Department of Pharmaceutical Sciences, Pharmacology/Toxicology Program, College of Pharmacy, Washington State University, Pullman, Washington 99164-6534
Received November 12, 2003; accepted January 28, 2004
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ABSTRACT |
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Key Words: immune memory; immune suppression; influenza virus; Ah receptor; dioxin.
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INTRODUCTION |
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Numerous AhR ligands have immunomodulatory properties. However, despite considerable study, the precise mechanism by which AhR activation affects immunity is not entirely clear. For example, although numerous descriptive studies indicate that the AhR ligand TCDD is one of the most immunosuppressive chemicals known, the direct cellular targets of TCDD within the immune system and the stage of activation at which they are impacted are not clearly defined. Our work (Vorderstrasse et al., 2003; Warren et al., 2000
) and that of many others (reviewed in 8,9) reveal two aspects of immune responsiveness that stand out as especially sensitive to perturbation by TCDD: lymphocyte function and host resistance. In vivo exposure of rodents to TCDD adversely affects clonal expansion, lymphocyte differentiation, cytokine and antibody production, and isotype switching (Ito et al., 2002
; Kerkvliet et al., 1996
; Lundberg et al., 1992
; Prell et al., 1995
, 2000
; Shepherd et al., 2000
; Tucker et al., 1986
). With regard to host resistance, exposure to TCDD impairs survival to many pathogens, including Salmonella sp., Streptococcus pneumoniae, Herpes virus, and influenza A virus (Burleson et al., 1996
; Hinsdill et al., 1980
; House et al., 1990
; Nohara et al., 2002b
; Thigpen et al., 1975
; Warren et al., 2000
). In the context of the immune response to influenza virus, TCDD treatment diminishes the production of virus-specific IgG1, IgG2a, IgG2b, and IgM while enhancing the amount of virus-specific IgA (Vorderstrasse et al., 2003
; Warren et al., 2000
). Additionally, we have shown that exposure to TCDD renders virus-specific CD8+ T lymphocytes hyporesponsive to antigen, resulting in an impaired CTL (cytotoxic T lymphocyte) response during a primary infection (Mitchell and Lawrence, 2003
). In spite of this large database describing immunotoxicity associated with exposure to TCDD in a primary immune response, very few studies have explicitly examined the effects of TCDD on immunological memory.
Antigen-specific immunological memory provides natural immunity, protecting humans and animals from the pathological and often fatal consequences of repeated infections by the same or related pathogens. Given the critical role immunological memory plays in human health, it is imperative that we better understand how exposure to immunosuppressive compounds affects the acquisition of antigen-specific memory. Indeed, it is logical to postulate that suppression of a primary immune response to an antigen by immunosuppressive chemicals, such as dioxins, would lead to a less robust or ablated recall response.
Given that many AhR ligands are abundant pollutants and that TCDD, the highest affinity AhR ligand, is one of the most immunosuppressive chemicals known, information on the effects of this family of chemicals on immunological memory is surprisingly scant. Epidemiological data imply that exposure to dioxins and PCBs may impair protective immunity in humans (Weisglas-Kuperus et al., 1995, 2000
), and it has been reported that exposure of mice to a single dose of TCDD suppresses secondary antibody responses to sheep red blood cells and tetanus toxin (Hinsdill et al., 1980
; Vecchi et al., 1980
). Likewise, the antibody response and T-cell-derived cytokines were suppressed in TCDD-treated mice following a secondary challenge with ovalbumin (Nohara et al., 2002a
). However, the hypothesis that exposure to AhR ligands impairs immunological memory has not been definitively tested in the context of host resistance, nor have the kinetics of a recall response been thoroughly examined.
