Overexpression of tumor necrosis factor-alpha produces an increase in lung volumes and pulmonary hypertension

Masaki Fujita1, John M. Shannon1, Charles G. Irvin1, Karen A. Fagan2, Carlyne Cool1, Andrei Augustin1, and Robert J. Mason1

1 Department of Medicine, National Jewish Medical and Research Center, Denver 80206; and 2 Cardiovascular Pulmonary Research Laboratory, University of Colorado Health Science Center, Denver, Colorado 80262


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor (TNF)-alpha is a key proinflammatory cytokine that is thought to be important in the development of pulmonary fibrosis, whereas its role in pulmonary emphysema has not been as thoroughly documented. In the present study, TNF-alpha was overexpressed in alveolar type II cells under the control of the human surfactant protein C promoter. In this report, we further characterized the pulmonary abnormalities and provided a physiological assessment of these mice. Histopathology of the lungs revealed chronic inflammation, severe alveolar air space enlargement and septal destruction, and bronchiolitis. However, pulmonary fibrosis was very limited and only seen in the subpleural, peribronchiolar, and perivascular regions. Physiological assessment showed an increase in lung volumes and a decrease in elastic recoil characteristic of emphysema; there was no evidence of restrictive lung disease characteristic of pulmonary fibrosis. In addition, the mice raised in ambient conditions in Denver developed pulmonary hypertension. Gelatinase activity was increased in the lavage fluid from these lungs. These results suggest that in these mice TNF-alpha contributed to the development of pulmonary emphysema through chronic lung inflammation and activation of the elastolytic enzymes but by itself was unable to produce significant pulmonary fibrosis.

emphysema; pulmonary fibrosis; pulmonary hypertension


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TUMOR NECROSIS FACTOR (TNF)-alpha is an important proinflammatory cytokine that has been implicated in a variety of pathological processes. TNF-alpha is produced mainly by activated monocytes or macrophages in response to inflammatory stimuli such as lipopolysaccharide (LPS) (32). TNF-alpha binds to receptors present on virtually all cells throughout the body. If TNF-alpha is released in large amounts systemically, TNF-alpha activates neutrophils, induces the release of other inflammatory cytokines, alters endothelial cell properties, and may lead to shock. On the other hand, a modest but persistent production of TNF-alpha may lead to chronic inflammation with fever, anemia, bone resorption, and wasting (3).

There are compelling reasons to believe that TNF-alpha is involved in pulmonary fibrosis. Pulmonary fibrosis is a devastating disease with a 5-yr mortality of 50% and is characterized pathologically by chronic inflammation and a fibroproliferative process in the lung interstitium. Although the pathogenesis of pulmonary fibrosis is not known, data from patients with idiopathic pulmonary fibrosis and animal models of pulmonary fibrosis suggest that a variety of growth factors and cytokines are involved in the development of pulmonary fibrosis. These include TNF-alpha (45), platelet-derived growth factor, transforming growth factor (TGF)-beta , and TGF-alpha . The importance of TNF-alpha in the pathogenesis of pulmonary fibrosis has been highlighted in recent studies. TNF-alpha mRNA is increased in lungs from idiopathic pulmonary fibrosis patients (37). Treatment with an anti-TNF-alpha antibody prevents the development of both bleomycin-induced pulmonary fibrosis (35) and silica-induced pulmonary fibrosis (36). In animal models, administration of a TNF-alpha soluble receptor prevents bleomycin-induced pulmonary fibrosis (38). Recently, a murine model of overexpression of TNF-alpha under control of the human surfactant protein (SP) C promoter (SP-C/TNF-alpha transgenic mice) has been developed (29), and these transgenic mice were reported to have severe alveolitis, alveolar distension, and progressive pulmonary fibrosis. However, their physiological phenotype has not been defined.

There is, however, also evidence that TNF-alpha by itself may not produce pulmonary fibrosis. The effects of TNF-alpha on collagen gene expression in vitro appear to be inhibitory (27). Although TNF-alpha causes acute lung inflammation (32), short-term TNF-alpha production by LPS or single-dose administration of TNF-alpha does not lead to pulmonary fibrosis. Furthermore, gene transfer of TNF-alpha induces limited pulmonary fibrosis compared with TGF-beta (41). Recently, Sulkowska et al. (43) reported that long-term TNF-alpha administration in rats causes emphysema-like pulmonary remodeling in addition to fibrosis. Although TNF-alpha has been associated with emphysema (24), TNF-alpha is not thought to be central to the development of emphysema and serves more of a marker of chronic inflammation.

