Eosinophils and CCR Regulate Interleukin- Transgene-Induced Pulmonary Remodeling

【摘要】  Interleukin (IL)-13 transgene overexpression in the lung induces features of chronic inflammatory lung disorders, including an eosinophil-rich inflammatory cell infiltration, airway hyper-reactivity, and remodeling of the airway (eg, subepithelial fibrosis, goblet cell metaplasia, and smooth muscle hypertrophy and hyperplasia). Here, we aimed to define the role of eosinophils and eosinophil signaling molecules in IL-13-mediated airway disease. To accomplish this, we mated IL-13-inducible lung transgenic mice with mice deficient in eosinophil chemoattractant molecules (eotaxin-1, eotaxin-2, and their receptor CCR3) and with mice genetically deficient in eosinophils (dbl-GATA). We report that in the absence of eotaxin-2 or CCR3, there was a profound reduction in IL-13-induced eosinophil recruitment into the lung lumen. In contrast, in the absence of eotaxin-1, there was a fourfold increase in IL-13-mediated eosinophil recruitment into the airway. IL-13 transgenic mice deficient in CCR3 had a 98% reduction in lung eosinophils. Furthermore, the reduction in pulmonary eosinophils correlated with attenuation in IL-13-induced mucus cell metaplasia and collagen deposition. Mechanistic analysis identified alterations in pulmonary protease and transforming growth factor-ß1 expression in eosinophil-deficient mice. Taken together, these data definitively identify a functional contribution by eosinophils on the effects of chronic IL-13 expression in the lung.
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Asthma, one of the most common serious chronic diseases of childhood, is an inflammatory lung disease characterized by airway wall remodeling and reduced respiratory function. The chronic inflammatory process, with an airway infiltrate composed primarily of CD4+ lymphocytes and eosinophils, contributes to airway remodeling, defined as altered lung structural changes.1 These structural changes in the airway include mucus cell metaplasia and increased deposition of collagen, proteoglycans, and other matrix proteins in the subepithelial layer.2 Importantly, these structural changes are implicated, at least partially, in the clinical manifestations of asthma.1,3,4
Animal studies have defined a central effector role for interleukin (IL)-13 in many pathological features of experimental asthma.5-12 Pulmonary overexpression of IL-13 results in inflammation, airway fibrosis, mucus metaplasia, airway hyper-responsiveness, and enhanced lung volumes and compliance.6,13 IL-13 receptors are expressed by a number of structural cells in the lung, including epithelial and smooth muscle cells, suggesting that direct signaling in these cells may be largely responsible for lung remodeling.14 However, IL-13 also induces a profound inflammatory infiltrate composed of numerous cell types, especially eosinophils; it has been proposed that IL-13-induced chemokine and protease expression and activity are at least partially responsible for cellular recruitment.15,16 IL-13 is known to be a potent stimulator of an array of chemokines, including the eotaxins (CCL11, CCL24, and CCL26), eosinophil-specific chemoattractants.15,17 The eotaxins act locally in tissues to promote eosinophil accumulation by enhancing and orchestrating migration to the site of inflammation by stimulating chemotaxis.18,19 Whereas chemokines are notorious for stimulating several receptors, the eotaxins are unusual in that they signal through a single chemokine receptor CC chemokine receptor (CCR) 3, a receptor abundantly expressed on eosinophils.20,21 In the mouse system, CCR3 expression is almost exclusively detected on eosinophils.22,23 Importantly, recent studies have demonstrated that CCR3 disruption impaired eosinophil recruitment in acute models of experimental asthma.24-26 In addition to regulating eosinophil trafficking into mucosal tissues, the eotaxins activate the respiratory burst apparatus, induce degranulation, and up-regulate adhesion molecule expression.27-29 Activated eosinophils have been shown to be a source of several molecules implicated in tissue remodeling processes, including IL-13-induced transforming growth factor (TGF)-ß1.30,31 Prior studies have shown that IL-13-induced eosinophil recruitment in the lung airway and lung tissue in an acute model are regulated by eotaxin-2 and eotaxin-1, respectively,32 but the role of these molecules in regulating other aspects of IL-13-induced pathology has not been addressed.
In this study, our aim was to elucidate the role of eosinophils in chronic IL-13-mediated lung pathology. In contrast to recent studies using genetically engineered eosinophil-deficient mouse strains that have primarily focused on examining the role of eosinophils in allergen-induced allergic lung disease,33,34 we aimed to test the role of eosinophils under conditions of a strong chronic model of experimental asthma elicited by overexpression of a IL-13 transgene in the lung. Although transgenic systems produce relatively high levels of cytokines compared with physiological concentrations, they may more closely model the human condition, which typically involves ongoing exposure to triggers (such as viral infections or perennial allergens). As such, we examined several independent genetically altered mouse lines with defects in eosinophil development or recruitment aiming to identify pathways downstream from eosinophils. Specifically, we used multiple independent genetic approaches by generating an inducible transgenic system that targets expression of IL-13 to the lung in wild-type mice and mice deficient in eosinophils (dbl-GATA), eotaxin-1, eotaxin-2, and CCR3. Herein, we report the consequences of these genetic engineering events on the outcome of chronic IL-13-mediated pulmonary disease. The data to be presented support a central regulatory role for eosinophils and the eotaxin/CCR3 axis in the development of multiple aspects of lung pathology resulting from chronic IL-13 exposure, including a marked effect on the development of airway remodeling. As such, these results draw attention to the therapeutic potential of anti-eosinophil-directed therapeutics.