The goal of this study was to characterize the anamnestic response to influenza A virus in mice exposed to TCDD. To test whether ligation of the AhR during the activation of naïve lymphocytes impairs the acquisition of immunological memory, we measured the memory pool of CD8+ T cells and virus-specific antibody levels prior to reinfection. We then monitored the kinetics of the response following homotypic infection; that is, reinfection with the same strain of virus. Our data are surprising and reveal that although exposure to TCDD suppresses the primary response, resulting in lower levels of virus-specific IgG and diminution of the memory CD8 pool, the secondary response to homotypic infection is nevertheless host-protective. TCDD-treated mice are able to clear the virus upon reinfection and show no significant signs of mortality or morbidity. These findings have implications for assessing the risks posed by exposure to AhR ligands, determining the mechanisms by which these chemicals adversely affect lymphocyte function, and understanding the mechanisms that control the acquisition of immunological memory.
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MATERIALS AND METHODS |
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Influenza A virus, strain HKx31, was grown in the allantoic cavity of 10-day old embryonated, pathogen-free chicken eggs and prepared as previously described (Warren et al., 2000). Anesthetized (Avertin, 2,2,2-tribromoethanol) mice were inoculated intranasally (i.n.) with 120 hemagglutinating units (HAU) of HKx31. In control mice this dose of virus does not typically cause mortality. Mock-infected vehicle- and TCDD-treated animals received an equivalent volume of sterile PBS (i.n.) and were used as negative controls. Animals were sacrificed by Avertin overdose at various times post infection and blood was obtained by cardiac puncture for the collection of plasma.
For studies in which animals were bled serially, blood was collected from the saphenous vein (www.uib.no/vivariet/mou_blood/Blood_coll_mice_html). Mediastinal lymph nodes (MLN) were removed aseptically and single-cell suspensions prepared as previously described (Warren et al., 2000). MLN cells were enumerated using a Coulter counter. In some studies, bronchoalveolar lavage (BAL) fluid was collected and stored at
80°C for the measurement of antibody levels in the lung (Warren et al., 2000
). Lungs were removed, snap-frozen in liquid nitrogen, and stored at
80°C until used for the assessment of pulmonary viral titers.
Immunophenotypic analysis and tetramer staining.
Freshly isolated MLN cells were stained with combinations of the following antibodies purchased from BD Pharmingen (San Diego, CA) or Caltag Laboratories (Burlingame, CA): biotinylated-anti-CD19, FITC-labeled anti-CD44, PE-labeled anti-CD62L, and Tricolor- or APC-labeled anti-CD8a. Spectral red streptavidin was purchased from Southern Biotech (Birmingham, AL). Appropriately labeled, isotype-matched antibodies were used to determine nonspecific fluorescence. NP366-specific CD8+ T cells were detected using PE-labeled DbNP366 tetrameric complexes (generously provided by Dr. Peter Doherty, University of Melbourne, Australia). To label the cells with tetramer, MLN cells were incubated for 1 h at room temperature with PE-DbNP366 prior to incubation with antibodies against other cell-surface markers. For all experiments, data were collected from 50,000 cells using a FACSort flow cytometer (Becton Dickinson, San Jose, CA) and analyzed using WinList software (Verity Software, Topsham, ME). Dead cells and debris were excluded using a combination of forward angle light scatter and 90° side scatter.
Virus-specific antibody levels.
Influenza virus-specific antibodies were analyzed by stacking enzyme-linked immunosorbant assay (ELISA). Ninety-six well plates were coated with 5 µ;g/ml sucrose gradient purified influenza virus (Charles River/Spafas, North River, CT). Plasma or BAL fluid were prepared in a series of four-fold dilutions ranging from 1:100 to 1:102,400 for plasma and from 1:10 to 1:10,240 for BAL fluid, and 100 µ;l of each dilution was added to the plate. Biotinylated anti-isotype-specific antibodies (Southern Biotech, Birmingham, AL) were used to assess the relative amount of specific antibody isotypes. For each isotype, a dilution of plasma that was in the linear range of absorbance was selected, and samples from vehicle- and TCDD-treated animals were compared at the same dilution.
Virus-specific antibody forming cells.
