* School of Pharmacy, West Virginia University, Morgantown, West Virginia 26506;
Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505;
Mechanical and Aerospace Engineering Department, West Virginia University, Morgantown, West Virginia 26506; and
School of Medicine, West Virginia University, Morgantown, West Virginia 26506
Received September 11, 2003; accepted October 31, 2003
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ABSTRACT |
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Key Words: diesel exhaust particles; inhalation; Listeria monocytogenes; alveolar macrophages; lymphocytes; cytokines.
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INTRODUCTION |
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Indeed, with diameters <2 µm, these particles can remain airborne for long periods of time and get deposited in great numbers deeply in the lungs. The air concentration of DEP nationwide is relatively low (~25 µg/m3), but in certain urban areas, the air level of DEP can be considerable higher. In the Los Angeles Basin, one estimate has placed the rate of DEP intake by humans at 300 µg/13 day(s) (Diaz-Sanchez, 1997). Also, exposure of truckers, railroad and construction workers, and engine mechanics to DEP is an occupational health concern. A report from the Department of Labor showed that the worst-case mean exposures to DEP in underground metal and nonmetal mines are about 2000 µg/m3, with maximum measurements as high as 3650 µg/m3 (Department of Labor, Mine Safety, and Health Administration, 1998
). To date, DEP exposure has been shown to induce pulmonary inflammation (Nagai et al., 1996
), increased susceptibility to bacterial infection (Yin et al., 2002
, 2003
), allergic asthma (Al-Humadi et al., 2002
; Takano et al., 1997
), pulmonary fibrosis, and lung cancer (Mauderly et al., 1994
) in experimental animals. Because DEP are a major component of particulate air pollution in most industrialized urban areas, their effect on pulmonary infections is of great environmental and occupational concern.
Increasing evidence shows that DEP may exert a strong effect on the pulmonary immune system. In allergic asthma, DEP exposure was shown to skew the immune response toward immunoglobulin E production and augment allergen-induced airway inflammation and eosinophil infiltration (Al-Humadi et al., 2002; Nel et al., 1998
, 2001
). While DEP enhance the T helper (Th) 2 responses to allergic sensitization, the same particles have been shown to suppress the host defense mechanism against bacterial infection, which may involve a downregulation of the CD4+ Th1 and CD8+ immunity (Yin et al., 2002
). The DEP-altered immune responses can be readily seen through changes in cytokine production by alveolar macrophages (AM) and lymphocytes. Under allergic sensitization, such as by ovalbumin, the adjuvant effect of DEP on interleukin- (IL-) 4 and IL-5 production by lymphocytes has been demonstrated (Steerenberg et al., 2003
; Takano et al., 1997
). On the other hand, DEP, through the organic components, have been shown to inhibit bacteria- or lypopolysacchraide- (LPS-) mediated secretion of tumor necrosis factor-
(TNF-
), IL-1ß, and IL-12 (Yang et al., 1997
, 1999
; Yin et al., 2002
, 2003
) by AM. IL-12 is rapidly produced by AM in response to bacterial infection and is known to play a key role in initiating and maintaining a Th1 response to clear the bacteria (Trinchieri, 1995
, 1998
). The suppression of production of IL-12 and other cytokines by AM exposed to DEP weakens the host defense and may lead to diminished development of bacteria-specific lymphocytes that secret interferon-
(IFN-
).
Although exposure of rats to DEP resulted in particle distribution in the alveolar region as well as in the lung-draining lymph nodes (LDLN) through particle translocation in the local lymphoid system (Chan et al., 1981; Yu and Yoon, 1991
), the in vivo effect of DEP exposure on the T lymphocytemediated immune responses against bacterial infection has not been clearly demonstrated. Studies from our laboratory have shown that in Brown Norway rats exposed to 100 mg/m3 DEP for 4 h and then to Listeria monocytogenes (Listeria), DEP strongly aggravated Listeria infection at 3 days postinfection, resulting in a 10-fold increase in bacterial count when compared to the air-exposed, Listeria-infected rats. But, at 7 days postinfection, the DEP-exposed rats showed a strong T cellmediated immunity and were able to clear the bacteria as efficiently as the air-exposed rats (Yin et al., 2002
, 2003
). This is despite the fact that DEP exhibit a direct inhibitory effect on lymphocyte production of key cytokines including IL-2 and IFN-
in cell culture.
