 MED [minimal erythemal dose]). This exposure caused greater than 80% suppression of the contact sensitivity response to dinitrofluorobenzene applied at a site distant from UVR (Sailstad et al., 2000).
Virus.
Mouse-adapted Influenza A/Hong Kong/68 (H3N2), was prepared from BALB/c mouse lung extracts (virus went through 18 passages in mice) and assessed for plaque-forming units (pfu) on Madin Darby canine kidney (MDCK) cell monolayers as previously described (Selgrade et al., 1988). Mice were anesthetized with metafane, and then inoculated intranasally (in) with 300 pfu (LD40) of virus in 0.05 ml Hanks' balanced salt solution without Mg++ or Ca++ (HBSS, GIBCO BRL Laboratories, Grand Island, NY). Sham or mock infection used an intranasal inoculation with 0.05 ml HBSS containing no virus.
Experimental design.
Four experiments were performed to address 3 hypotheses. In experiment 1, a dose-mortality response experiment was performed to confirm the hypothesis that UVR affects influenza-induced mortality in a dose-dependent manner. Experiment 2 varied the timing of UVR exposure (given at Days 0 and 3) relative to infections and measured body weight loss, a parameter associated with morbidity in influenza infection, to test the hypothesis that exposure of mice to UVR prior to infection was necessary to give time for the UVR to induce cytokines and other processes that could affect morbidity and mortality. Experiment 3 examined the effect of UVR exposure 3 days prior to infection with influenza virus on cytokine levels and cell morphology profiles in the bronchoalveolar lavage (BAL) fluid to test the hypothesis that preexposure to UVR altered the cell and cytokine profiles induced by influenza infection. Following lavage, lungs were homogenized and cytokine profiles in the lung homogenates (LH) were also determined, to screen LH for altered cytokine levels induced by UVR and/or virus. Once the virus-induced cytokines that were altered by UVR were identified, experiment 4 was performed to confirm these alterations using LH obtained from mice that had not been lavaged. Experiment 4 was performed identically with experiment 3, except that the lungs were not lavaged and mice were only sacrificed up to Day 5 after infection, because after Day 5, mice begin to die from the infection, and there is a selection for survivors. All LH data shown in the figures is from experiment 4. In experiments 2, 3 and 4, animals were exposed to 8.2 kJ/m2 UVR or light and infected with 300 pfu virus (LD40) or HBSS. The groups were designated light/sham-infected, UVR/sham-infected, light/virus-infected, and UVR/virus-infected. In experiment 3, only one light/sham-infected group from the same shipment of mice was examined, and the mice were light-treated and sham-infected after the infection of the other groups, and sacrificed immediately. These were labeled "control" on the graphs in Figures 3, 5A, 5C, 6A, and 7A. In this experiment, the two-way ANOVA was compared with this one control. It was assumed (based on previous data) that baseline control levels of inflammatory cells and cytokines in BAL from this group would be minimal (which was the case) and it would not be necessary to sacrifice a light/sham-infected group for each day following infection. However, in experiment 4, each day following infection had its own matched control for cytokines in LH, because LH does have varying baseline cytokine levels; this is reflected in Figures 5B, 5D, 6B, 6C, and 7B
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Body weight loss.
In experiment 2, animals were tagged for individual identification and exposed to 8.2 kJ/m2 UVR or light and infected either immediately or 3 days after irradiation with 300 pfu virus (LD40) (n = 12 for each of the 4 groups). Body weight for each individual animal was monitored for 14 days and compared with the initial body weight at the start of the experiment on Day 3. The percentage of body weight lost was calculated for each animal and the percentages were then averaged for each group.
Lung lavage and lung homogenization.
