* 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; and
School of Medicine, West Virginia University, Morgantown, West Virginia 26506
1 To whom correspondence should be addressed at School of Pharmacy, West Virginia University, 1 Medical Center Drive, Morgantown, WV 265069530. Fax: 304-293-2576. E-mail: jma{at}hsc.wvu.edu.
Received April 14, 2005; accepted July 12, 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: diesel exhaust particles; airway inflammation; airway hyperresponsiveness; glutathione; reactive oxygen species; nitric oxide.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have established an allergic asthma model using Brown Norway (BN) rats sensitized by aerosolized ovalbumin (OVA) administered once a week over a month-long period that produced significant levels of OVA-specific IgE and IgG (Al-Humadi et al., 2002). Exposure of rats to DEP (5 mg/kg) through intratracheal instillation prior to the OVA-sensitization enhanced production of antigen-specific antibodies at the end of allergen sensitization, and this enhancement correlated with an increase in interleukin (IL)-4 mRNA expression in lung tissue. Steerenberg and colleagues (1999
, 2003
) also reported the use of BN rats in elucidating the adjuvant effect of DEP on timothy grass pollen allergy after intranasal or intratracheal instillation of DEP in combination with the pollen allergen. In a recent study, we further showed that short-term exposure to DEP by inhalation (20 mg/m3, 4 h/day for 5 days) prior to OVA sensitization enhanced OVA-induced antibody production but attenuated the OVA-induced inflammatory responses that included inflammatory cell infiltration, lactate dehydrogenase (LDH) activity, albumin, and nitric oxide (NO) contents in bronchoalveolar lavage (BAL) fluid, the development of T lymphocytes and their CD4+ and CD8+ subsets in lung lymph nodes, and production of NO, IL-10, and IL-12 by alveolar macrophages (AM) (Dong et al., 2005
). The inhibition of OVA-induced airway inflammation by DEP at 30 days post-exposure contradicted the DEP effect reported previously, where DEP were administered to the already-sensitized animals or to those during the sensitization phase (Ichinose et al., 1997
, 2002
; Miyabara et al., 1998
; Takano et al., 1997
, 1998
). This suggests that there may be acute and delayed responses to DEP exposure that produce different effects on allergic immune/inflammatory reactions.
The aim of the present study was to examine the effects of DEP inhalation just prior to the last dose of OVA exposure on allergen-mediated immune responses in the BN rat model. We have shown previously that the organic component of DEP induces cellular generation of reactive oxygen species (ROS) that leads to an antioxidative response and a switch from T helper (Th)1 to Th2 immunity (Yin et al., 2004b). This would enhance the allergic responses in antibody production. Nitric oxide produced from lung cells, such as AM and epithelial cells, on the other hand, has been shown to induce eosinophil-mediated airway inflammation (Liu et al., 1997
). Both the reactive oxygen and nitrogen intermediates may deplete intracellular glutathione (GSH) and cause a change in the redox state of GSH, which has been shown to regulate cellular production of Th1/Th2 cytokines and T cell development (Murata et al., 2002
). To gain more insight into the underlying mechanism(s) through which DEP alter the asthmatic immune responses, the potential involvement of ROS, NO, and GSH depletion in relation to DEP, OVA, and the combined DEP and OVA exposures was investigated.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
OVA immunization.
A solution of OVA (Grade V, Sigma Chemical Co., St. Louis, MO) in endotoxin-free saline (1%) was aerosolized using a DeVilbiss-646 nebulizer (DeVilbiss, Somerset, PA). To achieve the desired concentration, filtered air was passed through the nebulizer and used as a diluent for the aerosolized OVA. The concentration of OVA in the chamber was determined by collecting samples onto 0.4 µm filters (Polycarbonate Membrane, Poretics Corporation, Livermore, CA) from a chamber side port at a rate of 1 l/min. Filters were washed with 10 ml of endotoxin-free saline and analyzed for protein using the Coomassie blue dye reagent (Bio-Rad Laboratories, Hercules, CA). Rats were sensitized to OVA at an average chamber concentration of 42.3 ± 5.7 mg/m3 for 30 min on days 1, 8, and 15, and challenged with OVA on day 29. Non-sensitized animals were exposed to aerosolized endotoxin-free saline following the same exposure schedule.
DEP exposure.