We hypothesized that the alteration of T cellmediated immunity depends not only on the pharmacological effect of DEP but also on a dynamic relationship between pulmonary responses and exposure conditions. It is possible that with intact host defense mechanism, a normal lung, can effectively respond to an acute toxic insult even when the exposure dose is high. Under this condition, the large increase in the number of bacteria may result in a strong development of bacteria-specific T cell responses to eliminate the bacteria, thus overcoming the inhibitory effect of DEP. Under repeated or chronic exposures at a lower dose, however, where DEP have a moderate effect on AM function, the inhibitory effect of DEP on the development of T lymphocytes may become a determining factor in regulating the pulmonary responses. For this reason, we have examined the effect of DEP on the pulmonary immune responses to Listeria infection using a repeated exposure protocol at 20 mg/m3 for 4 h/day for 5 days. Our objective was to show the inhibitory effects of DEP on AM and T lymphocytemediated immune responses, their correlation with the bacterial clearance and inflammatory lung injury, and the altered infection pattern via this exposure protocol in comparison to that of a previous study in which rats were exposed to DEP through a single exposure of 100 mg/m3 for 4 h (Yin et al., 2002, 2003
).
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MATERIALS AND METHODS |
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Animals.
Male Brown Norway rats [BN/CrlBR] weighing 200250 g were purchased from Charles River Laboratories (Wilmington, MA). They were housed in a clean-air and viral-free room with restricted access, given a conventional laboratory diet and tap water ad libitum, and allowed to acclimate for 1 week before use in an animal facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care. During the week before inhalation exposure, the animals were conditioned to the exposure unit. Animals were placed in the tubes of the exposure unit for increasing time periods from 14 h/day for 4 successive days.
Inhalation exposure of rats to DEP.
The inhalation exposure system and DEP exposure procedure used in this study have been previously described and characterized (Yin et al., 2002, 2003
). Briefly, rats were exposed to either filtered air or DEP (20.62 ± 1.31 mg/m3) for 4 h/day for 5 consecutive days using a nose-only directed flow exposure unit (CH Technologies, Inc., Westwood, NJ). DEP concentrations in the exposure unit were monitored by both gravimetric sampling of dust collected on a polycarbonate membrane filter (37 mm, 0.45 µm, Poretics Corporation, Livermore, CA) at a sampling rate of 1 l/min and a Grimm Model 1.108 portable dust monitor (GRIMM Technologies, Inc., Douglasville, GA), allowing simultaneous measurements of the particle concentration in the exposure unit in real time. The estimated mean lung deposit of DEP for the inhalation exposure, according to the calculation of Leong et al. (1998)
, was 389 ± 25 µg/rat.
Listeria culture and intratracheal instillation.
Listeria was cultured overnight in brain heart infusion broth (BHI, Difco Laboratories, Detroit, MI) at 37°C in a shaking incubator. Diluted solution of the Listeria culture was further cultured for 3 h to achieve log growth. Following incubation, the bacterial concentration was determined spectrophotometrically at 600 nm. For the preparation of heat-killed Listeria monocytogenes (HKLM), the bacteria were incubated at 80° C for 1 h, washed, and resuspended in sterile PBS. An aliquot of the HKLM was plated overnight on BHI plates to ensure that there were no viable bacteria. For animal infection, the culture was diluted with sterile saline to the desired concentration; 2 h after the last DEP exposure, rats were lightly anesthetized with methohexital sodium (25 mg/kg body weight, ip; Eli Lilly Co., Indianapolis, IN) and inoculated intratracheally with 100,000 colony-forming units (CFU) of Listeria in 500 µl of sterile saline or 500 µl of the vehicle alone, as described previously (Antonini et al., 2000). To ensure that the number of Listeria given to the rats was suitable, the bacterial sample used for animal infection was diluted and plated on BHI plates and the colonies were counted after being cultured overnight at 37°C. According to the combination of inhalation exposure and bacterial instillation, there were four different treatment groups in this study, namely, air + saline, DEP + saline, air + Listeria, and DEP + Listeria.
