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
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ABSTRACT |
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Tumor necrosis factor
(TNF)- 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-
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-
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
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INTRODUCTION |
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TUMOR NECROSIS
FACTOR (TNF)- is an important proinflammatory cytokine that
has been implicated in a variety of pathological processes. TNF-
is
produced mainly by activated monocytes or macrophages in response to
inflammatory stimuli such as lipopolysaccharide (LPS)
(32). TNF-
binds to receptors present on virtually all cells throughout the body. If TNF-
is released in large amounts systemically, TNF-
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-
may lead to chronic inflammation with fever, anemia, bone resorption, and wasting (3).
There are compelling reasons to believe that TNF- 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-
(45), platelet-derived growth factor,
transforming growth factor (TGF)-
, and TGF-
. The importance of
TNF-
in the pathogenesis of pulmonary fibrosis has been highlighted
in recent studies. TNF-
mRNA is increased in lungs from idiopathic
pulmonary fibrosis patients (37). Treatment with an
anti-TNF-
antibody prevents the development of both
bleomycin-induced pulmonary fibrosis (35) and
silica-induced pulmonary fibrosis (36). In animal models,
administration of a TNF-
soluble receptor prevents bleomycin-induced
pulmonary fibrosis (38). Recently, a murine model of
overexpression of TNF-
under control of the human surfactant protein
(SP) C promoter (SP-C/TNF-
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- by itself may not produce
pulmonary fibrosis. The effects of TNF-
on collagen gene expression
in vitro appear to be inhibitory (27). Although TNF-
causes acute lung inflammation (32), short-term TNF-
production by LPS or single-dose administration of TNF-
does not
lead to pulmonary fibrosis. Furthermore, gene transfer of TNF-
induces limited pulmonary fibrosis compared with TGF-
(41). Recently, Sulkowska et al. (43)
reported that long-term TNF-
administration in rats causes
emphysema-like pulmonary remodeling in addition to fibrosis. Although
TNF-
has been associated with emphysema (24), TNF-
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- 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.
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MATERIALS AND METHODS |
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Animals.
SP-C/TNF- 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-
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- ELISA.
The level of TNF-
was determined using a mouse TNF-
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. -Elastin zymography was also performed according
to a method described elsewhere (40). Briefly,
-elastin
(Elastin Products, Owensville, MO) was incorporated into SDS-
polyacrylamide gels. The concentration of
-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.
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RESULTS |
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Identification of SP-C/TNF- 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-
; 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).
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Air space enlargement and chronic inflammation in SP-C/TNF-
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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Pulmonary physiology of SP-C/TNF- 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).
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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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 -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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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-
mRNA expression as expected (Figs. 1C and
7A). In addition, the mRNAs
for IL-2, IL-10, interferon (IFN)-
, lymphotoxin (LT)-
, and
TNF-
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-
expression was extremely high, further increases in the
expression of IL-6, TGF-
1, and TGF-
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-
1 and TGF-
2 were confirmed, as reported by
others (42).
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DISCUSSION |
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The initial report using SP-C/TNF- transgenic mice indicated
that TNF-
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-
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- in the developing lung or acquired due to
the persistent chronic inflammation and chronic excessive protease
activity. The potential role of expression of TNF-
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-
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-
and
SP-C/platelet-derived growth factor mice do show air space enlargement
(19, 47). The SP-C/TGF-
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-
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-
could be
responsible for these changes. Administration of TNF-
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-
, was reported to induce emphysematous changes in the
lungs (4, 18). Thus we conclude that TNF-
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- 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-
1-trypsin leads to pulmonary emphysema. Since
>90% of pulmonary emphysema patients are smokers and do not have
anti-
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-
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- is well known to upregulate MMPs including
collagenase (33). Gelatinases A and B were upregulated in
the TNF-
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- overexpression may be due to alteration
in expression levels of other cytokines. For example, TNF-
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-
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-
(15). IL-10 was originally identified as an inhibitor of
IFN-
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-
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-
, 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- transgenic mice. The hypothesis that the
Th2 response leads to chronic inflammation and emphysema is supported
by the observation that overexpression of IFN-
, 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- overexpression mediated by an adenovirus vector
administration exhibited only limited pulmonary fibrosis. Although the
role of TNF-
has been well established in the development of
pulmonary fibrosis, our impression is that overexpression of TNF-
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- is known to affect the circulatory system (28). In
these SP-C/TNF-
transgenic mice, however, serum levels of TNF-
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-
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-
has also been reported to increase pulmonary
arterial pressure (23). TNF-
may also increase
expression of vasoconstrictors such as endothelin. Thus TNF-
itself
may, in part, be responsible for pulmonary hypertension. Further study
will be required to clarify the role of TNF-
as well as the
mechanism of pulmonary hypertension in TNF-
-overexpressing mice.
TNF- 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-
. Serum TNF-
levels are
reported to be higher in patients with COPD than in normal controls.
TNF-
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-
levels have been found in
induced sputum from patients with COPD (24). Moreover,
TNF-
has been observed in bronchial biopsies from COPD patients
using immunocytochemical techniques (31). Burnett and his
colleagues (5) reported that TNF-
, but not IL-1 and
IFN-
, augmented fibronectin digestion by neutrophils. Taken
together, these data suggest that TNF-
may be a significant contributor to the pathophysiology of emphysema.
In mice with targeted TNF--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-
could be an important factor in the
regulation of MMPs that can produce emphysema. We also have shown that
TNF-
expression produces a complex cytokine response, which has a
predominantly Th1 phenotype. However, the exact mechanism by which
TNF-
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.
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ACKNOWLEDGEMENTS |
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We thank Y. Miyazaki for providing the SP-C/TNF- transgenic
mice. We are appreciative of Karen Edeen for excellent technical assistance, Lynn Cunningham for the histology, and Karen Sheff for
statistical analysis.
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FOOTNOTES |
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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.
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