National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown, West Virginia 26505
Received June 25, 2002; accepted November 13, 2002
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ABSTRACT |
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Key Words: diesel exhaust particles; immunosuppression; antibody-forming cell response; respiratory exposure; cytokine modulation.
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INTRODUCTION |
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Numerous reports have shown that exposure to DEP may affect immunological function. Increased antigen-induced immunoglobin E (IgE) production following DEP exposure has been noted by many investigators; and it has been postulated that the adjuvant effects of DEP may contribute to the increased incidence and severity of asthma and other autoimmune diseases (Diaz-Sanchez et al., 1996; Miyabara et al., 1998; Takafuji et al., 1987
; Yoshino and Sagai, 1999
). Exposure to DEP has also been shown to decrease interferon production in response to viral infection (Hahon et al., 1982
), enhance influenza multiplication within the lungs (Hahon et al., 1985
), suppress phagocytic activity of macrophages (Castranova et al., 1985
; Jakab et al., 1990
), depress the lipopolysaccharide (LPS)-induced secretion of TNF-
and IL-1 by alveolar macrophages (Yang et al., 1997
), and decrease host resistance to pulmonary Listeria monocytogenes infection (Yang et al., 2001
). The production of T helper 1 (Th1) and Th2 cytokines in the lungs has been shown to be altered following exposure to DEP (Diaz-Sanchez et al., 1997
; van Zijverden et al., 2000
). Cytokines produced by Th cells play a critical role in regulating immune responses and determining the outcome of diverse immunologically mediated clinical syndromes, including infections, autoimmunity, and allergic diseases (reviewed in Romagnani, 1994
). The Th1 cytokines, such as IL-2, IFN-
, and lymphotoxin, are important in cell-mediated immunity; while Th2 cytokines, including IL-4, 5, 6, 9, 10, and 13 are important in the humoral response (reviewed in Abbas et al., 1996
; Mosmann 1992
). These data indicate that DEP may have dual effects on the pulmonary immune system, enhancing IgE-mediated responsiveness to various antigens on one hand and suppressing cell-mediated immunity on the other.
Few studies have evaluated the suppressive effects of DEP on systemic immunity. The present study was undertaken to investigate the immunosuppressive potential of DEP following pulmonary exposure of mice via intratracheal aspiration. Following a general toxicological assessment, the splenic antibody-forming cell (AFC) response to sheep red blood cells (sRBC) was used to evaluate the systemic immune status of exposed animals. This assay was chosen as a sensitive indicator of immune competence, requiring the coordinated interaction of antigen presenting, T, and B cells (Luster et al., 1988). The effects of in vivo DEP exposure on in vitro spleen cell function were also investigated to gain insight regarding the possible mechanisms by which DEP affected in vivo responses to sRBC.
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MATERIALS AND METHODS |
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Animals and exposures.
Female B6C3F1 mice (68 week-old), purchased from Taconic (Germantown, NY), were used in the study. B6C3F1mice are widely accepted for use in immunotoxicology studies and are the strain chosen for standard immunotoxicology evaluation by the NTP. Upon arrival, mice were housed in shoe-box cages, five animals per cage, in the NIOSH Association for Assessment and Accreditation of Laboratory Animal Care International (AALAC)-accredited animal facility. Room temperature and humidity were targeted at 2224°C and between 40 and 70%, respectively, and the light/dark cycle was set on 12-h intervals. Animals had access to certified rodent chow (LabDiet, PMI® Nutrition International TestDiet®, Richmond, IN) and water ad libitum. The mice were allowed to acclimate in the facility for at least 4 days before the start of treatment.