Bronchoalveolar lavage (BAL) and biochemical assays.
At 3, 7, and 10 days after bacterial inoculation, rats were deeply anesthetized with an overdose of sodium pentobarbital (50 mg/kg, ip; Butler, Columbus, OH) and euthanized by exsanguinations through the abdominal aorta. The lungs were lavaged with Ca2+/Mg2+-free, phosphate-buffered saline (PBS, pH 7.4) at a volume of 6 ml for the first lavage and 8 ml for the subsequent lavages until a total of 80 ml of BAL fluid was collected. The BAL fluid samples were centrifuged at 500 x g for 10 min at 4°C, and the cell-free supernatant from the first lavage was analyzed for various biochemical parameters. The cell pellets from all washes for each rat were combined, washed, and resuspended in 1 ml PBS. The numbers of AM and neutrophils in the BAL cell suspension were determined according to their unique cell diameters using an electronic cell counter equipped with a cell-sizing unit (Coulter Electronics, Hialeah, FL).
Albumin content, a measure to quantify increased permeability of the bronchoalveolar-capillary barrier, and lactate dehydrogenase (LDH) activity, an indicator of general cytotoxicity, were determined in the acellular BAL fluid from the first lavage. Measurements were performed with a COBAS MIRA auto-analyzer (Roche Diagnostic Systems, Montclair, NJ). Albumin content was determined colorimetrically at 628 nm based on albumin binding to bromcresol green using an albumin BCG diagnostic kit (Sigma Chemical Co., St. Louis, MO). LDH activity was determined by measuring the oxidation of lactate to pyruvate coupled with the formation of a reduced form of nicotinamide adenine dinucleotide at 340 nm using the Roche Diagnostic reagents and procedures (Roche Diagnostic Systems).
Isolation of lymphocytes.
All LDLN from each rat were collected and a single cell suspension was prepared as described previously (Yin et al., 2003). The cells were washed twice with PBS and lymphocytes were isolated by Histopaque (density, 1.083; Sigma Chemical Co.) gradient centrifugation. Briefly, the samples were centrifuged for 30 min at 2500 rpm and lymphocytes were collected, washed twice, and resuspended in 1 ml PBS. The number of lymphocytes was counted by a standard hemocytometer and the cell viability was assessed by the trypan blue dye exclusion technique. The cell samples thus prepared showed both the lymphocyte content and viability of greater than 98%.
Differential counts of T cell subsets.
The numbers of CD4+ and CD8+ T cell subsets in lymphocytes recovered at 7 and 10 days postexposure were determined by flow cytometry, as described previously (Yin et al., 2003). Lymphocytes were stained with the addition of FITC-labeled CD4+ or CD8+ monoclonal antibody (mAb, BD Pharmingen, San Diego, CA) for 30 min on ice in the dark. The flow cytometric data were collected with a Becton-Dickinson FACScan using FACScan Research software (version B; Becton-Dickinson Immunocytometry System, San Jose, CA), and analyzed using the PC-LYSYS (v 1.0) software (Becton-Dickinson). The absolute numbers of cells in each lymphocyte subpopulation were calculated by multiplying the total number of cells by the percentage of the total within each phenotype, as determined by flow cytometry.
Pulmonary clearance of Listeria.
The colony forming units (CFU), an index of viable bacteria per lung in all Listeria-infected rats, were determined as described previously (Yin et al., 2002). The lungs were removed from all Listeria-infected rats following BAL and homogenized in sterile water. The tissue homogenates or their dilutions were quantitatively plated in triplicate on brain heart infusion agar plates using an Autoplate 4000 spiral plater (Spriral Biotech, Inc., Norwood, MA). After incubation at 37°C overnight, the CFU in each plate were counted using a scanner. The counts were averaged and corrected for dilution to yield the CFU/ml by a computer-based program (CIA-BEN V2.2; Spiral Biotech, Inc., Norwood, MA), through which the CFU per lung from each treatment group were determined.
Cell culture and cytokine determination.
