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Department of Medicine, University of California San Diego, La Jolla, CA 92093
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and reduced the number of TGF-1-positive eosinophils and TGF-1-positive mononuclear cells recruited to the airway. In vitro studies demonstrated that ISS inhibited TGF-1 expression by macrophages (RAW 264.7 cell line and bone marrow-derived macrophages). In addition, ISS significantly reduces lung levels of expression of the chemokine thymus- and activation-regulated chemokine, as well as the number of peribronchial CD4+ lymphocytes that express Th2 cytokines that promote peribronchial fibrosis. Overall, these studies demonstrate that ISS can reverse features of airway collagen deposition by reducing levels of lung TGF-1, as well as by reducing levels of the chemokine thymus- and activation-regulated chemokine and the numbers of peribronchial CD4+ lymphocytes that drive the ongoing Th2 immune response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, , and 
), IL-6, IL-10, and IL-12 (10). ISS also up-regulates the expression of Th1 cytokine receptors such as the IFN- receptor (11). In addition to inducing Th1 responses, ISS also inhibits expression of Th2 cytokines in the lung including IL-4 (2, 12), IL-5 (1), IL-9 (12), and IL-13 (13), while also reducing levels of expression of Th2 cytokine receptors such as the IL-4R (11). The immunomodulatory effect of ISS is due to its effect on cells that express TLR-9 (14) the receptor for ISS, in particular dendritic cells, as well as macrophages, and B cells.
In mouse models ISS is effective in inhibiting Th2-mediated eosinophilic inflammation in the lung when administered either systemically or delivered directly to the mucosa of the airway (1). A single systemic dose of ISS inhibits Th2 responses to OVA inhalation challenge for at least 4 wk (5). ISS is as effective as corticosteroids in preventing (1), or reversing (7), eosinophilic inflammation and airway hyperreactivity in mouse models. More recently, studies in our (13) and other laboratories (15) have demonstrated that prophylactic administration of ISS can prevent airway remodeling when administered to mice before starting inhalation challenge with allergen. Prophylactic ISS administration significantly prevents features of airway remodeling including peribronchial fibrosis, as well as the allergen-induced increased thickness of the peribronchial smooth muscle layer (13). ISS administration not only prevented the development of these structural features of airway remodeling, but also inhibited the development of airway hyperreactivity, eosinophilic inflammation, airway mucus expression, and reduced levels of profibrotic growth factors including TGF- and IL-13 (13). In this study we have investigated whether ISS can reverse established airway remodeling, as opposed to the previous studies that demonstrated ISS can prevent airway remodeling (13, 15). In prior studies examining whether ISS can prevent airway remodeling we started the therapeutic intervention with ISS before the onset of the first of a series of 3 mo of repetitive OVA challenges (13). In contrast, in this study we first instituted the 3 mo of repetitive OVA challenges to induce airway remodeling, and at that time point instituted ISS therapy to determine whether airway remodeling can be reversed. Although a variety of pathologic features comprise airway remodeling in asthma (16), we have focused in this study in particular on examining the effect of ISS on reversing established peribronchial collagen deposition in the lung.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice (n = 16 mice/group; The Jackson Laboratory, Bar Harbor, ME), 8–10 wk of age, 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 alum (Sigma-Aldrich) in 200 µl of normal saline. Intranasal OVA challenges (20 ng/50 µl in PBS) were administered on days 27, 29, and 31 under isoflurane (Vedco, St. Joseph, MO) anesthesia. Intranasal OVA challenges were then repeated twice a week for 3 mo (see Fig. 1 for protocol) a time point that we have previously demonstrated is associated with airway remodeling (13, 17). Age- and sex-matched control mice were sensitized but not challenged with OVA during the 3-mo study. Following cessation of 3 mo of OVA challenges, mice were followed for a further 1 or 3 mo without receiving additional OVA challenges. Mice were sacrificed at the different time points and bronchoalveolar lavage (BAL) fluid and lungs were analyzed. All animal experimental protocols were approved by the University of California, San Diego Animal Subjects Committee.
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Different groups of mice (n = 16 mice/group) were administered i.p. endotoxin-free (<1 ng/mg DNA) phosphorothioate ISS-oligodeoxynucleotides (ODN) (5'-TGACTGTGAACGTTCGAGATGA-3') (Trilink, San Diego, CA) (100 µg in 100 µl of sterile, endotoxin-free PBS), or diluent control starting after completion of the 3-mo period of twice weekly intranasal OVA challenges. Therapy was continued every other week for 1 or 3 mo (see Fig. 1). Previous studies in our laboratory have demonstrated that ISS, but not mutated ODN (5'-TGACTGTGAAGGTTGGAGATGA-3'), which lacks the CpG motif present in ISS, prevents OVA-induced airway remodeling in this model (13). ISS inhibits OVA-induced eosinophilic inflammation and airway hyperreactivity when administered 1 day before OVA challenge (1), and this inhibitory effect lasts at least 4 wk (5).
