* Experimental Toxicology Division, NHEERL, ORD, U.S. EPA, Research Triangle Park, North Carolina; and
Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Received January 22, 2001; accepted April 18, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: dioxin; dust mite; allergy; immunosuppression; IgE; lung; rats.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In contrast, the antibody response to sheep erythrocytes is markedly suppressed by TCDD in mice, but is increased in rats exposed to TCDD one week before immunization (Smialowicz et al., 1996). Likewise, TCDD exposure prior to infection suppressed the mouse splenic lymphoproliferative response to parasite antigens (Luebke et al., 1994
) but enhanced the response in rats (Luebke et al., 1995
). Because dioxin exposure increases rat antigen-specific responses under certain conditions, exposure may also favor the development of an allergic response, or increase the severity of an allergic response to certain environmental antigens. These studies were conducted to test the hypothesis that TCDD exposure exacerbates the allergic-type immune responses to house dust mite antigen in rats.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TCDD exposure.
TCDD (purity >98% by gas chromatography-mass spectroscopy analysis, Radian Corp., Austin, TX) was dissolved in acetone and added to corn oil (Sigma Chemical Co., St. Louis, MO). The acetone was removed under vacuum and the stock solution of TCDD was diluted with corn oil to prepare dosing solutions containing 0.2, 2.0, or 6.0 µg TCDD/mL. Animals were dosed by intraperitoneal injection of 5 µl of dosing solution/g of body weight, providing doses of 0 (corn oil), 1, 10, or 30 µg TCDD/kg of body weight. One injection was given 7 days before the first sensitization (on day 21; see Fig. 1).
|
Next, experiments were done to evaluate early post-sensitization effects of TCDD exposure on cytokine message and functional endpoints 2 days after the second sensitization ("Series 2" in Fig. 1). This time point was chosen in order to evaluate influential early events (particularly cytokine responses and shifts in B and T lymphocyte populations) in the development of dust mite allergy that might be missed, if only day-of-challenge and later samples were collected.
Sensitization and challenge.
Purified house dust-mite (HDM) antigen from Dermatophagoides farinae was obtained from ground, whole-bodied mites after defatting, extraction in 0.125-M ammonium bicarbonate, and dialysis against distilled water (Greer Laboratories, Lenoir, NC). The resultant extract contained >75% of group-I allergen, as assessed by the vendor. Beginning one week after TCDD exposure, rats were sensitized by intratracheal (it) instillation of 10 µg HDM, followed by a second instillation 48 h later (days 14 and 12). Rats were given an it challenge (day 0 in Tables and Figs.) of 10 µg HDM 14 days after the first sensitization.
Immediate hypersensitivity.
The bronchoconstrictive response to HDM challenge, as measured by enhanced pause, was evaluated on day 0 (day of challenge with HDM) in 5 rats at each dose level, using methods previously described in detail (Dong et al., 1998; Lambert et al., 1998
). The enhanced pause is a unit-less measure of airflow obstruction, which reflects changes in pulmonary function related to quantitative differences in time and extent between inspiration and expiration (Lambert et al., 1998
). Briefly, rats were placed in a plethysmograph for 10 min to obtain a baseline, then removed, anesthetized with halothane, and challenged by intratracheal instillation of 10 µg HDM. Animals were returned to the plethysmograph and enhanced pause values were recorded at 1-min intervals for 20 min.
Sample collection.
On each assay day, animals were weighed, anesthetized by intraperitoneal injection of pentobarbitol, and weighed. Blood was collected by cardiac puncture. BALF was obtained from each animal as previously described (Gilmour et al., 1996). The spleen and mainstem bronchial lymph nodes were removed aseptically and placed in culture medium.
Serum immunoglobulin levels.
Serum was separated from clotted blood samples by centrifugation and aliquots of serum frozen at 80°C for later analysis. Total IgE plus HDM-specific IgG and IgE levels in thawed serum samples were measured by ELISA, as previously described (Lambert et al., 2000).
BALF analysis.
Viable cells were counted on a hemocytometer in the presence of trypan blue dye. Differential cell counts were performed on slides prepared by centrifugation (Cytospin Model II, Shandon, Pittsburgh, PA) of 50,000 cells onto glass slides, followed by staining with Diff Quik (American Scientific, Inc., Sewickley, PA). Cells were removed from the remaining BALF by centrifugation and the supernatant was assayed for protein using CoomassiePlus reagent (Pierce, Rockford, IL), and LDH (kit 228, Sigma, St Louis, MO) on a Cobas FARA instrument (Hoffman-La Roche, Branchburg, NJ)
Lymphocyte proliferation assay.