Influenza virus-specific antibody forming cells (AFC) were enumerated using isotype specific enzyme-linked immunosorbant spot-forming assay (ELISPOT) (28, and Technical Notes supplied by Millipore). Ninety-six well MultiscreenTM plates with PVDF membranes (Millipore, Bedford, MA) were coated overnight at 4°C with 10 µ;g/ml (100 µ;l) purified influenza A/HKx31 (Charles River/SPAFAS, North River, CT). Wells were blocked with complete RPMI media containing 5% FBS. MLN cells were plated in serial three-fold dilutions from 5 x 105 to 229 cells per well (100 µ;l/well) and the plates were incubated for 4 h in a humidified incubator (37°C, 5% CO2). Plates were washed thoroughly with PBS containing 0.05% Tween-20 and incubated overnight at 4°C with biotinylated goat anti-mouse isotype-specific antibodies (100 µ;l of 1:1000 dilution; Southern Biotechnology, Birmingham, AL). After washing, avidin-peroxidase (5 µ;g/ml; Sigma, St. Louis, MO) was added and plates were incubated for an additional hour at room temperature. Spots representing AFC were visualized by adding 3-amino-9-ethylcarbazole. Membranes were sent to Zellnet Consulting Inc. (New York, NY) for enumeration in blind. The number of AFC was calculated using spots enumerated from a dilution of cells within the linear range of the dilution series.
Viral burden.
Pulmonary virus titer was determined by amplification in MDCK cells followed by a standard hemagglutination assay, as previously described (Barrett and Inglis, 1985; Lawrence et al., 2000
). Briefly, frozen lungs from individual animals were disrupted and homogenates were clarified by centrifugation. Homogenates were added to 5 x 104 viable MDCK cells in eight serial dilutions. After a three-day incubation, culture supernatants were tested for the presence of viral hemagglutinin activity using chicken erythrocytes. As a positive control, lung samples collected during a primary infection were used, while lung homogenates from mock-infected mice served as negative controls.
Statistical methods.
All statistical analyses were performed using Statview statistical software (SAS, Cary, NC). Significant treatment effects were determined by two-way analysis of variance (ANOVA), followed by a Fishers PLSD to compare the mean values from each treatment group at a specific point in time. For the data presented in Figures 1A, 2, and 5, the mean values in a treatment group were also compared over time within that group (by ANOVA and Fishers PLSD). Values of p 0.05 serve as the basis for designation of statistical significance.
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RESULTS |
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The levels of virus-specific antibodies were measured in plasma from a cohort of vehicle- and TCDD-treated mice that were serially bled prior to, and on days 10 and 40 after primary infection with A/HKx31, and at various times following secondary infection (Fig. 1A). Primary infection with influenza virus caused a substantial increase in plasma IgM and IgG isotypes, and, as expected, treatment with TCDD impaired this primary response. Interestingly, when levels of residual antibodies were examined 40 days after primary infection, the suppression induced by exposure to TCDD was still evident.
Reinfection of vehicle-treated mice elicited little to no change in the levels of virus-specific antibodies in the plasma, even when examined 21 days after secondary challenge. This observation is consistent with other analyses of the kinetics of the antibody response to respiratory viral infection (Hocart et al., 1989; Wells et al., 1979
). With the exception of IgG1, reinfection of the cohort to which TCDD was administered prior to primary infection resulted in antibody levels that, relative to vehicle-treated mice, were decreased for at least seven days after reinfection. This decrease either reflects suppression of the memory response, or it reflects the fact that 40 days after a primary infection, the TCDD-treated mice simply have a lower base-line level of virus specific antibodies. The amount of IgM in the plasma was not changed following reinfection in either treatment group, and the levels in the TCDD-treated mice never approached the amount detected in vehicle-treated mice. However, virus-specific IgG antibodies were slightly, but significantly elevated following reinfection in the TCDD-treated mice, and eventually reached control levels by two to three weeks after reinfection.