A standardized DEP sample (standard reference material 2975), representing heavy-duty diesel engine with a mass median aerodynamic diameter of 0.5 µm, was purchased from the National Institute of Standards and Technology (Gaithersburg, MD). Diesel exhaust particles were suspended in endotoxin-free sterile saline (Baxter Healthcare Corporation, Deerfield, IL), followed by sonication for 2 min in an ultrasonic processor with a micro tip (Heat System-Ultrasonics, Plainview, NY) prior to use. The DEP inhalation exposure system used in this study has been described and characterized elsewhere (Yin et al., 2002, 2004a
). Rats were exposed to either filtered air or DEP (22.7 ± 2.5 mg/m3), 4 h/day for 5 consecutive days, on days 2428, 24 h prior to the last (challenge) dose of OVA, using a nose-only directed flow exposure unit (CH Technologies, Inc., Westwood, NJ). The DEP concentration in the exposure unit was 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 with a Grimm Model 1.108 portable dust monitor (GRIMM Technologies, Inc., Douglasville, GA), which allows continuous measurement of the particle concentration in the exposure unit in real time. The estimated mean lung deposition of DEP for the above-described inhalation exposure, calculated based on the method of Leong et al. (1998)
, was 402 ± 58 µg/rat.
The exposure groups (5 rats/group) for the present study were: non-sensitized/air-exposed/saline challenged (saline + air); non-sensitized/DEP-exposed/saline challenged (saline + DEP); OVA-sensitized/air-exposed/OVA challenged (OVA + air); and OVA-sensitized/DEP-exposed/OVA challenged (OVA + DEP). The animals were subjected to whole-body plethysmography on day 30, 24 h after the last OVA dose, and they were sacrificed on day 31 for biochemical and cellular measurements. All parameters were measured after saline/OVA challenge (the last dose) with and without DEP exposure.
Measurement of Airway Responsiveness
Airway responsiveness was assessed by inducing airflow obstruction with a methacholine (MCh) aerosol using a noninvasive method (Hamelmann et al., 1997). Minute volume, tidal volume, breathing frequency, and enhanced pause (Penh) were obtained from conscious rats placed in a whole-body plethysmograph (Buxco Electronics Inc., Troy, NY). In this system, rats were unrestrained and tolerated repetitive measurements. Measurements of MCh responsiveness were obtained by exposing rats for 3 min to aerosolized PBS and incremental doses (6.2525 mg/ml) of aerosolized MCh (Sigma) in PBS, and monitoring the breathing pattern for 3 min after each MCh challenge. The Penh values measured during each 3-min sequence were averaged and expressed, for each MCh concentration, as a percentage of baseline Penh values observed after PBS exposure.
Bronchoalveolar Lavage (BAL) and Determination of BAL Markers
BAL.
Rats were deeply anesthetized with an overdose of sodium pentobarbital (200 mg/kg, ip; Butler, Columbus, OH) and euthanized by exsanguination via the vena cava. After clamping off the right apical lobe, the remaining lung lobes were first lavaged with 6 ml Ca2+/Mg2+-free phosphate-buffered solution (PBS, 145 mM NaCl, 5 mM KCl, 1.9 mM NaH2PO4, 9.35 mM Na2HPO4, and 5.5 mM glucose; pH 7.4). The first BAL fluid sample was centrifuged at 500 x g for 10 min at 4°C, and the resultant cell-free supernatant (4 ml/rat) was analyzed for various biochemical parameters. The lungs were further lavaged with 6 ml aliquots of PBS until 80 ml of BAL fluid was collected. These samples were also centrifuged for 10 min at 500 x g and the cell-free BAL fluid discarded. The cell pellets from all washes for each rat were combined, washed, and resuspended in PBS and evaluated as described below.
BAL cell differentiation.
The BAL cell were numerated using a Coulter Multisizer II and AccuComp software (Coulter Electronics, Hialeah, FL). Cell suspensions (5 x 104 cells) were centrifuged for 5 min at 800 rpm and pelleted onto a slide using a Cytospin centrifuge (Shandon Life Sciences International, Cheshire, England). Three hundred cells per rat were identified and differentiated after labeling with Leukostat stain (Fisher Scientific, Pittsburgh, PA). The absolute numbers of cells differentiated were calculated by multiplying the total number of cells by the percentage of the total within each cell type.
Albumin and LDH.
The albumin content, which indicates injury to the bronchoalveolar-capillary barrier, and LDH activity, which indicates cytotoxicity, were determined in the first fraction of acellular BAL fluid, using 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). Lactate dehydrogenase activity was determined by measuring the oxidation of lactate to pyruvate coupled with the formation of reduced form of nicotinamide adenine dinucleotide at 340 nm using the Roche Diagnostic reagents and procedures (Roche Diagnostic Systems, Indianapolis, IN).
Chemiluminescence (CL).
The light generation as CL by resting or stimulated AM as a result of ROS production was determined in a total volume of 0.5 ml HEPES buffer. Resting CL was determined by incubating BAL cells containing 0.5 x 106 AM at 37°C for 10 min in 0.008 % (w/v) luminol (Sigma) followed by the measurement of CL for 15 min. Luminol was used as an amplifier to enhance detection of the light and was first dissolved in a small amount of ethanol before being brought up to its final concentration in HEPES buffer. To determine zymosan-stimulated CL, unopsonized zymosan (2 mg/ml, Sigma) was added immediately prior to the measurement of CL. Measurement of CL was performed with an automated Berthold Autolumat LB 953 luminometer (Wallace, Inc., Gaithersburg, MD) for 15 min, and the integral of counts versus time was calculated. Zymosan-stimulated CL was calculated as the total counts of stimulated cells minus the total counts of the corresponding resting cells. The zymosan-stimulated CL was attributed to AM only, as rat neutrophils do not respond to unopsonized zymosan in this system.
NO production.
The production of NO by AM was determined as follows. Cells were suspended in Eagle's minimum essential medium (MEM, Biowhittaker, Walkersville, MD) supplemented with 1 mM glutamine, 100 µg/ml streptomycin, 100 units/ml penicillin, and 10% heat-inactivated fetal bovine serum (FBS). Aliquots of 1 ml cell suspension containing 2 x 106 AM were incubated in a humidified incubator (37°C and 5% CO2) for 2 h to allow cell attachment to the culture plate. The non-adherent BAL cells were removed by rinsing the monolayer three times with culture medium. The remaining AM-enriched cells were incubated in 1 ml medium for 24 h at 37°C and 5% CO2. The level of nitrite produced from NO in the AM-conditioned media was measured colorimetrically with the Greiss reaction using sodium nitrite as a standard (Green et al., 1982). The levels of NO in the first fraction of acellular BAL fluids were also determined using the Greiss assay.
Western blot analysis.
The recovered AM-enriched cells were washed with PBS and then suspended in 100 µl of a lysis buffer (50 mM Tris-HCl, 1% NP-40, 2 mM EDTA, 100 mM NaCl, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride; pH 7.5) and left on ice for 10 min. Cytoplasmic extracts were separated from the nuclei by centrifugation at 14,000 rpm for 10 min at 4°C and determined for protein content using a BCA Protein Assay Kit (Pierce, Rockford, IL). An equal amount of protein (30 µg/well) for each sample was boiled for 5 min, loaded, and run for electrophoresis in a 420% Tris-Glycine gel (Invitrogen, Carlsbad, CA) at 125 V. The gel was transferred electrophoretically (Bio-Rad Laboratories, Hercules, CA) to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and the blots were blocked with 5% milk in TBST buffer (20 mM Tris-HCl, 100 mM NaCl, 0.1% Tween 20; pH 7.5) for 1 h at room temperature. Membranes were then probed with a polyclonal rabbit antibody against inducible NO synthase (iNOS) and a horseradish peroxidaseconjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were developed using commercially developed enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ). For quantification, bands in photographs were scanned by a densitometer linked to a computer system (Personal Densitometer SI, Amersham Biosciences, Piscataway, NJ).
Analysis of Alveolar Type II (ATII) Cells
Isolation of ATII cells.
Type II cells were isolated using a standard protocol (Dobbs et al., 1986). Briefly, after removal of AM and blood cells, elastase solution (MP Biomedicals, Inc., Irvine, CA) was instilled via the trachea to dissociate the cells from lung tissue. The lung tissue was then minced in the presence of DNase I (Sigma) and FBS, and the suspension was sequentially filtered through nylon mesh. The cell suspension was plated on bacteriological plastic dishes coated with rat IgG (Sigma). After 1 h at 37°C, the nonadherent ATII cells were removed from the plate to which AM and other immune cells were adherent. Cells obtained by this method contained
90% ATII cells and >90% excluded trypan blue.
Western blot analysis.
Expression of iNOS in ATII cells was determined by Western blot analysis using the same procedures for AM as described above, using cytoplasmic protein (30 µg/well) extracted from the freshly isolated ATII cells.
Flow cytometric analysis.
Freshly isolated ATII cells (5 x 105 cells) were washed with a washing buffer (PBS with 2% FBS and 0.02% NaN3, pH 7.4), and re-suspended in DMEM/F12 medium (Invitrogen, Carlsbad, CA) containing 10 µM of 4,5-diaminofluorescein diacetate (DAF, Sigma) or 5 µM dihydroethidium (DHE, Molecular Probes, Eugene, OR) at 37°C for 30 min. After washing, the flow cytometric data were immediately collected with a Becton-Dickinson FACScan using FACScan Research Software (Becton-Dickinson Immunocytometry System, San Jose, CA), and analyzed using the PC-LYSYS software (Becton-Dickinson).
Analysis of Lymphocytes from Lung-Draining Lymph Nodes (LDLN)
Isolation of lymphocytes.
Lymphocytes were isolated from LDLN as described previously (Yin et al., 2003). Briefly, LDLN were excised from each rat after BAL, teased apart, and homogenized with a glass pestle in a screen cup (Sigma). Single cell suspensions were obtained by passing the cell clumps through a 22-gauge needle attached to a 10-ml syringe, and washed twice with PBS. Lymphocytes were isolated by Histopaque (density 1.083; Sigma) gradient centrifugation. Samples were centrifuged for 30 min at 2500 rpm, and lymphocytes were collected, washed, re-suspended in 1 ml of PBS, and counted using a standard hemocytometer. The cell samples thus prepared showed predominance of lymphocytes and cell viability of greater than 98% as determined by the trypan blue exclusion technique.
Flow cytometric analysis.
The effects of OVA and/or DEP exposures on frequencies of T cell subsets in LDLN, i.e., their expressions of CD3, CD4, and CD8 cell surface markers, were examined with a flow cytometric method described elsewhere (Yin et al., 2003). Lymphocytes (106 cells) were stained with the addition of FITC-labeled conjugated antibodies against these cell surface markers (BD Pharmingen, San Diego, CA). The flow cytometric data were collected with a Becton-Dickinson FACScan using FACScan Research Software (Becton-Dickinson Immunocytometry System), and analyzed using the PC-LYSYS software (Becton-Dickinson).
Determination of Intracellular GSH
Alveolar macrophages or lymphocytes (2 x 105 cells) were plated in 96-well microplates, washed twice with PBS, and lysed with 240 µl of a cold lysing buffer (0.1% triton X-100 in 0.1 M sodium phosphate buffer, 5 mM EDTA, pH 7.5). The lysates were acidified with 0.1 N HCl (12 µl) and protein precipitated with 50% sulfosalicyclic acid (12 µl) followed by centrifugation at 4°C. Samples of the supernatants were assayed for total GSH according to the method of Buchmuller-Rouiller et al. (1995). Briefly, 50 µl of cell supernatants or GSH standards were distributed to each well of a 96-well microplate, followed by 50 µl of 2.4 mM 5,5'-dithio-bis(2-nitrobenzoic acid). After the mixture was incubated at room temperature for 10 min, 50 µl each of NADPH (0.667 mg/ml) and glutathione reductase (40 µg/ml) were added. The results of the GSH-specific reaction were monitored by OD readings at 405 nm every minute for 8 min with a Spectramax 250 plate spectrophotometer using Softmax Pro 2.6 software (Molecular Devices Corp., Sunnyvale, CA). One of the OD readings obtained with the most satisfied standard curve was selected as the final result.
Determination of OVA-Specific IgE and IgG
Blood samples were collected during exsanguination from vena cava of rats at sacrifice. The sera dilutions with 5% horse serum albumin (HOSA)/PBS of 1/50 were analyzed for OVA-specific IgE and IgG. Diluted sera (100 µl) were added to a 96-well plate (ICN Biomedicals, Horsham, PA) that had been previously coated with 200 µl of 1% OVA carbonate coating buffer and blocked with a 5% HOSA/coating buffer according to the method of Voller and Bidwell (1986). The plates were incubated overnight at 4°C and subsequently incubated with sheep anti-rat IgE (100 µl, 1:2500 dilution in HOSA/PBS, ICN Biomedicals, Costa Mesa, CA) which, according to manufacturer provided information, is specific for IgE class and does not cross-react with the other Ig classes, including IgG. The plates were then incubated with horseradish peroxidasebound donkey anti-sheep IgG (100 µl, 1:5000 dilution in HOSA/PBS, ICN Biomedicals) for 2 h each at room temperature. The plates were washed 3 times after each incubation, treated with tetramethylbenzidine (Sigma), and read at 630 nm. Ovalbumin-specific IgG was determined using goat anti-rat IgG (1:500 dilution in HOSA/PBS, Sigma) and peroxidase-labeled rabbit anti-goat IgG (1:12,500 dilution in HOSA/PBS, Sigma) as detection antibodies, following the same protocol described above. The serum from one animal exposed to OVA was assigned a value of 100 and used as a reference to obtain relative concentrations for the OVA-specific IgE and IgG in serum samples from each group.
Immunohistochemistry
Before BAL, the right apical lobe was clamped off to prevent entry of lavage fluid. Following BAL and excision of LDLN, the clamp was removed and all lobes were inflated intratracheally with 10% formalin. The right apical lobe was processed within 24 h and embedded in paraffin. Sections were cut at 5 µm, deparaffinized in xylene, rehydrated, and stained for iNOS expression (Porter et al., 2002). Briefly, microwave antigen retrieval with citrate buffer (pH 6.0) of rehydrated tissue was performed, followed by peroxidase blocking with a 1:1 mixture of 3% H2O2 and methanol. Slides were incubated overnight at 4°C with iNOS monoclonal antibody (N32020, Transduction Laboratories, Lexington, KY, 1:50 dilution). Localization was achieved using a streptavidin-biotin-peroxidase system for use on rat specimens (K0609, Dako, Carpinteria, CA), with diaminobenzidene (Zymed Laboratories, San Francisco, CA) as the chromogen. Tissues were counterstained with Mayer's hematoxylin, dehydrated and covered with a coverslip. Non-stained sections where the primary antibody was omitted were obtained as negative controls, and sections from rats that had been intratracheally instilled with lipopolysacchride (LPS, Sigma, 10 mg/kg) 24 h prior to sacrifice were stained for positive controls.
Statistical Analysis
Results are expressed as means ± standard error (SE). The significance of the interaction among different treatment groups for different parameters at each time point was assessed by analysis of variance (ANOVA). The significance of difference between individual groups was analyzed using the Tukey-Kramer's Honestly Significant Different Test. For all analyses, the criterion of significance was set at p < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanisms through which DEP alter the allergic reactions remain unclear in part because of the complex, time-dependent effects of DEP on allergen-induced immune responses. One of the cellular actions of DEP is the induction of intracellular ROS through the organic component-mediated activation of cytochrome P450 1A1 and other metabolic enzymes (Ma and Ma, 2002; Whitekus et al., 2002
; Yin et al., 2004b
). The imbalance of cellular antioxidative responses to ROS generation induced by DEP leads to oxidative stress and a reduction of total intracellular GSH in AM and lymphocytes (Al-Humadi et al., 2002
; Dong et al., 2005
). The present study shows that ROS generation in AM and ATII cells is an important feature of the combined DEP and OVA exposure. As shown by CL measurements, a marked increase in oxidant activity, which corresponds to a lowered total GSH concentration in AM and lymphocytes, is associated with the combined DEP and OVA exposure, which is greater than either DEP or OVA exposure alone. This suggests that DEP pre-exposure augments ROS production by cells from OVA-sensitized rats. GSH, in addition to its role in protecting cells from oxidative injury, is critical to macrophages and dendritic cells that act as antigen-presenting cells for the development of T-cellmediated immune responses. Depletion of GSH in these cells has been shown to skew the development of T cells from Th1 to Th2 type (Murata et al., 2002
; Peterson et al., 1998
), a manifestation of increased allergic asthmatic responses. That DEP augment OVA-specific antibody production is consistent with the fact that DEP facilitate depletion of GSH in AM and lymphocytes.
Nitric oxide has been considered an important marker in allergen-induced inflammatory responses. This reactive nitrogen intermediate can directly react with and deplete intracellular GSH (Folkes and Wardman, 2004) and may play a role in the development of eosinophilia and AHR in mouse and rat allergic models (Feder et al., 1997
; Liu et al., 1997
). It has been shown that NO derived from iNOS in epithelial cells promotes asthmatic inflammation by downregulating Th1 cells that secrete interferon-
and concomitantly upregulate Th2 cells that secrete IL-4 and IL-5 (Barnes and Liew, 1995
). On the other hand, the constitutive NO-synthase (cNOS)derived NO has been shown to exert bronchoprotective effects in asthma including airway smooth muscle relaxation and inhibition of smooth muscle proliferation (Ricciardolo et al., 2001
, 2003
). In fact, NO derived from cNOS and from iNOS may play different roles in the airways. The former seems to protect airways from excessive bronchoconstriction while the latter has a modulatory role in inflammatory disorders of the airways such as asthma. The effect of DEP pre-exposure, as shown in the present study, is to increase iNOS expression in AM and ATII cells of OVA-sensitized and challenged rats, suggesting that DEP interact directly with these lung cells. This interaction results in an acute response of increased production of NO that is known to mediate OVA-induced eosinophilic inflammation and AHR.
Our study shows an apparent linkage between the ROS and NO generation with increased responses of T lymphocytes. Both DEP and OVA exposure enhanced the numbers of T cells and their CD4+ and CD8+ subsets recovered from the LDLN in the BN rat model. But it was the combined DEP and OVA exposure that yielded a substantial increase in T cell responses, which correlate with increased production of ROS and NO and decreased level of GSH. Clinical investigations have observed that CD4+ T lymphocytes and their secretion of Th2 cytokines played a central role in initiating and sustaining asthmatic responses in the asthmatic airway (Robinson, 2000). Studies in animal models further showed that depletion of CD4+ T lymphocytes by administration of anti-CD4 antibody inhibited allergen-induced airway eosinophilia and AHR (Gavett et al., 1994
; Komai et al., 2003
). CD8+ T lymphocytes also play a role in allergic responses. Miyahara et al. (2004a
,b
) showed that CD8-deficient mice had a significantly lower AHR and eosinophilia in response to OVA sensitization and challenge comparing to the wild-type, but the allergic response was fully restored by adoptive transfer of antigen-primed effector CD8+ T cells.
In summary, this study demonstrated that short-term DEP exposure enhances OVA-induced airway inflammation and antigen-specific IgE and IgG production, and that it increases airway responsiveness in allergen-sensitized rats. The adjuvant effect was characterized by an increase in the responses of CD4+ and CD8+ T lymphocytes in LDLN, both of which are known to play a major role in allergic asthma, that was accompanied by an increase in ROS and NO production and iNOS expression in AM and ATII cells and a decrease in GSH levels in AM and lymphocytes.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barnes, P. J., and Liew, F. W. (1995). Nitric oxide and asthmatic inflammation. Immunol. Today 128, 128130.[CrossRef]
Buchmuller-Rouiller, Y., Corrandin, S. B., Smith, J., Schneider, P., Ransijn, A., Jongeneel, C. V., and Mauel, J. (1995). Role of glutathione in macrophage activation: Effect of cellular glutathione depletion on nitrite production and leishmanicidal activity. Cell. Immunol. 164, 7380.[CrossRef][ISI][Medline]
Dobbs, L. G., Gonzalez, R., and Williams, M. C. (1986). An improved method for isolating type II cells in high yield and purity. Am. Rev. Respir. Dis. 134, 141145.[ISI][Medline]
Dong, C. C., Yin, X. J., Ma, J. Y. C., Millecchia, L., Barger, M. W., Roberts, J. R., Zhang, X. D., Antonini, J. M., and Ma, J. K. H. (2005). Exposure of Brown Norway rats to diesel exhaust particles prior to ovalbumin (OVA) sensitization elicits IgE adjuvant activity but attenuates OVA-induced airway inflammation. Tox. Sci. doi:10.1093/toxsci/kfi298.
Feder, L. S., Stelts, D., Chapman, R. W., Manfra, D., Crawley, Y., Jones, H., Minnicozzi, M., Fernandez, X., Paster, T., Egan, R. W., Kreutner, W., and Kung, T. T. (1997). Role of nitric oxide on eosinophilic lung inflammation in allergic mice. Am. J. Respir. Cell. Mol. Biol. 17, 436442.
Folkes, L. K., and Wardman, P. (2004). Kinetics of the reaction between nitric oxide and glutathione: implications for thiol depletion in cells. Free Radic. Biol. Med. 37, 549556.[CrossRef][ISI][Medline]
Gavett, S. H., Chen, X., Finkelman, F., and Wills-Karp, M. (1994). Depletion of murine CD4+ T lymphocytes prevents antigen-induced airway hyperreactivity and pulmonary eosinophilia. Am. J. Respir. Cell Mol. Biol. 10, 587593.[Abstract]
Graham, L. M. (2004). All I need is the air that I breath: outdoor air quality and asthma. Paediatr. Respir. Rev. 5(Suppl A), S59S64.[CrossRef][Medline]
Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S., and Tannenbaum, S. R. (1982). Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem. 126, 131138.[CrossRef][ISI][Medline]
Hamelmann, E., Schwarze, J., Takeda, K., Oshiba, A., Larsen, G. L., Irvin, C. G., and Gelfand, E. W. (1997). Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am. J. Respir. Crit. Care Med. 156, 766775.
Ichinose, T., Takano, H., Miyabara, Y., Sadakano, K., Sagai, M., and Shibamoto, T. (2002). Enhancement of antigen-induced eosinophilic inflammation in the airways of mast-cell deficient mice by diesel exhaust particles. Toxicology 180, 293301.[CrossRef][ISI][Medline]
Ichinose, T., Takano, H., Miyabara, Y., Yanagisawa, R., and Sagai, M. (1997). Murine strain differences in allergic airway inflammation and immunoglobulin production by a combination of antigen and diesel exhaust particles. Toxicology 122, 183192.[CrossRef][ISI][Medline]
Komai, M., Tanaka, H., Masuda, T., Nagao, K., Ishizaki, M., Sawada, M., and Nagai, H. (2003). Role of Th2 responses in the development of allergen-induced airway remodeling in a murine model of allergic asthma. Br. J. Pharmacol. 138, 912920.[CrossRef][ISI][Medline]
Koren H. S. (1995). Associations between criteria air pollutants and asthma. Environ. Health Perspect. 103(Suppl 6), 235242.[ISI][Medline]
Leong, B. K. J., Coombs, J. K., Sabaitis, C. P., Rop, D. A., and Aaron, C. S. (1998). Quantitative morphometric analysis of pulmonary deposition of aerosol particles inhaled via intratracheal nebulization, intratracheal instillation or nose-only inhalation in rats. J. Appl. Toxicol. 18, 149160.[CrossRef][ISI][Medline]
Liu, S. F., Haddad, E. B., Adcock, I., Salmon, M., Koto, H., Gilbey, T., Barnes, P. J., and Chung, K. F. (1997). Inducible nitric oxide synthase after sensitization and allergen challenge of Brown Norway rat lung. Br. J. Pharmacol. 121, 12411246.[ISI][Medline]
Ma, J. Y. C., and Ma, J. K. H. (2002). The dual effect of the particulate and organic components of diesel exhaust particles on the alteration of pulmonary immune/inflammatory responses and metabolic enzymes. Environ. Carcinog. Ecotox. Rev. C 20, 117147.[CrossRef]
Melgert, B. N., Postma, D. S., Geerlings, M., Luinge, M. A., Klok, P. A., van der Strate, B. W., Kerstjens, H. A., Timens, W., and Hylkema, M. N. (2004). Short-term smoke exposure attenuates ovalbumin-induced airway inflammation in allergic mice. Am. J. Respir. Cell Mol. Biol. 30, 880885.
Miyabara, Y., Takano, H., Ichinose, T., Lim, H. B., and Sagai, M. (1998). Diesel exhaust enhances allergic airway inflammation and hyperresponsiveness in mice. Am. J. Respir. Crit. Care Med. 157, 11381144.
Miyahara, N., Swanson, B. J., Takeda, K., Taube, C., Miyahara, S., Kodama, T., Dakhama, A., Ott, V. L., and Gelfand, E. W. (2004a). Effector CD8+ T cells mediate inflammation and airway hyperresponsiveness. Nat. Med. 10, 865869.[CrossRef][ISI][Medline]
Miyahara, N., Takeda, K., Kodama, T., Joetham, A., Taube, C., Park, J. W., Miyahara, S., Balhorn, A., Dakhama, A., and Gelfand, E. W. (2004b). Contribution of antigen-primed CD8+ T cells to the development of airway hyperresponsiveness and inflammation is associated with IL-13. J. Immunol. 172, 25492558.
Murata, Y., Shimamura, T., and Hamuro, J. (2002). The polarization of T(h)1/T(h)2 balance is dependent on the intracellular thiol redox status of macrophages due to the distinctive cytokine production. Int. Immunol. 14, 201212.
Peterson, J. D., Herzenberg, L. A., Vasquez, K., and Waltenbaugh, C. (1998). Glutathione levels in antigen-presenting cells modulate Th1 versus Th2 response patterns. Proc. Natl. Acad. Sci. U. S. A. 95, 30713076.
Porter, D. W., Millecchia, L., Robinson, V. A., Hubbs, A., Willard, P., Pack, D., Ramsey, D., McLaurin, J., Khan, A., Landsittel, D., Teass, A., and Castranova, V. (2002). Enhanced nitric oxide and reactive oxygen species production and damage after inhalation of silica. Am. J. Physiol. Lung Cell Mol. Phys. 283, L485L493.[ISI]
Ricciardolo, F. (2003). Multiple roles of nitric oxide in the airways. Thorax 58, 75182.
Ricciardolo, F. L., Timmers, M. C., Geppetti, P., van Schadewijk, A., Brahim, J. J., Sont, J. K., de Gouw, H. W., Hiemstra, P. S., van Krieken, J. H., and Sterk, P. J. (2001). Allergen-induced impairment of bronchoprotective nitric oxide synthesis in asthma. J. Allergy Clin. Immunol. 108, 198204.[CrossRef][ISI][Medline]
Robinson, D. S. (2000). The Th1 and Th2 concept in atopic allergic disease. Chem. Immunol. 78, 5061.[ISI][Medline]
Steerenberg, P. A., Dormans, J. A., van Doorn, C. C., Middendorp, S., Vos, J. G., and van Loveren, H. (1999). A pollen model in the rat for testing adjuvant activity of air pollution components. Inhal. Toxicol. 11, 11091122.[CrossRef][ISI][Medline]
Steerenberg, P. A., Withagen, C. E., Dormans, J. A., van Dalen, W. J., van Loveren, H., and Casee, F. R. (2003). Adjuvant activity of various diesel exhaust and ambient particles in two allergic models. Toxicol. Environ. Health A 66, 14211439.[CrossRef]
Takano, H., Ichinose, T., Miyabara, Y., Shibuya, T., Lim, H. B., Yoshikawa, T., and Sagai, M. (1998). Inhalation of diesel exhaust enhances allergen-related eosinophil recruitment and airway hyperresponsiveness in mice. Toxicol. Appl. Pharmacol. 150, 328337.[CrossRef][ISI][Medline]
Takano, H., Yoshikawa, T., Ichinose, T., Miyabara, Y., Imaoka, K., and Sagai, M. (1997). Diesel exhaust particles enhance antigen-induced airway inflammation and local cytokine expression in mice. Am. J. Respir. Crit. Care Med. 156, 3642.
Voller, A., and Bidwell, D. (1986). Enzyme-linked immunosorbent assay. In Manual of Clinical Laboratory Immunology, 3rd ed. (Rose, N. R., Friedman, H., Fahey, J. L., Eds.), pp. 99110. American Society for Microbiology, Washington, DC.
Whitekus, M. J., Li, N., Zhang, M., Wang, M., Horwitz, M. A., Nelson, S. K., Horwitz, L. D., Brechun, N., Diaz-Sanchez, D., and Nel, A. E. (2002).Thiol antioxidants inhibit the adjuvant effects of aerosolized diesel exhaust particles in a murine model for ovalbumin sensitization. J. Immunol. 168, 25602567.
Yin, X. J., Dong, C. C., Ma, J. Y. C., Antonini, J. M., Roberts, J. R., Stanley, C. F., Schafer, R., and Ma, J. K. H. (2004a). Suppression of cell-mediated immune responses to Listeria infection by repeated exposure to diesel exhaust particles in Brown Norway rats. Toxicol. Sci. 77, 263271.
Yin, X. J., Ma, J. Y. C., Antonini, J. M., Castranova, V., and Ma, J. K. H. (2004b). Roles of reactive oxygen species and heme oxygenase-1 in modulation of alveolar macrophage-mediated pulmonary immune responses to Listeria monocytogenes by diesel exhaust particles. Toxicol. Sci. 82, 143153.
Yin, X. J., Schafer, R., Ma, J. Y. C., Antonini, J. M., Weissman, D. N., Siegel, P. D., Barger, M. W., Roberts, J. R., and Ma, J. K. H. (2002). Alteration of pulmonary immunity to Listeria monocytogenes by diesel exhaust particles (DEP). I. Effects of DEP on early pulmonary responses. Environ. Health Perspect. 110, 11051111.[ISI][Medline]
Yin, X. J., Schafer, R., Ma, J. Y. C., Antonini, J. M., Weissman, D. N., Siegel, P. D., Barger, M. W., Roberts, J. R., and Ma, J. K. H. (2003). Alteration of pulmonary immunity to L. monocytogenes by diesel exhaust particles (DEP). II. Effect of DEP on T-cell mediated immunity. Environ. Health Perspect. 111, 524530.[ISI][Medline]