Department of Pharmaceutical Sciences, Pharmacology/Toxicology Graduate Program, College of Pharmacy, Washington State University, Pullman, Washington
Received December 13, 1999; accepted March 16, 2000
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ABSTRACT |
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Key Words: dioxin; lymphocyte; mouse; pulmonary; lymph node; immune suppression; host resistance; anti-viral immunity; cytokine.
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INTRODUCTION |
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While the precise mechanism of immunotoxicity remains unclear, exposure of mice to TCDD suppresses both humoral and cell-mediated immune responses to a variety of antigens (reviewed by Kerkvliet 1998). Numerous studies have shown that in vivo exposure to TCDD specifically leads to the dose-dependent suppression of T lymphocyte function, including proliferation, differentiation, cytokine production, and T cell-dependent B-cell responses (Kerkvliet et al., 1990, 1996
; Lundberg et al., 1992
; Neumann et al., 1993
; Prell et al., 1995
; Tomar and Kerkvliet, 1991
). The immune response to influenza virus is T-cell dependent, relying on the activation of both CD4+ and CD8+ T lymphocytes. Following virus entry and infection of lung epithelial cells, antigen-presenting cells (APC) migrate to the regional lymph nodes, where they present viral antigens and activate virus-specific T lymphocytes. CD4+ T cells produce cytokines such as interleukin (IL)-2 and interferon (IFN)-
, which drive both B-cell activation and the clonal expansion and differentiation of CD8+ T cells, leading to the creation of virus-specific cytotoxic T lymphocytes (CTL) and neutralizing antibodies. Following activation and differentiation in the lymph node, CTL migrate to the lung and kill virus-infected cells. Based on numerous studies using athymic mice, MHC class I-deficient mice, and neutralizing antibodies, it is generally accepted that a primary infection with influenza virus is resolved by the contact-dependent cytolytic activity of CD8+ T lymphocytes (Allan et al., 1990
; Baumgarth et al., 1994
; Eichelberger et al., 1991
; Lukacher et al., 1984
; Topham et al., 1997
; Yap et al., 1978
).
Given the importance of T cells in the clearance of influenza virus and the sensitivity of T cells to perturbation by exposure to TCDD, we hypothesized that decreased host resistance to influenza virus is due, at least in part, to suppression of T cell-dependent adaptive immunity. To test this, we examined whether exposure to TCDD suppresses the expansion and cytolytic activity of T cells in the regional lymph node, and we measured the production of influenza virus-specific antibodies and cytokines that are important for the differentiation and proliferation of T cells. Additionally, we examined TCDD-induced alterations in the pulmonary immune response to infection with influenza virus, including cellular recruitment to the lung, cytolytic activity, and production of IFN and IL-12.
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MATERIALS AND METHODS |
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TCDD exposure.
TCDD (Cambridge Isotope Laboratories, Inc., Woburn, MA) was dissolved in anisole and diluted in peanut oil to 1 µg/ml. Mice were given a single oral dose of 1, 5, or 10 µg/kg body weight by gavage one day prior to infection with influenza virus. Control mice received peanut oil-anisole vehicle in the same manner as described above.
Influenza virus.
Influenza virus (A/HKx31; H3N2) was received as a gift from Dr. Michael Coppola (Argonex, Charlottesville, VA). A/HKx31 is a murine-adapted recombinant strain that bears the internal components of A/PR8/34 (H1N1) and the external components of A/Aichi (H3N2). A/HKx31 was propagated according to methods described by Barrett and Inglis (1985). Briefly, viable fertilized chicken eggs (Spafas, Preston, CT) were inoculated with 0.05 hemagglutinating units (HAU) influenza virus in 100 µl of Hanks balanced salt solution, 10 mM HEPES, on gestational day 10. Infected eggs were incubated for 48 h at 37°C followed by refrigeration overnight at 4°C. Under aseptic conditions, allantoic fluid was harvested, centrifuged, and immediately frozen at 80°C until just prior to use. The titer of the allantoic fluid was determined by hemagglutination of avian erythrocytes. Mice were intranasally infected under anesthesia (Avertin) with 120 HAU influenza virus in 31 µl of allantoic fluid. One HAU of influenza virus is defined as the amount of virus that agglutinates 50% of the erythrocytes when a solution containing virus and a 0.5% solution of erythrocytes are combined in equal volumes.
Collection and preparation of bronchoalveolar lavage (BAL) cells.
A catheter attached to a 1-ml syringe was inserted into an incision in the trachea immediately posterior to the larynx. The respiratory tract was washed with RPMI 1640 containing 1% BSA and 10 mM HEPES using 3 separate 1-ml aliquots, each of which was infused and withdrawn 3 times. Cells were enumerated using a Coulter Counter (Beckman Coulter Corp., Miami, FL). Isolated BAL cells were used immediately for assessment of cytolytic activity, differential cell staining (Leukostat; Fisher Scientific, Santa Clara, CA), or were stained for flow cytometric analysis.
Collection and preparation of mediastinal lymph node (MLN) cells.
Single-cell suspensions were prepared under aseptic conditions by pressing both MLN from a single animal between the frosted ends of 2 microscope slides. MLN cells were suspended in cold RPMI 1640 containing 2.5% FBS (Hyclone, Logan, UT) and 10 mM HEPES. Cellular debris was removed by sedimentation and the cells were enumerated using a Coulter Counter. MLN cells were either used immediately for analysis by flow cytometry or were transferred to cultures for re-stimulation. For ex vivo re-stimulation, MLN cells (2 x 106) were suspended in RPMI 1640 containing 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µg/ml gentamicin, and 50 µM 2-mercaptoethanol, and were incubated at 37°C for 24 h or 5 days with 1 x 106 influenza virus-infected, irradiated antigen-presenting cells (DC2.4 dendritic cells; a gift from Dr. Ken Rock, Dana Farber Cancer Institute, Boston, MA).
CTL assay.
Influenza virus-specific cytolytic activity was assessed using a standard 5-h 51Cr-release assay, as described by Nonacs et al. (1992). Briefly, BAL cells or ex vivo re-stimulated MLN cells were incubated with 51Cr-labeled, influenza virus-infected MC57G fibroblasts at E:T ratios from 100:1 to 6.25:1. Released radioactivity was measured after a 5-h incubation at 37°C. Total releasable 51Cr was determined by lysing target cells with 0.5% SDS, and spontaneous release was obtained by incubating target cells in media only. Specific lytic activity was calculated as follows:
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Immunophenotypic analyses.
Freshly isolated cells were incubated with previously determined optimal concentrations of fluorochrome-conjugated antibodies. Appropriately labeled, isotype-matched immunoglobulins were used as controls for non-specific fluorescence. The following were used as primary antibodies: Tricolor-labeled anti-CD8a, FITC-labeled anti-CD44, PE-labeled anti-Vß8.3 from Caltag Laboratories (Burlingame, CA); and FITC-labeled anti-CD4, FITC-labeled anti-Vß8.3, and PE-labeled anti-CD62L from PharMingen (San Diego, CA). Data were collected from 25,000 (MLN) or 100,000 (BAL) cells by listmode acquisition, using a FACScan flow cytometer (Becton Dickenson, San Jose, CA). Dead cells, clumps, and debris were excluded from analysis, using a combination of forward angle and 90° light scatter and propidium iodide exclusion. Data were analyzed using WinList software (Verity Software, Topsham, ME).
Differential cell analyses.
BAL cells from individual animals were transferred to microscope slides using a cytological centrifuge, and were stained with hematoxylin and eosin (LeukoStat; Fisher Scientific, Pittsburgh, PA). Monocytes/macrophages, neutrophils, or lymphocytes were enumerated by differential counts of 200 cells on coded slides.
Cytokine analyses.
Cytokines were analyzed using matched antibody pairs in a sandwich enzyme-linked immunosorbant assay (ELISA). ELISA reagents were supplied by the following sources: IL-2 and IFN, PharMingen (San Diego, CA); IL-12, R&D Systems (Minneapolis, MN), and Genzyme Diagnostics (Cambridge, MA). ELISAs were conducted according to the manufacturers' recommended protocols.
Antibody analyses.
Influenza virus-specific antibody levels were analyzed using a stacking ELISA. Purified influenza virus (x31, Spafas, Preston, CT) was bound to 96-well plates. A volume of 100 µl of each sample (e.g., plasma) was added to the ELISA in 4-fold serial dilutions from 1:25 to 1:25,600. Vehicle- and TCDD-treatment groups were compared at a plasma dilution in which both groups were in the linear range of absorbance. Anti-isotype-specific antibodies (Southern Biotechnology Associates, Inc., Birmingham, AL) were used to assess specific isotypes, according to the manufacturer's recommended procedure.
Statistical analyses.
The results presented are representative of at least 3 to 5 independent experiments. Statistical analyses were performed using Statview (version 4.01, Abacus Concepts, Berkeley, CA). Using a one-way ANOVA, followed by post hoc tests (Fisher PLSD), differences between independent variables were compared over time and between each treatment group. Differences were considered significant when p was less than 0.05.
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RESULTS |
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TCDD alters cytokine production and decreases the influx of CTL cells into the lung.
Based on our observations that exposure to TCDD diminishes T-cell expansion and CTL activity in the MLN and suppresses lymphocyte recruitment to the lung, we hypothesized that CTL recruitment to the lung would likewise be suppressed. If this were true, we would expect to find alterations in the pulmonary cytokine profile, reduced numbers of CD8+ T cells, and diminished CTL activity in lungs from mice exposed to TCDD. Two cytokines that are particularly important for a CTL response are IL-12 and IFN (Kos and Engleman, 1996
; Monteiro et al., 1998
). We examined the levels of these two cytokines in the lung over the course of infection. In vehicle-treated mice, IFN
was first detected in the BAL fluid 5 days after infection, but rapidly decreased and was undetectable 3 days later, while IL-12 increased steadily throughout the course of infection (Fig. 6
, open circles). In mice treated with TCDD, the concentration of IFN
in BAL fluid reached a maximum on post-infection day 7, 2 days after the vehicle-treated group, and was nearly 10-fold greater at that time point. In contrast, IL-12 in lung lavage fluid from the TCDD-treated group declined steadily beginning on day 4 (Fig. 6B
). By day 9 post infection, the concentration of IL-12 in lung lavage fluid from vehicle-treated mice was nearly 10-fold greater than the level in TCDD-treated mice.
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We further characterized CD8+ T cells and identified CTL in the lung. Virus-specific CTL have been characterized as a subset of CD8+ T cells that express high levels of CD44 and low levels of CD62L (Doherty et al., 1996; Hou and Doherty, 1993
). Based on the expression of CD44 and CD62L, we assessed the presence of an effector population of CD8+ T cells in the lung. In vehicle-treated mice, 95% of the CD8+ BAL cells bear the CTL phenotype (Fig. 8A
). Measuring CTL over time revealed that these cells comprise about 20% of the total population of BAL cells on day 8 post infection, and 30% on day 9 post infection (Fig. 8C
). In virus-infected mice treated with TCDD, there was a 20% decrease in the mean channel fluorescence of CD44 on CD8+ BAL cells on day 9 post infection (Fig. 8B
) and the percentage of all BAL cells bearing a CTL phenotype was reduced more than 2-fold compared to vehicle-treated mice.
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DISCUSSION |
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Several laboratories have reported that in vivo treatment of rats and mice with TCDD increases mortality following respiratory infection with influenza A virus (Burleson et al., 1996; House et al., 1990
; Yang et al., 1994
). However, few studies have been performed to determine the mechanisms by which exposure to TCDD decreases host resistance to influenza virus. In particular, the effects of TCDD on T-cell function following in vivo infection with influenza virus have not been ascertained. Thus, one goal of this study was to determine whether exposure to TCDD suppresses T-cell function following respiratory viral infection. The effects of TCDD on T-cell responses reported here are consistent with numerous reports that exposure to TCDD suppresses T cell-dependent immunity. For example, our results are quite similar to findings of Kerkvliet et al. (1996), who reported a diminished percentage of CD4+ and CD8+ T cells and suppression of ex vivo splenic IL-2 and IFN
in a tumor allograft model. In this same model, exposure to TCDD also decreased the expansion and differentiation of CD8+ T cells, as defined by the altered expression of CD44 and CD62L, phenotypic markers of CTL (Kerkvliet et al., 1996
). Our findings concur with other studies, in which activation-induced T cell expansion and the production of IL-2 and IFN
were reduced following in vivo exposure to TCDD (Lundberg et al., 1992
; Prell et al., 1995
). Likewise, treatment with TCDD suppresses splenic CTL responses to tumor cell challenge in mice (De Krey and Kerkvliet, 1995
; Kerkvliet et al., 1990
) and disrupts cell-mediated immunity to cytomegalovirus infection in rats (Ross et al., 1997
).
Further evidence that exposure to TCDD disrupts cell-mediated immunity is provided by our observation that TCDD treatment suppresses IL-12 levels. Perhaps more than any other cytokine studied to date, IL-12 plays an obligatory role in the activation and maintenance of cell-mediated immunity to infectious diseases (Seder et al., 1996; Trinchieri, 1997
). IL-12 induces the expression of co-stimulatory molecules on APC and directly drives the generation of CTL (Trinchieri, 1997
). The specific role of IL-12 in cell-mediated immunity to influenza A virus was recently examined (Monteiro et al., 1998
). In vivo depletion of IL-12 in BALB/c mice decreased virus-specific CTL activity and viral clearance from the lung, confirming that IL-12 contributes to protective immunity to influenza virus. Consistent with a role for altered IL-12 levels in TCDD-mediated suppression of cell-mediated immunity, decreased IL-12 levels were reported in mice exposed to TCDD and challenged with P815 mastocytoma cells (Shepherd et al., manuscript submitted). However, a causal relationship between altered IL-12 and TCDD-mediated immunotoxicity remains to be formally examined.
Another T cell-dependent response to infection with influenza virus is the generation of antibodies (Gerhard et al., 1997). IgM, IgG1, IgG2a and IgG2b, and IgA isotypes have been implicated as important effectors of viral clearance and protective immunity in mice (Hocart et al., 1989
; Jones and Ada, 1986
; Justewicz et al., 1995
). Consistent with an earlier report, we found that exposure to TCDD suppresses the level of influenza virus-specific IgM and IgG in plasma and lung lavage fluid (House et al., 1990
). In contrast to IgM and IgG levels, TCDD treatment enhances the amount of influenza virus-specific IgA in plasma, a finding that is consistent with reports that exposure to TCDD increases IgA in rats and humans (Moran et al., 1986
; USAF, 1991
). This effect of TCDD on IgA is quite interesting and may reveal an effect of TCDD on cytokines that regulate isotype switching to IgA. However, it is worth noting that recent studies using IgA-deficient mice have demonstrated that IgA is not required to prevent infection, generate neutralizing antibodies, or effectively clear virus and virally infected cells (Mbawuike et al., 1999
).
While the significance of increased IgA remains unclear, our findings that exposure to TCDD suppresses influenza-specific IgM and IgG, impairs cytokine production and CTL activity in the MLN, and decreases CD8+ cells and IL-12 levels in the lung indicate that TCDD impairs both the cell-mediated and humoral immune responses to influenza A virus. This interpretation is consistent with the deleterious effects of TCDD observed in other experimental systems and supports the conclusion that TCDD suppresses the generation of an adaptive immune response. However, the idea that exposure to TCDD prevents adequate development of adaptive immunity and that this defect underlies the impaired host resistance to infection with influenza virus is likely to be overly simplistic. While exposure to TCDD clearly impairs T-cell responses, lung lavage cells isolated from TCDD-treated mice are able to lyse virus-infected target cells as effectively as BAL cells from vehicle-treated controls. Furthermore, when we compared the virus titer of lungs obtained on post infection day 9 from vehicle- and TCDD-treated mice, we detected no infectious virus in either group (Lawrence et al., in press). Thus, in addition to equivalent cytolytic activity, this demonstrated that the TCDD-treated mice that survived to day 9 were able to clear the virus from their lungs.
The ability of TCDD to suppress adaptive immunity and enhance mortality following an otherwise non-lethal infection, yet at the same time not to diminish lung cell cytolytic activity or the clearance of virus from the lung, was surprising to us. This finding raises several very interesting questions: what cell or molecule is responsible for the cytolytic activity and viral clearance in lungs of mice treated with TCDD, and, in the absence of an increased pulmonary virus burden, what mechanism is responsible for the enhanced mortality observed in mice exposed to TCDD? Possible explanations include the activation of another population of cells that compensates for the diminished number and activity of CD8+ T cells in the lung, or the excessive production of cytokines such as IFN or tumor necrosis factor (TNF)-
. Alternatively, it is possible that the fewer CTL in the lung are simply more efficient killers. This last scenario seems unlikely. The overwhelming evidence that exposure to TCDD suppresses T-cell function, makes it difficult to put forth a plausible explanation for how fewer T cells could have much greater cytolytic activity than T cells from vehicle-treated mice. Instead, it seems more probable that another mechanism is responsible.
In addition to contact-mediated lysis, cells infected with influenza virus can be killed by TNF-induced apoptosis (Liu et al., 1999
), and TNF
is found in the lung following influenza virus infection (Hennet et al., 1992; our unpublished observations). Furthermore, in other model systems, exposure to TCDD has been reported to increase TNF
levels (Clark and Taylor, 1994
; Fan et al., 1997
; Moos et al., 1994
). Excessive production of TNF
has been correlated with adverse outcomes following infection, often resulting in increased mortality (Nguyen and Biron, 1999
; Zhang et al., 1996
). Thus, overproduction of TNF
in the lung would provide an explanation for both virus clearance and increased mortality. Infection with influenza virus stimulates the rapid production of TNF
in the lung. However, in both vehicle- and TCDD-treated mice, TNF
levels declined rapidly and were undetectable after about 72 h (our unpublished observations). Therefore, it seems unlikely that excessive pulmonary TNF
is a mechanism for cytolytic activity, virus clearance, or enhanced mortality observed in mice exposed to TCDD and infected with influenza virus.
On the other hand, the elevated level of IFN in lung lavage fluid from mice exposed to TCDD provides a possible explanation for the destruction of virus-infected cells and enhanced mortality. The pivotal role of IFN
in host resistance to intracellular pathogens is well-documented (Farrar and Schreiber, 1993
; Young and Hardy, 1995
). In the case of anti-viral immunity, IFN
enhances T-cell expansion and differentiation, influences antibody isotype switching, and induces proteins and enzymes that alter viral replication. Moreover, via augmentation of the killing activity of macrophages, neutrophils, and NK cells, IFN
facilitates the destruction of virus-infected cells. A cause-and-effect relationship between excessive IFN
in the lung, cytolytic activity, and the immunotoxic effects of TCDD is not yet clear. When specifically considering a role for IFN
in the context of host resistance to influenza virus, it is important to point out that IFN
has no known direct activity against influenza virus (Baumgarth and Kelso, 1996
; Graham et al., 1993
). Thus, a direct IFN
-mediated mechanism for virus clearance appears unlikely. Instead, one possible relationship is that exposure to TCDD enhances the activity of NK cells or increases the recruitment of NK cells to the lung.
NK cells are very effective at recognizing and destroying lung epithelial cells infected with influenza virus, and NK cells and IFN produced by NK cells are necessary for the clearance of a primary infection with influenza virus (Kos and Engleman, 1996
; Monteiro et al., 1998
). Reports of the effects of TCDD on NK cells are contradictory. Exposure of rats and mice to TCDD has been found to have no effect (House et al., 1990
; Montovani et al., 1980
), decrease (Yang et al., 1994
), and increase (Funseth and Ilback, 1992
) NK-cell activity. Nevertheless, in addition to promoting viral clearance and compensating for diminished CTL in mice exposed to TCDD, overly activated NK cells, via the production of IFN
, may damage healthy tissue and cause mortality.
In addition to enhancing NK-cell activity, increased IFN may also lead to the over-activation of macrophages and neutrophils in the lung. In fact, the increased percentage and number of neutrophils in BAL cells from TCDD-treated mice provides circumstantial evidence for excessive inflammation as a mechanism for tissue damage. Interestingly, when observed, mortality in the TCDD-treated group consistently occurred between days 5 to 8 after infection with influenza virus: the same days in which increased IFN
and increased neutrophils are found. Evidence for an IFN
-mediated increase in pathological effects following infection with influenza virus has recently been demonstrated by Karupiah et al. (1998). They reported an association between elevated pulmonary nitric oxide following infection of mice with influenza A virus. Furthermore, using metabolic inhibitors of nitric oxide synthase (NOS) and NOS2-deficient mice, they found NOS2 induction by IFN
is detrimental to host survival following infection with influenza A virus. Thus, one possible reason for mortality is that exposure to TCDD increases IFN
levels in the lung, leading to excessive inflammation and tissue damage.
The relationship between increased IFN in the lung and TCDD-mediated immunotoxicity has yet to be established. One possible mechanism is that exposure to TCDD increases IFN
by a direct AhR-mediated mechanism. In support of this theory, a putative dioxin response element has been identified in the murine IFN
gene (Lai et al., 1996
). Further study is needed to fully understand how exposure to TCDD enhances pulmonary IFN
levels, while IFN
production by MLN cells is suppressed. Nevertheless, increased IFN
in the lung offers a mechanistic explanation for the enigmatic finding that lung cells from mice exposed to TCDD and infected with influenza virus exhibit equivalent cytolytic activity. Moreover, through the over-activation of inflammatory mediators, IFN
-induced tissue damage may explain the enhanced mortality observed in mice treated with TCDD.
In summary, we have found that IFN levels in the MLN are suppressed while IFN
levels in the lung are greatly enhanced, indicating that exposure to TCDD affects cytokine production in a tissue-specific manner. We have also presented evidence that treatment with TCDD suppresses the adaptive immune response to infection with influenza virus. However, it is not clear whether this effect of TCDD on T cell-dependent immunity underlies the enhanced mortality because lung cells isolated from mice exposed to TCDD have cytolytic activity against influenza virus-infected cells. While the mechanisms for the presence of cytolytic activity and decreased host resistance to influenza virus remain to be determined, it is clear that there are differences in the effects of TCDD on immune cells and cytokine production in the lymph node and lung.
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ACKNOWLEDGMENTS |
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NOTES |
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