ToxSci Advance Access originally published online on May 12, 2004
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Toxicological Sciences 79, 211-213 (2004)
Toxicological Sciences vol. 79 no. 2 © Society of Toxicology; all rights reserved.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and Long Term Immunologic Memory
Department of Environmental Health, Boston University School of Public Health, 715 Albany Street (R-408), Boston, Massachusetts 02118
Received April 5, 2004; accepted April 6, 2004
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The highlighted article by B. Paige Lawrence and Beth Vorderstrasse addresses an oft forgotten aspect of immunotoxicity, the effects of environmental toxins on immunologic memory. Here, the authors take a step towards filling that information gap by evaluating the effects of a prototypic environmental toxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on memory responses to a real-world pathogen, influenza A virus, presented to an animal model in a physiologically relevant manner. Multiple outcomes are evaluated, the vast majority of which suggest important and long-term TCDD-induced changes in the immune system after both primary and secondary exposure to this pathogen. The implications of these studies with regard to the immuno-competence of TCDD-exposed individuals are far reaching.
The article highlighted in this issue of Toxicological Sciences, written by B. Paige Lawrence and Beth Vorderstrasse of Washington State University, addresses an oft forgotten aspect of immunotoxicity, the effects of environmental toxins on the web of cellular interactions that collectively mediate immunologic memory. That this aspect of immunotoxicity has been neglected is surprising given the critical surveillance function performed by memory cells of both the B- and T-lymphocyte compartments. Here, Drs. Lawrence and Vorderstrasse take a step toward filling that information gap by evaluating the effects of a prototypic environmental toxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on memory responses to a real-world pathogen, influenza A virus, presented to an animal model in a physiologically relevant manner, i.e., intranasaly. At a minimum, this article demonstrates that secondary immune responses to viral pathogens are likely to be significantly compromised by TCDD exposures occurring prior to or at the time of primary infection. Whether or not this immune alteration results in increased susceptibility to viral infections is uncertain. No increase in mortality was seen in the present study following secondary challenge to homotypic influenza virus. However, as discussed below, the failure to demonstrate increased susceptibility here, in the face of altered B-cell and CD8+ T-cell responses, may lead to an underestimate of the long-term immunologic effects of TCDD.
The backdrop for these highlighted studies is provided in part by published work from Drs. Lawrence and/or Voderstrasse, performed in collaboration with Dr. N. Kerkvliet's laboratory, in which several aspects of primary immune T-cell responses, e.g., CD8+ T-cell responses, antigen presentation, and cytokine production were shown to be diminished following acute TCDD exposure (Kerkvliet et al., 2002
; Vorderstrasse et al., 2003b). As shown by Drs. Lawrence and Voderstrasse (Neff-LaFord et al., 2003), and by other investigators (Burleson et al., 1996; House et al., 1990), the apparent outcome of this immunosuppression is increased mortality following primary viral infection. While one might presume that this result necessitates an accompanying decrease in anamnestic, i.e., secondary responses, the experiments to confirm this presumption had not been performed. Furthermore, the intrinsic differences between naïve and memory T lymphocyte responses to influenza (Crowe et al., 2003) suggest that TCDD may differentially regulate primary and secondary influenza-specific immune responses.
One of the difficulties in carrying out this type of immunologic study is the need to assay multiple immunologic endpoints to fairly assess the protective value of the immune system. Drs. Lawrence and Voderstrasse addressed this need by assaying multiple immune parameters, including production of virus-specific IgM, IgG2a, IgG2b, IgG1, and IgA in the plasma and, in what one would presume is the more critical location, bronchial fluid. Previous studies from several laboratories demonstrated that all of these antibody isotypes play a role in influenza immunity. IgA in particular was of interest given that the requirements for influenza-specific IgA responses are less stringent than the requirements for influenza-specific IgG responses in that the former do not require cognate B-cell-T-cell interactions (Sangster et al., 2003). Similarly, analysis of CD8+ T-cell responses was necessitated by the clear role that these cells play in primary and secondary responses to influenza (Kedzierska et al., 2004.).
The pattern of responsiveness (or nonresponsiveness) that emerged from these comprehensive studies is remarkable. First, while TCDD suppressed primary responses of IgG2a, IgG2b, and IgG1, as measured by virus-specific antibody levels in the plasma, secondary responses were only temporarily (3–7 days) suppressed. The decrease in plasma IgG isotypes paralleled a decrease in the number of IgG antibody-secreting draining lymph node B cells, suggesting that the suppressive effects of TCDD were mediated at least in part by inhibition of B-cell activation and/or differentiation into antibody secreting cells. IgM responses were profoundly suppressed by TCDD exposure during both primary and secondary responses. In contrast, the levels of all antibody isotypes detected in bronchial fluid, with the exception of IgA, were lower in TCDD-treated animals during both primary and secondary influenza-specific responses. Remarkably, an increase in plasma-borne virus-specific IgA was observed in TCDD-treated mice following both an initial viral infection and secondary challenge. Finally, the number of virus-specific CD8+ T cells, detected with fluorescent viral peptide/MHC I/ß2 microglobulin tetramers, was significantly reduced during both primary and secondary responses in TCDD-treated mice.