The aim of this study was to characterize the physiological abnormalities of SP-C/TNF-alpha transgenic mice (29). We anticipated that this transgenic mouse would be useful for investigating the pathogenesis of slowly progressive pulmonary fibrosis. However, the physiology and histopathology of older mice resembled that of emphysema more than that of pulmonary fibrosis. We found that the pulmonary alterations in these transgenic mice were characterized by increased lung volumes and decreased recoil. In addition, these mice reared at moderate altitude (Denver, CO; 5,280 feet) developed pulmonary hypertension and right ventricular hypertrophy.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. SP-C/TNF-alpha transgenic mice were a kind gift of Y. Miyazaki (Department of Clinical Immunology, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan). Since the severity of disease in offspring from the Tg-10 line was more consistent and correlated better with the levels of TNF-alpha mRNA in the lungs than the other line, Tg-2, the Tg-10 line was used in this study (29). The transgenic mice were crossed with C57BL/6 mice and bred in an animal facility documented to be free of murine-specific pathogens. All transgenic mice were identified by PCR analysis of genomic DNA isolated from the toe, using primers as described by Miyazaki et al. (29). We studied mice at 2 days, 1 wk, 2 wk, 1 mo (4-6 wk), 2.5 mo (9-11 wk), 6 mo (24-28 wk), 8 mo (32-36 wk), and 12 mo of age. Littermate transgene-negative mice were used as controls, since these animals are the most closely matched genetically. This study was approved by the Institutional Animal Care and Use Committee at the National Jewish Medical and Research Center, Denver, CO.

Histological analysis. Mice were killed by intraperitoneal injection of pentobarbital sodium. Lungs were inflated at 25 cmH2O static pressure by intratracheal instillation of 4% paraformaldehyde in phosphate-buffered saline (PBS). Then lungs were taken and immersed in the same buffer overnight at 4°C, washed with PBS, and then immersed in 70% ethanol. In the case of 2-day-old and 1-wk-old mice, the lungs were inflated with 4% paraformaldehyde in PBS until fully inflated under direct observation. Tissue sections were stained by hematoxylin and eosin, sirius red, trichrome, and pentachrome methods.

Morphometry was done by methods previously reported (10). Mean linear intercept, an indicator of air space size, was calculated for each mouse from 10 randomly selected fields at 200 power by means of a 21-line counting grid. Mean linear intercept was determined from sections of five transgenic mice and five littermate controls at 1, 2.5, and 6 mo.

RNA extraction and Northern hybridization. RNA was prepared by the guanidine isothiocyanate-cesium chloride gradient method. Total RNA was electrophoresed under denaturing conditions, transferred to a nylon membrane, and hybridized with 32P-labeled murine probes of interleukin (IL)-12 p40 (39) and type I procollagen (16).

RNase protection assay. For RNase protection assay (RPA), mouse cytokine multiprobe template sets (mCK-1b and mCK-3) were purchased from PharMingen (San Diego, CA). RPA was performed according to the manufacturer's protocol. Briefly, antisense transcripts were synthesized by using T7 RNA polymerase and [32P]CTP (ICN, Costa Mesa, CA). Samples of 5 µg of total lung RNA were hybridized with radiolabeled probe mix at 56°C overnight. Hybrids were digested with RNases A and T1. Protected fragments were separated on 5% polyacrylamide-8 M urea gels and analyzed by autoradiography. The increases of mRNA levels were quantified with ImageQuant (Molecular Dynamics, Sunnyvale, CA).

Pulmonary physiology. Mice were anesthetized by intraperitoneal administration of pentobarbital sodium (70 mg/kg). After anesthesia, a tracheostomy was performed with an 18-gauge metal cannula. Mechanical ventilation was initiated with a volume ventilator (model 683; Harvard Apparatus, South Natick, MA) at a rate of 160 breaths/min, tidal volume of 150 µl, and a positive end-expiratory pressure of 2 cmH2O. After tracheal cannulation, the animals were placed within a pressure plethysmograph where lung mechanics were measured as previously described (21). Transpulmonary pressure (PTP) was determined as the pressure difference between a side arm of a four-way connection attached to the tracheal cannula and pressure with the plethysmograph as determined with a differential pressure transducer (MP45; Validyne Instruments, Northridge, CA). Volume (V) was determined as the pressure difference between the plethysmograph and a reference jar.

After a period of mechanical ventilation, the lungs were inflated to total lung capacity (TLC; 30 cmH2O PTP), and a 30-s period PTP and V were collected to obtain pulmonary resistance and dynamic compliance (Cdyn) (21). To obtain the static pressure-volume characteristic, the following procedure was used. The mice were first inflated twice to TLC, and on the third inflation, the pressure-volume characteristics were determined. Slow inflations (~1 ml in 2-3 min) were performed, which included static interruptions of flow. Both inspiratory and expiratory curves were performed. This pressure-volume procedure was repeated five more times or until visibly reproducible curves were obtained.

PTP and V were digitally converted (National Instruments, Austin, TX) and processed with a custom-built program (Labview; National Instruments). This program allows the operator to select static pressure-volume plot points from either the inspiratory or expiratory limbs of the pressure-volume curves. The data from each pressure-volume curve were collected, and pooled mean pressure-volume parameters were obtained for each animal. Static expiratory compliance (Cstat) was then determined as the slope of the linear regression of the data over the initial volume range [functional residual capacity (FRC) + 0.5 ml].