Processing of lung for image analysis
Lungs in the different groups of mice were equivalently inflated with an intratracheal injection of a similar volume of 4% paraformaldehyde solution (Sigma-Aldrich) to preserve the pulmonary architecture. The inflated lungs were embedded in paraffin, stained with H&E, periodic acid Schiff (PAS), trichrome stain, or processed for immunohistochemistry. Lungs from the different experimental groups were processed as a batch for either histologic staining or immunostaining under identical conditions. Stained and immunostained slides were all quantified under identical light microscope conditions, including magnification (x20), gain, camera position, and background illumination. The quantitative histologic and image analysis of all coded slides was performed by research associates blinded to the coding of all the slides.
Effect of ISS on peribronchial fibrosis
Three methods (trichrome stain, total lung collagen content, peribronchial collagen III and V immunostaining) were used to quantify peribronchial fibrosis.
Peribronchial trichrome staining. The area of peribronchial trichrome staining in paraffin-embedded lung was outlined and quantified using a light microscope (Leica DMLS; Leica Microsystems, Rochester, NY) attached to an image analysis system (Image-Pro Plus; Media Cybernetics, Silver Spring, MD) as previously described (13, 17). Results are expressed as the area of trichrome staining per micrometer length of basement membrane of bronchioles 150–200 µm of internal diameter. At least 10 bronchioles were counted in each slide.
Lung collagen assay. The amount of lung collagen was measured using a collagen assay kit that uses a dye reagent that selectively binds to the (Gly-X-Y)n tripeptide sequence found within the triple helix sequence of mammalian collagens (Bicolor, Newtownabbey, Ireland, U.K.) as previously described in this laboratory (13, 17) and other laboratories (18). Measurement of lung collagen bound dye was made using a Microwell plate reader (Bio-Rad, Hercules, CA) set at 550 nm absorbance. In all experiments a collagen standard was used to calibrate the assay.
Peribronchial collagen III and collagen V immunostaining. Lung sections were processed for immunohistochemical detection of collagen III or collagen V using a primary mAb directed against either mouse collagen III or V (Polysciences, Warrington, PA) as previously described (13, 17). The area of peribronchial collagen III or collagen V staining was outlined and quantified using a light microscope attached to an image analysis system as previously described (13, 17). Results are expressed as the area of peribronchial III or peribronchial V staining per micrometer length of basement membrane of bronchioles 150–200 µm of internal diameter. At least 10 bronchioles were counted in each slide.
Fibroblast TLR-9 expression
Fibroblast TLR-9 mRNA expression was determined by RT-PCR using the murine fibroblast cell line PA317 (American Type Culture Collection, Manassas, VA). Total cellular RNA was isolated from fibroblasts using TRIzol reagent (Invitrogen Life Technologies, Gaithersburg, MD) as previously described in this laboratory (12). Mouse bone marrow-derived mast cells, which we have recently demonstrated to express TLR-9 (12), were used as a positive control. The expression of TLR-9 was assessed with the following sense and antisense 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' (12). The PCR products (611 bp) were electrophoresed in a 1.5% agarose gel and were visualized with ethidium bromide.
Effect of ISS on levels of TGF-1
Levels of TGF-1 were quantitated by ELISA and by immunohistochemistry.
TGF-1 ELISA.
The concentrations of TGF-1 (sensitivity of assay 5 pg/ml) in BAL and homogenized lung supernatants were assayed by ELISA (R&D Systems, Minneapolis, MN) as previously described in this laboratory study (13, 17). Before the TGF-1 assay, the BAL and lung supernatant samples were treated with 2.5 N acetic acid to activate any latent TGF-1 (13, 17). The concentrations of the lung supernatant protein was assayed using a Micro BCA TM protein assay reagent kit (Pierce, Rockford, IL), which has a sensitivity of 0.5 µg/ml. Levels of active TGF-1 in lung supernatants are expressed as picograms of TGF-1 per milligram of lung protein.
TGF-1 immunohistochemistry.
Serial sequential lung sections were processed for TGF-1 immunohistochemistry in a similar manner as described earlier for lung collagen III and collagen V immunostaining. The primary monoclonal anti-TGF-1 Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The number of individual cells staining positive for TGF-1 in the peribronchial space were counted using a light microscope. Results are expressed as the number of peribronchial cells staining positive for TGF-1 per bronchiole of bronchioles 150–200 µm of internal diameter. At least 10 bronchioles were counted in each slide.