【关键词】  eosinophils regulate interleukin- transgene-induced pulmonary remodeling

Materials and Methods

Inducible IL-13 Lung Transgenic Mice

Bitransgenic mice (CC10-iIL-13) were generated in which IL-13 was expressed in a lung-specific manner that allowed for external regulation of transgene expression, as previously described.35 CC10-iIL-13 mice deficient in eosinophils, CCR3, eotaxin-1, and eotaxin-2 were generated by breeding the CC10-iIL-13 (FVB/n) with the dbl-GATA (BALB/c), CCR3 (BALB/c), eotaxin-1 (SVEV), and eotaxin-2 (SVEV) gene-targeted mice for three generations. CCR3 gene-targeted (CCR3KO) and dbl-GATA (dbl-Gata) mice were generously provided by Drs. Alison Humbles and Craig Gerard (Children??s Hospital, Boston, MA). For all experiments, wild-type mice with the appropriate mixed backgrounds were used as controls. Transgene expression was induced by feeding bitransgenic mice doxycycline-impregnated (dox) food (625 mg/kg; Purina Mills, Richmond, IN). Animals were housed under pathogen-free conditions in accordance with institutional guidelines.

Bronchoalveolar Lavage Fluid Collection and Analysis

Mice were sacrificed by an intraperitoneal injection of Ketaject (ketamine hydrochloride; 0.2 mg/kg; Phoenix Pharmaceuticals, St. Joseph, MO). A midline neck incision was made, and the trachea was cannulated. The lungs were lavaged two times with 1.0 ml of lavage buffer . The recovered bronchoalveolar lavage fluid (BALF) was centrifuged at 400 x g for 5 minutes at 4??C and resuspended in 200 µl of lavage buffer. Lysis of red blood cells was performed using RBC lysis buffer (Sigma, St. Louis, MO) according to the manufacturer??s recommendations. Total cell numbers were counted with a hemacytometer. Cytospin preparations of 1 x 105 cells were stained with the Hema 3 Staining System (Fisher Diagnostics, Middletown, VA), and differential cell counts were determined.

Northern Blot Analysis

RNA was electrophoresed in an agarose-formaldehyde gel, transferred to Gene Screen transfer membranes (NEN, Boston, MA) in 10x sodium chloride and sodium citrate, and cross-linked by UV radiation. The cDNA probes, generated by polymerase chain reaction (PCR) or from commercially available vectors , were sequence confirmed, radiolabeled with 32P, and hybridized using standard conditions.

Eosinophil Quantitation

Lung tissue eosinophils were identified by anti-major basic protein (MBP) staining. The lungs were inflation-fixed in 10% neutral buffered formalin at 25 cm H2O, embedded in paraffin, cut into 5-µm sections, and fixed to positively charged slides. Endogenous peroxidase in the tissues was quenched with 0.5% hydrogen peroxide in methanol. Lung sections were digested (10 minutes, 37??C) with pepsin (Zymed, San Francisco, CA) and blocked by incubation at room temperature in 3% normal goat serum in PBS for 2 hours. The blocked sections were treated with rabbit anti-mouse MBP at 1:10,000 dilution (a kind gift of James and Nancy Lee, Mayo Clinic, Scottsdale, AZ) in 3% normal goat serum/PBS overnight at 4??C. The slides were subsequently washed free of primary antibody with several changes of PBS, followed by incubation with biotinylated goat anti-rabbit IgG (1:250 dilution) and avidin-peroxidase complex (Vector Laboratories, Burlingame, CA) for 2 hours and 45 minutes, respectively. These slides were developed with nickel diaminobenzidine-cobalt chloride solution to form a black precipitate and counterstained with nuclear fast red. Replacing the primary antibody with normal rabbit serum ablated the immunostaining. Quantification of immunoreactive cells was performed by counting the positively stained cells under low-power magnification of longitudinal sections, and eosinophil levels are expressed as the number of eosinophils per square millimeter.

Enzyme-Linked Immunosorbent Assay Measurements

Cytokine levels were measured in the BALF using enzyme-linked immunosorbent assay kits specific for murine IL-13, eotaxin-1, and eotaxin-2 (R&D Systems, Minneapolis, MN) and TGF-ß1 (Promega Corporation, Madison, WI). Detection limits for the enzyme-linked immunosorbent assays were 8, 10, 12, and 15 pg/ml for IL-13, eotaxin-1, eotaxin-2, and TGF-ß1, respectively.

Lung Histopathological Changes

Mice were sacrificed by an intraperitoneal injection of Ketaject (ketamine hydrochloride; 0.2 mg/kg; Phoenix Pharmaceuticals). Lungs were inflation fixed with 10% neutral-buffered formalin at 25 cm H2O and immersed in the same fixative. The inflated lungs were embedded in paraffin and stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), or Masson??s trichrome stain. PAS-stained airway goblet cells were enumerated by light microscopy examination (magnification, x400). To quantitate the level of mucus expression in the airway, the number of PAS+ and total epithelial cells in individual bronchioles was counted. At least three medium-sized bronchioles (defined by having approximately 90 to 150 luminal airway epithelial cells) were evaluated in each slide. Results are expressed as the percentage of PAS+ cells per bronchiole, which is calculated from the number of PAS+ epithelial cells per bronchiole divided by the total number of epithelial cells in each bronchiole. Quantification of collagen was performed by morphometric analysis using the Metamorph Imaging System (Universal Imaging Corporation, West Chester, PA). The lung sections were taken from the same position in each set of mice, and at least three to four random sections/mouse were analyzed. Using digital image capture, collagen-stained areas surrounding large airways were quantified (square micrometers) and normalized to length of basement membrane (micrometers).