Lymph-node cell suspensions were prepared using ground-glass homogenizers (Gilmour et al., 1996) and spleen-cell suspensions prepared as described by Luebke et al. (1992). The proliferation assay was performed as previously described (Gilmour et al., 1996
). Lymphocyte responsiveness was expressed as net CPM; i.e., the CPM of incorporated tritiated thymidine (3HTdR) by cells incubated with HDM, minus the CPM of cells incubated with media alone.
Cytokine evaluation.
Approximately 100 mg of caudal lung lobe tissue was removed, weighed, snap-frozen on dry ice, and stored at 70°C in 1.0 ml of TRI Reagent (Sigma, St. Louis, MO); the tissue was later homogenized and total RNA was extracted according to the manufacturer's instructions. Semiquantitative PCR assays were performed as previously described (Dong et al., 1998; Lambert et al., 1999
). Briefly, RNA concentrations were measured by a GeneQuant spectrophotometer (Pharmacia Biotech, Inc., Piscataway, NJ) set at 260 nM wavelength, and RNA purity was assessed by the ratio of absorbance at 260 and 280 nM. Complimentary DNA (cDNA) was synthesized using primers for interleukin 4 and 5 as described by Siegling et al. (1994). TNF
, IFN
, and G3PDH cDNAs were synthesized according to the instructions accompanying commercial primers (Clontech, Palo Alto, CA). Amplifications were performed in thermal cycler strip tubes using a 9600 GeneAmp PCR System (Perkin-Elmer Corporation, Norwalk, CT). Concentrations of specific cDNA sequences were quantified by separating 10 µl of each amplification mixture through a 2% agarose gel. Integrated optical densities (IOD) of amplification products that resolved as single bands of a predicted size were divided by the IOD of the G3PDH DNA band to normalize for any differences in the amount of input RNA.
Flow cytometry.
Phenotypic analysis of bronchial lymph node cells was done 2 days after the second sensitization and 2 days after challenge, on animals in the second series of experiments. Cells were dual-labeled with monoclonal antibodies to CD5 (OX19, phycoerythrin-tagged, PharMingen, San Diego, CA) and IgG kappa light chain (OX12, FITC-tagged, Serotec, Raleigh, NC) to identify T and B cells, respectively. T cells were subtyped by staining with a cocktail of monoclonal PE-labeled anti-CD4 and FITC-labeled anti-CD8 (PharMingen). Appropriate isotype controls were included. Details of the procedure can be found in Ladics et al. (1998).
Statistical analysis.
Data are expressed as mean ± SEM. Immediate bronchoconstriction data were analyzed by comparing all groups, using repeated-measures analysis of variance (ANOVA, SAS Institute, Cary, NC). Group differences were considered significant if the test statistical type I error was <0.05. Other endpoints were evaluated using Dunnett's t-test for comparing multiple groups to a control.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
The BALF concentration of leukocytes was greater in sensitized control rats than in non-sensitized animals, and exposure to TCDD decreased the sensitization-related cellular influx (Fig. 7 insert). TCDD exposure decreased the percentage of eosinophils in the BALF of both sensitized and non-sensitized rats (Fig. 7
). Although the percentage of eosinophils was similar in sensitized and non-sensitized controls, the absolute number of eosinophils was increased (data not shown) by the increased cellularity of sensitized rat BALF.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Suppression of HDM-specific immune responses by exposure to TCDD reduced the subsequent immune-mediated lung disease following intratracheal allergen challenge. Previous adoptive transfer studies using this model had demonstrated that serum IgE antibody conferred the ability to produce allergen-induced bronchospasm, while activated lymphocytes were responsible for inducing pulmonary inflammation, acute lung injury and airway hyper-responsiveness (Lambert et al., 1998). Thus, it was not surprising that these pathological features were reduced in animals with suppressed T-cell and antibody responses. Because total IgE in serum and splenic mitogen responses were unaffected by TCDD exposure, suppression appeared to be an antigen-specific event rather than a generalized reduction in immune competence.
Although our results did not support the hypothesis, the outcome of these studies is noteworthy in that suppression of antibody production occurred at doses of TCDD reported by others to enhance antigen-specific immune function. For example, Smialowicz et al. (1996) reported an increase in numbers of PFCs at doses of 30 µg TCDD/kg in F344 rats 7 days before immunization. Increased responses do not appear to be rat strain-dependent, because similar results were observed in other strains, including the BN, following exposure to 3 µg TCDD/kg (Ralph Smialowicz, personal communication). Also, Fan et al. (1996) observed increased SRBC-specific IgG titers, as well as increased delayed hypersensitivity responses to keyhole limpet hemocyanin in Sprague-Dawley rats at TCDD exposure levels of 20 µg/kg. Thus, this paper is the first to report suppression of thymus-dependent antibody production in adult rats exposed to TCDD.
