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Department of Medicine, University of California at San Diego, La Jolla, CA 92093
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a key cytokine-regulating fibrosis, is also generated by mast cells (15). Coculture of mast cells and fibroblasts in vitro results in both fibroblast proliferation and mast cell proliferation, again suggesting a role for mast cells in fibrosis (16). The potential importance of mast cells to AHR and inflammation has also been studied extensively in mice exposed to acute OVA challenge. Studies of AHR and eosinophilic inflammation in mast cell-deficient mice demonstrate an important role for mast cells in AHR to methacholine (17). Mast cell-deficient WBB6F1-W/Wv mice sensitized to OVA without adjuvant, followed by exposure to repetitive intranasal Ag challenge, have less AHR and airway eosinophilia compared with congenic +/+ controls (18, 19). Furthermore, reconstitution of mast cell-deficient WBB6F1-W/Wv mice with bone marrow-derived mast cells re-establishes AHR to aerosolized Ag, establishing the importance of mast cells in acute Ag-induced bronchial hyperreactivity (18). The importance of the mast cell in chronic as opposed to acute mouse models of asthma has not been as extensively investigated. Our laboratory4 and others (20) have developed mouse models in which repetitive allergen administration is associated with sustained lung Th2 responses for up to 6 mo of allergen challenge. Because Th2-derived cytokines such as IL-4 (21, 22) and IL-9 (23, 24, 25, 26) are known to serve as mast cell growth factors, we sought to determine whether repetitive airway allergen challenge is associated with the accumulation of peribronchial mast cells as well as the expression of these lung mast cell growth factors. In addition, because our laboratory (27) and others (28, 29) have demonstrated that immunostimulatory DNA sequences (ISS) containing CpG-rich motifs down-regulate the Th2 inflammatory response in mouse models of asthma, we sought to determine whether ISS could inhibit both the accumulation of peribronchial mast cells and the expression of mast cell growth factors in a murine model of repetitive airway allergen challenge for periods of 1–6 mo.
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Materials and Methods |
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Female BALB/c mice (Harlan Laboratories, Indianapolis, IN; n = 12 mice/group) were used for in vivo experiments when they reached 8–10 wk of age. Mouse bone marrow-derived mast cells (MBMMCs) were generated from the bone marrow of BALB/c mice for in vitro experiments. All animal experimental protocols were approved by the University of California at San Diego Animal Subjects Committee.
Acute and chronic OVA challenge protocols
In both the acute and chronic Ag challenge experimental protocols, mice were immunized s.c. on days 0, 7, 14, and 21 with 25 µg of OVA (grade V; Sigma-Aldrich, St. Louis, MO) adsorbed to 1 mg of aluminum hydroxide (Sigma-Aldrich) in 200 µl of PBS, as previously described in our laboratory (27).
In the acute protocol, intranasal OVA challenges (20 µg in 50 µl of PBS) were administered on days 26, 28, 30, and 35, with mice being sacrificed on day 36. In the chronic protocol, mice completed the same intranasal OVA challenges on days 26, 28, and 30 as in the acute protocol and then continued to receive twice weekly intranasal OVA challenges for an additional 1, 3, or 6 mo. To facilitate intranasal administration of OVA, mice were briefly anesthetized with isoflurane (Isosol TM; Abbott Laboratories, North Chicago, IL).
Administration of ISS (50 µg in 200 µl of PBS i.p.) in the acute Ag challenge protocol was started on day 24 before the first intranasal OVA challenge on day 26 and repeated on day 34 before the final OVA challenge on day 35. In the chronic Ag challenge protocol, the first dose of ISS (50 µg in 200 µl of PBS i.p.) was also administered on day 24 before the first intranasal OVA challenge on day 26. The same dose of ISS was subsequently administered i.p. every other week until the chronic OVA protocol was terminated after 1, 2, 3, and 6 mo of repetitive airway OVA challenge. Previous studies in our laboratory have demonstrated that ISS inhibits OVA-induced eosinophilic inflammation and AHR when administered 1 day before OVA challenge (27) and that this inhibitory effect lasts at least 4 wk (30).
Determination of airway responsiveness in vivo
Airway responsiveness was analyzed on the day of sacrifice using a single-chamber, whole-body plethysmograph obtained from Buxco (Troy, NY), as previously described (27). The enhanced pause (Penh), a dimensionless value that correlates well with pulmonary resistance measured by conventional two-chamber plethysmography in ventilated mice in this laboratory (31) and other laboratories (32), was used to detect airway responsiveness.
In the chronic Ag exposure protocol, airway responsiveness was assessed on the day of sacrifice. Mice were exposed for 3 min to nebulized PBS to establish baseline Penh values, and they were subsequently exposed to increasing concentrations of nebulized methacholine (Sigma-Aldrich) in PBS using an Aerosonic ultrasonic nebulizer (DeVilbiss, Somerset, PA). After each nebulization, recordings were taken for 3 min. The Penh values measured during each 3-min sequence were averaged, and In Stat (San Diego, CA) software was used to perform linear regression analysis of the results to determine the provocative dose of methacholine required to cause a 200% increase in baseline Penh values (PC200) for each mouse.
Enumeration of peribronchial lung mast cells
On the day of sacrifice, mouse lungs were tied off at the trachea with surgical suture and were preserved in 10% buffered formalin (Sigma-Aldrich, St. Louis, MO) before being embedded in paraffin. The paraffin-embedded lungs were sectioned at 5 µm onto microscope slides. The paraffin was removed from the lung sections using alcohol gradients and Citrosolv (Fisher, Pittsburgh, PA). The lung sections were stained with 1% toluidine blue (Sigma-Aldrich) to detect metachromatic mast cell cytoplasmic granules as previously described in this laboratory (33).
Light microscopy was used to count the number of mast cells per airway in lung sections of naive mice, as well as mice acutely or chronically exposed to OVA Ag challenge. To standardize the quantification of peribronchial mast cells, airways were characterized by size using an image analysis system as large (125–250 µm), medium (60–125 µm), or small (<60 µm) by measuring the diameter of the long axis of the airway. The peribronchial area was defined as the circumferential area extending from the lumenal surface of the airway epithelium to 8 µm beneath the basement membrane of the airway epithelium in small airways, to 15 µm beneath the basement membrane of the airway epithelium in medium-sized airways, and to 20 µm beneath the basement membrane of the airway epithelium in large airways. The distance beneath the basement membrane chosen to define the peribronchial space was based on pilot experiments demonstrating the area in which the majority of non-mast cell peribronchial inflammatory cells were recruited after Ag challenge. The number of mast cells per airway were counted in 10 randomly selected airways of each size in each mouse.
In addition to detecting mast cells with toluidine blue staining, anti-IgE immunostaining was performed on selected lung sections using a rat anti-mouse IgE mAb. Endogenous peroxidase activity was quenched by incubation of in-lung sections for 30 min in 0.3% H2O2 in methanol. The lung sections were then incubated overnight with the primary Ab (rat anti-mouse IgE mAb; BD PharMingen, San Diego, CA) at a concentration of 1/20 at 4°C. After washing with PBS, the lung sections were incubated with the biotinylated secondary Ab (mouse anti-rat IgG1/2a; BD PharMingen), at a concentration of 1/100 for 30 min. The lung sections were then sequentially incubated with ABC reagent (Avidin, biotinylated HRP; Pierce, Rockford, IL), 3,3'-diaminobenzidine (DAB) buffer (DAB substrate kit; Vector Laboratories, Burlingame, CA), and DAB substrate reagent before briefly counterstaining with hematoxylin.
Lung cytokine analysis
The concentrations of IL-4, IL-9, and stem cell factor were assayed in lung tissue by ELISA, as previously described in our laboratory (34). Lungs homogenized in lysis buffer (0.5% Triton X-100, 150 mM NaCl, 15 mM Tris, 1 mM CaCl2, and 1 mM MgCl2) were centrifuged at 10,000 x g for 20 min. After the lung supernatant was passaged through a 0.8-µm pore size filter, the lung supernatant was assayed for cytokines by ELISA (R& D Systems, Minneapolis, MN). The sensitivity of the IL-4 assay was 7.8 pg/ml, the IL-9 assay was 61 pg/ml, and the stem cell factor assay was 31 pg/ml.
ISS and rhodamine-labeled ISS
Endotoxin-free (<1 ng/mg DNA) phosphorothioate ISS (5'-TGACTGTGAACGTTCGAGATGA-3') and mutated oligodeoxynucleotides (M-ODN) (5'-TGACTGTGAAGGTTGGAGATGA-3') (Trilink, San Diego, CA) were synthesized as previously described (27) for use in in vitro and in vivo experiments. For in vitro experiments, rhodamine was conjugated to the 5' end of ISS or M-ODN by Solulink (San Diego, CA) to provide fluorescently labeled ISS (5'-rhodamine-ISS-3') or M-ODN (5'-rhodamine-M-ODN-3') for studies of ISS binding to mast cells.
MBMMCs
MBMMCs were generated from the bone marrow of BALB/c mice. The bone marrow cells were cultured in medium containing 20% WEHI supernatant as a source of IL-3, as previously described in this laboratory (35). After 3 wk, the viability of MBMMCs in the bone marrow culture was >95% as assessed by trypan blue staining, and the purity of MBMMCs was >95% as assessed by toluidine blue staining (35).
Expression of Toll-like receptor-9 (TLR-9) by MBMMCs assessed by RT-PCR
MBMMC TLR-9 mRNA expression was determined by RT-PCR. Total cellular RNA was isolated from MBMMCs using TRIzol reagent (Life Technologies, Gaithersburg, MD) as previously described in this laboratory (36). As a negative control, naive CD4+ T cells derived from mouse spleen were positively selected using a MACS CD4+ T cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. The expression of TLR-9 was conducted with the following sense and anti-sense oligonucleotide sequence primers: sense primer, 5'-AGG CTG TCA ATG GCT CTC AGT T-3'; antisense primer, 5'-TGA ACG ATTTCC AGT GGT ACA AGT-3' (37). PCR amplification was conducted in a 50-µl reaction volume containing 50 pmol of each primer, 50 mM KCl, 20 mM Tris-HCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 1 µl of formamide, and 1 U of Taq polymerase (BRL, Gaithersburg, MD). The reaction mixture was denatured at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s and 60°C for 30 s and extended at 72°C for 30 s followed by an extension of 8 min at 72°C. The PCR products (611 bp) were electrophoresed in a 1.5% agarose gel and were visualized with ethidium bromide.
Effect of ISS on MBMMC proliferation in vitro
To assess whether ISS directly influences mast cell proliferation in vitro, 1 x 105 MBMMCs were cultured in triplicate for 1 or 7 days in a 96-well plate in the presence or absence of ISS (1–10 µg/ml) and 20% WEHI supernatant. After 1 or 7 days in culture, wells containing MBMMCs were washed and aspirated with a pipette three times to recover all of the MBMMCs in each well as previously described in this laboratory (38). The total number of MBMMCs in each well was counted using a light microscope.
Binding of rhodamine-labeled ISS to MBMMCs
To assess whether mast cells can bind ISS, MBMMCs (5 x 105/ml) were incubated with ISS conjugated to rhodamine (2 µM) or as a negative control with 2 µM rhodamine alone for 6 h at 37°C, and the binding was visualized using fluorescence confocal microscopy. To determine whether pretreatment with unlabeled ISS would inhibit the rhodamine-labeled ISS from binding to MBMMCs, MBMMCs were pretreated with 10 µM unlabeled ISS 30 min before rhodamine-labeled ISS was added to the MBMMCs. At the end of the 6-h incubation period, MBMMCs were aspirated, washed with cold PBS/BSA, cytospun onto slides that were fixed with 1% paraformaldehyde in PBS, and were examined by confocal microscopy.
Confocal microscopy
The mean fluorescence intensity (MFI) of 10 randomly selected fields of MBMMCs was measured using a DeltaVision deconvolution microscope system (Applied Precision, Issaquah, WA). A charge-coupled device (Roper Scientific, Tucson, AZ) mounted on a Nikon microscope (Melville, NY) was used to capture images from the slides, using a rhodamine filter and a 20x lens with 1 s of exposure time. The data sets were analyzed using SoftWorx software (Applied Precision) on a Silicon Graphics Octane workstation (Mountain View, CA).
Effect of ISS on Ag/IgE-mediated MBMMC mediator release
In these experiments, 1 x 106 MBMMCs were cultured in triplicate for 4–6 h in the presence or absence of ISS (1–10 µg/ml). MBMMCs were then sensitized with anti-DNP IgE (1 µg/106 MBMMCs) for 30 min, washed, and challenged with DNP-BSA Ag (200 ng) at 37°C for 20 min in Tyrodes buffer containing 1.8 mM CaCl2 and 1 mM MgCl2 (38). Levels of histamine in cell pellets and supernatants were measured 20 min after Ag stimulation, whereas supernatant IL-6 levels were measured 12 h after Ag stimulation. Histamine and IL-6 were measured with immunassays having a sensitivity of 0.5 pg/ml and 7.8 pg/ml, respectively.
Statistical analysis
Statistical analysis was performed with ANOVA, and individual groups were compared using an unpaired Mann-Whitney test. All statistical analyses were performed with a statistical software package (In Stat). Values of p < 0.05 were considered statistically significant. Results are expressed as the mean ± SEM unless otherwise indicated.
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Results |
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Peribronchial mast cells were quantitated using toluidine blue staining (Fig. 1A) in lung sections of mice challenged with OVA allergen for varying time periods. As depicted in Fig. 1, the majority of the accumulated mast cells were intraepithelial (
90%), with the remainder being subepithelial. Preliminary studies demonstrated that the number of toluidine blue-positive staining cells (Fig. 1A) were equivalent to the number of cells that stained positive for IgE (Fig. 1B), and therefore only the number of toluidine blue-positive cells were quantitated. Peribronchial mast cells were not detected in unchallenged mice (0 ± 0 mast cells per large, medium, and small airways; n = 8 mice/group). A very small number of peribronchial mast cells were occasionally detected after acute OVA challenge (0.05 ± 0.04 mast cells per large airway and 0 ± 0 mast cells per medium and small airways; n = 8 mice/group).
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Mice pretreated with ISS before repetitive OVA challenges had fewer peribronchial mast cells in airways of all sizes compared with OVA-challenged mice that had not received ISS. ISS-treated mice repetitively challenged with OVA had a significantly lower number of peribronchial mast cells in the large airways after 1 mo of repetitive OVA challenge (6.8 ± 0.8 vs 10.5 ± 1.0 mast cells per airway; ISS + OVA vs OVA; p = 0.006), after 2 mo of OVA challenge (5.5 ± 0.7 vs 8.7 ± 1.0 mast cells per airway; ISS + OVA vs OVA; p = 0.006), after 3 mo of OVA challenge (5.5 ± 0.8 vs 9.5 ± 1.1 mast cells per airway; ISS + OVA vs OVA; p = 0.02), and after 6 mo of OVA challenge (2.4 ± 0.6 vs 7.3 ± 0.6 mast cells per airway; ISS + OVA vs OVA; p < 0.0001) (Fig. 2A). ISS also significantly reduced the number of peribronchial mast cells in the medium-sized airways (Fig. 2B) after repetitive OVA challenge for 3 mo (ISS + OVA vs OVA; p = 0.007) and 6 mo (ISS + OVA vs OVA; p = 0.0001), as well as in the small airways at 2 mo (ISS + OVA vs OVA; p = 0.001), 3 mo (ISS + OVA vs OVA; p = 0.03), and 6 mo (ISS + OVA vs OVA; p = 0.001) (Fig. 2C).
Effect of chronic Ag challenge on expression of mast cell growth factors (IL-4, IL-9, stem cell factor)
To investigate the potential mechanism(s) by which repetitive OVA challenge induced an increase in the number of peribronchial mast cells, we measured levels of cytokines in the lung, which are known to stimulate mast cell proliferation (IL-4, IL-9, stem cell factor). Increased levels of lung IL-4 were detected after repetitive OVA challenge (141.2 ± 17.7 pg/ml vs 21.9 ± 7.1 pg/ml; 3 mo OVA vs no OVA; n = 12; p < 0.0001; Fig. 3A). Similarly, increased levels of lung IL-9 were detected after repetitive OVA challenge (229.5 ± 75.1 pg/ml vs 7.9 ± 7.9 pg/ml; 3 mo OVA vs no OVA; n = 10; p < 0.0001; Fig. 3B). Levels of lung stem cell factor were not increased after repetitive OVA challenge (521.0 ± 31.1 pg/ml vs 538.7 ± 20.5 pg/ml; 3 mo OVA vs no OVA; n = 12; p = NS).
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ISS inhibits airway hyperreactivity to methacholine induced by repetitive OVA challenge
In the absence of ISS therapy, mice developed significant airway hyperreactivity to methacholine after repetitive OVA exposure for up to 6 mo. Repetitively OVA-challenged mice had lower PC 200 values compared with non-OVA-challenged mice at 1 mo (4.3 ± 0.9 mg/ml vs 22.2 ± 2.5 mg/ml; p = 0.0005), 2 mo (3.5 ± 0.4 mg/ml vs 11.3 ± 0.8 mg/ml; p = 0.0007), 3 mo (4.5 ± 0.7 mg/ml vs 28.2 ± 4.5 mg/ml; p < 0.0001), and 6 mo (4.9 ± 0.7 mg/ml vs 14.3 ± 1.8 mg/ml; p < 0.0001) (Fig. 4). Pretreatment of repetitively OVA-challenged mice with ISS significantly reduced airway hyperreactivity, as evidenced by the higher PC200 values in ISS-treated mice at 1 mo (ISS + OVA vs OVA; p < 0.0001), 3 mo (ISS + OVA vs OVA; p = 0.004), and 6 mo (ISS + OVA vs OVA; p = 0.0009; Fig. 4). Overall, these results suggest that ISS significantly reduced the increase in airway hyperreactivity to methacholine induced by repetitive airway OVA exposure.
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RT-PCR studies demonstrated that MBMMCs strongly expressed the TLR-9 receptor (Fig. 5).
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Having demonstrated that MBMMCs express TLR-9, the receptor for ISS, studies were performed to determine whether fluorescently labeled ISS bound to MBMMCs. Confocal microscopy studies demonstrated that MBMMCs bound rhodamine-labeled ISS but not rhodamine alone (Fig. 6). The MFI of MBMMCs incubated with control rhodamine alone (93.2 ± 2.; n = 29) was similar to the background MFI of MBMMCs, which ranged from 80 to 100 (Fig. 7). The MFI of MBMMCs incubated with rhodamine-labeled ISS was significantly greater than that of MBMMCs incubated with rhodamine alone (997.7 ± 58.6 vs 93.2 ± 2.1; n = 29; p < 0.0001). Pretreatment of MBMMCs with 10 µM ISS for 30 min significantly decreased the MFI of MBMMCs incubated with rhodamine-labeled ISS (148.1 ± 8.0; n = 29; p < 0.0001 compared with no pretreatment with ISS), suggesting that the uptake of rhodamine-labeled ISS was inhibited by the unlabeled ISS (Fig. 7). A rhodamine-labeled M-ODN (which only differs in not having the CpG motif present in rhodamine-labeled ISS) did not bind to MBMMCs (data not shown).
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To determine whether the binding of ISS by MBMMCs induced any functional changes in MBMMCs, MBMMC proliferation assays were performed in the presence or absence of ISS in vitro for 1 or 7 days. In the 1 day experiments, the number of MBMMCs in the cultures containing 1 µg/ml ISS (1.00 ± 0.04 x 105 cells) and 10 µg/ml of ISS (1.06 ± 0.07 x 105) were not significantly different from the number of MBMMCs in cultures without ISS (1.02 ± 0.03 x 105)(n = 9)(p = NS).
There was a significant increase in the number of MBMMCs after 7 days of in vitro culture of MBMMCs without ISS (1.0 x 105 vs 1.48 ± 0.10 x 105; MBMMC time, 0 h, vs MBMMC No ISS for 7 days; p = 0.007). After 7 days of incubation of MBMMCs with ISS, there was no significant difference in the number of MBMMCs in cultures containing 1 µg/ml ISS (1.65 ± 0.08 x 105 cells) compared with MBMMC cultures without ISS (1.48 ± 0.10 x 105 cells; n = 9; p = NS).
Effect of ISS on Ag/IgE-mediated mast cell activation in vitro
To determine whether ISS inhibits mast cell activation in vitro, MBMMCs were cultured in the presence or absence of ISS (1 µg/ml) and then activated by IgE receptor cross linking with Ag. MBMMC release of preformed granule mediators (histamine) was not inhibited by ISS (Fig. 8A). Although ISS did not induce histamine release from unstimulated MBMMCs, it did induce significant release of IL-6 from unstimulated MBMMCs (Fig. 8B). The levels of IL-6 released by MBMMCs in response to ISS stimulation were significantly greater than the levels of IL-6 induced by Ag/IgE stimulation of MBMMCs (240.7 ± 63.6 pg/ml vs 70.2 ± 18.7 pg/ml; ISS vs Ag; p = 0.001; Fig. 8B). There was no synergy in IL-6 release when MBMMCs were stimulated with Ag/IgE in combination with ISS (Fig. 8B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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600/10,000 mononuclear cells, with fewer detected in lung (150/10,000 mononuclear cells) (40). Additional evidence against circulating mast cells being recruited to the airway is derived from the histologic analysis of the lung distribution of mast cells in repetitively OVA-challenged mice in which there is an absence of a gradient of mast cells being detected in lung sections from pulmonary blood vessels to the airway. Whereas eosinophils can readily be detected in lung sections in and around blood vessels, in the interstitial space, and in peribronchial regions, this pattern of distribution was not noted for mast cells. We did not note mast cells adherent to pulmonary blood vessels, diapedesing between endothelial cells, or accumulating outside of blood vessels. Because mast cells proliferate in response to a variety of cytokines and/or growth factors, we have investigated whether mast cell growth factors are expressed in the lung after repetitive OVA challenge. Initial studies focused on detecting mast cell growth factors released by Th2 cells because the repetitive OVA challenge model we have developed is associated with chronic expression of Th2 cytokines (IL-5, IL-13) and eosinophilic inflammation.4 Th2 cytokines known to induce mast cell proliferation include IL-4 (21, 22) and IL-9 (23, 24, 25, 26). In vitro IL-4 enhances the growth of bone marrow-derived mast cells (21, 22), and IL-9 promotes mast cell proliferation and differentiation (22, 23, 24, 25, 26). IL-9 expression in transgenic mice causes mast cell hyperplasia and bronchial hyperresponsiveness (24, 25). Repetitive OVA challenge was associated with increased expression of these Th2 cytokines (IL-4, IL-9), which can both affect mast cell proliferation. We also measured levels of stem cell factor (SCF), whose predominant cellular source is not Th2 cells, to determine whether repetitive OVA challenge induced expression of this mast cell growth factor (41). Repetitive OVA challenge did not induce increased levels of SCF, suggesting that SCF was not responsible for the increased numbers of peribronchial mast cells noted.
Because we have previously demonstrated that ISS inhibits Th2 cell-dependent accumulation of eosinophils in the lung (27), we performed experiments to determine whether ISS inhibited Th2 cytokines (IL-4 and IL-9) with mast cell growth factor activity, as well as mast cell accumulation in the lung. Although previous studies in an acute OVA challenge model have demonstrated that ISS inhibits IL-4 expression (28), this study is the first to demonstrate that ISS inhibits allergen-induced IL-9 expression. IL-9 expression is increased in asthmatic airways compared with normal subjects, and IL-9 mRNA expression has been correlated with FEV1 and AHR to methacholine, identifying it as an important cytokine in allergic asthma (26). Although this study demonstrates an association between the ability of ISS to inhibit the expression of mast cell growth factors in the lung and the inhibition of mast cell lung accumulation, this study is not able to demonstrate whether this is a cause and effect relationship or only an association.
To determine whether ISS could be having any direct effect on mast cell proliferation, we initially determined whether mast cells express TLR-9 the receptor for ISS (42). RT-PCR studies demonstrated that MBMMCs strongly express TLR-9. Additional in vitro experiments demonstrated that MBMMCs were able to bind rhodamine-labeled ISS, and this binding was inhibited by pretreatment with unlabeled ISS. Although mast cells express TLR-9, ISS does not directly inhibit mast cell proliferation in vitro. ISS did not inhibit Ag/IgE-mediated MBMMC degranulation, but did it induce IL-6 generation. Previous studies using unstimulated MBMMCs have demonstrated that ISS induces MBMMC expression of TNF and IL-6 (43). The generation of IL-6 in response to ISS stimulation is likely to be a beneficial effect of ISS in allergic inflammation. Studies with IL-6 transgenic mice have demonstrated an important anti-inflammatory role for IL-6 in mouse models of asthma (44).
Overall, these studies demonstrate that mice exposed to repetitive OVA challenge, but not acute OVA challenge, have an accumulation of peribronchial mast cells and express mast cell growth factors including IL-4 and IL-9 in the lung. Although mast cells express TLR-9, ISS does not directly inhibit mast cell proliferation in vitro, suggesting that ISS inhibits accumulation of peribronchial mast cells in vivo by indirect mechanism(s) that include inhibiting the lung expression of Th2 cell-derived mast cell growth factors.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David H. Broide, University of California at San Diego, Basic Science Building, Room 5090, 9500 Gilman Drive, La Jolla, CA 92093-0635. E-mail address: dbroide{at}ucsd.edu
3 Abbreviations used in this paper: AHR, airway hyperreactivity; ISS, immunostimulatory sequence; MBMMC, mouse bone marrow-derived mast cell; Penh, enhanced pause; DAB, 3,3'-diaminobenzidine; M-ODN, mutated oligodeoxynucleotide; MFI, mean fluorescence intensity; SCF, stem cell factor.
4 J. Y. Cho, M. Miller, K. J. Baek, J. W. Han, J. Nayar, M. Rodriguez, S. Y. Lee, K. McElwain, S. McElwain, E. Raz, and D. H. Broide. Immunostimulatory DNA inhibits TGF- expression and airway remodeling. Submitted for publication.
Received for publication April 10, 2003. Accepted for publication August 28, 2003.
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1 and TNF
secreted by mast cells stimulated via the Fc
RI activate fibroblasts for high-level production of monocyte chemoattractant protein-1 (MCP-1). Cell. Immunol. 201:42.[Medline]
1 selectively inhibits IL-3-dependent mast cell proliferation without affecting mast cell function or differentiation. J. Immunol. 143:1591.[Abstract]
7 (47 integrin) for tissue-specific homing. J. Exp. Med. 194:1243. and IL-6 production but not degranulation from murine bone marrow-derived mast cells. J. Leukocyte Biol. 69:253.Related articles in The JI:
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 M. Miller, J. Y. Cho, K. McElwain, S. McElwain, J. Y. Shim, M. Manni, J. S. Baek, and D. H. Broide Corticosteroids prevent myofibroblast accumulation and airway remodeling in mice Am J Physiol Lung Cell Mol Physiol, January 1, 2006; 290(1): L162 - L169. [Abstract] [Full Text] [PDF]
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E. M. Hessel, M. Chu, J. O. Lizcano, B. Chang, N. Herman, S. A. Kell, M. Wills-Karp, and R. L. Coffman Immunostimulatory oligonucleotides block allergic airway inflammation by inhibiting Th2 cell activation and IgE-mediated cytokine induction J. Exp. Med., December 5, 2005; 202(11): 1563 - 1573. [Abstract] [Full Text] [PDF]
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J. A. Boyce and K. F. Austen No audible wheezing: nuggets and conundrums from mouse asthma models J. Exp. Med., June 20, 2005; 201(12): 1869 - 1873. [Abstract] [Full Text] [PDF]
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C. J. Youn, M. Miller, K. J. Baek, J. W. Han, J. Nayar, S. Y. Lee, K. McElwain, S. McElwain, E. Raz, and D. H. Broide Immunostimulatory DNA Reverses Established Allergen-Induced Airway Remodeling J. Immunol., December 15, 2004; 173(12): 7556 - 7564. [Abstract] [Full Text] [PDF]
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 J. Y. Cho, M. Miller, K. J. Baek, J. W. Han, J. Nayar, M. Rodriguez, S. Y. Lee, K. McElwain, S. McElwain, E. Raz, et al. Immunostimulatory DNA Inhibits Transforming Growth Factor-{beta} Expression and Airway Remodeling Am. J. Respir. Cell Mol. Biol., May 1, 2004; 30(5): 651 - 661. [Abstract] [Full Text] [PDF]
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