Groups of mice were euthanized with an intraperitoneal (ip) injection of 0.1 ml Nembutal sodium (250 mg sodium pentobarbital, Abbott Laboratories, North Chicago, IL/kg) at daily intervals 3 days prior to infection and on days 07 postinfection. In experiment 3, lungs of mice were lavaged; total and differential cell counts and cytokine measurements on BAL fluid were made as described below. The lungs were then immediately homogenized in 10% w/v HBSS with sterile sand using a mortar and pestle and the homogenates stored at 700C for later determination of virus titers and preliminary cytokine determinations, as described below (n = 5 per group). In experiment 4, lungs of mice were not lavaged, but were snap frozen in liquid nitrogen and stored at 700C until they were homogenized (n = 5 per sham-infected group; n = 10 per virus-infected group) (Smith et al., 1994). Lungs were homogenized using a polytron homogenizer at 40C in PBS-based antiprotease lysis buffer containing a protease inhibitor cocktail (complete, Boehringer-Mannheim, Indianapolis, IN, 1 tablet/50 ml PBS). Samples were sonicated in this buffer for 15 s, then filtered through a 1.2 µm syringe filter. Lung homogenates (LH) were aliquoted and stored at 700C until assayed for cytokines. No sample aliquot was thawed more than twice prior to assay.
Lung differential cell counts.
Mouse tracheas were cannulated and lungs were lavaged twice with 1.0 ml of sterile HBSS. The resulting BAL fractions for each mouse were pooled and centrifuged at 600 x g for 10 min. The BAL supernatant was stored at 700C for cytokine assay. The cell pellet was resuspended in 0.5 ml RPMI cell culture medium containing 10% fetal bovine serum (GIBCO). Cell viability was assessed by dilution in 0.4% trypan blue dye (GIBCO) and counted on a hemocytometer. BAL cell morphology was determined by pelleting cells on a slide, using a cytocentrifuge operating at 1000 rpm for 5 min (Shandon, Pittsburgh, PA) and using a differential stain for leukocytes (LeukoStat, Fisher Scientific, Pittsburgh, PA).
Lung virus titers.
Virus titers were ascertained from LH extracts in HBSS using the MDCK plaque assay as previously described (Selgrade et al., 1988). Virus titers were monitored on days 17 postinfection in experiment 3 only.
Cytokine assays.
Cytokine release in BAL fluid and in LH was assessed by murine ELISA kits (Genzyme, Cambridge, MA unless indicated otherwise) for detection of the following cytokines, with detection limits shown in parenthesis. BAL fluid from Experiment 3: TNF- (<35 pg/ml), IL-1
(<15 pg/ml), IL-1ß (<15 pg/ml), IFN-
(<20 pg/ml), IL-6 (<8.8 pg/ml), and IL-10 (<30 pg/ml). LH from lavaged lungs in experiment 3: IL-1
, IL-1ß, GM-CSF (<7.8 pg/ml, R&D Systems, Minneapolis, MN), TNF-
, IFN-
, IL-4 (Endogen, Woburn, MA, <5 pg/ml), IL-5 (Endogen, <7.7 pg/ml), IL-10, IL-12 (total (<60 pg/ml) and p70 (<20 pg/ml)). LH from snap-frozen lungs in experiment 4: IL-5, IL-6, IL-10, IL-12 (total), IFN-
and TNF-
. All samples from an experiment were assayed on the same day for each cytokine.
Statistics.
Data was analyzed using a two-way analysis of variance, followed by pairwise comparisons. The significance level of these comparisons was adjusted for multiple comparisons of the means using a Bonferroni correction. Group means were considered to be significantly different at p < 0.05.
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RESULTS |
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BAL cell counts.
Differential cell counts were performed (experiment 3) in order to assess any differences in the influenza-induced inflammatory response that might result from UVR exposure before deaths occurred (Fig, 3). Lavage fluid was collected from mice that were exposed to UVR or light at 3 days prior to infection. Both UVR/virus-infected and light/virus-infected animals showed an increase in numbers of recovered alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) early in the infection (Day 2); there were no significant differences in numbers of AM or PMN between UVR/virus-infected and light/virus-infected animals (Figs. 3A and 3B). Total BAL cells also reflected the lack of significant change in PMN and AM (Fig. 3C
). However, 5 days after infection there was an influx of lymphocytes in the BAL of UVR/virus-infected animals that was significantly less than the lymphocyte influx in BAL from light/virus-infected mice (Fig. 3D
). There were no inflammatory changes in the lungs of UVR/sham-infected animals; lungs from these mice resembled those lungs from light/sham-infected mice (Fig. 3A3D
). Day 7 data is not shown in Figure 3
; it represents the BAL cell counts of surviving mice, because the mice began to die on Day 6 in experiment 3. There were no differences between the light/virus-infected and UVR/virus-infected groups in any cell type on Day 7.