On the first day of the experiment, mice were assigned to experimental groups and ear punched for identification. Mice were exposed to particles by intratracheal aspiration as follows: under light isoflurane anesthesia (Abbott Laboratories, North Chicago, IL), mice were held vertically by their incisor teeth against an angled restraining device. The tongue was gently extended to prevent swallowing, and the particle solution was pipetted directly into the oropharynx. The tongue was maintained in extension until the DEP had been aspirated into the lungs. The mice were exposed to 1, 5, or 15 mg DEP/kg of body weight 3 times in a period of 2 weeks, i.e., Monday and Friday of the first week and Wednesday of the second week, or 6 times over 4 weeks. For the AFC experiment, 2 additional doses, 0.05 and 0.2 mg/kg, were included. The volume of instillation was 25 µl/10 g of body weight. Control animals received the same volume of sterile saline. Except for experiments evaluating T-cell function, another group of mice, which served as the positive control group, was given 25 mg/kg/day of cyclophosphamide (CP, Sigma Chemical Co., St. Louis, MO), a well characterized immunosuppressant, by intraperitoneal injection for the 4 consecutive days prior to sacrifice.
Body and tissue weights.
Two or 4 weeks after the initial particle exposure, mice were euthanized by CO2 asphyxiation and body weights were recorded. The wet weights of the lungs, thymus, spleen, and liver were recorded after dissection.
Hematology.
On the day of sacrifice, blood was collected into EDTA tubes by cardiac puncture. Selected hematological parameters were evaluated using an automatic hematology analyzer (Abbott CELL-DYN 3500 system, Abbott Diagnostics, Abbott Park, IL), including peripheral erythrocyte and leukocyte counts, leukocyte differential (lymphocytes, neutrophils, monocytes, and eosinophils), platelet count, hematocrit, hemoglobin, mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).
Spleen IgM AFC response to a T-dependent antigen, sRBC.
The primary IgM response to sRBC (Rockland, Gilbertsville, PA) was determined using a modified hemolytic plaque assay (Jerne and Nordin, 1963). Four days before sacrifice, the particle- or saline-exposed mice were sensitized by intravenous injection of 7.5 x 107 sRBC in 0.2 ml saline. On the day of sacrifice, 2 or 4 weeks after the initial DEP instillation, mice were euthanized by CO2 asphyxiation and spleens were aseptically removed. Single cell suspensions in Hanks balanced salt solution (HBSS, Gibco BRL, Grand Island, NY) were prepared from individual animals by pressing spleens between the frosted ends of microscopic slides. An aliquot of single cells (100 µl), at 1:30 or 1:120 dilutions of total spleen cells (in 3 ml HBSS), was added to a test tube containing a 0.5 ml warm agar/dextran mixture (0.5% Bacto-Agar, DIFCO Laboratories, Detroit, MI; and 0.05% DEAE dextran, Sigma Chemical Co.), 25 µl of 50% sheep blood cell solution, and 25 µl of 1:4 dilution guinea pig complement (Gibco BRL). The mixtures were poured into petri dishes, covered with microscope coverslips, and incubated at 37°C for 3 h. The plaques (representing antibody forming cells) were viewed and quantified using a Bellco magnification viewer (Vineland, NJ). The results were expressed as specific activity (IgM AFC/106 splenocytes) and total activity (IgM AFC per spleen), respectively.
Enumeration of total spleen cells and splenic lymphocyte subpopulations.
One aliquot of spleen single-cell suspensions was placed in 10 ml Isoton, two drops of Zap-O-globin (Beckman Coulter, France) were added, and the total number of splenic nucleated cells was counted using an electronic cell counter (Coulter Electronics Inc., Hialeah, FL). To enumerate B cells, T cells, and CD4+ and CD8+ T-cell subsets, spleen cells were collected by centrifugation and suspended in phosphate-buffered saline (PBS), pH 7.4, containing 1% bovine serum albumin and 0.1% sodium azide, to a cell density of 1.5 x 106/ml. The cells were incubated with Fc Block (clone 2.4G2, Pharmingen, San Diego, CA) for 5 min to prevent nonspecific binding and then labeled with an appropriate monoclonal antibody (mAb) conjugated to a fluorescent probe for visualization, using flow cytometry. The antibodies were obtained from Pharmingen. B cells were enumerated using antimouse CD45R/B220 antibodies (clone RA3-6B2) conjugated to fluorescein isothiocyanate (FITC). Antimouse CD3 mAb (clone 1452C11) conjugated to FITC was used to enumerate T cells. For the T-cell subsets, the cells were identified by using antimouse CD4+ mAb (clone H129.19) conjugated to FITC and antimouse CD8+ mAb (clone 536.7) conjugated to phycoerythrin (PE). An isotype control was used for each antibody. Cell suspensions were incubated with labeling antibodies on ice, in the dark, for at least 30 min, washed, and then resuspended in 0.1 ml of a 1 µg/ml solution of propidium iodide (PI). After a 5-min incubation with PI, cells were washed, resuspended in PBS, and enumerated using a FacsVantage Flow Cytometer (Becton Dickinson, San Jose, CA). PI-stained cells (dead cells) were eliminated from the analysis. The forward scatter threshold was set to eliminate red blood cells. The results were expressed as the percentage of gated live cells with the corresponding cell surface marker and as the absolute numbers of cells calculated using the total numbers of splenic nucleated cells.
