American Journal of Respiratory Cell and Molecular Biology

The production of the inflammatory cytokine interleukin (IL)-1 is increased in lungs of patients with chronic obstructive pulmonary disease (COPD) or asthma. To characterize the in vivo actions of IL-1 in the lung, transgenic mice were generated in which human IL-1β was expressed in the lung epithelium with a doxycycline-inducible system controlled by the rat Clara cell secretory protein (CCSP) promoter. Induction of IL-1β expression in the lungs of adult mice caused pulmonary inflammation characterized by neutrophil and macrophage infiltrates. IL-1β caused distal airspace enlargement, consistent with emphysema. IL-1β caused disruption of elastin fibers in alveolar septa and fibrosis in airway walls and in the pleura. IL-1β increased the thickness of conducting airways, enhanced mucin production, and caused lymphocytic aggregates in the airways. Decreased immunostaining for the winged helix transcription factor FOXA2 was associated with goblet cell hyperplasia in IL-1β–expressing mice. The production of the neutrophil attractant CXC chemokines KC (CXCL1) and MIP-2 (CXCL2), and of matrix metalloproteases MMP-9 and MMP-12, was increased by IL-1β. Chronic production of IL-1β in respiratory epithelial cells of adult mice causes lung inflammation, enlargement of distal airspaces, mucus metaplasia, and airway fibrosis in the adult mouse.

Chronic obstructive pulmonary disease (COPD) is a major cause of chronic morbidity and mortality throughout the world. It is currently the fourth leading cause of death in the world and is projected to rank fifth as a worldwide burden of disease by 2020 (1). Although cigarette smoking is the most important risk factor for the development of this illness, exposure to other noxious agents can also initiate an inflammatory reaction in the lung that then leads to COPD.

COPD is characterized by chronic inflammation throughout the airways, parenchyma, and pulmonary vasculature (1). The inflammatory response, where neutrophils, macrophages, and lymphocytes are increased in the airways and parenchyma, does not resolve after smoking cessation (2). Enlargement of mucus-secreting glands and increases in the number of goblet cells are associated with mucus hypersecretion in COPD. Chronic inflammation in the bronchi leads to remodeling of the airway wall, with increasing collagen content. Destruction of the lung parenchyma leads to emphysema, defined as abnormal enlargement of the distal airspaces. The early changes of emphysema include disruption of elastic fibers that then leads to the loss of the alveolar septa (3). At the same time, collagen synthesis takes place, leading to increased collagen in the septa (4).

Inflammation is also a cornerstone of asthma, a major chronic lung disease whose prevalence has increased rapidly in the recent years. The main pathologic features of asthma include mucus metaplasia, subepithelial fibrosis, airway wall thickening, and smooth muscle cell hypertrophy (3). The distinction between COPD and asthma is not absolute, because many patients manifest features of both of these disorders (3).

Interleukin (IL)-1 is one of the major cytokines involved in the initiation and persistence of inflammation (5). Enhanced production of IL-1 is documented in stable COPD, with further increase during exacerbations of the illness (6). Alveolar macrophages and peripheral blood mononuclear cells from smokers produce more IL-1 than those from nonsmokers (7, 8). When exposed to cigarette smoke, human bronchial epithelial cells from patients with COPD respond with a greater increase of IL-1 production than those of nonsmokers (9). Bronchoalveolar lavage (BAL) fluid from smokers contains higher levels of IL-1β than BAL fluid from nonsmokers, and the levels of IL-1β are related to the smokers' lung function (10). Moreover, haplotypes of IL-1β and of the naturally occurring IL-1 antagonist IL-1 receptor antagonist (IL-1ra) are different in smokers with rapidly declining lung function than in smokers with normal lung function (11), suggesting that IL-1 participates in the progression of lung disease in smokers. Increased expression of IL-1 by bronchial epithelium, increased numbers of IL-1–producing macrophages in the bronchial submucosa, and elevated levels of IL-1 in bronchoalveolar lavage fluid as well as in tracheal biopsy samples have been reported in asymptomatic and symptomatic individuals with asthma (12, 13). The roles of IL-1 in COPD or asthma are not defined, however, and the actions of sustained IL-1 production in the lung in vivo have not been studied in an experimental model.