The BAL cells and lymphocytes were suspended in RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) containing 2 mM glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, and 10% heat-inactivated fetal bovine serum. Aliquots of 1 ml cell suspensions, adjusted to 2 x 106 AM or lymphocytes, were added to each well of a 24-well tissue culture plate. Before the stimulation, the BAL cells were incubated in a humidified incubator (37°C and 5% CO2) for 2 h to allow cell attachment to the culture plate. The nonadherent BAL cells were then removed by rinsing the monolayer three times with culture medium. The remaining AM-enriched cells or lymphocytes were then treated with either LPS (1 µg/ml, Sigma Chemical Co.), concanavalin A (ConA, 2 µg/ml, Sigma Chemical Co.), or HKLM (107/ml) for 24 or 48 h. The AM- and lymphocyte-conditioned media were collected, centrifuged (1200 x g for 4 min), and aliquots of the supernatants were stored at -70°C until assayed.
The amounts of TNF-, IL-1ß, and IL-10 produced by AM and IL-2, IL-6, IL-10, and IFN-
produced by lymphocytes in cell cultures under various exposure conditions were quantified by the enzyme-linked immunosorbent assay (ELISA) using the OptEIA ELISA sets according to the manufacturers instructions (BD Pharmingen). Briefly, 96-well ELISA plate (Corning, Corning, NY) was coated with a purified antirat mAb and blocked with an assay diluent (BD PharMingen) before use. Recombinant standards (BD Pharmingen) and samples were added to the plate and incubated for 2 h at room temperature. The plate was then incubated with biotinylated mAb for 1 h and avidinhorseradish peroxidase conjugate for 30 min at room temperature. The plate was developed with tetramethylbenzidine with 50% H2O2 in the dark, and color reaction was stopped with 2 N H2SO4 and then analyzed at 450 nm with a SpectraMax 250 plate spectrophotometer using Softmax Pro 2.6 software (Molecular Devices Co., Sunnyvale, CA). The levels of IL-12 in the culture media were quantified by ELISA using a commercial ELISA kit (BioSource International, Inc., Camarillo, CA). The range of detection was: 31.32000 pg/ml for IL-1ß, IL-2, IL-6, and IFN-
, 15.61000 pg/ml for IL-10 and TNF-
, and 7.8500 pg/ml for IL-12.
Statistical analysis.
The experimental results are expressed as means ± standard error (SE) of multiple measurements. Statistical analyses were carried out with the JMP IN statistical program (SAS Institute, Inc., Cary, NC). Values were expressed as means ± SE. The significance of the interaction among the different treatment groups for the different parameters at each time point was assessed using an analysis of variance (ANOVA). The significance of difference between individual groups was analyzed using the Tukey-Kramers Honestly Significant Difference (HSD) test. For all analyses, the criterion of significance was set at p < 0.05.
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RESULTS |
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DISCUSSION |
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The present study was carried out to elucidate how DEP may affect the development of T cellmediated immunity under an exposure condition that would not lead to a strong acute pulmonary inflammatory response. Brown Norway rats were exposed to DEP at 20.62 ± 1.31 mg/m3 for 4 h/day for 5 days. This exposure protocol gave a total DEP inhalation equivalent to that of a single dose exposure at 100 mg/m3 for 4 h but induced no inflammatory lung injury in noninfected rats, as indicated by the recovered AM, neutrophils, LDH activity, and the albumin content in the BAL fluid. The dose may appear to be high in comparison to the reported environmental and occupational concentrations, but it results in a lung deposit that is relevant to both nonoccupational and occupational exposure settings, as discussed previously (Yin et al., 2002). Based on the reported values of minute ventilation (0.16 l/min) and percentage of deposition (~10%) for rats (Leong et al., 1998
), the estimated lung deposit of DEP for the current exposure is 389 µg, which is equivalent to a deposit of about 97,000 µg in the human lungs according to the ratio of lung surface area of humans and rats (about 1:250). Although the latter seems to be a large amount, it is reachable through chronic exposure to low doses. For example, using a percentage of deposition of 25% for humans, a resident of the Los Angeles Basin area can arrive at a daily intake of 75 µg and an accumulative value of 97,000 µg in 3.5 years, according to one estimate that has placed the rate of DEP intake by humans there at 300 µg/13 day(s) (Diaz-Sanchez, 1997
). This suggests that even at a considerable rate of pulmonary clearance, it is still possible that, in urban areas where a high concentration of DEP is found, there is a significant accumulation of DEP in the lungs of long-time residents. In occupational settings such as in certain underground mining sites, the air DEP concentration has reached as high as 3.65 mg/m3 (Department of Labor, Mine Safety, and Health Administration, 1998
). Even at 1 mg/m3, the daily deposit of DEP would be 2400 µg (percentage of deposit, 25%; minute ventilation, 20 l/min). At this rate, the accumulated lung deposit of DEP would reach the value of 97,000 µg in 40 working days. These calculations demonstrate that the lung deposits of DEP from doses used in the current study are within the potential concentration range for both nonoccupational and occupational settings.