Protease Expression by Real-Time PCR Analysis

RNA samples were subject to reverse transcription using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) according to manufacturer??s instructions. Genes of interest were quantitated by real-time PCR using the LightCycler instrument in conjunction with the ready-to-use LightCycler FastStart DNA Master SYBR Green I reaction mix (Roche Diagnostics, Indianapolis, IN). Primer pairs and annealing temperatures for each gene of interest can be found in Table 1 . Results (picograms of cDNA) were normalized to glyceraldehyde-3-phosphate dehydrogenase (picograms of cDNA) amplified from the same cDNA mix.

Table 1. Primer Sequences and LightCycler Conditions for Pulmonary Proteases

Statistical Analysis

Data are expressed as mean ?? SEM. Statistical significance comparing different sets of mice was determined by Student??s t-test.

Results

Effect of IL-13 on Eotaxin and CCR3 Expression

We initially assessed the levels of the eosinophil-specific chemokines, eotaxin-1 and eotaxin-2, and CCR3 in an inducible transgenic system that targets expression of IL-13 to the lung. At 6 weeks of age, CC10-iIL-13 mice were fed normal food pellets or food pellets impregnated with doxycycline (dox). After 4 weeks, markedly increased levels of eotaxin-1, eotaxin-2, and CCR3 mRNA were detectable by Northern blot analysis in the lungs of CC10-iIL-13 mice fed dox food compared with control double transgenic mice fed normal food (Figure 1A) . Eotaxin-1 and eotaxin-2 protein levels were also strikingly elevated (16- and 277-fold, respectively) in the BALF from dox-fed CC10-iIL-13 mice (P 0.001; Figure 1B ).

Figure 1. Expression of CCR3 and the eotaxin chemokines is induced by lung IL-13 transgenic overexpression. A: Northern blot analysis of total lung RNA from CC10-iIL13 mice that were fed normal (NO DOX) or dox-impregnated (DOX) food for 4 weeks reveals IL-13-induced expression of eotaxin-1, eotaxin-2, and CCR3. Each lane represents a separate mouse. Ethidium bromide (EtBr)-stained gel is also shown. B: The levels of eotaxin-1 and eotaxin-2 protein were assessed in the BALF of CC10-iIL13 mice that were fed normal (NO DOX) or dox-impregnated (DOX) food for 4 weeks. Protein levels represent the mean ?? SEM of six to seven mice per group (derived from two experiments). *P < 0.001.

To identify the role of eosinophils and CCR3 in IL-13-induced inflammation, we generated CC10-iIL-13 mice with a severe defect in eosinophil development (dbl-GATA) and mice that were gene-targeted for CCR3, eotaxin-1, or eotaxin-2. We first compared protein levels of IL-13 and the eotaxin chemokines in lungs from wild-type double-transgenic mice and double-transgenic mice that are deficient in eosinophils, eotaxin-1, eotaxin-2, or CCR3. After 4 weeks on dox food, BALF IL-13 protein levels increased dramatically in wild-type CC10-iIL13 mice (from 0.18 ?? 0.02 to 112 ?? 21 ng/ml, P 0.001, n = 5 to 8 mice/group). No significant differences in IL-13 protein levels were detected in the gene-altered CC10-iIL-13 mice compared with their wild-type control mice (data not shown). Thus, expression of IL-13 was not attributed to the eotaxins and/or eosinophils, consistent with the transgene being expressed in respiratory Clara cells.33,34 Lung protein levels of the eotaxin chemokines were also examined. Similar levels of IL-13-induced eotaxin-1 and eotaxin-2 proteins were detected in the CCR3 and the eotaxin gene-targeted mice with no difference in either eotaxin chemokine accumulation between the gene-targeted mice and their wild-type control mice, demonstrating that there is no compensatory overexpression of the remaining eotaxin chemokine in either of the gene-targeted mice (data not shown).

Effect of CCR3 and Its Ligands on IL-13-Induced Airway Eosinophilia

IL-13-induced BALF eosinophilia was profoundly inhibited (84 ?? 1.7%, n = 2 experiments, with 3 to 4 mice/group/experiment, P = 0.03) in the absence of eotaxin-2 (Figure 2A) . However, eotaxin-1 deficiency resulted in an increase (fourfold, P = 0.002) in IL-13-induced airway eosinophilia when compared with wild-type controls (23.7 ?? 4.3 versus 7 ?? 1.7 x 104 eosinophils, respectively; Figure 2B ). Notably, IL-13-induced airway eosinophilia was markedly reduced in CCR3-deficient mice (95 ?? 3%, n = 3 experiments, with 4 to 5 mice/group/experiment, P 0.007) and absent in the dbl-GATA mice when compared with wild-type controls (Figure 2C ; data not shown).

Figure 2. IL-13 expression results in airway eosinophilia that is eotaxin-2 and CCR3-dependent and tissue eosinophilia that is CCR3 and eotaxin-1-dependent. Eosinophils in BALF from wild-type (WT) and eotaxin-2-deficient (ETX2KO; A), eotaxin-1-deficient (ETX1KO; B), or CCR3-deficient (CCR3KO; C) CC10-iIL13 mice fed dox food for 4 weeks. A representative experiment is shown (mean ?? SEM, n = 3 with three to six mice per group per experiment). *P = 0.03. Quantitation of eosinophil infiltration, as detected by anti-MBP immunohistochemistry, in the lungs in wild-type (WT, white bars) and CCR3- (CCR3KO, black bars; D), eotaxin-1- (ETX1KO, gray bars E), and eotaxin-2-deficient (ETX2KO, black bars) mice is shown before (NO DOX) and after (DOX) 4 weeks on dox food. Data are presented as mean ?? SEM (n = 4 to 10 mice per group). *P 0.05. F: H&E-stained lungs from wild-type (WT) and dbl-GATA (dbl-Gata) mice fed dox food for 4 weeks are shown. Arrow, eosinophils.