Functional residual capacity (FRC) was then measured by saline displacement. TLC was calculated as the sum of FRC and the volume needed to reach 30 cmH2O pressure. In all cases, littermate mice were used as controls.

Cardiovascular physiology. In a separate group of animals, after induction of anesthesia (ketamine-xylazine; 15 mg/kg), a 26-gauge needle connected to a pressure transducer (Gould-Statham, Costa Mesa, CA) was inserted percutaneously into the left ventricular (LV) and right ventricular (RV) chambers via a subxyphloid approach (11). The quality of the pressure wave was monitored during procedure. Blood was obtained by a cardiac puncture and hematocrit was measured. After euthanasia, the heart was carefully dissected and divided into RV wall and LV wall with septum (LV+S), and each was weighed separately.

Bronchoalveolar lavage. In animals with a tracheal tube inserted, the lungs were lavaged with 1-ml aliquots of PBS five times for a total of 5 ml. Total cell counts were determined with a hemocytometer. Differential counts of bronchoalveolar lavage (BAL) fluid were performed on 200 cells from a smear stained with a modified Wright's stain (DiffQuik; American Scientific Products, McGaw Park, IL).

TNF-alpha ELISA. The level of TNF-alpha was determined using a mouse TNF-alpha ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol.

Zymography. For gelatin zymography, 40 µl of BAL fluid were electrophoresed in SDS-polyacrylamide gels containing 1% gelatin under nonreducing conditions. After electrophoresis, the gel was washed with 2.5% Triton X-100 in H2O followed by 10 mM Tris · HCl, pH 7.4. The gel was then incubated in an activation buffer, 100 mM Tris · HCl, pH 7.4, 10 mM CaCl2, and 50 mM NaCl2 at 37°C for 24 h. As the control study, either 20 mM EDTA as metalloproteinase inhibitor or 2 mM phenylmethylsulfonyl fluoride (PMSF) as serine proteinase inhibitor was added to the activation buffer. kappa -Elastin zymography was also performed according to a method described elsewhere (40). Briefly, kappa -elastin (Elastin Products, Owensville, MO) was incorporated into SDS- polyacrylamide gels. The concentration of kappa -elastin was 1.2 mg elastin/ml gel. After electrophoresis at 4°C, the gel was washed with 2.5% Triton X-100 in H2O followed by 50 mM Tris · HCl, pH 8.0, containing 5 mM CaCl2. The gel was then incubated in an activation buffer (addition of 500 µl of 1 mM zinc chloride in 10 mM acetic acid into 100 ml of Tris buffer, 50 mM Tris · HCl, pH 8.0, and 5 mM CaCl2) at 37°C for 48 h. After incubation, the gel was stained with Coomassie brilliant blue R250 and destained.

Hydroxyproline assay. A hydroxyproline assay was performed as previously described elsewhere (23) with some modification. Briefly, after death, mouse lungs were removed, snap-frozen, and lyphophilized for at least 48 h. Then 10 mg of ground lung were added to 500 µl of 6 N HCl and incubated overnight at 120°C. Five microliters of the samples and standards were applied to an ELISA plate. Fifty microliters of citric-acetate buffer (5% citric acid, 7.24% sodium acetate, 3.4% NaOH, and 1.2% glacial acetic acid, pH 6.0) and 100 µl of chloramine T solution (564 mg of chloramine T, 4 ml of H2O, 4 ml of n-propanol, and 32 ml of citrate-acetate buffer) were added and incubated for 20 min at room temperature. Then 100 µl of Ehrlich's solution (4.5 g of 4-dimethylaminobenzaldehyde, 18.6 ml of n-propanol, and 7.8 ml of 70% perchloric acid) were added and incubated at 65°C for 15 min. Reaction product was read at optical density of 550 nm. Hydroxyproline (Sigma, St. Louis, MO) standard solutions of 0-200 µg/ml were used to construct the standard curve.

Statistics. Data are expressed as means ± SE. The statistical analyses were performed using a computer program (SAS software, version 6). ANOVA was performed, and then the Tukey-Kramer method was applied to adjust for multiple comparison. P < 0.05 was considered to indicate a significant difference.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of SP-C/TNF-alpha transgenic mice. We identified transgenic mice by two different methods. One was by PCR using primers based on human SP-C promoter and mouse TNF-alpha ; the other was by Southern hybridization with a human SP-C promoter probe. Transgenic mouse lungs also had a very characteristic and distinctive phenotype of large lungs with orange-colored pleural surfaces, as reported previously (29).

As shown in Fig. 1A, there was an increase in neutrophils, lymphocytes, and macrophages in BAL fluid from transgenic mice. There were high levels of TNF-alpha protein in the lavage fluid from all mice evaluated, which ranged from 1 to 6 mo of age (Fig. 1B). Serum levels of TNF-alpha were slightly increased [6-mo-old transgenic mice, 35.6 ± 1.5 pg/ml; 6-mo-old controls, 17.5 ± 1.8 pg/ml; not significant (NS), P = 0.056]. The mRNA for TNF-alpha , which was assessed by a RPA, remained highly elevated and relatively constant from 1 to 6 mo of age, which was the range of the ages of the animals tested for these analyses.