Brown Norway rats were used because of their applicability to investigations involving immunological reactions such as pulmonary allergic sensitization. These rats exhibited very high resistance to Listeria infection and survived at initial inoculation doses as high as 600,000 CFU of Listeria/rat (data not shown). In the present study, rats exposed to DEP or clean air for 5 days were inoculated with 100,000 CFU of bacteria and maintained for up to 10 days. All rats, including those exposed to DEP and showed elevated lung burden of bacteria (42.96 x 106 CFU of Listeria/lung) at 3 days postinfection, survived without marked symptoms during the entire experimental period. The repeated exposure protocol resulted in a significant retardation of Listeria clearance up to 7 days postinfection. In comparison, rats exposed to 100 mg/m3 for 4 h showed strongly aggravated infection at day 3, but the bacteria count returned to control level at 7 days postinfection (Yin et al., 2002). This indicates the dynamic nature of pulmonary responses with respect to the severity of the toxic stimulation and the potential of a delayed effect by the accumulation of inhaled DEP on the pulmonary immune system. For this reason, we have examined the effect of repeated DEP exposure on AM production of cytokines and the development of T cellmediated immune responses against Listeria infection.
Numerous AM-derived cytokines are known to be necessary for the generation of a protective immune response against Listeria (Bancroft et al., 1989; Czuprynski et al., 1992
). Both IL-1ß and TNF-
activate NK cells to release IFN-
, which activates macrophages to kill the bacteria. These cytokines are also T cell activators (Akira et al., 1990
; Hsieh et al., 1993
).
IL-10, on the other hand, is a potent immunosuppressive factor that downregulates macrophage bactericidal activity (Fleming et al., 1999). The effect of DEP exposure on the production of IL-10 by AM is of interest because some intracellular pathogens, including Listeria, specifically target macrophages for infection and use IL-10 to dampen the host immune response and, thus, prolong their survival (Redpath et al., 2001
). Also, IL-12 has been shown to play a key role in the initiation of T cellmediated immunity (Trinchieri, 1995
, 1998
). This cytokine is produced rapidly by AM following infection to initiate the development of Th1 responses (Park and Scott, 2001
). Our study shows that the repeated DEP exposure resulted in a diminished ability of Listeria-infected AM to secret IL-1ß, TNF-
, and IL-12 up to 7 days postinfection. These results are consistent with those obtained from a single dose exposure at 100 mg/m3 (Yin et al., 2002
). Furthermore, we showed that DEP strongly augmented Listeria-induced IL-10 production by AM up to 10 days postinfection. This clearly indicates a prolonged effect of the inhaled DEP on macrophage function and suggests that the inhibition of the innate immune responses is responsible for the impairment of early bacterial clearance.
Although the innate immunity is efficient in limiting the initial spread of infection, sterilization of Listeria infection depends on the later development of acquired T cell responses involving CD4+ Th1 and CD8+ cells, (Kaufmann, 1993; Unanue, 1997
; Shen et al., 1998
). In this aspect, the results of our study demonstrated a striking difference between the effects of repeated DEP exposure at a lower dose and the single high-dose exposure (with the same total lung burden of DEP) on the development of T cellmediated immune responses. Under repeated low-dose exposure, DEP was found to suppress the development of bacteria-induced T lymphocytes in LDLN at 7 and 10 days postinfection (Fig. 3
) and markedly inhibit the development of the CD8+ T cell subset that is closely associated with Listeria-induced T cell responses in the Brown Norway rats (Yin et al., 2003
). The effect of repeated DEP exposure on the pattern of cytokine secretion by lymphocytes also indicates a diminished T cellmediated immune responses. For up to 10 days postinfection, DEP exposure inhibited the Listeria-induced lymphocyte secretion of IL-2, which promotes T cell proliferation, and of IFN-
, a key cytokine in cell-mediated immunity for bacterial defense (Fig. 4
). Although lymphocytes from the combined DEP and Listeria exposure showed a moderate increase in IL-6 production, they were significantly less responsive to HKLM in the production of IL-10 (Fig. 5
). In rats exposed to 100 mg/m3, the DEP exposure augmented the development of T cellmediated immune responses (characterized by an elevation of the CD4+ and CD8+ cell counts and the CD8+/CD4+ ratio), increased IL-2 responsiveness, and increased lymphocyte production of IL-2 and IFN-
(Yin et al., 2003
).