Effect of ISS on peribronchial inflammatory cell infiltration (MBP+, F4/80+) and number of TGF-1-positive cells.
We have previously used double immunostaining to TGF-1 in combination with either immunostaining to major basic protein (MBP) or F4/80, to demonstrate that eosinophils and mononuclear cells account for the majority of nonepithelial peribronchial cell expression of TGF-1 in the remodeled airway following 3 mo of repetitive OVA challenge using this mouse model (17). Therefore, to determine whether ISS induced a decrease in the total number of TGF-1-positive cells, as well as a parallel reduction in the number of peribronchial cells staining positive for either MBP or F4/80, we counted the total number of peribronchial cells staining positive for TGF-1, as well as the total number of peribronchial cells staining positive for either MBP and F4/80 in ISS treated or control mice. Results are expressed as the total number of peribronchial cells staining positive for TGF-1/bronchiole, and as the total number of peribronchial cells staining positive for either MBP or F4/80/bronchiole, of bronchioles 150–200 µm of internal diameter. At least 10 bronchioles were counted in each slide.
Effect of ISS on macrophage TGF-1 and IL-6 expression in vitro
Previous studies have not reported whether ISS modulates macrophage TGF-1 expression that was the focus of these experiments. In these experiments, 5 x 105 macrophage RAW 264.7 cells were incubated with LPS 100 ng/ml in triplicate for 24 h in the presence or absence of ISS or mutated ODN (10 µg/ml). Supernatants were collected and assayed for TGF-1 by ELISA. As a control, we examined the effect of ISS on RAW 264.7 cell line expression of IL-6 (measured by ELISA), which several groups have demonstrated is induced by ISS (19). We also performed experiments assessing the effect of ISS on macrophage TGF-1 expression using purified populations of macrophages derived from the culture of mouse bone marrow using culture methods previously described in this laboratory (20).
Effect of ISS on levels of lung thymus-and activation-regulated chemokine (TARC) expression and peribronchial CD4+ T lymphocyte accumulation
As T lymphocytes produce cytokines such as IL-5 and IL-13 that contribute to airway remodeling (16, 17), we measured levels of lung expression of the T lymphocyte chemoattractant TARC, as well as the number of peribronchial CD4+ lymphocytes. Previous studies have not examined whether ISS effects the lung expression of chemokines such as TARC that bind to CCR4 receptors on Th2 cells, as well as whether ISS effects the number of peribronchial CD4+ lymphocytes in the remodeled airway.
Measurement of TARC. Lungs were homogenized as described, and the lung supernatant was assayed for TARC by ELISA.
Number of peribronchial CD4+ lymphocytes. Serial sequential lung sections were processed for CD4 immunohistochemistry in a similar manner as already described for lung MBP immunostaining. The primary monoclonal anti-CD4 Ab was obtained from Santa Cruz Biotechnology. Results are expressed as the number of peribronchial cells staining positive for CD4 per bronchiole of bronchioles 150–200 µm of internal diameter. At least 10 bronchioles were counted in each slide.
Effect of ISS on macrophage TARC expression in vitro
As TARC is expressed by macrophages we measured levels of TARC in supernatants derived from RAW 264.7 cells stimulated with LPS 100 ng/ml in triplicate for 24 h in the presence or absence of ISS or mutated ODN (10 µg/ml).
Effect of ISS on area of peribronchial -smooth muscle actin immunostaining
Lung sections for -smooth muscle actin immunostaining were processed in a similar manner as described for lung collagen immunostaining. The primary anti--smooth muscle actin mAb was obtained from Sigma-Aldrich. The area of peribronchial -smooth muscle actin immunostaining was outlined and quantified using a light microscope attached to an image analysis system as earlier described.
Effect of ISS on airway mucus expression
To quantitate the level of mucus expression in the airway, the number of PAS-positive and PAS-negative epithelial cells in individual bronchioles were counted as previously described in this laboratory study (13, 17). At least 10 bronchioles were counted in each slide. Results are expressed as the percentage of PAS-positive cells per bronchiole that is calculated from the number of PAS-positive epithelial cells per bronchus divided by the total number of epithelial cells of each bronchiole.