The implications of this work are considerable and varied. First to be considered is the long-term effect of a single TCDD exposure on IgM and CD8+ T-cell responses in animal models. This long-term immunosuppression, like that observed in humans (Tonn et al., 1996.), may be a function of the long biologic half-life of TCDD (e.g., approximately 7.6 years in humans (Michalek and Tripathi, 1999), a propensity for lymphocytes in TCDD-treated animals to become anergic to foreign antigens (Mitchell and Lawrence, 2003), and/or a TCDD-mediated, irreversible loss of precursor lymphocytes (Fine et al., 1990.). Any one of these elements would bode ill for the maintenance of a competent immune system. Whatever the mechanism(s), these results suggest TCDD-mediated susceptibility to a variety of pathogens, if not homotypic influenza reinfection. In this regard, the authors correctly point out that TCDD-induced decreases in the number of influenza epitope (NP366)-specific CD8+ T cells, which presumably exhibit lytic activity, probably parallels a decrease in killer T cells specific for other viral epitopes. Since memory CTL are critical for resistance to secondary infections with related but antigenically distinct influenza subtypes (i.e., heterosubtypes), TCDD-treated mice may be susceptible for many months to infections with other viral heterosubtypes.
Secondly, the sheer complexity of the response to TCDD demonstrates how difficult it is to predict the effects of this toxin on any given immunologic outcome. For example, if one were to posit that TCDD immunosuppression was due exclusively to its effect on antigen presenting cells, then the remarkable increase in IgA production would not have been predicted.
Third, the increase in virus-specific plasma IgA in TCDD-treated mice is consistent with the relatively unique requirements for production of this isotype, most notably the ability to generate IgA isotype switching by interactions between B cells and dendritic cells directly in the absence of T cells (Litinskiy et al., 2002.). Indeed, the sparing of only the IgA response and not the more CD4 T cell-dependent IgG and CD8+ T-cell responses, could reflect preferential targeting of CD4 T cells by TCDD (Kerkvliet et al., 2002.).
Fourth, the notable demonstration that TCDD does not affect a secondary IgG2a or IgA response to viral antigen when TCDD is administered only at the time of secondary infection emphasizes the qualitative differences between primary and secondary immune responses. The requirements for naïve T-cell activation are generally considered to be more stringent than the requirements for memory T-cell activation. This result also underscores the difficulty of predicting the effectiveness of an immune response when the precise time at which TCDD exposure has occurred, relative to viral infection (or vaccination), is not known.
Collectively, the results demonstrate profound effects of TCDD on primary antiviral responses, and more subtle effects upon secondary infection. What then can account for the apparently normal resistance of TCDD-treated mice to secondary viral infection? The authors entertain several possibilities. First, while somewhat delayed, the antibody and CD8+ T-cell response may "catch up" with the secondary infection in time to rescue the mice. While the authors suggest that this is not likely given that vehicle- and TCDD-treated mice clear virus from the lungs with equal kinetics, it remains to be seen if the same would be true had the secondary viral load been higher. Secondly, the reduced levels of IgG and CD8+ T cells may have been sufficient for viral clearance. As above, a higher secondary viral load may have revealed a relative susceptibility of TCDD-treated mice. Third, the ability of TCDD to increase IgA levels in plasma and its failure to suppress IgA levels in the affected tissue, i.e., the lung, are reasonably proposed as the most likely mechanism of host resistance.
While the lack of IgA inhibition seems to be the most likely explanation for the survival of TCDD-treated mice following secondary influenza infection, there may be other mechanisms in play. At first glance, the more obvious suppression of antibody levels in bronchial fluid as compared with plasma might be interpreted as an indicator of immuno-incompetence in the affected organ leading to increased viral susceptibility. However, the affinities of the respective bronchial fluid and plasma antibodies were not assessed. It was shown in the late 1970's that antigen-specific antibodies produced by B cells in distant lymph nodes are of higher affinity than those of B cells in draining nodes close to the site of antigen exposure. This selection of high affinity antibody-producing clones is due to a "microevolutionary" process in which a lower concentration of antigen at the distant site increases the selective pressure for high affinity antigen-specific B cell clones (Sherr and Siskind, 1977). Therefore, the relative affinities of the antibodies induced by viral antigen encounter in the collective of distant lymph nodes may have been higher than the affinities of B cells activated in draining lymph nodes where antigen was abundant. It would be postulated then that it is the higher affinity plasma antibodies, which remained relatively unaffected by TCDD, rather than the lower affinity bronchial fluid antibodies, which confer resistance to secondary viral challenges.
In addition, previous work from Drs. Lawrence and Vorderstrasse demonstrated an increase in pulmonary neutrophilia in TCDD-treated mice (Vorderstrasse et al., 2003a). It may be that this neutrophilia compensates for the loss of antibody- and CD8+ T cell-mediated immunity during a secondary response, even though it is insufficient to ameliorate an initial infection.
Finally, the authors use survival after a secondary infection as a marker for immune competence. Importantly, a large fraction of TCDD-treated mice (55–64%) die after the first infection. In essence, the mice that make it to the second infection are selected for their survivability, which may or may not depend on the elements of the immune system assayed in the current study. This pathogen-dependent selection of "healthy" animals may explain the difference between this report and others in which both primary and secondary antibody responses to nonpathogens (e.g., sheep red blood cells and ovalbumin) were profoundly suppressed by TCDD (Vecchi et al., 1980; Nohara et al., 2002). Had a lower initial dose of virus been used in the present study, one that did not result in significant death after an initial viral insult, then reduced survival of mice exposed to TCDD and experiencing a second viral infection may have been more evident. As the authors point out, this is not to say that the significance of this report on primary and secondary responses in TCDD-treated mice is diminished. To the contrary, the results highlighted here may underestimated the impact of dioxin on pathogen resistance since the changes in immune parameters were observed in mice with relatively competent immune systems which enabled their survival following an initial and formidable viral challenge.
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1 For correspondence via fax: 617-638-6463. E-mail: dsherr{at}bu.edu
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