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Fig. 1.   Time-dependent differences in lavage cell counts, tumor necrosis factor-alpha (TNF-alpha ) protein in bronchoalveolar lavage fluid (BALF), and TNF-alpha mRNA levels in lungs from surfactant protein (SP) C/TNF-alpha transgenic and control mice. A: cell counts. Mac, macrophages; Neu, neutrophils; Lym, lymphocytes. Tg(-), control mice; Tg(+), SP-C/TNF-alpha transgenic mice. Each lung was lavaged 5 times with 1-ml aliquots of PBS. B: TNF-alpha protein levels in BALF measured by ELISA are shown with their SE. C: TNF-alpha mRNA levels were measured by RNase protection assay (RPA). Data were obtained from least 3 mice in each group.

Air space enlargement and chronic inflammation in SP-C/TNF-alpha transgenic mice. Histopathological findings were consistent with those with a lymphocytic interstitial lung disease (Fig. 2). Inflammatory cells consisting of lymphocytes, macrophages, and some neutrophils infiltrated the interstitium. The infiltration tended to be localized to areas adjacent to the pleura and bronchioles. Bronchiolitis was consistently noted in transgenic animals. Macrophage accumulations were also observed in alveolar spaces, and some appeared to be hemosiderin laden and others multinuclear. These findings were seen from 1 mo of age and appeared to progress gradually until 2.5 mo of age. However, the chronic inflammation tended to be resolved significantly, especially in the alveolar septum by 6 and 12 mo of age, and air space enlargement became the dominant abnormality. Air space enlargement was not seen in mice killed at 2 or 7 days of age but was noticeable in mice at 14 days of age.


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Fig. 2.   Lung histology from the SP-C/TNF-alpha transgenic and control mice. The mice were killed and lungs were instilled with 4% paraformaldehyde in PBS. The lungs were from 2.5-mo-old SP-C/TNF-alpha transgenic (A) and control (B) mice; from 6-mo-old SP-C/TNF-alpha transgenic (C) and control (D) mice; and from 12 mo-old SP-C/TNF-alpha transgenic (E) and control (F) mice. Note the marked alveolar enlargement in the lung of transgenic mice at all ages. Arrows point to clubbed alveolar septa indicative of alveolar wall destruction. All panels are at the same magnification. Original magnification, ×35.

The extent of pulmonary fibrosis was evaluated by histochemical methods and quantitation of hydroxyproline. Sirius red staining was used to demonstrate fibrillar collagen. However, the lungs from transgenic mice did not show an abundance of red fibers indicative of interstitial collagen (data not shown). Only limited scattered red fibers were seen in the area adjacent to the pleura as well as in the peribronchial and perivascular regions. In addition, lungs stained by trichrome or pentachrome methods demonstrated only minimal fibrosis. The hydroxyproline data are expressed per lung and per milligram of dry lung weight. Hydroxyproline per lung was increased, as previously reported and anticipated, since the lungs were nearly twofold larger (29). However, the hydroxyproline per milligram of dry lung weight of transgenic mice was less than that of control littermates (Fig. 3). This suggests that the increase in total lung hydroxyproline in transgenic mice was related to an increase in the size of the lungs and not to fibrosis per se. Furthermore, type I procollagen mRNA levels of transgenic mice were lower than those of controls (data not shown). Although transgenic mice exhibited persistent pulmonary inflammation, this chronic inflammation did not eventually lead to significant pulmonary fibrosis.


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Fig. 3.   Differences in hydroxyproline between SP-C/TNF-alpha transgenic and control mice. Hydroxyproline content was determined in lungs of 6-mo-old transgenic [Tg (+)] and littermate mice [Tg(-)]. Data are presented per total lung (top), which reflects lung size, or per dry lung weight (bottom), which normalizes the data for the amount of lung tissue. *Statistical significance (P < 0.05); mean ± SE for 6 animals in each group is shown.

The dominant histopathological and physiological findings in older mice were alveolar air space enlargement and not fibrosis (Figs. 2 and 4). Clubbed ends of alveolar septa indicated actual alveolar wall destruction. These emphysematous-like changes progressed with age and were most noticeable in the older mice. The clubbed ends are thought to represent alveolar septal destruction and are indicated by the arrows in Fig. 2. They were a common occurrence and indicate that the alterations were due to a pathological process and not to an artifact of overdistension. Quantitative morphometry was used to measure the extent of alveolar enlargement. Mean linear intercept scores increased with age in the transgenic mice and were higher from the transgenic mice than those from controls at all ages studied (Fig. 5).


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Fig. 4.   Age-dependent changes in pressure-volume (PV) curves from SP-C/TNF-alpha transgenic and control mice. A: 1 mo old (n = 7). B: 2.5 mo old (n = 6). C: 6 mo old (n = 7). Solid line, data from the SP-C/TNF-alpha transgenic mice; dashed line, data from control mice; arrowheads, directions of inspiratory phase and expiratory phase of the PV curve. Results are the means of 4 transgenic and 4 controls at each time point. PTP, transpulmonary pressure; V, volume.



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Fig. 5.   Lung function in SP-C/TNF-alpha transgenic (solid bars) and control (open bars) mice. Comparison of total lung capacity (TLC), functional residual capacity (FRC), static expiratory compliance (Cstat), and mean linear intercept (Lm) between SP-C/TNF-alpha transgenic and control mice is shown. *Statistical difference (P < 0.05); number of animals in each group is the same as in Fig. 4.

Pulmonary physiology of SP-C/TNF-alpha transgenic mice. Total body weight (BW) was not significantly different between the two groups (6-mo-old transgenic mice, 24.9 ± 0.6 g; 6-mo-old controls, 23.9 ± 0.7 g; NS). However, wet lung weight (in mg) per body weight (BW in g) showed significant differences. Transgenic mice had more than a twofold increase in lung weight [6-mo-old transgenic mice, 20.6 ± 0.6 mg/BW (g); 6-mo-old controls, 9.8 ± 0.7 mg/BW (g); P < 0.001]. Measurement of lung volumes also demonstrated that the transgenic mice had larger lungs than controls (Fig. 5). The FRC of 6-mo-old transgenic mice was 0.91 ± 0.04 ml, whereas the FRC of 6-mo-old controls was 0.46 ± 0.02 ml (P < 0.001). The TLC of 6-mo-old transgenic mice was 2.32 ± 0.09 ml, whereas the TLC of 6-mo-old controls was 1.42 ± 0.06 ml (P < 0.001). Pulmonary resistance was similar between the two groups (6-mo-old transgenic mice, 0.80 ± 0.02 cmH2O · ml-1 · s; 6-mo-old controls, 0.87 ± 0.06 cmH2O · ml-1 · s; NS).

The pressure-volume curves showed an increase in lung volume and a decrease in elastic recoil (Fig. 4). The pressure-volume curves in terms of lung volumes and recoil at a given volume are statistically increased in the transgenic mice. At 1 and 2.5 mo of age, the differences in the pressure-volume curves seemed to be best accounted for by changes in their lung volumes. Cdyn was not significantly different (1-mo-old transgenic mice, 0.032 ± 0.003 ml/cmH2O; 1-mo-old controls, 0.030 ± 0.002 ml/cmH2O; NS; 2.5-mo-old transgenic mice, 0.036 ± 0.003 ml/cmH2O, 2.5 mo-old-controls; 0.040 ± 0.002 ml/cmH2O, NS). At these ages, the static expiratory compliances (Cstat) of transgenic mice were similar to the controls, as shown in Fig. 5. However, at 6 mo of age, the differences in pressure-volume curves became more apparent. Besides the changes in lung volume, the slopes of the inspiratory and expiratory limbs of the pressure-volume curve were steeper than those of controls. The changes were similar whether the results are expressed as absolute volumes or as a percentage of predicted littermate control lung volumes. Cstat differed significantly between transgenic mice and controls (Fig. 5). Cdyn also became higher in 6-mo-old transgenic mice (6-mo-old transgenic mice, 0.071 ± 0.004 ml/cmH2O; 6-mo-old controls, 0.052 ± 0.002 ml/cmH2O; P < 0.001).

Pulmonary hypertension and RV hypertrophy. RV hypertrophy was characteristic of these transgenic mice raised in Denver. We measured pressures in the RV and LV walls as well as the weights of RV and LV+S walls, which are summarized in Tables 1 and 2. The ratio of RV wall to BW demonstrates that RV hypertrophy was occurring as early as 1 mo of age. The ratio of LV+S wall to BW revealed significant differences at 6 mo of age. The systolic and mean RV pressures were significantly higher in the transgenic animals at 8 mo of age (Table 2). LV pressure was similar between the two groups. The hematocrit also differed significantly between the two groups (8-mo-old transgenic mice, 52.1 ± 1.4%; 8-mo-old controls, 45.7 ± 0.8%; P < 0.05). Histopathology revealed no inflammatory cell infiltration or fibrosis in the hearts from transgenic mice (data not shown).

                              
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Table 1.   Comparison of the weights of RV and LV+S, the weights normalized to BW, and the ratio of RV to (LV+S) in SP-C/TNF-alpha transgenic and control mice


                              
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Table 2.   Comparison of right and left ventricular pressures between the SP-C/TNF-alpha transgenic and control mice

Increased activity of elastolytic enzymes. Since the relationship between emphysema and matrix metalloproteinase (MMPs) and other proteases is thought to be critical to the development of alveolar wall destruction and emphysema (13, 14, 34), we measured proteolytic activity by zymography on BAL fluid samples from the transgenic and control mice. As shown in Fig. 6, clear bands, areas of enzymatic activity, were detected at estimated molecular sizes of 66 and 160 kDa in transgenic mice at 6 mo. Similar results were obtained at 1 mo (data not shown). Although BAL from control mice demonstrated bands at 66 kDa, they were very faint compared with those from the transgenic mice. These bands were inhibited by treatment with EDTA but not with PMSF. In the case of kappa -elastin zymography, we obtained clear bands at 28 and 70 kDa, as well as faint ~92-kDa clear bands. The 28-kDa band was seen in both transgenic and control mice. This 28-kDa band is probably neutrophil elastase, based on its molecular mass. The 70- and ~92-kDa bands were only observed in transgenic mice. Macrophage metalloelastase (45 and 22 kDa) was not detected in any experiments. The ~66-70 kDa band is thought to be gelatinase A (MMP-2, 72-kDa gelatinase), whereas the ~92 kDa is thought to be gelatinase B (MMP-9, 92-kDa gelatinase). Murine gelatinase B migrates slightly slower than 92 kDa and has a predicted molecular mass of 105 kDa (25, 44). Results of Western blotting using antibody against gelatinase A or gelatinase B confirmed that the 160-kDa bands were complexes of gelatinase A and B (data not shown).


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Fig. 6.   Difference in elastolytic and gelatinase activities between SP-C/TNF-alpha transgenic and control mice. Bronchoalveolar lavage fluid from the SP-C/TNF-alpha transgenic and control mice was analyzed for enzymatic activity by gelatin and kappa -elastin zymography. A: kappa -elastin zymography. B: gelatin zymography. Tg(+), BAL fluids from the SP-C/TNF-alpha transgenic mice. Tg(-), BAL fluids from control mice. Areas of gelatin or elastin degradation are seen as clear zones. Molecular mass markers are shown in left lane (in kDa).

Elevated Th1 cytokine expressions. The cytokine profile in the lungs of the transgenic animals was assessed by RPA during the period of chronic lung inflammation and alveolar wall destruction. There was a sustained increase of TNF-alpha mRNA expression as expected (Figs. 1C and 7A). In addition, the mRNAs for IL-2, IL-10, interferon (IFN)-gamma , lymphotoxin (LT)-beta , and TNF-beta were increased. Other cytokines such as IL-4 and IL-5 were expressed below the level of detection by this RPA (Fig. 7B). Northern hybridization showed an increase of IL-12 p40 mRNA expression only in transgenic mice (Fig. 7C). Since TNF-alpha expression was extremely high, further increases in the expression of IL-6, TGF-beta 1, and TGF-beta 2 could not be clearly detected because of the high background. However, when the background was subtracted, the increased expression of IL-6 and the unchanged expression of both TGF-beta 1 and TGF-beta 2 were confirmed, as reported by others (42).


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Fig. 7.   Differences in cytokine mRNA expression between SP-C/TNF-alpha transgenic and control mice. A: cytokine mRNA expression was measured by RPA mCK-1b and mCK-3 kit from PharMingen using total lung RNA. Tg(+), RNA from SP-C/TNF-alpha transgenic mice; Tg(-), RNA from controls. B: RPA results are shown normalized to the expression of the constitutive genes L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). *Statistical significance compared with controls. C: Northern hybridization with interleukin (IL)-12 p40. Ten micrograms of total RNA extracted from lungs were electrophoresed and transferred to nylon membrane. Tg(+), RNA from three 6-mo-old SP-C/TNF-alpha transgenic mice; Tg(-), RNA from 3 controls. 28S rRNA Northern hybridization results are shown as controls for equivalence of RNA loading. LT, lymphotoxin; IFN, interferon; TGF, transforming growth factor.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The initial report using SP-C/TNF-alpha transgenic mice indicated that TNF-alpha overexpression resulted in interstitial pneumonia and pulmonary fibrosis (29). However, our physiological and histopathological data demonstrated that the transgenic mice 1 and 2.5 mo old exhibited features of both interstitial lung disease and emphysema. At 6 mo of age, increased lung volumes and overdistension became the dominant physiological characteristics. The physiological data were consistent with the histopathological appearance. The initial description of these mice had also noted alveolar enlargement and an increase in the mean linear intercept (29). These results revealed that SP-C/TNF-alpha transgenic mice have a phenotype that is more similar to pulmonary emphysema than to pulmonary fibrosis. In addition, these animals raised in Denver at moderate altitude develop pulmonary hypertension and marked RV hypertrophy.