Statistical analysis
Results in the different groups of mice were compared by ANOVA using the nonparametric Kruskal-Wallis test followed by posttesting using Dunn’s multiple comparison of means. All results are presented as mean ± SEM. A statistical software package (Graph Pad Prism, San Diego, CA) was used for the analysis. Values of p < 0.05 were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice exposed to repetitive OVA challenge for 3 mo had a significant increase in the levels of peribronchial fibrosis compared with non-OVA-challenged mice as assessed by several parameters including lung collagen (Fig. 2A), trichrome stain (B), as well as collagen III (C), and collagen V (D) immunostaining. The non-OVA-challenged mice in all experiments now described were sensitized to OVA as described in Materials and Methods. Mice challenged with OVA for 3 mo, and then followed for an additional 1 mo, or for 3 mo without further OVA challenges maintained the increase in peribronchial fibrosis compared with non-OVA-challenged mice at 1 mo of follow up, and at 3 mo of follow up (Fig. 2, A–D). Although levels of peribronchial trichrome staining and collagen V immunostaining were still significantly elevated at 1 and 3 mo following discontinuation of OVA challenges, these levels did decline following discontinuation of OVA challenges (Fig. 2, B and D). In contrast, there was no decline in levels of lung collagen or collagen III when OVA challenges were discontinued (Fig. 2, A and C).
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Fibroblasts have not thus far been reported to express TLR-9. To determine whether ISS could directly modulate fibroblast function via TLR-9 on fibroblasts we performed RT-PCR studies to detect whether TLR-9 is expressed by fibroblasts using a fibroblast cell line. RT-PCR studies demonstrated that the fibroblast cell line PA317 did not express TLR-9 the receptor for ISS (data not shown). Control mouse bone marrow-derived mast cells strongly expressed TLR-9 as previously described in this laboratory study (12).
Effect of ISS on lung TGF-1 expression
As the fibroblast cell line did not express TLR-9, we examined whether ISS reduced expression of TGF-1 a growth factor associated with peribronchial fibrosis (16, 20). We measured levels of TGF-1 in BAL fluid, lung, as well as the number of peribronchial cells expressing TGF-1. Levels of BAL TGF-1 (Fig. 2E), lung TGF-1 (F), and the number of peribronchial TGF-1-positive cells (G) were significantly increased in mice exposed to repetitive OVA challenge for 3 mo compared with non-OVA-challenged mice. One month after the 3 mo of OVA challenges were discontinued, BAL levels of TGF-1 (Fig. 2E), lung levels of TGF-1 (F), and the number of peribronchial TGF-1-positive cells (G) were still significantly elevated compared with non-OVA-challenged mice, although levels had declined from the time of the last OVA challenge. At 3 mo after the OVA challenges were discontinued, BAL levels of TGF-1 (Fig. 2E), and the number of peribronchial TGF-1-positive cells (G) were still significantly elevated compared with non-OVA-challenged mice, although levels had declined from the time of the last OVA challenge. Lung levels of TGF-1 (Fig. 2F) were no longer elevated 3 mo after OVA challenges were discontinued.
Institution of ISS treatment after completion of the OVA challenges significantly reduced the BAL levels of TGF-1 (Fig. 2E), lung levels of TGF-1 (F), and the number of peribronchial TGF-1-positive cells (G) in mice following 1 mo of ISS treatment. Similar declines in BAL levels of TGF-1 (Fig. 2E), and the number of peribronchial TGF-1-positive cells (G) were noted following 3 mo of ISS treatment.
Mechanisms by which ISS inhibits TGF-1 expression
ISS can theoretically reduce levels of lung TGF-1 by either decreasing the total number of lung cells that express TGF-1 (e.g., the number of MBP+ eosinophils, or F4/80+ mononuclear cells) in the airway, and/or by decreasing levels of expression of TGF-1 by individual cells in the airway. We therefore examined whether ISS reduced the number of TGF-1-positive cells in vivo, and whether it directly inhibited macrophage TGF-1 expression in vitro.
Effect of ISS on the number of MBP+ and F4/80+ peribronchial cells
In previous studies, using double immunostaining to TGF-1 and MBP or F4/80, we have demonstrated that mononuclear cells and eosinophils are the predominant peribronchial cellular source of TGF-1 following 3 mo of repetitive OVA challenge (17). We therefore determined the effect of ISS on reducing the total number of peribronchial mononuclear cells and eosinophils, as well as the effect of ISS on reducing the number of peribronchial TGF-1-positive cells. Repetitive OVA challenge for 3 mo in untreated mice induced a significant increase in the number of MBP+ peribronchial cells (35.7 ± 2.3 vs 0.3 ± 0.1 peribronchial MBP+ cells) (OVA vs no OVA; p = 0.0001) (Fig. 2H), and F4/80+ peribronchial cells (36.7 ± 3.8 vs 0.5 ± 0.1 peribronchial F4/80+ cells) (OVA vs no OVA; p = 0.0001) (I) compared with non-OVA challenged mice. After discontinuing OVA challenges at 3 mo, the number of peribronchial eosinophils declined to a greater degree than did the number of peribronchial mononuclear cells (Fig. 2, H and I). However, mice challenged with OVA for 3 mo, and then followed for up to 3 mo, without further OVA challenges maintained a significant increase in the number of peribronchial MBP+ and F4/80+ cells compared with non-OVA-challenged mice at 1 mo of follow up (p = 0.001; MBP, Fig. 2H) (p = 0.001; F4/80; I), and at 3 mo of follow up (p = 0.001; MBP, H) (p = 0.001; F4/80, I). Institution of ISS treatment at the time of discontinuing OVA challenges significantly reduced the number of peribronchial MBP+ and F4/80+ cells in mice following 1 mo of ISS treatment (p = 0.001; MBP, Fig. 2H; p = 0.001; F4/80, I), as well as following 3 mo of ISS treatment (p = 0.001; MBP, H; p = 0.001; F4/80, I).
ISS induced a significant decrease in the total number of peribronchial cells that express TGF-1 (TGF-1-positive cells per bronchus), as well as a parallel decline in the combined total number of eosinophils and mononuclear cells per bronchus. Following 3 mo of OVA challenges, therapy with ISS for 1 mo, compared with treatment with diluent, reduced both the number of peribronchial TGF-1 positive cells (9.1 ± 0.6 vs 18.5 ± 1.0 peribronchial TGF-1-positive cells per bronchus) (p = 0.001), and the total combined number of peribronchial eosinophils plus mononuclear cells (5.8 ± 1.4 vs 27.9 ± 1.9 MBP+ and F4/80+ cells per bronchus) (p = 0.001).
Effect of ISS on macrophage TGF-1 and IL-6 levels
To determine whether ISS inhibits release of TGF-1 by macrophages, macrophages (RAW 264.7 cell line) were incubated in vitro in the presence or absence of ISS. Supernatants were assayed for both TGF-1, and as a control IL-6 a cytokine known to be regulated by ISS. Incubation of macrophages with ISS induced a significant release of IL-6 into the supernatant (628.2 ± 2.4 vs 9.2 ± 1.6 pg/ml IL-6) (ISS vs medium; p = 0.008) (Fig. 3A), whereas ISS did not induce macrophages to release significant amounts of TGF-1 compared with cells incubated in medium alone (B) (ISS vs medium). A mutated ODN did not induce significant IL-6 or TGF-1 release from macrophages (mutated ODN vs medium) (Fig. 3, A and B). Although ISS induced macrophages to release significantly more IL-6 than TGF-1, LPS differed from ISS in that it stimulated both IL-6 release from macrophages (1066.9 ± 53.8 vs 9.2 ± 1.6 pg/ml IL-6) (LPS vs medium; p = 0.001) (Fig. 3A), and TGF-1 release from macrophages (279.0 ± 52.4 vs 68.6 ± 18.9 pg/ml TGF-1) (LPS vs medium; p = 0.001) (B). To determine whether ISS modulated TGF-1 expression by macrophages, macrophages were incubated with LPS to release TGF-1 in the presence or absence of ISS. ISS significantly inhibited LPS-stimulated macrophage release of TGF-1 (45.1 ± 18.5 vs 279.2 ± 52.4 pg/ml TGF-1) (LPS plus ISS vs LPS; p = 0.001) (Fig. 3B), reducing TGF-1 to levels similar to that noted in macrophages incubated with medium (45.1 ± 18.5 vs 68.6 ± 18.9 pg/ml TGF-1) (LPS plus ISS vs medium) (B). In contrast, ISS had a slight effect on inhibiting IL-6 release from LPS stimulated macrophages (LPS plus ISS vs LPS; p = 0.05) (Fig. 3A).
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1 release from a macrophage cell line, ISS also inhibited the LPS-induced release of TGF-1 from bone marrow-derived macrophages (52.3 ± 13.8 vs 86.4 ± 10.9 pg/ml TGF-1) (LPS plus ISS vs LPS; p = 0.05) (Fig. 3C).
Effect of ISS on reversing area of peribronchial -smooth muscle actin immunostaining
In addition to determining whether ISS reversed peribronchial collagen deposition, we also investigated whether ISS reversed the allergen induced increase in the area of peribronchial -smooth muscle actin immunostaining. Repetitive OVA challenge for 3 mo in untreated mice induced a significant increase in peribronchial -smooth muscle actin immunostaining compared with non-OVA-challenged mice (1.28 ± 0.06 vs 0.30 ± 0.02 µm2/µm) (OVA vs no-OVA; p = 0.0001) (Fig. 4). Mice challenged with OVA, and then followed without further OVA challenges maintained a significant increase in the area of peribronchial -smooth muscle actin immunostaining compared with non-OVA-challenged mice at 1 mo of follow up (p = 0.001), as well at 3 mo of follow up (p = 0.001) (Fig. 4). However, discontinuation of OVA challenges for 3 mo following the 3 mo OVA challenge period significantly reduced levels of peribronchial -smooth muscle actin immunostaining (Fig. 4).
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-smooth muscle actin immunostaining in mice following 1 mo of ISS treatment (0.26 ± 0.02 vs 1.27 ± 0.17 µm2/µm; p = 0.001) (Fig. 4), as well as following 3 mo of ISS treatment (0.33 ± 0.03 vs 0.59 ± 0.05 µm2/µm; p = 0.01) (Fig. 4). ISS inhibits lung TARC expression and the accumulation of CD4+ peribronchial cells
As previous studies have demonstrated that ISS inhibits expression of Th2 cytokines (1, 2, 12, 13) but does not act directly on T lymphocytes, we investigated whether one mechanism by which ISS may inhibit the expression of Th2 cytokines is through inhibition of expression of T cell chemoattractants (e.g., TARC) and resultant inhibition of recruitment of CD4+ lymphocytes to the airway.
Effect of ISS on levels of lung TARC
Levels of lung TARC were significantly increased in mice exposed to repetitive OVA challenge for 3 mo compared with non-OVA-challenged mice (652.1 ± 35.2 vs 59.9 ± 16.9 pg TARC/mg lung protein) (OVA vs no-OVA; p = 0.01) (Fig. 5A). Mice challenged with OVA and then followed for an additional 1 mo (p = 0.05) (Fig. 5A), or 3 mo (p = 0.05) (A) maintained an increase in the lung levels of TARC compared with non-OVA-challenged mice. However, the levels of lung TARC at 1 and 3 mo after discontinuing OVA challenges were significantly less than that noted following 3 mo of OVA challenges (Fig. 5A).
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Effect of ISS on the accumulation of CD4+ peribronchial cells
Repetitive OVA challenge for 3 mo in untreated mice induced a significant increase in the number of CD4+ peribronchial cells compared with non-OVA-challenged mice (13.2 ± 1.5 vs 0.3 ± 0.1 peribronchial CD4+ cells) (OVA vs no OVA; p = 0.001) (Fig. 5B). Interestingly, mice challenged with OVA for 3 mo, and then followed for up to 3 mo, without further OVA challenges maintained the increase in the number of CD4+ peribronchial cells compared with non-OVA-challenged mice at 1 mo of follow up (p = 0.001) (Fig. 5B), and at 3 mo of follow up (p = 0.001) (B). Institution of ISS treatment after completion of OVA challenges significantly reduced the number of peribronchial CD4+ cells in mice following 1 mo of ISS treatment (3.4 ± 0.6 vs 14.3 ± 1.4 peribronchial CD4+ cells; p = 0.001) (Fig. 5B), as well as following 3 mo of ISS treatment (2.1 ± 0.4 vs 11.4 ± 0.9 peribronchial CD4+ cells; p = 0.001) (B).
Effect of ISS on macrophage TARC expression
To determine whether ISS inhibits expression of TARC by macrophages, macrophages (RAW 264.7 cell line) were stimulated in vitro in the presence or absence of ISS. Neither ISS nor mutated ODN alone induced expression of TARC by macrophages (Fig. 5C). LPS stimulated significant TARC release from macrophages (69.7 ± 11.2 vs 2.0 ± 0.8 pg/ml TARC) (LPS vs unstimulated; p = 0.001) (Fig. 5C). ISS had no inhibitory effect on LPS induced TARC release from macrophages (Fig. 5C).
Effect of ISS on airway mucus expression
Repetitive OVA challenge for 3 mo in untreated mice induced a significant increase in the percentage of airway epithelium that stained positive with PAS compared with non-OVA-challenged mice (39.6 ± 1.8 vs 0.3 ± 0.01% PAS-positive cells per bronchus) (OVA vs non-OVA; p = 0.0001) (Fig. 6). Mice challenged with OVA and then followed without further OVA challenges maintained a significant increase in the percentage of airway epithelium that stained positive with PAS compared with non-OVA-challenged mice at 1 mo of follow up (p = 0.001), or at 3 mo of follow up (p = 0.001) (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 expression by macrophages), as well as by reducing the number of TGF-1 positive eosinophils and mononuclear cells recruited to the airway. In addition, ISS significantly reduces levels of expression of the chemokine TARC, as well as the number of peribronchial CD4+ lymphocytes. CD4+ lymphocytes are likely to play an important role in orchestrating the airway remodeling response by generating Th2 cytokines such as IL-5 (17) and IL-13 (16), which have been implicated in airway remodeling. ISS induced reduction in the number of peribronchial CD4+ lymphocytes is therefore likely to contribute to reducing peribronchial fibrosis by reducing levels of cytokines such as IL-5 and IL-13 that contribute to fibrosis. Previous studies from this laboratory have demonstrated that 3 mo of OVA challenge is associated with significantly increased levels of expression of Th2 cytokines including IL-4 (12), IL-5 (17), IL-9 (12), and IL-13 (13), and that ISS administered prophylactically for 3 mo, inhibits Th2 cytokine expression (12, 13). Overall, this study demonstrates that ISS can reverse features of airway collagen deposition, and that this reversal is associated with significant inhibitory effects on lung TGF-1 expression. However, we cannot exclude additional direct effects of ISS on lung fibroblasts to inhibit collagen synthesis. The absence of TLR-9 expression (as assessed by RT-PCR) on a fibroblast cell line suggests that ISS may not exert direct effects on fibroblasts, but as fibroblasts are heterogenous cells further studies with primary lung fibroblasts are needed to exclude this possible direct inhibitory effect of ISS on lung fibroblast collagen synthesis.
In humans, the basement membrane of airway epithelium is comprised of two layers, the basal lamina (referred to as the true basement membrane and of normal thickness in asthma) and the lamina reticularis (comprised of collagen type I, type III, type V, and fibronectin), which is thickened in asthma (16). As peribronchial deposition of type III and type V collagen is a characteristic feature of remodeling in asthma, we were interested to determine whether ISS could not only prevent deposition of collagen as demonstrated in previous studies (12, 15), but was also able to reverse the collagen already deposited in the remodeled airway. In this study we demonstrate that ISS can reverse allergen-induced peribronchial collagen deposition of types III and type V collagen by reducing accumulation of peribronchial CD4+ cells that drive ongoing eosinophil and macrophage accumulation, expression of TGF-, and collagen deposition. Although discontinuation of OVA challenge at 3 mo is associated with some reduction in collagen deposition over the subsequent 3 mo follow up period, ISS significantly accelerates the reduction in peribronchial collagen deposition. This study therefore suggests that although allergen avoidance can reduce levels of peribronchial collagen deposition, ISS administration is significantly better than allergen avoidance in reversing peribronchial collagen deposition. Stopping exposure to allergen also leads to reduced levels of peribronchial inflammatory cells (eosinophils, mononuclear cells, but not CD4+ cells), reductions in levels of TGF- (lung TGF- more than BAL TGF-) and reductions in collagen deposition (collagen III rather than collagen V), suggesting that switching off the inflammatory response reduces ongoing collagen deposition whether it is due to avoidance of allergen (a small reduction in fibrosis), or due to ISS (more effective and therefore a larger reduction in fibrosis). At present it is not clear whether stopping exposure to allergen activates all the same pathways (and to the same degree) as that induced by ISS to reduce levels of peribronchial collagen deposition.
One potential mechanism by which ISS can reduce fibroblast collagen synthesis is through an effect on cell types that secrete TGF-1, a cytokine that stimulates fibroblasts to synthesize collagen (21). ISS reduces the number of peribronchial cells expressing TGF-1, suggesting that ISS either inhibits the expression of TGF-1 and/or modulates the number of TGF-1-positive cells in the airway. To address whether ISS can modulate cellular TGF-1 expression, we performed in vitro studies with macrophages, which express TLR-9 to determine whether ISS could modulate levels of TGF-1 expression by macrophages. Although previous studies have demonstrated that ISS induces macrophages to express cytokines such as IL-6, TNF, and IL-12 (19), these studies have not focused on whether ISS modulates macrophage TGF-1 expression. Our in vitro studies demonstrated that ISS directly inhibits macrophage TGF-1 expression, suggesting that ISS may inhibit levels of lung TGF-1 by reducing the levels of TGF-1 expressed by peribronchial macrophages. Thus, ISS may reduce levels of TGF-1 through several mechanisms including inhibiting macrophage TGF-1 expression, as well as by reducing the number of peribronchial cells that express TGF-1 (mononuclear cells, eosinophils). Indeed, our studies demonstrated that ISS induced a parallel decline in the total number of peribronchial TGF-1-positive cells, as well as the total number of MBP+ and F4/80+ peribronchial cells that are significant sources of TGF-1. In studies with IL-5 deficient mice we have previously demonstrated that there is a significant reduction in both the number of MBP+ eosinophils and TGF-1-positive cells detected in the airway of mice subjected to repetitive OVA challenge for 3 mo (17), suggesting that eosinophils are a significant source of TGF-1 in this model of airway remodeling. The mechanism by which ISS reduces the number of peribronchial eosinophils is likely due to decreased production in the number of bone marrow eosinophils (1) and decreased trafficking of eosinophils into the lung, rather than increased apoptosis of eosinophils as ISS does not directly induce apoptosis of eosinophils in vitro (1).