Effect of CCR3 and Its Ligands on IL-13-Induced Lung Tissue Eosinophilia

Pulmonary tissue accumulation of eosinophils is a prominent feature of IL-13-induced inflammation; we therefore examined the effect of eotaxin-1, eotaxin-2, and CCR3 deficiencies on eosinophil infiltration into the lung tissue. IL-13 expression resulted in a marked increase in peribronchial eosinophils in wild-type mice (Figure 2, DCF) . However, there was a dramatic reduction (98%) in eosinophil accumulation in peribronchial lung tissue in CCR3-deficient mice when compared with wild-type mice . Notably, eotaxin-2 deficiency had no effect on IL-13-induced peribronchial eosinophil recruitment when compared with wild-type mice (474 ?? 64 versus 473 ?? 177 eosinophils/mm2; Figure 2E ). Examination of dbl-GATA mice revealed lung tissue devoid of eosinophils, as measured by the absence of MBP+ cells (Figure 2F ; data not shown).

Effect of Eosinophils and CCR3 on IL-13-Induced Airway Inflammation

Total BALF cells recovered from the airways of CC10-iIL13 wild-type mice after 4 weeks of transgene expression were significantly increased (ninefold) compared with controls (Figure 3) . Cell recruitment into the airway in response to IL-13 transgene expression was impaired in the absence of CCR3 expression or eosinophils (Figure 3) . The reduction in total inflammatory cell accumulation suggested a role, either directly or indirectly, for eosinophils and CCR3 signal transduction in chemoattraction of leukocytes other than eosinophils in IL-13-induced airway disease. As such, we next examined the effect of eosinophil and CCR3 deficiency on IL-13-induced chemoattraction of other cell types. Neutrophil and lymphocyte accumulation in the airway in response to IL-13 transgene expression was unchanged in the CCR3KO or dbl-GATA mice when compared with wild-type control mice (data not shown). However, airway macrophage accumulation after 4 weeks of transgene expression was significantly reduced in both the CCR3KO and the dbl-GATA mice when compared with the wild-type control mice (3.3 ?? 0.6 x 105 wild-type versus 1.8 ?? 0.3 x 105 CCR3KO BAL macrophages, n = 3 experiments).

Figure 3. IL-13 transgene expression results in leukocyte recruitment that is partially regulated by eosinophils and CCR3. IL-13 transgene-induced total leukocyte accumulation (mean ?? SEM) in the airways of wild-type (WT, white bars), CCR3KO (black bars), and dbl-GATA (dbl-Gata, gray bars) mice; n = 3 experiments. *P 0.05 when compared with wild-type control mice.

Effect of Eosinophils and CCR3 on IL-13-Induced Mucus Production

We next examined the role of eosinophils and CCR3 on IL-13-induced mucus production. Examination of PAS+ cells in the bronchial epithelium of CCR3-deficient mice revealed a significant decrease (40 ?? 5%, n = 3 experiments, P < 0.001) in mucus production after 4 weeks of IL-13 transgene expression compared with wild-type control mice (Figure 4, A and B) . Notably, a marked reduction in PAS+ epithelium was also observed in the dbl-GATA mice (37 ?? 9%, n = 3 experiments; Figure 4, A and B ).

Figure 4. IL-13 transgene expression results in mucus cell metaplasia that is partially eosinophil and CCR3-dependent. PAS staining for mucus (A) and quantitation of PAS+ cells (mean ?? SEM; B) in airways of CC10-iIL13 wild-type (WT), CCR3-deficient (CCR3KO), and dbl-GATA (dbl-Gata) mice after 4 weeks on dox food; n = 3 experiments with four to six mice per group per experiment. *P 0.001 when compared with wild-type control mice.

Effect of Eosinophils and CCR3 on IL-13-Induced Collagen Deposition

To examine the effect of eosinophil and CCR3 deficiency on IL-13-induced lung fibrosis, we quantitated the area of collagen deposition surrounding large airways in the lungs of CC10-iIL13 wild-type, CCR3KO, and dbl-GATA mice. Four weeks of IL-13 transgene expression resulted in a threefold increase in area of collagen deposition surrounding large airways in the lungs of CC10-iIL13 wild-type mice (from 26.2 ?? 8.5 to 77.9 ?? 13 µm2/µm basement membrane). The absence of CCR3 or eosinophils resulted in a 47 to 50% reduction in IL-13-induced collagen deposited around large airways (Figure 5, A and B) . The fibrogenic effects of IL-13 have been proposed to be mediated by TGF-ß1; eosinophils, along with macrophages and epithelial cells, are sources of TGF-ß1.31 As such, we next determined whether protein levels of active BAL TGF-ß1 were altered in the absence of CCR3 or eosinophils. CCR3 deficiency had no effect on the protein levels of IL-13-induced active BAL TGF-ß1 compared with wild-type controls (Figure 5C) . In contrast, eosinophil deficiency resulted in a significantly reduced accumulation of active TGF-ß1 in the BALF of dbl-GATA mice (Figure 5C) .