The pathogenesis of the air space enlargement could be developmental due to expression of TNF-alpha in the developing lung or acquired due to the persistent chronic inflammation and chronic excessive protease activity. The potential role of expression of TNF-alpha in the developing or newborn lung is extremely difficult to disprove. In the mouse lung, alveolar septation proceeds until 1-2 wk of age (2). It is difficult to exclude the possibility that there may be subtle changes in alveolarization before birth or shortly after birth. SP-C is expressed early in gestation, and it is highly likely that TGF-alpha is expressed in utero. We examined lung histology 2 and 7 days after birth and found no abnormalities. However, our methods are probably too insensitive to detect subtle changes. The earliest alterations in the lung histopathology were noticeable at 14 days. The major histopathological changes in terms of interstitial inflammation occurred at 1 mo. The inflammation appeared to accompany the air space enlargement. We do not believe that the air space enlargement is due to the SP-C promoter and the gene insertion per se. Other genes under the control of the SP-C promoter such as the human IL-1 receptor antagonist do not show alveolar enlargement, whereas SP-C/TGF-alpha and SP-C/platelet-derived growth factor mice do show air space enlargement (19, 47). The SP-C/TGF-alpha mice have an increased compliance and alveolar enlargement. However, the total lung volume as measured at 30 cmH2O is similar to control mice (19). The SP-C/TNF-alpha mice in our study show the largest lung volumes produced over expression of any gene by the SP-C promoter to our knowledge. As reported in other studies, TNF-alpha could be responsible for these changes. Administration of TNF-alpha has previously been shown to cause emphysema-like lung remodeling (43). In addition, repeated LPS administration, which is known to be a strong inducer of TNF-alpha , was reported to induce emphysematous changes in the lungs (4, 18). Thus we conclude that TNF-alpha and the resultant chronic inflammation likely produce the alveolar enlargement and emphysema-like changes and that the alveolar enlargement is not due solely to abnormal septation. However, abnormalities in lung growth and alveolar septation cannot be excluded.

The protease-antiprotease hypothesis is well established as a mechanism for developing pulmonary emphysema (4, 20, 22). In SP-C/TNF-alpha transgenic mice, we have shown that there is increased activity of gelatinases A and B. Proteases instilled into rodent animals produce emphysema, and it is well known that genetic deficiency of anti-alpha 1-trypsin leads to pulmonary emphysema. Since >90% of pulmonary emphysema patients are smokers and do not have anti-alpha 1-trypsin deficiency, additional mechanisms for developing pulmonary emphysema are being investigated. The role of chronic alveolar inflammation has been given recent attention as a mechanism for development of pulmonary emphysema as part of the inflammatory injury and repair hypothesis (6). Smokers' lungs before the development of emphysema show an accumulation of macrophages, lymphocytes, and neutrophils in the walls and adjacent air spaces of the respiratory bronchioles, alveolar ducts, and alveoli (6, 12). The TNF-alpha transgenic mice have chronic mononuclear inflammation in terminal bronchioles and alveolar units at 1 and 2.5 mo of age and an increase in inflammatory cells recovered by lavage. We believe that it is this chronic mononuclear inflammation that leads to emphysema-like changes and air space enlargement.

MMPs are thought to be important for the development of pulmonary emphysema (33). Collagenase-overexpressing transgenic mice demonstrate emphysematous changes (7). In clinical studies, expression of MMPs is increased in alveolar macrophages, BAL fluids, and tissue extracts from emphysema patients (13, 14, 34). TNF-alpha is well known to upregulate MMPs including collagenase (33). Gelatinases A and B were upregulated in the TNF-alpha transgenic animals compared with controls from 1 mo of age through 6 mo of age, and these MMPs are known to have elastinolytic activity (40). Activation of MMPs is thought to contribute to the destruction of alveolar walls, which may explain the loss of static elastic recoil seen in our mice. Gelatinases A and B are produced mainly by macrophages and neutrophils, and thus activity of these MMPs is thought to be depend on the extent and type of inflammation. The protease-antiprotease imbalance hypothesis and chronic inflammation likely both play a role in the development of air space enlargement in this model.

The observed effects of TNF-alpha overexpression may be due to alteration in expression levels of other cytokines. For example, TNF-alpha was reported to stimulate pulmonary fibrosis via an increase of IL-5, which mediates eosinophil recruitment and fibrogenic cytokine production (48). We therefore investigated the expression of a number of cytokines in the lungs of transgenic mice. In this model, IL-12, IL-2, IL-10, LT, and IFN-gamma were upregulated. However, it is important to note that IL-4 and IL-5 were not detected in either the transgenic mouse or control lungs. IL-4 is considered to play an important role in determining the Th2 phenotype (30). BAL also did not show any increases in eosinophils. Increased IL-12 production may have caused a shift to the Th1 phenotype. IL-10 is well known to be produced by lymphocytes, macrophages, and other cells in response to TNF-alpha (15). IL-10 was originally identified as an inhibitor of IFN-gamma and IL-2 synthesis in Th1 cells. However, IL-10 has been found to be a general inhibitor of both Th1 and Th2 cytokines (1). In the SP-C/TNF-alpha transgenic mouse, the increase in IL-10 was observed at all times examined and was not further elevated at 6 mo of age when the inflammatory response subsided. Our data indicate that the Th1 pattern of cytokine production (IL-2, IF-gamma , and IL-10) was the dominant pattern in these mice.

As described previously, it was surprising to us that this chronic interstitial pneumonia did not lead to pulmonary fibrosis but instead produced the phenotype of pulmonary emphysema. Several studies have stressed the importance of Th2 cytokine production in pulmonary fibrosis (17, 26). In our experiments, the Th1 response was observed in SP-C/TNF-alpha transgenic mice. The hypothesis that the Th2 response leads to chronic inflammation and emphysema is supported by the observation that overexpression of IFN-gamma , which plays an important role in the Th1 response, leads to emphysematous changes (46). Determining the validity of the hypothesis that the Th1 response leads to the development of emphysema will require additional studies and other models.

As shown by Miyazaki et al. (29), some fibrosis was present in the transgenic mouse lungs. Fibrosis was not severe in these mice as shown by sirius red staining of the lungs, hydroxyproline content, and type I procollagen mRNA expression. There was no increase in hydroxyproline when the values were normalized to milligrams of dry weight, which reflects lung size. Sime et al. (41) also showed that TNF-alpha overexpression mediated by an adenovirus vector administration exhibited only limited pulmonary fibrosis. Although the role of TNF-alpha has been well established in the development of pulmonary fibrosis, our impression is that overexpression of TNF-alpha itself is not sufficient to produce pulmonary fibrosis and a second factor is required for the development of pulmonary fibrosis.

Pulmonary hypertension is an additional part of the physiological phenotype noted in our study in Denver. This finding was not reported in the original description of this transgenic mouse (29). TNF-alpha is known to affect the circulatory system (28). In these SP-C/TNF-alpha transgenic mice, however, serum levels of TNF-alpha were only slightly increased, and there was no change in LV pressure. By contrast, RV pressure and size showed significant increases. Although we did not measure pulmonary pressure directly, the peak RV pressure should reflect pulmonary artery pressure since there was no evidence of pulmonary stenosis. This SP-C/TNF-alpha mouse provides an opportunity of investigating chronic interstitial inflammation that ultimately leads to alveolar enlargement, large lung volumes, and severe pulmonary hypertension. However, the extent of pulmonary hypertension may depend on the altitude at which the animals are raised. In addition to the high hematocrits, some transgenic mice appeared cyanotic. Since no morphological changes were seen in the left heart or pulmonary veins, we consider that pathological changes in the lungs produced secondary pulmonary hypertension and RV hypertrophy. Infusion of TNF-alpha has also been reported to increase pulmonary arterial pressure (23). TNF-alpha may also increase expression of vasoconstrictors such as endothelin. Thus TNF-alpha itself may, in part, be responsible for pulmonary hypertension. Further study will be required to clarify the role of TNF-alpha as well as the mechanism of pulmonary hypertension in TNF-alpha -overexpressing mice.

TNF-alpha has not received much attention as a causative factor in the development of pulmonary emphysema. There are, however, several reports that suggest a relationship between emphysema, chronic obstructive pulmonary disease (COPD), and TNF-alpha . Serum TNF-alpha levels are reported to be higher in patients with COPD than in normal controls. TNF-alpha levels are correlated with body weight loss among COPD patients and might be correlated with the severity of COPD (8, 9). Increased numbers of neutrophils and TNF-alpha levels have been found in induced sputum from patients with COPD (24). Moreover, TNF-alpha has been observed in bronchial biopsies from COPD patients using immunocytochemical techniques (31). Burnett and his colleagues (5) reported that TNF-alpha , but not IL-1 and IFN-gamma , augmented fibronectin digestion by neutrophils. Taken together, these data suggest that TNF-alpha may be a significant contributor to the pathophysiology of emphysema.

In mice with targeted TNF-alpha -increased expression in the lungs, we found an increase in lung volume and alveolar enlargement, loss of recoil, and pulmonary hypertension and RV hypertrophy. This phenotype is more consistent with emphysema than with pulmonary fibrosis. TNF-alpha could be an important factor in the regulation of MMPs that can produce emphysema. We also have shown that TNF-alpha expression produces a complex cytokine response, which has a predominantly Th1 phenotype. However, the exact mechanism by which TNF-alpha alters cytokine expression is unclear and awaits further investigation. These mice provide the opportunity of further defining the relationship between chronic inflammation and the development of alveolar wall destruction.


    ACKNOWLEDGEMENTS

We thank Y. Miyazaki for providing the SP-C/TNF-alpha transgenic mice. We are appreciative of Karen Edeen for excellent technical assistance, Lynn Cunningham for the histology, and Karen Sheff for statistical analysis.


    FOOTNOTES

The study was supported by National Heart, Lung, and Blood Institute Grants HL-56556, HL-56638, HL-58464, and HL-60793 and by the Environmental Lung Agency (R825702).

Present address of C. G. Irvin: Dept. of Medicine, University of Vermont, Colchester, VT 05446.

Address for reprint requests and other correspondence: R. J. Mason, Dept. of Medicine, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206 (E-mail: masonb{at}njc.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 November 1999; accepted in final form 21 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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