In this study, we also made the novel observation that ISS reduces the number of peribronchial CD4+ lymphocytes. As CD4+ lymphocytes do not express the TLR-9 receptor, this reduction in the number of CD4+ lymphocytes is unlikely to be due to a direct effect of ISS on CD4+ lymphocytes. As the chemokines TARC binds to CCR4 receptors on Th2 lymphocytes, we explored whether ISS modulated levels of expression of this chemokine. Mouse models of allergic airway inflammation have shown that neutralization of TARC cannot only inhibit T cell and eosinophil infiltration into the lung but can also inhibit bronchial hyperresponsiveness (22). In addition, BAL fluid from asthmatic subjects after allergen challenge contains significantly increased levels of TARC (23). Interestingly, ISS induced a significant decrease in levels of lung TARC that was associated with a significant reduction in peribronchial CD4+ T lymphocytes. As CD4+ lymphocytes orchestrate the inflammatory response including the recruitment of TGF-1+ eosinophils, ISS-mediated reduction in the number of peribronchial CD4+ lymphocytes will also contribute to reductions in TGF-1 levels. As TARC is expressed by macrophages, epithelium, and T lymphocytes (22, 23), we explored whether ISS directly modulated levels of TARC expression by macrophages. In vitro ISS did not inhibit TARC release by macrophages. Therefore, ISS mediated reductions in TARC levels in vivo could either be due to direct effects of ISS on mononuclear cells (less likely based on our in vitro studies), or due to effects of ISS on other TARC-expressing cells such as epithelial cells. In addition, ISS may modulate levels of TARC in vivo through effects on mediators that subsequently effect TARC production by these cells and/or inhibit recruitment of TARC-producing cells. Immunostaining lung sections with an Ab to TARC demonstrated increased staining of peribronchial mononuclear cells in OVA-challenged mice, with significant reductions in TARC staining in ISS treated mice (data not shown). In addition to ISS having effects on CD4+ T cell recruitment to the lung, we have recently demonstrated that ISS can induce Th2 cell death in the lung through induction of IDO (24). In these studies, OVA specific in vitro generated Th2 cells (DO11.10) were adoptively transferred into SCID mice that were challenged acutely with OVA (this in vivo SCID mouse model is deficient in T regulatory cells) (24). Results from these studies demonstrated that ISS induction of IDO in the lung plays a key role in mediating inhibition of Th2 cell responses using an acute OVA Ag challenge protocol. This effect of ISS as an inducer of Th2 cell death was inhibited by coadministration of an IDO inhibitor (24). These adoptive transfer experiments of Th2 cells in SCID mice indicate that ISS induction of IDO, rather than ISS induction of T regulatory cells, is a key mechanism mediating inhibition of Th2 cell responses in this acute adoptive transfer OVA model. In the chronic model of airway remodeling, the reductions in levels of lung TGF- induced by ISS suggest that ISS is not inducing TGF- expressing T regulatory cells. However, further studies are needed to determine whether T regulatory cells are induced by ISS in the chronic model of airway remodeling.
In summary, in this study we have demonstrated that systemic administration of ISS to mice that had already developed established airway remodeling significantly reduced the degree of airway collagen deposition. The ability of ISS to reduce lung collagen expression in vivo may be mediated through its effects on both reducing TGF-1 expression by macrophages, as well as by reducing the number of TGF-1-positive eosinophils and mononuclear cells recruited to the airway. In addition, ISS significantly reduces levels of expression of the chemokine TARC, as well as the number of peribronchial CD4+ lymphocytes further reducing signals for peribronchial recruitment of TGF-1 positive eosinophils.
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1 This study was supported by National Institutes of Health Grants AI 33977 and AI 38425 (to D.B.) and National Institutes of Health Grant AI 40682 (to E.R.). 
2 Address correspondence and reprint requests to Dr. David H. Broide, Department of Medicine, Basic Science Building, Room 5090, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0635. E-mail address: dbroide{at}ucsd.edu
3 Abbreviations used in this paper: ISS, immunostimulatory sequence; TARC, thymus- and activation-regulated chemokine; ODN, oligodeoxynucleotide; BAL, bronchoalveolar lavage; PAS, periodic acid Schiff; MBP, major basic protein.
Received for publication January 5, 2004. Accepted for publication October 15, 2004.
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expression and airway remodeling. Am. J. Respir. Cell Mol. Biol. 30:651.1. J. Exp. Med. 194:809. in human disease. N. Engl. J. Med. 342:1350.This article has been cited by other articles:
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