Figure 5. IL-13 transgene expression results in collagen deposition that is partially eosinophil and CCR3-dependent. A: Collagen deposition as shown in Masson??s trichrome-stained lungs from wild-type (WT), CCR3-deficient (CCR3KO), and dbl-GATA (dbl-Gata) CC10-iIL13 mice after being fed dox food for 4 weeks. Each panel is an individual mouse. Magnification, x100. B: Quantitation (mean ?? SEM) of area of collagen staining surrounding airways of CC10-iIL13 wild-type (WT), CCR3-deficient (CCR3KO), and dbl-GATA (dbl-Gata) mice after 4 weeks on dox food; n = 2 to 3 experiments. *P 0.04 when compared with wild-type control mice. C: TGF-ß1 protein levels (mean ?? SEM) were measured in the BALF of CC10-iIL13 wild-type (WT), CCR3-deficient (CCR3KO), and dbl-GATA (dbl-Gata) mice after 4 weeks on dox food; n = 8 to 14 mice/group (derived from two to three experiments). *P 0.02 when compared with wild-type control mice.

Effect of Eosinophils and CCR3 on IL-13-Induced Protease Expression

Previous studies have shown that IL-13 induces the expression of a number of proteases that contribute to IL-13-induced inflammation and lung remodeling.6,16 We were next interested in the contribution of eosinophils to IL-13-induced protease expression. The levels of mRNA encoding cathepsin B, cathepsin K, cathepsin S, matrix metalloproteinase (MMP)-12, MMP-13, MMP-19, and Adam-8 were increased after 4 weeks of IL-13 expression (Table 2) . Eosinophil deficiency resulted in significantly decreased levels of mRNA encoding for cathepsin B, cathepsin S, and MMP-13 (Table 2) .

Table 2. Quantitative Analysis of Protease Expression (pg x 10C3) in Lung (Normalized to Glyceraldehyde-3-Phosphate Dehydrogenase)

Discussion

We generated an inducible transgenic system that targets IL-13 expression to the lungs of wild-type mice and mice deficient in eosinophils, CCR3, eotaxin-1, or eotaxin-2. First, we demonstrated that airway eosinophilia induced by IL-13 is CCR3- and eotaxin-2-dependent. Second, we established a role for eotaxin-1 in negatively regulating airway eosinophil accumulation, as demonstrated by an increase in eosinophils in the lung lumen in the absence of eotaxin-1. Third, we established that IL-13-induced lung tissue eosinophilia is CCR3-dependent and partially eotaxin-1-dependent. Fourth, we reveal a role for eosinophils and CCR3 in amplifying IL-13-induced mucus and collagen production. Finally, we identified a contributory role for eosinophils in regulating IL-13-induced TGF-ß1 and protease expression.

IL-13-induced inflammation has been shown to be regulated by a number of different mediators, including chemokines. Signal transduction through CCR1 and CCR2 is important in IL-13 effector pathways because deficiency in these cytokine receptors resulted in decreased IL-13-induced pulmonary inflammation associated with macrophages.15,36 Mechanistic analysis revealed attenuated IL-13-induced chemokine expression in the absence of CCR1 expression.36 Although IL-13 has been shown to be a potent inducer of the eosinophil chemoattractants, CCL11/eotaxin-1 and CCL24/eotaxin-2, and lung eosinophilia, the contribution of these chemokines, their receptor CCR3, and eosinophils in IL-13-mediated lung inflammation has remained largely unexplored.15 Our studies addressed this deficiency by demonstrating that chronic IL-13 exposure induces production of CCR3 and its ligands, resulting in eosinophil recruitment, and that these events have a central role in IL-13-mediated lung remodeling in vivo. In addition, because eosinophils have been shown to produce IL-13,37,38 these observations allow for speculation that an IL-13- and CCR3-feedback loop exists in IL-13-mediated responses. In these responses, IL-13 would induce CCR3 ligand elaboration by resident lung cells and macrophages; this would, in turn, induce tissue eosinophilia and production of eosinophil-derived IL-13. These amplification events could contribute to the intensity and persistence of the inflammatory response in chronic lung diseases such as asthma and chronic obstructive pulmonary disease.

Our current study establishes that IL-13-induced lung eosinophilia is differentially regulated by eotaxin-1 compared with eotaxin-2. In particular, we demonstrate that eotaxin-2 is crucial for IL-13-induced airway eosinophilia. We also demonstrate that eotaxin-1 is more important for lung tissue eosinophilia because peribronchial eosinophil accumulation was reduced in eotaxin-1-deficient mice. Interestingly, there was a significant increase in airway eosinophil accumulation in the absence of eotaxin-1, suggesting that eotaxin-1 may be critical for retention of eosinophils in the lung tissue. This finding is compatible with a prior study from our laboratory in which acute IL-13-induced lung eosinophilia, but not airway eosinophilia, was shown to be dependent on eotaxin-1. It is interesting to note that in that model, the relative level of eosinophils in the lung of eotaxin-1-deficient mice was increased compared with wild-type mice.32 Our current study describes a mechanism by which airway eosinophilia is regulated by localized expression of the eotaxin chemokines. Notably, CCR3 was required for both airway and peribronchial eosinophilia in our transgenic mice, demonstrating a cooperative mechanism for CCR3 and its ligands in directing the recruitment and localization of pulmonary eosinophils. Further studies are needed to identify the downstream mechanism, such as alteration in adhesion molecules on resident lung cells by the eotaxin chemokines, in the cooperative orchestration of eosinophil localization in the asthmatic lung.39,40 Because lung tissue eosinophilia was only partially eotaxin-1-dependent and was independent of eotaxin-2 expression, other CCR3 ligands must contribute to the peribronchial eosinophil accumulation. Taken together, these results support an essential role for CCR3 signaling in orchestrating IL-13-mediated leukocyte recruitment and spatial localization into the lung.