Virus titers.
Figure 4 shows the lung virus titers in these same mice (experiment 3) over the course of infection in both UVR/virus-infected and light/virus-infected groups. Upon infection with 300 pfu of influenza virus, lung virus titers multiplied from the instilled 300 pfu to their maximum of 107 pfu by 24 h after infection and were sustained over the next few days in both groups. By Day 5, they began to decline slightly. Mice began to die on Day 6. Preexposure to UVR did not affect virus titers significantly on any day after infection (Ryan et al., 2000
).
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In the light/virus-infected group, IL-6 was elevated early in infection (Day 2) and showed a biphasic pattern of induction in both BAL and in LH, rising again on Days 56. In the UVR/virus-infected group, UVR significantly elevated IL-6 beyond the levels induced by influenza early in the infection in BAL, and produced a trend in elevated IL-6 levels late in the infection on Day 6 (Fig. 5C). This trend also occurred in the late phase of the infection on Day 5 in LH (Fig. 5D
). No IL-1
or IL-ß was found in BAL after UVR exposure or at any time point following infection, regardless of whether or not the animals received UVR pre-exposure. In contrast to BAL, IL-1
was induced by influenza virus in LH of light/virus-infected mice, and the levels were significantly decreased by UVR on Day 5 in LH of UVR/virus-infected mice (data not shown). IL-1ß was also detected in LH of all groups, but the virus did not significantly elevate it, and UVR had no effect on any day (data not shown). No GM-CSF was detected at any time or in any group in LH.
Th1 versus Th2 cytokine induction.
Figure 6 shows the kinetics of expression of Th1 cytokines, IFN-
in BAL and LH, and IL-12 in LH, of mice treated with UVR or control light 3 days prior to infection or sham infection. UVR changed the pattern of influx of IFN-
into BAL following influenza virus infection (Fig. 6A
). The response to influenza alone (the light/virus-infected group) was biphasic: IFN-
was significantly increased on Day 2, absent on Days 3 and 4, and elevated on Days 5, 6, and 7. In UVR/virus-infected animals, the kinetics of the response appeared to shift to the right by one day, excepting the initial two-day time point. IFN-
increased in the UVR/virus-infected group also on Day 2, but remained elevated on Day 3, and was absent on Day 4. When the second peak of IFN-
occurred on Day 5 in the light/virus-infected group, IFN-
was significantly suppressed in the UVR/virus-infected group, but was not significantly different from the light/virus-infected group on subsequent days. IFN-
in LH was significantly enhanced on Days 2 and 3 and significantly depressed on Day 5 postinfection in UVR/virus-infected mice as compared to the light/virus-infected mice (Fig. 6B
).
Virus-induced total IL-12 (a combination of biologically active IL-12p70 and its inhibitor, IL-12p40) in LH was not significantly dampened by UVR pretreatment on Day 5, although there was a trend towards a decrease (Fig. 6C, UVR/virus-infected versus light/virus-infected groups). This trend would be consistent with the Th1 response (represented by IFN-
) being significantly downregulated only on Day 5 in both experiments. The low level of biologically active IL-12 p70 also mirrored this effect (data not shown). Total IL-12 (but not IL-12p70) was also the only cytokine induced in LH by UVR alone (in the UVR/sham-infected group) one day following UVR exposure (experiment 3, data not shown), implying that the IL-12 inhibitor was induced by UVR exposure alone.
In BAL, the IL-10 induction observed in the light/virus-infected group was dampened in the UVR/virus-infected group on Days 4 and 5 (Fig. 7A). In contrast, when comparing the UVR/virus-infected group to the light/virus-infected group, UVR did not appear to affect the pattern of Th2 cytokine induction of IL-10 by influenza virus in LH from experiment 4 or the virus-induced decrease in the levels of IL-10, IL-4, or IL-5 later in the course of infection after Day 4 (IL-10, Fig. 7B
) or Day 5 (IL-4 and IL-5, data not shown). However, in experiment 3, IL-10 levels in LH mirrored the BAL results in Figure 7A
.
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DISCUSSION |
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In addition to mortality, influenza causes other physiological effects such as body weight loss and hypothermia in mice (Wong et al., 1997). Our previous study demonstrated accelerated and enhanced morbidity as measured by body weight loss in UVR/virus-infected mice. In the present study we confirmed this result. Using body weight loss as an indicator of enhanced morbidity offers several advantages over mortality as an endpoint. The onset and development of disease could be assessed before death occurred and effects on body weight loss were more consistent and reproducible. The body weight data suggest that enhanced mortality following UVR was not a catastrophic event that occurred as the result of release of toxic mediators late in the infection but was the culmination of a process that started early in infection. The three-day delay between UVR exposure and influenza infection that is required to see enhanced body weight loss suggests that mediators produced locally by UVR exposure need to be distributed systemically before an effect is observed. A three-day delay between UVR exposure and application of hapten is also necessary for the systemic immunosuppression of CHS by UVR (Noonan and De Fabo, 1990
).
Given that immunosuppression does not appear to be the mechanism responsible for the enhanced mortality in influenza-infected mice treated with UVR or dioxin (Burleson et al., 1996; House et al., 1990
; Ryan et al., 2000
; Selgrade et al., 1988
; Warren et al., 2000
), other mechanisms must be considered. Certain evidence suggests that the lethal effect of influenza virus infection in mice is mediated by the host's response to infection rather than the direct cytopathic effect of viral replication (Hurd and Heath, 1975
; Shimomura et al., 1982
; Sullivan et al., 1976
). Cytokines, reactive oxygen species (ROS), and reactive nitrogen species (RNS) all have been shown to be mediators involved in the pathological process of influenza virus-induced lung damage (Akaike et al., 1996
; Knobil et al., 1998
; Oda et al., 1989
; Suliman et al., 2001
). We began our search for a mechanism of enhanced mortality by looking for potential differences in cytokine responses following infection of UVR-exposed and control mice by measuring cytokine levels in BAL and LH.
TNF- and IL-1 are proinflammatory cytokines that have been associated with fever and other physiological responses to influenza in mice and humans (Conn et al., 1995
). They are involved in maintaining homeostasis and may influence the activation of the hypothalamic-pituitary-adrenal (HPA) axis of infected mice and increase the concentrations of MHPG (3-methoxy,4-hydrophenylethyleneglycol, a major catabolite of norepinephrine) and tryptophan in the brain (Dunn et al., 1989
). Therefore, influenza, in elevating these proinflammatory cytokines, can act as a behavioral stressor. Major symptoms of infection are decreased body temperature and motor activity, changes in sleep responses (Fang et al., 1995
), and loss of appetite (Swiergel and Dunn, 1999
) contributing to cachexia and dehydration. Thus, TNF and IL-1 were likely candidates for causing the enhanced morbidity and mortality by influenza and it was surprising that preexposure to UVR did not significantly impact the rise in TNF-
or IL-1
induced by the virus.
Overproduction of IL-6 can be detrimental to the host and has been associated with viral pathogenesis (Kishimoto et al., 1992). In this study, influenza alone caused significant increases in IL-6 levels, and there was a trend toward increased IL-6 in UVR/virus-infected mice toward the end of the time course. However, at this point in time differences in cytokine release could be the effect of enhanced pathogenesis rather than the cause. Early in the infection, LH from UVR/virus-infected mice had significantly decreased IL-6 levels compared with the light/virus-infected group, and the BAL from these mice had significantly increased IL-6. These opposing effects in different compartments of the lung are difficult to interpret and may not be related to pathological effects. Others have shown that antagonism of IL-6, TNF-
, and IL-1 was not sufficient to prevent the decreases in feeding and loss of body weight induced by influenza infection (Swiergel and Dunn, 1999
). Results from our study do not suggest a role for proinflammatory cytokines in enhanced morbidity and mortality in mice exposed to UVR prior to infection. However, these results do not preclude effects on cytokine sensitivity, activity, receptors, and inhibitors, which could play a role in the UVR-induced exacerbations of influenza pathogenesis.
In mice, the balance of Th1 cells and Th2 cells appears to affect the pathological consequences of influenza virus infection. Whereas Th1 cells provided protection against a lethal influenza challenge in mice, Th2 cells did not protect mice against influenza-induced lethality and actually exacerbated the pulmonary pathology induced by this virus (Graham et al., 1994). Influenza virus infection in mice also caused a significant decrease in thymus weight that was enhanced by UVR preexposure, which may influence the immune response (Burleson et al., 1996
; Dunn et al., 1989
; Ryan et al., 2000
). Our cytokine responses following influenza infection were similar to previously published results and indicate that influenza does not present a rigid Th1 phenotype, for IL-10 was present along with IFN-
in both BAL (Hennet et al., 1992
; Peper and Ferguson, 1996
) and mediastinal lymph node cytokine profiles (Sarawar and Doherty, 1994
). However, we were again unable to clearly associate changes in these cytokines with enhanced disease in UVR/virus-infected mice. The data also does not indicate that UVR stimulates Th2 responses. Although suppression of Th1 is the mechanism responsible for UVR suppression of DTH and CHS (Ullrich, 1998
), others have shown that UVR suppression of Th1 responses is not necessarily associated with enhanced Th2 responses (Garssen et al., 1999
; Van Loveren et al., 2000
). To confirm whether or not the downregulation of the Th1 response has a role in the enhanced influenza pathogenesis induced by UVR, IL-12 could be supplemented to restore the Th1 response prior to infection. IL-12 supplementation has been shown to enhance the Th1 response and has been successfully utilized to restore CHS in mice exposed to UVR (Schmitt et al., 1995
).
Prior exposure to UVR enhanced (LH) or prolonged (BAL) the early IFN- response. The source of the IFN-
early in infection may have been natural killer cells. UVR possibly induced other cytokines or transcription factors that may have enhanced the early induction of IFN-
. Whether or not the alterations in IFN-
production by UVR are associated with the enhanced morbidity and mortality of influenza-infected mice deserves further investigation. It is interesting to note that similar alterations of influenza-induced IFN-
and IL-12 also occurred in the dioxin-exposed mouse influenza model (Warren et al., 2000
). One possible mechanism for the increased body weight loss in the UVR/virus-infected group is that IFN-
could be synergizing with TNF-
to enhance body weight loss early in the infection. Recently, IFN-
was shown to be required along with TNF-
(but not IL-1ß or IL-6) in increasing skeletal muscle loss in mice via an NF-
B-induced downregulation of MyoD, a transcription factor that regulates skeletal muscle differentiation and repair of damaged tissue (Guttridge et al., 2000
). The study suggested that the combined effects of these cytokines would lead to the inability to repair damaged skeletal muscle, contributing to the overall wasting process associated with cachexia. Late in the infection, the decreased levels of in IFN-
and IL-10 in the BAL and the IFN-
decrease in the LH in mice previously exposed to UVR may be explained by the impaired influx of lymphocytes into the lung. (The rebound on Day 6 in LH may have been a survivor effect since some mice had died by this point).
There are other possible mechanisms for UVR-enhanced pathogenesis of influenza. We have shown in a previous study that ROS and RNS play a role in the pathogenesis of our influenza virus in B6C3 mice (Suliman et al., 2001) and hence could be involved in the enhanced pathogenesis of influenza by UVR. Increased oxidative stress observed in influenza infection in mice can cause indirect pathogenesis through influencing the cytokine profile (Han and Meydani, 2000
). Differences in neuroendocrine responses should also be explored. The mechanisms involved in UVR-enhanced morbidity and mortality following influenza infection may also be involved in enhanced mortality seen with dioxin and other toxicants and are therefore worth pursuing. In any case, it is clear that care must be taken in interpreting experiments involving influenza as a host resistance model. Enhanced mortality is not always the result of classical suppression of adaptive immunity, even when the test agent is a known immunosuppressant.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be sent at present address: Department of Pathology and Laboratory Medicine, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103. Fax: (973) 972-7293. Email: ryanlk{at}umdnj.edu.
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