The difference in pulmonary responses observed between the single and repeated exposure protocols may be attributed to the competing effect of Listeria induction of T cellmediated immunity and the inhibition of the innate immunity by DEP. Exposure to a high dose of DEP can strongly inhibit the early defense mechanism, resulting in severely aggravated infection. In this case, pulmonary response may be dominated by an increase in T cellmediated immunity. The Brown Norway rats appear to be very effective in mounting a CD8+ T cellmediated immunity against Listeria. Under repeated or chronic exposure to DEP at a low dose, the suppression of the initial immune responses can be moderate, and the development of T cellmediated immune responses will likely reflect the pharmacological effect of DEP. The mechanism through which DEP may alter production of cytokines by AM has not been reported. Studies have shown that the organic component of DEP was able to induce the generation of intracellular reactive oxygen species (ROS) in AM through, at least in part, increased expression of cytochrome P450 1A1 and the interaction of the organic compounds with the microsomal enzymes (Bonvallot et al., 2001; Takano et al., 2002
; Ma and Ma, 2002
). The ROS-mediated oxidative stress was, in turn, shown to induce an increased expression of heme oxygenase-1 (HO-1; Rensing et al., 1999
), a stress-responsive protein that has been shown to enhance cellular production of IL-10 but downregulate TNF-
(Inoue et al., 2001
; Tullius et al., 2002
). Preliminary studies from our laboratory showed that the DEP-mediated changes in the production of IL-12, TNF-
, and IL-10 by Listeria-infected AM were regulated through ROS generation and HO-1 activity, and the same results can be replicated in cells directly stimulated with superoxide anion.
It should be noted that despite the suppressive effect of DEP on the immune responses, substantial clearance of the bacteria from lung tissue occurred at 7 and 10 days postinfection. It is possible that the cell-mediated immunity to Listeria developed by the Brown Norway rats was strong and eventually overcame the suppressive effect of DEP. Listeria, as an intracellular pathogen, may also find a home in AM and lymphocytes by inducing cellular production of IL-10 to prolong their survival. As can be seen from Figures 2 and 5
, the Listeria infection had an inductive effect on the production of IL-10 by AM and lymphocytes up to 10 days postinfection. DEP exposure strongly enhanced the Listeria-induced IL-10 production by AM. Interestingly, the combined DEP and Listeria exposure yielded lymphocytes that secreted less IL-10 in response to HKLM than cells from Listeria-infected rats. The reason for this effect is not yet clear. One possibility is that the number or percentage of Listeria-specific T cells in lymphocytes from the combined exposure was much reduced compared to cells from rats exposed to Listeria only. Consequently, lymphocytes from the combined exposure are less responsive to HKLM. DEP moderately enhanced lymphocyte production of IL-6 at 3 and 7 days postexposure, which may have enhanced T cell proliferation. This cytokine is known to enhance IL-2 responsiveness and IL-2 secretion by lymphocytes (Akira et al., 1990
; Ford et al., 1991
).
In summary, this study demonstrates that DEP, following a repeated exposure protocol, suppressed the host defense against Listeria by downregulating the innate and bacteria-induced T lymphocyte responses in Brown Norway rats. The results on T cell development are strikingly different than those obtained from rats receiving a single high-dose exposure, suggesting that pulmonary responses to DEP may vary depending on the severity of exposure and that inhaled DEP and their accumulation may have a delayed effect on the pulmonary immune system.
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ACKNOWLEDGMENTS |
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NOTES |
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