Our data suggest that eosinophils and CCR3 have a fundamental role in amplifying IL-13-mediated lung remodeling in a chronic model of cytokine exposure. To identify the causative relationship between eosinophils and the onset of IL-13-induced lung pathologies, we used a mouse line harboring a deletion of a high-affinity double palindromic GATA binding site in the GATA-1 promoter (dbl-GATA), which led to the specific ablation of the eosinophil lineage.41 After chronic IL-13 exposure, we did not find MBP+ cells in the lungs of the dbl-GATA mice, suggesting that the lungs were devoid of eosinophils, because the granule protein MBP has been shown to be the core of eosinophil granules42,43 ; however, we cannot exclude the possibility of a granule-less eosinophil in these genetically altered mice. In this study, we focused on the role of eosinophils in lung remodeling; future studies are warranted to examine airway hyper-reactivity induced in our chronic model. We observed a significant decrease in the percentage of PAS+ airway epithelial cells in response to chronic IL-13 expression in the CCR3-deficient and the dbl-GATA mice. Although our data are consistent with prior studies, a recent study using the dbl-GATA mice in an allergen-induced experimental asthma model showed no reduction in mucus staining with eosinophil deficiency.33,34,44,45 The differences in experimental protocol (chronic and continuous cytokine exposure versus allergen challenges) may explain the divergent results. Neutrophils have been implicated in IL-13-induced mucin production through activation of epidermal growth factor receptor signaling.46,47 Our studies offer a parallel example of how myeloid cells regulate mucus production in the lung. We also demonstrate a significant reduction in IL-13-mediated collagen deposition surrounding the large airways in CCR3-deficient and dbl-GATA mice. TGF-ß1 has been shown to be a critical mediator of the fibrotic effects of IL-13.31 Although we found significantly reduced levels of active TGF-ß1 in the BAL of dbl-GATA mice, there were no significant differences between wild-type and CCR3-deficient mice, suggesting that multiple mechanisms, including those that do not use TGB-ß1-induced fibrosis, are operational in IL-13-mediated remodeling the lung. Expression of MMP-13, cathepsin S, and cathepsin B was significantly attenuated in the absence of eosinophils. Apart from digesting components of the ECM, metalloproteinases modulate the activity of other proteases and cytokines, including chemokines and TGF-ß1, which are important in lung diseases.48 In addition, cathepsin S and MMP-13 have collagenase activity, suggesting an important role for eosinophils in regulating fibrotic tissue responses.49,50

Taken together, we provide multiple lines of evidence that eosinophils are prominent effector cells involved with multiple parameters of IL-13-associated diseases, including goblet cell metaplasia and collagen deposition. Our chronic model uses an inducible transgenic system that results in expression of high levels of cytokine in the lung compared with levels that are likely operational in patients; such analyses have been shown to be useful for establishing pathophysiological paradigms and uncovering molecular insight into possible disease pathogenesis, as documented by a variety of similar transgenic systems.6,31,51-53 As such, our results highlight the need to develop effective clinical reagents (such as CCR3 antagonists substantiated by our present findings) that block or deplete lung eosinophils to test the compelling eosinophil hypothesis in asthma.

Acknowledgements

We thank Dr. Fred Finkelman and Nikolaos Nikolaidis for helpful discussions; Drs. Jeffrey Whitsett, Jamie Lee, Nancy Lee, Allison Humbles, and Craig Gerard for critical reagents; and Andrea Lippelman for assistance with preparation of this manuscript.

【参考文献】
  Jeffery PK: Remodeling in asthma and chronic obstructive lung disease. Am J Respir Crit Care Med 2001, 164:S28-S38

Fahy JV, Corry DB, Boushey HA: Airway inflammation and remodeling in asthma. Curr Opin Pulm Med 2000, 6:15-20

Bousquet J, Chanez P, Lacoste JY, White R, Vic P, Godard P, Michel FB: Asthma: a disease remodeling the airways. Allergy 1992, 47:3-11

Beckett PA, Howarth PH: Pharmacotherapy and airway remodelling in asthma? Thorax 2003, 58:163-174

Wills-Karp M, Luyimbazi J, Xu X, Schofield B, Neben TY, Karp CL, Donaldson DD: Interleukin-13: central mediator of allergic asthma. Science 1998, 282:2258-2261

Zheng T, Zhu Z, Wang Z, Homer RJ, Ma B, Riese RJ, Jr, Chapman HA, Jr, Shapiro SD, Elias JA: Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsin-dependent emphysema. J Clin Invest 2000, 106:1081-1093

Blease K, Jakubzick C, Westwick J, Lukacs N, Kunkel SL, Hogaboam CM: Therapeutic effect of IL-13 immunoneutralization during chronic experimental fungal asthma. J Immunol 2001, 166:5219-5224

Yang M, Hogan SP, Henry PJ, Matthaei KI, McKenzie AN, Young IG, Rothenberg ME, Foster PS: Interleukin-13 mediates airways hyperreactivity through the IL-4 receptor-alpha chain and STAT-6 independently of IL-5 and eotaxin. Am J Respir Cell Mol Biol 2001, 25:522-530

Kumar RK, Herbert C, Yang M, Koskinen AM, McKenzie AN, Foster PS: Role of interleukin-13 in eosinophil accumulation and airway remodelling in a mouse model of chronic asthma. Clin Exp Allergy 2002, 32:1104-1111

Kuperman DA, Huang X, Koth LL, Chang GH, Dolganov GM, Zhu Z, Elias JA, Sheppard D, Erle DJ: Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nat Med 2002, 8:885-889