IL-1 denotes two related polypeptides, IL-1α and IL-1β, that are products of separate genes and have different amino acid sequences, but share the same receptors and have similar activities. The macrophage-monocyte is a primary source of IL-1, but many other cells, including fibroblasts, T cells, neutrophils, and bronchial and alveolar epithelial cells, also produce IL-1 (5, 14, 15). IL-1β is synthesized as 31-kD precursors, proIL-1β. ProIL-1β essentially lacks biological effects. The active, mature IL-1β is produced upon cleavage of proIL-1β by a specific IL-1β–converting enzyme (ICE or caspase-1) (5) or by proteases such as matrix metalloprotease-9 (MMP-9 or gelatinase B) (16).

To determine the in vivo effector functions of IL-1β in the lung, we developed a transgenic mouse expressing mature human IL-1β in the lung epithelium in an externally regulatable manner. In this model, production of IL-1β is induced in the lung at chosen time points by doxycycline administration. These studies demonstrate that IL-1β is sufficient to induce lung inflammation, enlargement of distal airspaces, mucus metaplasia, and airway thickening and fibrosis in the adult mouse. IL-1β enhanced the production of neutrophil-attractant CXC chemokines KC (CXCL1) and MIP-2 (CXCL2) as well as of matrix metalloproteases MMP-9 and MMP-12 in the lungs.

Transgenic Mice

Human IL-1β cDNA (0.58 kb), coding for the active, mature IL-1β protein, was generated by RT-PCR from total RNA isolated from LPS-stimulated peripheral blood leukocytes. Protein translation start codon and 22 nucleotides of the 5′ untranslated region flanking it were incorporated into the forward PCR primer. The primer sequences (5′ to 3′) used were: forward primer, for the first 10 PCR cycles, TGC TCA AGT GTC TGA AGC AGC CAT GGC ACC TGT ACG ATC ACT GA, and reverse primer, TCC ACA TTC AGC ACA GGA CTC TCT G. Then 1 μl of the 25-μl reaction was further amplified using the forward primer TGC TCA AGT GTC TGA AGC AGC CA and the above reverse primer. The PCR product was gel purified and cloned using the pCR2.1-TOPO cloning system (Invitrogen, Carlsbad, CA) and sequenced. The insert was subcloned into the EcoRV site of the construct vector (17) between the (tet-O)7CMV minimal promoter and bovine growth hormone (bGH) polyadenylation signal sequence. The construct was verified by restriction enzyme digestion and sequencing. The (tetO)7CMV-hIL-1β-bGH DNA fragment was then isolated from the plasmid by digestion with AscI, and used for generation of transgenic mice by microinjection into mouse oocytes. Transgenic founder mice were identified by Southern analysis, and the results confirmed by PCR as described below.

To produce bitransgenic mice with regulatable IL-1β production, IL-1β transgene–positive mice were mated with mice bearing the CCSP-rtTA activator transgene (18) that express the reverse tetracycline transactivator (rtTA) under the control of the rat Clara cell secretory protein (rCCSP) promoter. Genotyping was done by PCR using primers specific for transgene constructs as follows. CCSP-rtTA transgene primers: forward primer in rCCSP promoter: ACT GCC CAT TGC CCA AAC AC, reverse primer in rtTA coding region: AAA ATC TTG CCA GCT TTC CCC ; (tetO)7CMV-hIL-1β-bGH transgene: forward primer in CMV minimal promoter: CCA TCC ACG CTG TTT TGA CC, reverse primer in IL-1β coding region: ACG GGC ATG TTT TCT GCT TG.

Animal Use and Administration of Doxycycline

The mice were housed in pathogen-free conditions according to protocols approved by Institutional Animal Care and Use Committee at Cincinnati Children's Hospital Research Foundation and the Animal Research Ethics Committee at Goteborg University. To induce IL-1β transgene expression in bitransgenic animals, the mice were given doxycycline (Sigma, St. Louis, MO) in drinking water at a concentration of 0.5 mg/ml for specified time periods; the solution was changed three times per week.

RNA Extraction and RT-PCR

Total RNA from lung and other tissue was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions, and treated with RNase-free DNase. RNA was reverse transcribed and cDNA was analyzed by PCR and agarose gel electrophoresis for human IL-1β, using transgene-specific primers described above, and β-actin mRNA. For quantification of MMP-9, MMP-12, KC, and MIP-2 mRNA levels in total lung RNA, RT-PCR was performed on 100 ng of total RNA in a single-tube reaction using Tth DNA polymerase (RNA Master SYBR Green I kit), according to the manufacturer's instructions (Roche, Mannheim, Germany) and Light Cycler real-time PCR instrument (Roche). Relative quantitation of starting amounts of mRNA was performed from amplification curves with Light Cycler software using standard curves from dilution series of RNA. The results were normalized to β-actin mRNA levels. Primer sequences (forward and reverse, 5′ to 3′) used were as follows: MMP-9: TCC GCA GAC CAA GAG GGT TTT C, AAG ATG TCG TGT GAG TTC CAG GGC; MMP-12: CCT GGA CCT GGT ATT CAA GGA GAT G, TTT GTC AAG GAT GGG GGT TTC AC; KC: GCA CCC AAA CCG AAG TCA TAG C, TTG TCA GAA GCC AGC GTT CAC C; MIP-2: CCC CCT GGT TCA GAA AAT CAT C, AAC TCT CAG ACA GCG AGG CAC ATC.

Histology and Immunohistochemistry

Mice were anesthesized by intraperitoneal injection of a mixture of ketamine, xylazine, and acepromazine, the abdomen was opened, and the animals were exsanguinated by transection of the abdominal aorta. The chest cavity was opened and the trachea exposed. A blunt cannula was inserted and tied to the trachea, and the lungs were inflated by instillation of 4% phosphate-buffered paraformaldehyde at a pressure of 25 cm H2O. After overnight fixation at 4°C, the tissue was processed through conventional paraffin embedding. Lung volume of selected animals was measured before tissue dehydration by volume displacement. Five-micrometer tissue sections were stained with hematoxylin and eosin, Masson's trichrome stain, periodic acid Schiff, or orcein stain (Sigma), using methods described (19). For immunohistochemistry, the primary antibodies were: chicken anti-mouse MUC5AC (20), goat anti-mouse MMP-9 (R&D Systems, Abingdon, UK), goat anti-mouse MMP-12 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse KC (Abcam, Cambridge, UK), goat anti-mouse MIP-2 (Abcam), and sheep anti-mouse FOXA2 (21). Biotinylated secondary antibodies and avidin–biotin peroxidase with DAB-nickel chloride (Vectastain Elite ABC) were used according to manufacturer's instructions (Vector Laboratories, Burlingame, CA). After staining for FOXA2, lung sections were counterstained for acid mucins with Alcian Blue pH 2.5 method.

Morphometric Analysis

Quantitation of distal airspace was performed from hematoxylin and eosin–stained lung tissue sections, using the mean chord length as a measure of alveolar size as described (22). A minimum of 10 representative fields from lungs of three mice of each genotype and treatment group were acquired in 8-bit grayscale at a final magnification of 1.89 pixels per micrometer using a Nikon Eclipse E800 microscope and DXM1200 digital camera (Nikon, Tokyo, Japan). Areas of bronchiolar airways and blood vessels were excluded from the analysis. Chord length analysis was performed using the public domain program NIH Image with a chord length macro (available from the US National Institutes of Health at http://rsb.info.nih.gov/nih-image). Binarized, inverted lung micrographs were subjected to a logical “AND” operation with horizontal and vertical grids of straight lines. The length of lines overlying alveolar space was then averaged as the mean chord length.

BAL and Lung Homogenate Studies

Mice were killed and a cannula attached to the trachea as described above. Lungs were lavaged with three 1-ml aliquots of sterile PBS, instilled, and aspirated with syringe three times for each aliquot. The aliquots were pooled and the volume was measured. Cells were separated from BAL fluid by centrifugation and resuspended in sterile PBS. Viable cells were counted by trypan blue exclusion using a hemacytometer and cells were cytocentrifuged on microscope slides. Differential cell counts were performed on slides stained with Diff-Quik (Scientific Products, McGaw Park, IL). Lungs were harvested and lung tissue was homogenized in PBS. Concentration of human IL-1β in lung homogenate and BAL fluid was measured using Quantikine ELISA assay kit according to the manufacturer's instructions (R&D Systems). The assay has detection level 1 pg/ml and does not cross-react with murine IL-1β. Total protein concentration was measured using the bicinchoninic acid method (Sigma).

Statistics

Student's t test was used for comparisons of group averages. Measurement values are expressed as mean ± SEM; P values < 0.05 were considered statistically significant.

Production and Characterization of Transgenic Mice

Three stable lines of IL-1β transgenic mice were generated (denoted in the following as lines K, L, and M). At 6 wk of age, bitransgenic mice were provided water containing doxycycline (0.5 mg/ml) for 8 d and the levels of human IL-1β were measured in BAL fluid and lung homogenates. The total amounts of human IL-1β in the BAL fluid were 46.5 ± 5.6 ng (n = 3), 33.5 ± 9.2 ng (n = 3), and 7.0 ± 2.0 ng (n = 3) in mice from lines K, L, and M, respectively. The levels of human IL-1β in the lung homogenates were 846 ± 150 ng/lung, 523 ± 75 ng/lung, and 40 ± 12 ng/lung in mice from lines K, L, and M, respectively. Thus, only ∼ 5.5–17.5% of the total hIL-1β was in the lavage fluid, suggesting that most of the protein remained intracellular.

IL-1β Increased Inflammatory Cells in BAL Fluid

To determine whether IL-1β induced migration of inflammatory cells into the airways, adult IL-1β bitransgenic mice and transgene negative littermates were given doxycycline for 8 d and inflammatory cells were counted in cytocentrifuge preparates from BAL fluid. IL-1β increased the total number of inflammatory cells 6- to 12-fold compared with control mice (Table 1)

TABLE 1. Inflammatory cells in bronchoalveolar lavage fluid




IL-1β Transgene Line

Control
K
L
M
Total number of cells24,300 ± 2,800298,500 ± 35,700185,300 ± 47,400126,600 ± 23,100
***
Neutrophils680 ± 220212,500 ± 11,000*90,400 ± 8,900*49,000 ± 5,300*
(%)(2.8 ± 0.9)(71.2 ± 3.6)(48.8 ± 4.8)(38.7 ± 4.2)
Alveolar macrophages23,100 ± 60080,900 ± 10,70091,900 ± 8,50074,800 ± 4,600
(%)(95.0 ± 2.3)(27.1 ± 3.5)(49.6 ± 4.6)(59.1 ± 3.6)
Lymphocytes530 ± 1505,100 ± 1,2002,800 ± 1,5002,700 ± 800
(%)
(2.2 ± 0.6)
(1.7 ± 0.4)
(1.5 ± 0.8)
(2.1 ± 0.6)

*P < 0.001 compared with control group.

P < 0.01 compared with control group.

Adult bitransgenic mice of three transgenic lines and controls (wild-type or CCSP-rtTA single transgenic) were given doxycycline water for 8 d and lungs were lavaged. Differential counting was performed on cytocentrifuge preparates of BAL cells. Four animals in each group were studied; the data are expressed as mean ± SEM.

. This was mainly due to a strong influx of neutrophils into the lung; the percentages of neutrophils in the BAL cytospins were 71.2 ± 3.6%, 48.8 ± 4.8%, and 38.7 ± 4.2% in lines K, L, and M, respectively (Table 1). Most of the remaining cells were macrophages. Because of the increased severity of inflammation, line K mice were used for further analyses.

Inducibility of hIL-1β Expression

To study whether production of hIL-1β was induced in the mice by doxycycline administration, adult bitransgenic mice from line K were maintained on water supplemented with doxycycline (0.5 mg/ml) for 2, 4, 6, 8, or 14 d. Levels of hIL-1β mRNA and protein in lung homogenate were determined by RT-PCR and ELISA, respectively (Figure 1A)

. Human IL-1β was not detectable in the lungs of bitransgenic mice drinking normal water. A rapid increase in hIL-1β mRNA expression and protein production occurred between Days 0 and 2 of doxycycline administration and a maximum was reached by 6 d (Figure 1A).

Reversibility of hIL-1 mRNA Expression and Protein Production

Bitransgenic adult mice (line K) were given doxycycline-containing water (0.5 mg/ml) for 8 d, followed by normal water for 1, 3, or 5 d before hIL-1β mRNA and protein were measured. hIL-1β mRNA expression declined rapidly after doxycycline withdrawal (Figure 1B). Three days after doxycycline was removed, hIL-1β protein level in lung homogenate had decreased by > 90% and was undetectable by Day 7 (Figure 1B).

Organ Specificity of hIL-1β mRNA Expression

To assess whether doxycycline-induced hIL-1β expression was lung-specific, bitransgenic animals (line K) were maintained on doxycycline for 1 wk and the presence of hIL-1β mRNA in pulmonary and various other tissues was compared using RT-PCR (Figure 1C). Human IL-1β transgene mRNA was readily detected in lung but not in liver, heart, brain, kidney, or stomach.

Body Weights and Lung Volumes of IL-1–Expressing and Control Mice

Body weights of adult IL-1β–expressing mice did not differ from those of control (wild-type or CCSP-rtTA single transgenic) littermates, as seen in Table 2

TABLE 2. Body weights and lung volumes (mean ± sem) in bitransgenic mice and their (wild-type or ccsp-rtta single transgenic) control littermates




Body Weight (g)

Lung Volume (ml)

Lung Volume/Body
 Weight (ml/kg)
Bitransgenic24.5 ± 0.80.87 ± 0.0435.8 ± 5.6
Control24.4 ± 1.20.71 ± 0.0329.4 ± 3.4
P value
0.94
0.01
0.03

The mice were given doxycycline water from the age of 3 wk until sacrifice at 8 wk. Six animals in both groups were studied. Lung volumes as measured by fluid displacement were significantly higher in bitransgenic mice.

. Lung volumes and lung volume/body weight ratios were significantly increased in bitransgenic mice after 5 wk of doxycycline administration (Table 2).

IL-1β Caused Pulmonary Inflammation, Elastin Degradation, and Collagen Deposition

To study the effects of IL-1 on lung histology, bitransgenic mice (line K) and littermate (wild-type or CCSP-rtTA single transgenic) controls were provided doxycycline water or regular water from 3 wk of age until killing at the age of 8 wk. Increased numbers of inflammatory cells were observed in the alveoli and parenchyma of lungs from bitransgenic mice given doxycycline (Figure 2)

. The alveoli in the lungs of the control mice were normal in size and appearance. In contrast, severe distal airspace enlargement was seen in bitransgenic mice given doxycycline. The mean alveolar cord length, 61.6 ± 4.7 μm in IL-1β–expressing mice, was significantly larger than the chord length in lungs from control animals, 36.6 ± 3.5 μm (Figure 2). Lung histology in bitransgenic mice that were not given doxycycline appeared normal, and the cord length was similar to that of control mice (35.6 ± 2.8 μm versus 34.3 ± 3.9 μm in bitransgenic mice that had not received doxycycline and in control mice, respectively), demonstrating that airspace enlargement did not develop in bitransgenic mice in the absence of doxycycline.

The effect of IL-1β on lung extracellular matrix components was assessed. Elastin fibers in IL-1β–expressing mice were disrupted, frayed, or short compared with those in the lungs of wild-type mice, as seen on orcein stain (Figure 3)

. To assess lung fibrosis, trichrome stain was performed. Lesions that stained intensely for collagen was observed on pleural surfaces of mice expressing IL-1β (Figure 4B), but not in controls. Increased collagen staining was also seen in some alveolar walls (Figure 4D).

IL-1β Caused Airway Thickening, Lymphocytic Nodules, Subepithelial Fibrosis, and Mucus Metaplasia

Airway histology was examined in mice that had been maintained on doxycycline-containing drinking water from 3 wk of age until killing at 8 wk. Airway histology in CCSP–IL-1β bitransgenic mice showed bronchiolar thickening and peribronchiolar lymphocytic nodules, whereas these changes were absent in control mice (Figures 5A and 5B)

. Extensive subepithelial fibrosis was also prominent in IL-1β–expressing mice, whereas only small amounts of collagen was present in airways of controls (Figures 5A and 5B).

To determine whether IL-1β caused abnormalities in airway epithelial cells, lungs of bitransgenic and control mice receiving doxycycline water were stained for MUC5AC and PAS. Whereas MUC5AC or PAS staining cells were rarely seen in wild-type mice (Figures 5C and 5E), MUC5AC- and PAS-positive cells lined the conductive airways of IL-1–expressing mice, as seen in Figures 5D and 5F, showing that IL-1β is a potent inducer of airway mucus metaplasia. Alcian Blue–reactive, mucus-producing cells lacked FOXA2 staining, whereas FOXA2 was detected in nuclei of adjacent, nongoblet, Alcian Blue–negative epithelial cells (Figure 5G).

IL-1β Enhanced the Production of MMP-9 and MMP-12

Because MMP-12 and MMP-9 are involved in the development of emphysema in other models (23, 24), the production of these proteases was studied. Adult bitransgenic mice and littermate controls were given doxycycline water for 1 wk. IL-1β increased MMP-9 and MMP-12 mRNA expression 3.6- and 8.9-fold, respectively, as determined by real-time RT-PCR, and protein production (Figure 6)

. MMP-12–immunopositive material was detected in macrophages within alveolar spaces and within alveolar walls in mice expressing IL-1β, whereas MMP-9 staining was seen in neutrophils in alveoli and airways as well as in areas of subepithelial fibrosis in airway walls (Figure 6).

IL-1β Increased the Production of CXC Chemokines KC and MIP-2

Because neutrophils were a major inflammatory cell infiltrating the lungs of IL-1–expressing mice, we studied the production of neutrophil chemotactic ELR+ CXC chemokines KC and MIP-2 in the lungs of bitransgenic mice and in control littermates given doxycycline for 1 wk. KC and MIP-2 mRNA expression were increased 2.6- and 14.2-fold, respectively, in bitransgenic mice compared with control littermates (Figure 7)

. Increased immunostaining for KC and MIP-2 was observed in IL-1β–expressing mice (Figure 7).

IL-1, COPD, and Asthma

The present results demonstrate that IL-1β is sufficient to induce a phenotype that recapitulates many of the features of COPD, including emphysema, inflammation with neutrophils and macrophages, airway fibrosis, lymphocyte nodules in the airways, and mucus cell metaplasia (2, 3). Several of these features, such as airway wall thickening, subepithelial fibrosis, and mucus metaplasia, are also part of the structural alterations in chronic asthmatic inflammation (3). Clinical studies have previously shown that increased IL-1 activity is associated with both COPD (6, 9, 11) and asthma (12, 13, 25). The present results support the notion that IL-1 has an important or primary role in the pathogenesis of chronic inflammatory diseases of the lung.

Timing of Transgene Induction

In the present study, the animals were treated with doxycycline from 3 wk to 8 wk of age to induce IL-1 production in the lung. By that time, the most active period of alveolarization, which takes place from Postnatal Day 4 until Day 14 in the mouse (26), is already complete. Alveolar wall thinning occurs between Days 14 and 21, after which the lung continues to grow. Growth is most active until ∼ 4 wk of age, but because rodent epiphyses never close, thoracic growth and alveolarization persist throughout life (26). Starting the expression of IL-1 during a period of active growth might have influenced the severity of the lung phenotype in the present study. A similar phenotype, however, resulted when the IL-1 transgene was activated from 5–10 wk of age (data not shown).

Mucus Metaplasia and FOXA2 in Airways of IL-1β–Expressing Mice

Mucus metaplasia is commonly associated with chronic inflammatory lung diseases, including COPD, asthma, cystic fibrosis, and bronchopulmonary dysplasia (BPD). In our model, IL-1β caused extensive mucus metaplasia in the airways. Consistent with the present results, IL-1β has been recently shown to increase MUC5AC secretion in bronchial epithelial cells in culture (27). Deletion of the winged helix transcription factor FOXA2 in airway epithelial cells also caused goblet cell hyperplasia in mice (21). Immunostaining for FOXA2 was decreased or absent in goblet cells in the airways of IL-1β–expressing mice, whereas FOXA2 staining was maintained in nongoblet bronchiolar epithelial cells. A similar association of goblet cell hyperplasia and decreased FOXA2 expression has also been shown in other mouse models with goblet cell hyperplasia as well as in human lung tissue in bronchiectasis and BPD (21), supporting the idea that FOXA2 inhibits mucin gene expression.

Elastin Decrease and Fibrosis in IL-1–Expressing Mice

Elastin was decreased in the lungs of IL-1–expressing mice. IL-1 may cause the degradation of elastin by enhancing the production of elastolytic enzymes, such as MMP-9 and MMP-12, or by recruiting neutrophils that produce neutrophil elastase. At the same time, however, IL-1β directly decreases elastin production by myofibroblasts (28), suggesting that the observed decrease in elastin may be a consequence of impaired deposition rather than destruction.

In human emphysema and in experimental emphysema caused by cigarette smoke exposure, collagen deposition and disruption of elastin occur in parallel (29, 30). In the present model, loss of elastin was accompanied by fibrosis in subpleural, peribronchiolar, and perivascular regions of the lung as well as in alveolar walls. IL-1 has both pro- and antifibrotic effects. On the one hand, IL-1β has been shown to increase the mRNA expression of the profibrotic cytokine TGF-β1 and the production of active TGF-β by endothelial cells (31). In addition, the IL-1 antagonist IL-1ra reduces pulmonary fibrosis caused in mice by bleomycin or silica (32), suggesting that IL-1 can be important in the pathogenesis of fibrosis. Moreover, IL-1 enhances fibroblast proliferation (33). On the other hand, IL-1 may limit fibrosis by inhibiting collagen production (33) and by enhancing the production of collagenases (5). Transient overexpression of IL-1 in rats by adenoviral gene transfer by intratracheal admininistration leads to increased production of TGF-β1 and to severe and progressive interstitial pulmonary fibrosis after loss of transgene expression (34). The possibility that profibrotic cytokines, such as TGF-β1, cause progressive fibrosis after IL-1 expression is terminated remains to be studied in the present model.

Neutrophils and Neutrophil Elastase in Inflammatory Lung Diseases

Induction of IL-1 production in the lungs caused a neutrophil influx into the lungs of transgenic mice. Neutrophils are increased in the airways of smokers and in the airways and bronchial tissue of patients with COPD (35). The number of neutrophils in the bronchial tissue is related to the severity of airway obstruction (35). Neutrophils are also increased in the lungs of patients with chronic refractory asthma (36).

Neutrophils produce elastolytic enzymes, including neutrophil elastase and MMP-9. Lack of the neutrophil elastase inhibitor α-1-antitrypsin is associated with early-onset emphysema in humans (35). Furthermore, experimental application of neutrophil elastase induces emphysema in animals (35). This protease is a potent secretagogue contributing to excess mucus production in COPD (35). Neutrophil elastase contributes to cigarette smoke–induced emphysema in mice (37) and is likely to participate in tissue destruction and mucus metaplasia also in IL-1–expressing mice. Mice lacking neutrophil elastase have reduced levels of IL-1β production in response to zymosan compared with wild-type mice (38), suggesting that neutrophil elastase promotes IL-1 production. IL-1, in turn, enhances neutrophil influx, thereby increasing elastase availability. Interestingly, while cigarette smoke–induced recruitment of neutrophils and macrophages is impaired in the absence of neutrophil elastase (37), lack of neutrophil elastase has no effect on neutrophil migration induced by IL-1 (38).

CXC Chemokines in Lung Injury

Neutrophil recruitment is regulated by neutrophil-attractant ELR+ CXC chemokines. Several ELR+ CXC chemokines exist in humans, including IL-8 and the growth-related oncogene family. Smoking enhances the production of IL-8 (35). In the mouse, KC and macrophage inflammatory protein-2 (MIP-2) are major ELR+ CXC chemokines expressed at sites of tissue inflammation. ELR+ CXC chemokine receptors have been characterized and found to be involved in neutrophil recruitment and activation. CXCR2 is the exclusive receptor for these ligands in mice (39). The production of KC and MIP-2 can be induced by inflammatory stimuli in different cell types, including macrophages and neutrophils as well as nonmyeloid cells such as endothelial cells and fibroblasts (40). In the present model, IL-1β enhanced the production of KC and MIP-2 in vivo. These chemokines, which have recently been shown to participate in the pathogenesis of ventilator-induced lung injury (41) and to regulate respiratory syncytial virus–induced mucus overproduction (42), may be crucial downstream mediators of IL-1–induced lung injury.

IL-1, MMPs, and Lung Remodeling

Both clinical and experimental studies provide convincing evidence that MMPs, particularly MMP-9 and MMP-12, are associated with emphysema (43, 44). IL-1β increased the production of both MMP-9 and MMP-12 in the present model. MMP-9 and MMP-12 can solubilize many extracellular matrix proteins, including elastin, and can therefore regulate IL-1–induced remodeling responses. MMP-9 and MMP-12 can also enhance neutrophil elastase activity by degrading α-1-antitrypsin (43, 44). Alveolar macrophages from patients with COPD release greater amounts of elastolytic enzymes in response to IL-1 than alveolar macrophages from nonsmokers or smokers with no COPD (45). Neutrophils are a major source of MMP-9, but many other cells, including macrophages and intrinsic lung cells (bronchial epithelial cells, Clara cells, and alveolar type II cells, fibroblasts, smooth muscle cells, and endothelial cells) are also able to produce MMP-9 (43). IL-1β has been previously shown to increase MMP-9 production in alveolar macrophages and bronchial epithelial cells in vitro (7, 46). In the present model, MMP-9 immunopositivity was seen in neutrophils and in fibrotic areas in airway walls. MMP-9 may be involved in fibrosis by activating TGF-β and increasing collagen synthesis by fibroblasts (43). MMP-9 can regulate IL-1β activity by processing IL-1β precursor into biologically active forms (16) and thereby increasing IL-1β activity, or by degrading IL-1β protein (47), thus limiting IL-1β action.

Studies in MMP-12–deficient mice have demonstrated that MMP-12 has an essential role in the pathogenesis of cigarette smoke–induced emphysema in mice (23). Emphysema in mice deficient in integrin α(v)β6 is also MMP-12–dependent (48). Lack of MMP-9 or MMP-12 partially protects mice from IL-13–induced emphysema (49). On the other hand, emphysema in SP-D–deficient mice is not dependent on MMP-9 or MMP-12, although the production of both metalloproteases is increased in these mice (50). The contributions of MMP-9 and MMP-12 to the tissue injury and remodeling in the present model remain to be assessed.

IL-1 and Tumor Necrosis Factor in Models of Emphysema

IL-1 and tumor necrosis factor (TNF) are proinflammatory cytokines with a different structure and different receptors but with overlapping biological properties. Infectious and inflammatory agents (such as endotoxin) induce the production of both of these cytokines. IL-1 and TNF can directly activate signal transduction pathways that affect connective tissue matrix production and synthesis of proteolytic enzymes (5). Moreover, TNF and IL-1 often act synergistically (5). Both constitutive and inducible TNF overexpression cause emphysema in transgenic mice (51, 52). MMP-12 mediates smoke-induced inflammation by releasing TNF from macrophages (53). Moreover, mice lacking TNF receptors I and II are to a significant extent protected from cigarette smoke–induced emphysema (53), further suggesting that TNF is an important factor in the events that lead to emphysema after smoke exposure. The possible contribution of IL-1 to smoke-induced emphysema has not been studied. However, a synergistic contribution of IL-1 and TNF to porcine elastase-induced emphysema is suggested by a study by Lucey and coworkers showing that deletion of both IL-1β type I receptor and type I and II TNF receptors protected mice from emphysema to a much greater extent than deletion either of IL-1β type I receptor or of types I and II TNF receptors (54).

In summary, the present study shows that the early-response inflammatory cytokine IL-1 causes an inflammatory lung disease with features of both COPD and asthma in transgenic mice. IL-1 induces the production of CXC chemokines that mediate neutrophil infiltration. Increased neutrophil elastase activity and activation of MMP-9 and MMP-12 probably contribute to tissue injury and remodeling in the present model.

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Correspondence and requests for reprints should be addressed to Kristina Bry, Goteborg University, Department of Pediatrics, The Queen Silvia Children's Hospital, 41685 Goteborg, Sweden. E-mail:

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