The levels of virus-specific antibody isotypes in BAL fluid were assessed in a separate study where mice were sacrificed 9 days and 60 days after primary infection and on days 3 and 5 following secondary infection (Fig. 1B). Consistent with observations of antibody levels measured in plasma, treatment with TCDD suppressed the amount of each antibody isotype detected in the lung following primary infection, and amounts generally remained depressed both prior to, and through day 5 following reinfection.
In contrast to the TCDD-induced suppression of IgM and IgG isotypes, virus-specific IgA is enhanced in plasma from TCDD-treated mice following primary infection with influenza virus (Vorderstrasse et al., 2003; Warren et al., 2000
). As shown in Figure 2A, IgA levels remained elevated in the plasma of TCDD-treated animals at five weeks following primary infection, as well as following reinfection. However, unlike the other isotypes examined, IgA levels measured in BAL fluid were unaffected by exposure to TCDD (Fig. 2B).
Detection of Antibody-Forming Cells in the Mediastinal Lymph Nodes
To investigate whether the altered antibody levels observed in TCDD-treated mice resulted from differences in the number of plasma cells, we compared the number of virus-specific antibody forming cells (AFC) in lymph nodes from vehicle- and TCDD-treated mice prior to and following reinfection. Mediastinal lymph nodes (MLN) were selected for these studies because they drain the lower respiratory tract (Wiley et al., 2001). Moreover, AFC in the MLN have been characterized in the context of respiratory infection with influenza viruses and other related viruses (Hyland et al., 1994
; Justewicz et al., 1995
; Sangster et al., 2000
; Topham et al., 1996
). As shown in Figures 3A and 3B, prior to reinfection (day 0) the total number of virus-specific IgG- and IgA-producing cells was not different between the two treatment groups. Reinfection resulted in an increase in the number of virus-specific AFC in both groups (Figs. 3A and 3B); however, the number of AFC per lymph node was not different when we compared cells from vehicle- and TCDD-treated mice. Consistent with the low levels of IgM antibodies detected during the secondary response (Fig. 1), no IgM-specific AFCs were detected prior to, or following reinfection (data not shown). The number of IgG- and IgA-specific AFC present following primary infection (inset in Figs. 3A and 3B) were substantially higher than in the recall response, and the effects of TCDD exposure at this time point were consistent with observed effects on plasma antibody levels.
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Exposure to TCDD Prior to Secondary Infection Has No Effect on Antibody Production
Given that adults have accumulated immune memory to a large variety of antigens, it is also important to know whether exposure of previously activated B cells to TCDD affects antibody production. To test this, we administered a single dose of TCDD one day prior to reinfection and monitored the levels of virus-specific antibodies in the plasma. As shown in Figure 4, exposure to TCDD directly before secondary challenge did not alter the magnitude or the kinetics of the IgG2a response relative to that observed in vehicle control animals. Likewise, the IgA response in mice given TCDD just prior to reinfection was not different during the first two weeks following reinfection, although the mice did have slightly (1015%) higher levels of virus-specific IgA at the latest time point examined. In a separate study, we validated these observations and examined other IgG subclasses, which were likewise unaffected by exposure to TCDD (data not shown). Taken together, these data suggest that previously activated B cells are less sensitive to modulation by TCDD.
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To monitor the expansion of virus-specific effector/memory CD8+ T cells we used CD44, a marker for effector and memory CD8+ T cells (Doyle et al., 1999; Hamilton-Easton and Eichelberger, 1995
). As shown in Figure 5C, when we compared the response in each treatment group, there was a profound difference in the total number of CD44hiDbNP366+CD8+ cells (i.e., virus-specific effector/memory CD8+ T cells). Specifically, during primary infection (day 8), the number of virus-specific CD8+ effector cells increased about 5-fold in the vehicle-treated mice, and the resulting population of virus-specific memory CD8+ T cells, measured on day 60, was 2.5-times higher than the number prior to infection. In contrast, treatment with a single dose of TCDD abrogated the expansion of CD44hiDbNP366+CD8+ cells during the primary response, and the number of virus-specific memory CD8+ cells detected 60 days after infection was 50% lower than the number in vehicle-treated mice. In fact, the number of virus-specific CD8+ cells in the TCDD-treated mice was not different from the number detected in uninfected mice.
Upon reinfection, there was a rapid expansion of CD44hiDbNP366+CD8+ cells in the vehicle group, consistent with the previously reported kinetics of the response of influenza virus-specific memory CD8+ T cells (Belz et al., 1998, 2000
; Flynn et al., 1998
). In contrast, the expansion of virus-specific memory CD8+ cells in TCDD-treated mice was delayed, such that three days after reinfection, there were 70% fewer CD44hiDbNP366+CD8+ cells in mice given TCDD compared to the vehicle treatment group. However, despite this initial suppression, the virus-specific memory CD8+ T cells did expand in the TCDD-treated mice, and the recall response was equivalent to that of the vehicle-treated animals by five days after reinfection. These results indicate that the response of virus-specific memory CD8+ T cells is delayed in mice treated with TCDD.
Host Resistance
To determine whether the suppressed antibody response and delayed expansion of virus-specific CD8+ T cells correlated with impaired host-resistance to homotypic infection, we monitored the reinfected mice for morbidity and mortality in two separate studies (Table 1). In the context of a primary infection, 4050% of TCDD-treated mice died from an otherwise sublethal infection with A/HKx31. Furthermore all mice, regardless of treatment, lost weight and demonstrated visible signs of illness. In contrast, there was no mortality in either vehicle- or TCDD-treated mice following secondary infection, and none of the mice exhibited any signs of morbidity. Based upon these observations, we conclude that host resistance to secondary infection was not compromised in the TCDD-treated mice.
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DISCUSSION |
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Despite the diminished magnitude of the recall response, we must reconcile the decreased levels of the various parameters measured with the fact that, upon reinfection, the TCDD-treated mice rapidly cleared influenza virus from their lungs. Moreover, these mice showed no overt signs of morbidity or mortality in the context of homotypic viral challenge. Given that, for the most part, the antibody and cell-mediated arms of anti-influenza immunity are suppressed by exposure to a single dose of TCDD, how then do these mice clear the infection?
There are several possible explanations for the ability of the TCDD-treated mice to successfully resist homotypic infection in the context of greatly diminished virus-specific CTL and IgG. One possibility is that the delayed CTL and antibody responses "catch up" in time to protect the host. However, this explanation is not logical as the virus is cleared from the lungs very rapidly in both vehicle- and TCDD-treated mice. That is, the virus was completely cleared by three days following reinfection, consistent with the rapidity expected in a recall response (as opposed to a primary infection, where viral clearance is not achieved until 79 days after infection). However, the IgG and CTL responses in the TCDD-treated mice do not achieve the same levels as the vehicle-treated controls until at least five days following reinfection. Thus, it is unlikely that resistance to reinfection in the TCDD-treated mice relies on the fact that the IgG and CTL responses eventually attain the levels observed in the control group.
Other mechanistic explanations therefore seem more probable. For instance, it is possible that although reduced, the residual IgG and CD8+ T cells are sufficient for host protection. Alternatively, the excess IgA in plasma and equivalent IgA levels in BAL fluid in mice treated with TCDD may serve an important host-protective role. While IgA is not considered to play a particularly important role in host protection during a primary infection with influenza A virus (Mbawuike et al., 1999), it forms a very important component of host resistance in the context of a secondary response to infection (Arulanandam et al., 2001
). Thus, given its well-established role in mucosal immunity, and the fact that exposure to TCDD enhances the levels of virus-specific IgA in mice treated with TCDD, neutralizing anti-viral IgA offers a plausible means by which the TCDD-treated mice clear the virus and survive reinfection.
Interestingly, although the mechanisms by which exposure to TCDD leads to enhanced IgA are not clear, this effect of TCDD on circulating IgA levels is not unique to the immune response to influenza virus. Exposure to TCDD has been reported to enhance IgA levels in rats and humans (Moran et al., 1986; USAF, 1991
). Likewise, while the levels of ovalbumin-specific IgG classes were suppressed by exposure of mice to TCDD (Shepherd et al., 2000
), plasma levels of ovalbumin-specific IgA were enhanced (Shepherd, personal communication). This indicates that measurement of a single antibody isotype may not be sufficient when examining the effects of TCDD on antibody production.
It seems logical that profound suppression of the response of naïve lymphocytes would correlate with impaired immunological memory. Indeed, the persistent reduction in IgG levels and the impaired recall response of CD8+ T cells make sense in the context of the suppressed primary response. These alterations in the antibody and CTL response may reflect a residual defect that arose during the activation of naïve lymphocytes, or may be due to defects in the ability of the antigen-specific cells to respond during a secondary challenge. Alternatively, this may simply reflect a smaller starting pool of memory cells. The answer to this may depend upon the type of lymphocyte examined. For instance, the number of virus-specific AFC present after resolution of a primary infection and in response to reinfection was not affected by exposure to TCDD. This suggests that, in contrast to naïve B cells, the function of previously activated B cells may be less sensitive to perturbation by TCDD. Therefore, the decreased levels of virus-specific IgM and IgG (and enhanced levels of IgA) observed in this study likely result from defects in the activation of naive B cells.
In contrast to B cells, exposure to TCDD suppressed the generation of virus-specific memory CD8+ T cells, as the number of DbNP366+CD44hiCD8+ cells was reduced in the TCDD-exposed group prior to reinfection. That exposure to TCDD suppresses the clonal expansion and differentiation of naïve T cells is well-established (Ito et al., 2002; Kerkvliet et al., 1996
; Lundberg et al., 1992
; Nohara et al., 2002b
; Warren et al., 2000
). With regard to CD8+ cells, exposure to TCDD diminishes differentiation into CTL, impairs clonal expansion in vivo, and decreases the production of interleukin-2 (IL-2) and interferon gamma (IFN
; Kerkvliet et al., 1996
; Lundberg et al., 1992
; Prell et al., 2000
; Warren et al., 2000
). Exposure to TCDD also affects the expression of costimulatory molecules on antigen presenting cells (Prell and Kerkvliet, 1997
; Shepherd et al., 2001
; Vorderstrasse and Kerkvliet, 2001
), suggesting that improper T-cell activation may underlie impaired CTL responses in TCDD-treated mice. Thus, given that activation of naïve T cells is profoundly suppressed in mice treated with AhR ligands, the observed decrease in the number of residual DbNP366+CD44hiCD8+ cells is not unexpected.
What is rather surprising is the differential kinetics of the response of this population of cells following reinfection. The response is faster than the response of naïve cells, but slower than the response of virus-specific memory CD8+ cells from mice vehicle-exposed group. This delay may simply reflect a reduced pool of memory cells resulting from impaired activation of naïve cells discussed above, or it may reflect effects of TCDD on T cell survival or commitment to the memory lineage. For example, in the absence of complete T cell activation, antigen-specific T cells either die or enter a state of antigen-specific nonresponsiveness (Hildeman et al., 2002). Enhancement of either event (death or anergy) could adversely affect the generation of memory T cells. There is growing evidence that exposure to TCDD drives antigen-activated T cells into a state of anergy (Mitchell and Lawrence, 2003
; Prell et al., 2000
). Additionally, many AhR ligands are reported to affect apoptotic regulatory mechanisms in T cells (Burchiel et al., 1992
; Kamath et al., 1997
; Pryputniewicz et al., 1998
; Rhile et al., 1996
; Yoo et al., 1997
), indicating that activation of the AhR may influence survival mechanisms crucial to the maintenance of a memory population.
Alternatively, defects in the response of memory CD8+ T cells may reflect the impaired response of virus-specific CD4 cells. Exposure to TCDD affects the activation and clonal expansion of CD4+ T cells (Lundberg et al., 1992; Shepherd et al., 2000
; Warren et al., 2000
). Based on recent reports (Belz et al., 2002
; Janssen et al., 2003
; Sun and Bevan, 2003
), it is clear that the role of CD4+ T cells in antiviral immunity and the activation of memory CD8+ T cells has likely been underestimated. These reports demonstrate that secondary CTL expansion is dependent upon the presence of CD4+ T cells during a primary immune response. Therefore, AhR-mediated defects in the function of CD4+ T cells may result in the reduced number and delayed response of virus-specific CD8+ T cells observed in mice exposed to TCDD prior to primary infection.
Additionally, AhR activation may selectively impair the generation of particular subsets of memory T cells. For instance, memory CD8+ T cells have been subdivided into two distinct subtypes: effector memory T cells (TEM) that respond immediately following reexposure to antigen, and central memory T cells (TCM), which are important for sustaining the memory response (Masopust et al., 2001; Sallusto et al., 1999
; Walzer et al., 2002
; Yajima et al., 2002
). Based on the idea that upon reexposure to antigen TCM differentiate into more TEM (Sallusto et al., 1999
), our data suggest that exposure to TCDD may hinder the development of TEM but not affect the generation of TCM. Thus, the delay in the response of memory CD8+ T cells may reflect the time required for differentiation of TCM into TEM.
All of the findings presented here need to be interpreted in the context of the fact that although a sublethal dose of virus is administered, about 50% of the TCDD-treated mice fail to survive the primary infection. Therefore, the memory response can be examined in only those mice that survive. This is not, however, a fatal flaw in this model system because, in the mice that live, both the cell-mediated and humoral responses to infection are severely affected by exposure to TCDD, even when examined many weeks following resolution of the primary infection. Thus, we can examine the kinetics and magnitude of the recall response in this cohort of mice, and based on these data, draw conclusions about how a suppressed primary response affects immunological memory.
While the underlying mechanisms remain to be defined, our data demonstrate that exposure to TCDD during the activation of naïve lymphocytes affects not only the primary response but leads to anomalies in the magnitude and nature of the recall response. In a model of homotypic infection, these alterations in the recall response did not result in negative consequences to the host. However, we posit that the decreased number of memory CTL would likely have a negative impact on host resistance during a heterosubtypic influenza virus infection. This idea stems from the fact that circulating antibodies play a very important role in protection during homotypic infection, whereas CTL are more critical for host resistance in the context of heterosubtypic infection (Doherty et al., 1997; Gerhard et al., 1997
). The underlying reason for this distinction is that different subtypes of influenza virus express different isoforms of certain proteins on their surface, and the different forms are immunologically distinct (Levine, 1992
). Therefore, antibodies that recognize one subtype of virus do not cross react with other subtypes, termed heterotypes or heterosubtypes. Instead, heterosubtypic immunity relies on antigens derived from internal components of the virus, which are presented by MHC class I molecules to CD8+ T cells. In other words, for heterosubtypic infection, CTL are the most important mediators of host resistance. Therefore, in the case of heterosubtypic infection, the diminished pool of antigen-specific memory CD8+ T cells observed in mice exposed to TCDD would likely have a significant and negative impact on host resistance. Additionally, the excess levels of IgA (e.g., neutralizing antibodies) that are host-protective in the context of a homotypic infection would not be effective in a heterosubtypic infection.
Taken together, the new information presented here has broader implications than just understanding how TCDD affects the immune response to infection with influenza virus. TCDD represents a large group of exogenous compounds that share a common mechanism of action. Therefore, the idea that activation of the AhR causes persistent defects in antibody levels and diminishes the number of antigen-specific memory T cells has health-related implications as humans rely on vaccination and immunological memory for protection from disease. While our data suggest that animals treated with TCDD can overcome homotypic viral infection, it will be important to determine whether the suppressive effects of exposure to TCDD adversely affect host resistance in the context of heterosubtypic immunity.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (509) 335-5902. E-mail: bpl{at}wsu.edu
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