Kumar RK, Herbert C, Webb DC, Li L, Foster PS: Effects of anticytokine therapy in a mouse model of chronic asthma. Am J Respir Crit Care Med 2004, 170:1043-1048

Leigh R, Ellis R, Wattie JN, Hirota JA, Matthaei KI, Foster PS, O??Byrne PM, Inman MD: Type 2 cytokines in the pathogenesis of sustained airway dysfunction and airway remodeling in mice. Am J Respir Crit Care Med 2004, 169:860-867

Zhu Z, Homer RJ, Wang Z, Chen Q, Geba GP, Wang J, Zhang Y, Elias JA: Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest 1999, 103:779-788

Hershey GK: IL-13 receptors and signaling pathways: an evolving web. J Allergy Clin Immunol 2003, 111:677-690

Zhu Z, Ma B, Zheng T, Homer RJ, Lee CG, Charo IF, Noble P, Elias JA: IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13-induced inflammation and remodeling. J Immunol 2002, 168:2953-2962

Lanone S, Zheng T, Zhu Z, Liu W, Lee CG, Ma B, Chen Q, Homer RJ, Wang J, Rabach LA, Rabach ME, Shipley JM, Shapiro SD, Senior RM, Elias JA: Overlapping and enzyme-specific contributions of matrix metalloproteinases-9 and -12 in IL-13-induced inflammation and remodeling. J Clin Invest 2002, 110:463-474

Shinkai A, Yoshisue H, Koike M, Shoji E, Nakagawa S, Saito A, Takeda T, Imabeppu S, Kato Y, Hanai N, Anazawa H, Kuga T, Nishi T: A novel human CC chemokine, eotaxin-3, which is expressed in IL-4-stimulated vascular endothelial cells, exhibits potent activity toward eosinophils. J Immunol 1999, 163:1602-1610

Rothenberg ME: Eotaxin: an essential mediator of eosinophil trafficking into mucosal tissues. Am J Respir Cell Mol Biol 1999, 21:291-295

Yang M, Hogan SP, Mahalingam S, Pope SM, Zimmermann N, Fulkerson P, Dent LA, Young IG, Matthaei KI, Rothenberg ME, Foster PS: Eotaxin-2 and IL-5 cooperate in the lung to regulate IL-13 production and airway eosinophilia and hyperreactivity. J Allergy Clin Immunol 2003, 112:935-943

Gao JL, Sen AI, Kitaura M, Yoshie O, Rothenberg ME, Murphy PM, Luster AD: Identification of a mouse eosinophil receptor for the CC chemokine eotaxin. Biochem Biophys Res Commun 1996, 223:679-684

Daugherty BL, Siciliano SJ, DeMartino JA, Malkowitz L, Sirotina A, Springer MS: Cloning, expression, and characterization of the human eosinophil eotaxin receptor. J Exp Med 1996, 183:2349-2354

Grimaldi JC, Yu NX, Grunig G, Seymour BW, Cottrez F, Robinson DS, Hosken N, Ferlin WG, Wu X, Soto H, O??Garra A, Howard MC, Coffman RL: Depletion of eosinophils in mice through the use of antibodies specific for C-C chemokine receptor 3 (CCR3). J Leukoc Biol 1999, 65:846-853

Justice JP, Borchers MT, Crosby JR, Hines EM, Shen HH, Ochkur SI, McGarry MP, Lee NA, Lee JJ: Ablation of eosinophils leads to a reduction of allergen-induced pulmonary pathology. Am J Physiol 2003, 284:L169-L178

Humbles AA, Lu B, Friend DS, Okinaga S, Lora J, Al Garawi A, Martin TR, Gerard NP, Gerard C: The murine CCR3 receptor regulates both the role of eosinophils and mast cells in allergen-induced airway inflammation and hyperresponsiveness. Proc Natl Acad Sci USA 2002, 99:1479-1484

Ma W, Bryce PJ, Humbles AA, Laouini D, Yalcindag A, Alenius H, Friend DS, Oettgen HC, Gerard C, Geha RS: CCR3 is essential for skin eosinophilia and airway hyperresponsiveness in a murine model of allergic skin inflammation. J Clin Invest 2002, 109:621-628

Pope SM, Zimmermann N, Stringer KF, Karow ML, Rothenberg ME: The eotaxin chemokines and CCR3 are fundamental regulators of allergen-induced pulmonary eosinophilia. J Immunol 2005, 175:5341-5350

Elsner J, Hochstetter R, Kimmig D, Kapp A: Human eotaxin represents a potent activator of the respiratory burst of human eosinophils. Eur J Immunol 1996, 26:1919-1925

Tenscher K, Metzner B, Schopf E, Norgauer J, Czech W: Recombinant human eotaxin induces oxygen radical production, Ca(2+)-mobilization, actin reorganization, and CD11b upregulation in human eosinophils via a pertussis toxin-sensitive heterotrimeric guanine nucleotide-binding protein. Blood 1996, 88:3195-3199

Kampen GT, Stafford S, Adachi T, Jinquan T, Quan S, Grant JA, Skov PS, Poulsen LK, Alam R: Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases. Blood 2000, 95:1911-1917

Robinson DS, Kay AB, Wardlaw AJ: Eosinophils. Clin Allergy Immunol 2002, 16:43-75

Lee CG, Homer RJ, Zhu Z, Lanone S, Wang X, Koteliansky V, Shipley JM, Gotwals P, Noble P, Chen Q, Senior RM, Elias JA: Interleukin-13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor beta(1). J Exp Med 2001, 194:809-821