In evaluating these apparently contradictory results, it must be considered that the enhanced systemic immune responses reported in TCDD-exposed rats followed parenteral immunization, rather than local (pulmonary) sensitization. It has been shown that both primary and secondary antibody responses to sheep erythrocytes can be induced by intravenous injection or intratracheal instillation of equal numbers of red cells (Stein-Streilein et al., 1979), indicating that either route of immunization can be effective. However, the route of antigen (allergen) exposure may still be pivotal in controlling whether suppression or enhancement is observed in xenobiotic-exposed animals. For example, Dong et al. (1998) reported an enhanced pulmonary allergic response to HDM in BN rats exposed to the pesticide carbaryl prior to systemic sensitization (i.e., subcutaneous injection) but found that the response was suppressed if HDM was given by intratracheal instillation (Wumin Dong, personal communication). One possible interpretation of these results, assuming that humans share this response pattern, is that the apparent increase in human allergic asthma is not the result of systemic antigen exposure. Rather, xenobiotic-induced exacerbation of allergic lung disease may be a special situation mediated by inhaled allergen and reactive gases (e.g., NO2; Gilmour et al., 1996
) or inhaled particulates in rodents (Lambert et al., 1999
, et al., 2000) and humans (Diaz-Sanchez et al., 1997
). Future experiments will address how strongly the route of immunization influences the outcome of antigen exposure following xenobiotic exposure, and how the nature of the xenobiotic influences the process.
The route of antigen administration is apparently not the only factor dictating whether TCDD will cause immune system enhancement or suppression. It is also possible that the chemical or immunological properties of the administered antigen influenced the outcome of antibody synthesis. For example, Smialowicz et al. (1996) reported suppression of antibody production in rats exposed to 30 µg TCDD/kg after immunization with the type 1 T-independent antigen TNP-LPS.This finding is in accord with earlier studies suggesting that a B-cell maturational defect underlies suppression of humoral immunity by TCDD in mice (Tucker et al., 1986). It remains to be determined whether HDM antigen is processed and responded to by the rat immune system in such a way that pre-sensitization exposure to TCDD has a suppressive, rather than stimulatory effect.
Enhanced antibody responses to SRBCs in TCDD-exposed rats were accompanied by a dose-related decrease in CD4-/CD8+ lymphocytes and a dose-related increase in IgM+ lymphocytes (Smialowicz et al., 1996). We found no differences in lymph node B- and T-lymphocyte populations 2 days after the second sensitization or 2 days after challenge, suggesting that exposure-related loss of lymphocyte populations was not responsible for the observed effects. It is unlikely that the disparity between the 2 studies are simply due to kinetic differences, since the length of time between TCDD exposure and sensitization (7 days) and sensitization and assay (4 days) were similar in both studies.
When the results of these and other published experiments are considered together, it appears that the combination of xenobiotic exposure, the immunologic microenvironment where antigen is first encountered, and possibly the type of immunogen that is injected influence whether suppression or enhancement of the immune response will occur. Clearly, TCDD exposure does not have a similar effect in rats under all conditions of immunization. Further studies are required to determine how widespread these disparate effects are, both in terms of immunosuppression and development of pulmonary hypersensitivity.
It is interesting to note that Weisglas-Kuperus et al. (2000) reported an inverse relationship between the odds ratio for developing allergic disease and total maternal and toddler TCDD toxic equivalents. While the biological basis for reduced allergic responses in humans and rodents may differ, the similarity of results suggest that immunosuppression is common to both events. From the standpoint of human risk assessment, it is important to determine whether the route of immune system sensitization, coupled with previous xenobiotic exposure, affects the development of allergy and possibly asthma. This is particularly important if subsequent studies suggest that contradictory results will likely be obtained in the xenobiotic-exposed host immunized locally instead of systemically.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
This report has been reviewed by the Environmental Protection Agency's Office of Research and Development, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CDC (1998). Forecasted state-specific estimates of self-reported asthma prevalence: United States. Morbid. Mortal. Weekly Rep. 47, 10221025.[Medline]
Diaz-Sanchez, D., Tsien, A., Fleming, J., and Saxon, A. (1997). Combined diesel exhaust particulate and ragweed allergen challenge markedly enhances human in vivo nasal ragweed-specific IgE and skews cytokine production to a T helper cell 2-type pattern. J. Immunol. 158, 24062413.[Abstract]
Dong, W., Gilmour, M. I., Lambert, A. L., and Selgrade, M. K. (1998). Enhanced allergic responses to house-dust mite by oral exposure to carbaryl in rats. Toxicol. Sci. 44, 6369.[Abstract]
Fan, F., Wierda, D., and Rozman, K. K. (1966). Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on humoral and cell-mediated immunity in Sprague-Dawley rats. Toxicology 106, 221228.
Gilmour, M. I., Park, P., and Selgrade, M. J. (1996). Increased immune and inflammatory responses to dust mite antigen in rats exposed to 5 ppm NO2. Fundam. Appl. Toxicol. 31, 6570.[ISI][Medline]
Institute of Medicine (1993). Report on Indoor Allergens. (A.M. Pope, R. Patterson, and H. Burge, Eds.). National Academy Press, Washington, DC.
Ladics, G. S., Smith, C., Loveless, S. E., Green, J. W., Flaherty, D., Gross, C., Shah, R., Williams, W., and Smialowicz, R. (1998). Phase two of an interlaboratory evaluation of the quantification of rat splenic lymphocyte subtypes using immunofluorescent staining and flow cytometry. Toxicol. Methods 8, 87104.[ISI]
Lambert, A. L., Dong, D. W., Selgrade, M. K., and Gilmour, M. I. (2000). Enhanced allergic sensitization by residual oil fly ash is mediated by soluble metal constituents. Toxicol. Appl. Pharmacol. 165, 8493.[ISI][Medline]
Lambert, A. L., Dong, W., Winsett, D. W., Selgrade, M. K., and Gilmour, M. I. (1999). Residual oil fly ash exposure enhances allergic sensitization to house dust mite. Toxicol. Appl. Pharmacol. 158, 269277.[ISI][Medline]
Lambert, A. L., Winsett, D. W., Costa, D. L., Selgrade, M. K., and Gilmour, M. I. (1998). Transfer of allergic airway responses with serum and lymphocytes from rats sensitized to dust mites. Amer. J. Resp. Crit. Care Med. 157, 19911999.
Luebke, R. W., Copeland, C. B., and Andrews, D. L. (1995). Host resistance to Trinchinella spiralis infection in rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fundam. Appl. Toxicol. 24, 285289.[ISI][Medline]
Luebke, R. W., Copeland, C. B., Andrews, D. L., Riddle, M. M,. and Smialowicz, R. J. (1992). Host resistance to Trichinella spiralis infection in rats and mice: Species-dependent effects of cyclophosphamide exposure. Toxicology 73, 305321.[ISI][Medline]
Luebke, R. W., Copeland,C. B., Diliberto, J. J., Akubue, P. I., Andrews, D. L., Riddle, M. M., Williams, W. C., and Birnbaum, L. S. (1994). Assessment of host resistance to Trichinella spiralis in mice, following pre-infection exposure to 2,3,7,8-TCDD. Toxicol. Appl. Pharmacol. 125, 716.[ISI][Medline]
Royce, S., Wald, P., Sheppard, D., and Balmes, J. (1993). Occupational asthma in a pesticides manufacturing worker. Chest 103, 295296.[Abstract]
Senthilselvan, A., McDuffie, H. H., and Dosman, J. A. (1992). Association of asthma with use of pesticides: Results of a cross-sectional survey of farmers. Amer. Rev. Respir. Dis. 146, 884887.[ISI][Medline]
Shelton, D., Urch, B., and Tarlo, S. M. (1992). Occupational asthma induced by a carpet fungicide, tributyl tin oxide. J. Allergy Clin. Immunol. 90, 274275.[ISI][Medline]
Siegling, A., Lehmann, M., Platzer, C., Emmrich, F., and Volk, H.-D. (1994). A novel multi-specific competitor fragment for quantitative PCR analysis of cytokine gene expression in rats. J. Immunol. Methods 177, 2328.[ISI][Medline]
Smialowicz, R. J., Williams,W. C., and Riddle, M. M. (1996). Comparison of the T cell-independent antibody response of mice and rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fundam. Appl. Toxicol. 32, 293297.[ISI][Medline]
Stein-Streilein, J., Gross, G. N., and Hart, D. A. (1979). Comparison of intratracheal and intravenous inoculation of sheep erythrocytes in the induction of local and systemic immune responses. Infect. Immun. 24, 145150.[ISI][Medline]
Takenaka, H., Zhang, K., Diaz-Sanchez, D., Tsien, A. and Saxon, A. (1995). Enhanced human IgE production results from exposure to the aromatic hydrocarbons from diesel exhaust: Direct effects on B-cell IgE production. J. Allergy Clin. Immunol. 95, 103115.[ISI][Medline]
Tucker, A. N., Vore, S. J., and Luster, M. I. (1986). Suppression of B-cell differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Molec. Pharmacol. 29, 372377.[Abstract]
Weisglas-Kuperus, N., Patandin, S., Berbers, G. A., Sas, T. C., Mulder, P. G., Sauer,P. J., and Hooijkaas, H. (2000). Immunologic effects of background exposure to polychlorinated biphenyls and dioxins in Dutch preschool children. Environ. Health Perspect. 108, 12031207.[ISI][Medline]