Cell culture and measurement of cell proliferation.
To evaluate the effects of in vivo exposure to DEP on T-cell function, we measured the proliferative activity of splenocytes in response to different ex vivo stimulants. Single spleen cells (2 x 106/ml) were suspended in culture medium, RPMI 1640 (Gibco BRL) supplemented with 2 mM glutamine, 100 µg/ml streptomycin, 100 units/ml penicillin, 10% heat-inactivated calf serum, and 2 x 105 M mercaptoethanol. Aliquots of spleen cells (2 x 105 in 100 µl culture medium) were transferred in triplicate to a 96-well, flat-bottom tissue-culture plate and cocultured with different stimulants. For each experiment, the final volume was 200 µl in each well. One set of spleen cells was stimulated with ConA (Simga Chemical Co.) at the final concentrations of 0, 0.5, 1, 2, 4, or 8 µg/ml. Another set of spleen cells was cocultured with either medium alone or plate-bound antimouse CD3 mAb (clone 1452C11, Pharmingen). One hundred µl anti-CD3 mAb (20 µg/ml in PBS) was added to each well of a tissue-culture plate and incubated at 4°C overnight. The plate was then washed and the spleen cells were added. All cells were incubated at 37°C in a 5% CO2 atmosphere for 72 h. During the final 1824 h of incubation, [3H]thymidine (1 µCi/well, ICN Biomedicals, Inc., Irvine, CA) was added. The cells were collected using an automated cell harvester (Tamtec Harvester 96®, Tamtec, Organge, CN) and the radioactivity was measured using a Wallac 1450 MicroBeta liquid scintillation counter (Wallac Inc., Gaithersburg, MD). The incorporation of [3H]thymidine into cells was measured as an indicator of cell proliferation. Results were expressed as counts per min (cpm) per 2 x 105 splenocytes.
Cell culture and measurement of cytokine production.
Single cell suspensions (2 x 106 splenocytes/ml) from individual saline or DEP-exposed mice were cultured with 2 µg/ml of ConA at 37°C, 5% CO2, for 72 h under the same conditions as were used for evaluating the proliferative activity described above. After incubation, the supernatants were collected and stored at -75°C until analysis of interleukin (IL)-2, IL-4, and interferon-gamma (IFN-), using a sandwich enzyme-linked immunosorbent assay (ELISA). All of the matched pairs of mAb and the mouse recombinant standards for individual cytokines were purchased as kits from Endogen (Woburn, WA). Assays were conducted following the procedures recommended by the manufacture. Briefly, a 96-well, flat-bottom immunoplate (Nunc F8 MaxiSorp, Nunc Ltd, Roskilde, Denmark) was coated with purified anticytokine mAb in PBS (pH 7.4) at room temperature overnight. After overnight incubation and blocking with 2% bovine serum albumin in PBS (pH 7.27.4), test supernatants and serial dilutions (1:2) of cytokine standards were added in duplicate and incubated at room temperature overnight. The standards for individual cytokines were run on every plate. Biotinylated anti-cytokine mAb, followed by streptavidin-conjugated horseradish peroxidase (dilution 1:8000, Southern Biotechnology Associates, Inc., Birmingham, AL) and tetramethylbenzidine substrate (DAKO Corporation, Carpinteria, CA) were used for cytokine detection. Color reactions were stopped by H2SO4. Absorbance of each sample was measured at both 450 and 550 nm. The readings of 550 nm were subtracted from the readings at 450 nm to correct for optical imperfections in the plates. The levels of individual cytokines in culture supernatants were calculated from the corresponding standard curve derived from the same plate. The detection limits of the assays were 15 pg/ml for IL-2, 100 pg/ml for IFN-
, and 10 pg/ml for IL-4. The results were expressed as ng of cytokines produced by 2 x 106 splenocytes.
Statistics.
With the exception of cytokine analysis that represents a single study, data presented are the combined results from at least 2 studies. The data were analyzed using JMPs, a statistical package obtained from SAS Institute, Inc. (Cary, NC). Data were expressed as the mean ± the standard error of the mean (SE) of experimental values. All data were tested for homogeneity of variance prior to analysis of variance (ANOVA) using Bartletts test; wherever the variance was heterogeneous, data were log-transformed. One-way ANOVA was used to analyze differences among the treatments; for multiple comparisons among means, Tukey-Kramers honestly significant difference (HSD) was used. For testing the trend of dose-dependency, the data for the spleen IgM AFC response to sRBC and the proliferation and cytokine data from splenocytes in response to ConA were also analyzed, using the Jonckheere-Terpstra test (Lehmann and DAbrera, 1975). The significance was set at p
0.05.
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RESULTS |
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DISCUSSION |
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Immune suppression occurred in mice with no obvious clinical signs of systemic toxicity. The only organ toxicity observed was an increase in lung weight. This increased lung weight is consistent with other studies in which pulmonary inflammation and edema have been demonstrated in humans and animals following respiratory exposure to DEP (Ichinose et al., 1995; Nightingale et al., 2000
). The mice exposed to the high dose of DEP (15 mg/kg) for 4 weeks had a decrease in platelet counts, although most of these values were still within the normal range for mice (8001100 x 103/mm3, Harkness and Wagner 1995
). These findings, however, are contrary to the published data for humans. An increase in platelet counts has been shown following exposure of human volunteers to DEP (Salvi et al., 1999
) and an increase in airborne particles has been associated with increased platelet counts in the U.S. population (Schwartz, 2001
).
Up to a 40% reduction in the IgM AFC response was observed in mice exposed to DEP for 2 weeks, compared to the saline controls. Earlier published studies, where rats and mice were chronically exposed to DEP by inhalation for 6 to 24 months, showed no significant differences in the IgM AFC response in test animals, compared to the air-exposed controls (Bice et al., 1985; Mentnech et al., 1984
). These discrepancies may be due to the differences in animal age, the methods of exposure employed in the studies (intratracheal aspiration vs. inhalation), and/or the length of exposures. Intratracheal instillation as an alternative to inhalation exposure has several advantages, including introduction of measured amounts of toxins into the lungs in a short period of time. However, the disposition, clearance, retention, and bioavailability of particles are different when administered by instillation compared to inhalation (reviewed in Brain et al., 1976
; Driscoll et al., 2000
). In addition, using inhalation, animals may be exposed to not only diesel particles, but also diesel fumes, which are a mixture of particulate matter and gaseous components, including carbon monoxide, nitric oxides, sulfur dioxide, aldehydes, hydrocarbons, etc.
Length of exposure has also been shown to modulate the immunotoxicity of chemicals due to enzyme induction and metabolism (Munson et al., 1991). DEP consists of hundreds of different organic compounds, including various polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs (IARC, 1989
). The immunotoxicity of PAHs has been related to chemical metabolic activation (Davila et al., 1995
; Diaz-Sanchez, 1997
). PAHs are metabolized by cytochrome P450 to form intermediates, such as epoxides, quinones, and phenols. These active metabolites are believed to be responsible for PAH-induced toxicity. On the other hand, these intermediates may then conjugate with glutathione (GSH) to form less reactive, water-soluble conjugates. The balance of bioactivation and detoxification is a key determinant of the toxicity of PAHs. Exposure of animals to DEP has been shown to increase the activity of CYP1A1 and CYP1B1, the P450s specifically involved in PAH metabolism (Bonvallot et al., 2001
; Hatanaka et al., 2001
) as well as increase GSH in lungs (Henderson et al., 1988
). The mutagenicity potential of DEP has been shown to decrease in the presence of S9 mixture (Hughes et al., 1997
). It is possible that the time-dependent process of PAH bioactivation and detoxification plays a role in the immune suppressive response of mice to DEP for 2 or 4 weeks.
The AFC assay is a sensitive indicator of the hosts ability to mount an antibody response to a specific antigen. When the T-dependent antigen sRBC is used, this response requires the coordinated interaction of antigen-presenting cells, T cells, and B cells. Any alteration in antigen processing and presentation by macrophages and/or dendritic cells, cytokine synthesis and release by T cells, or proliferation and/or differentiation of T cells and B cells could affect the final outcome (Luster et al., 1988). There is a vast amount of evidence demonstrating an increase in infectious disease in immune-compromised individuals. Luster et al. (1993)
demonstrated a high percentage of concordance between a reduction in the AFC response and decreased host resistance in mice. Although the cell(s) responsible for the decreased IgM AFC response following exposure to DEP is not yet known, several lines of evidence suggest that the B cells are not targeted. In this study, the number of spleen B cells was not significantly changed following DEP exposure. In contrast to the decreased IgM response observed in this study, the levels of IgE and IgG1 in response to ragweed or ovalbumin have been shown to increase following exposure to DEP (Heo et al., 2001
; Suzuki et al., 1996
). The adjuvant effect of DEP on enhancing B-cell function, such as IgE production, has been well documented in other studies (Muranaka et al., 1986
; Miyabara et al., 1998
; Takenaka et al., 1995
). On the other hand, T cells may be of importance in DEP-induced immunosuppression. In this study, the numbers of total, CD4+, and CD8+ T cells in spleens were reduced following DEP exposure. The proliferative response of splenocytes to the T-cell mitogen, ConA, was decreased dose-dependently along with the suppressed production of IL-2 and IFN-
. This decreased production of Th1 cytokines by DEP has been observed by other investigators (Diaz-Sanchez et al., 1997
; Fujimaki et al., 2001
; van Zijverden et al., 2000
). In addition, it has been demonstrated that DEP exposure decreased the pulmonary clearance of Listeria (Yang et al., 2001
), and the activation of T cells is essential in eliminating this intracellular pathogen. Therefore, the current evidence supports the concept that exposure to DEP can affect T-cell function, and this contributes to the suppressed systemic IgM response.
Macrophages may also play a role in DEP-induced immunosuppression. In contrast to the decreased proliferation of T cells in response to ConA, the splenocytes from DEP-exposed mice retained the same ability to proliferate in response to plate-bound anti-CD3 mAb. One of the differences in the mechanisms by which ConA and plate-bound anti-CD3 mAb activates T cells is the requirement for the participation of accessory cells. In the absence of accessory cells, ConA alone is not sufficient for T-cell proliferation (Ahmann et al., 1978; Chatila et al., 1987
); while the presence of accessory cells is not essential for T-cell proliferation stimulated by plate-bound anti-CD3 mAb (Jenkins et al., 1990
). DEP may impair antigen presenting-cell function and thereby indirectly render a decreased function of T cells. Although there was no direct evidence from this study, others have shown that macrophage function is decreased following DEP exposure, e.g., decreased macrophage phagocytosis (Castranova et al., 1985
; Jakab et al., 1990
) and suppressed responsiveness of alveolar macrophages to LPS and Listeria monocytogenes stimulation (Yang et al., 1997
, 2001
).
In contrast to the reported augmentation of the IgE response by DEP, the results of these studies have demonstrated that respiratory exposure of mice to DEP decreased the splenic IgM AFC response to the T-cell antigen, sRBC. This suppression was less severe following longer exposure, suggesting a role of enzyme induction and metabolism of chemicals attached to particles. Based on the results of these studies and previous reports, T cells and possibly macrophages are the likely cellular targets in this DEP-induced immunosuppression.
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NOTES |
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