Pope SM, Fulkerson PC, Blanchard C, Akei HS, Nikolaidis NM, Zimmermann N, Molkentin JD, Rothenberg ME: Identification of a cooperative mechanism involving interleukin-13 and eotaxin-2 in experimental allergic lung inflammation. J Biol Chem 2005, 280:13952-13961

Lee JJ, Dimina D, Macias MP, Ochkur SI, McGarry MP, O??Neill KR, Protheroe C, Pero R, Nguyen T, Cormier SA, Lenkiewicz E, Colbert D, Rinaldi L, Ackerman SJ, Irvin CG, Lee NA: Defining a link with asthma in mice congenitally deficient in eosinophils. Science 2004, 305:1773-1776

Humbles AA, Lloyd CM, McMillan SJ, Friend DS, Xanthou G, McKenna EE, Ghiran S, Gerard NP, Yu C, Orkin SH, Gerard C: A critical role for eosinophils in allergic airways remodeling. Science 2004, 305:1776-1779

Fulkerson PC, Fischetti CA, Hassman LM, Nikolaidis NM, Rothenberg ME: Persistent effects induced by IL-13 in the lung. Am J Respir Cell Mol Biol 2006, 35:337-346

Ma B, Zhu Z, Homer RJ, Gerard C, Strieter R, Elias JA: The C10/CCL6 chemokine and CCR1 play critical roles in the pathogenesis of IL-13-induced inflammation and remodeling. J Immunol 2004, 172:1872-1881

Schmid-Grendelmeier P, Altznauer F, Fischer B, Bizer C, Straumann A, Menz G, Blaser K, Wuthrich B, Simon HU: Eosinophils express functional IL-13 in eosinophilic inflammatory diseases. J Immunol 2002, 169:1021-1027

Gessner A, Mohrs K, Mohrs M: Mast cells, basophils, and eosinophils acquire constitutive IL-4 and IL-13 transcripts during lineage differentiation that are sufficient for rapid cytokine production. J Immunol 2005, 174:1063-1072

Burke-Gaffney A, Hellewell PG: A CD18/ICAM-1-dependent pathway mediates eosinophil adhesion to human bronchial epithelial cells. Am J Respir Cell Mol Biol 1998, 19:408-418

Tachimoto H, Kikuchi M, Hudson SA, Bickel CA, Hamilton RG, Bochner BS: Eotaxin-2 alters eosinophil integrin function via mitogen-activated protein kinases. Am J Respir Cell Mol Biol 2002, 26:645-649

Yu C, Cantor AB, Yang H, Browne C, Wells RA, Fujiwara Y, Orkin SH: Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo. J Exp Med 2002, 195:1387-1395

Larson KA, Horton MA, Madden BJ, Gleich GJ, Lee NA, Lee JJ: The identification and cloning of a murine major basic protein gene expressed in eosinophils. J Immunol 1995, 155:3002-3012

Peters MS, Rodriguez M, Gleich GJ: Localization of human eosinophil granule major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin by immunoelectron microscopy. Lab Invest 1986, 54:656-662

Cohn L, Homer RJ, MacLeod H, Mohrs M, Brombacher F, Bottomly K: Th2-induced airway mucus production is dependent on IL-4Ralpha, but not on eosinophils. J Immunol 1999, 162:6178-6183

Shen HH, Ochkur SI, McGarry MP, Crosby JR, Hines EM, Borchers MT, Wang H, Biechelle TL, O??Neill KR, Ansay TL, Colbert DC, Cormier SA, Justice JP, Lee NA, Lee JJ: A causative relationship exists between eosinophils and the development of allergic pulmonary pathologies in the mouse. J Immunol 2003, 170:3296-3305

Shim JJ, Dabbagh K, Ueki IF, Dao-Pick T, Burgel PR, Takeyama K, Tam DC, Nadel JA: IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils. Am J Physiol 2001, 280:L134-L140

Takeyama K, Dabbagh K, Lee HM, Agusti C, Lausier JA, Ueki IF, Grattan KM, Nadel JA: Epidermal growth factor system regulates mucin production in airways. Proc Natl Acad Sci USA 1999, 96:3081-3086

Shapiro SD, Senior RM: Matrix metalloproteinases: matrix degradation and more. Am J Respir Cell Mol Biol 1999, 20:1100-1102

Wolters PJ, Chapman HA: Importance of lysosomal cysteine proteases in lung disease. Respir Res 2000, 1:170-177

Parks WC, Shapiro SD: Matrix metalloproteinases in lung biology. Respir Res 2001, 2:10-19

Lee JJ, McGarry MP, Farmer SC, Denzler KL, Larson KA, Carrigan PE, Brenneise IE, Horton MA, Haczku A, Gelfand EW, Leikauf GD, Lee NA: Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma. J Exp Med 1997, 185:2143-2156

Rankin JA, Picarella DE, Geba GP, Temann UA, Prasad B, DiCosmo B, Tarallo A, Stripp B, Whitsett J, Flavell RA: Phenotypic and physiologic characterization of transgenic mice expressing interleukin 4 in the lung: lymphocytic and eosinophilic inflammation without airway hyperreactivity. Proc Natl Acad Sci USA 1996, 93:7821-7825

Zhang L, Ikegami M, Dey CR, Korfhagen TR, Whitsett JA: Reversibility of pulmonary abnormalities by conditional replacement of surfactant protein D (SP-D) in vivo. J Biol Chem 2002, 277:38709-38713

作者单位：From the Departments of Molecular Genetics, Biochemistry, and Microbiology,* and the Division of Allergy and Immunology, Cincinnati Children??s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio