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*
First Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan;
Miyagi Red Cross Blood Center, Sendai, Japan;
Biopharmaceutical Department, Fuji Chemical Industries, Takaoka, Japan; and
§
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent endopeptidases capable of proteolytically degrading many of the components of extracellular matrix (9). MMPs are produced by not only structural cells such as fibroblasts, endothelial cells, and epithelial cells (10, 11), but also inflammatory cells such as macrophages (12), lymphocytes (13), neutrophils (14), and eosinophils (15). They are secreted as latent forms followed by proteolytic processing to active forms (9), and involved in the physiologic processes such as development, angiogenesis, and wound healing, and in the pathologic conditions such as tumor invasion and inflammation (10, 16). The proteolytic activation of latent forms and enzymatic activities by active forms of MMPs is inhibited by endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs) that form a 1:1 complex with MMPs (9). The balance between the levels of MMPs and TIMPs is thought to be a critical factor in regulating the breakdown of connective tissues by MMPs, which is the case in pulmonary emphysema (17). Of the MMP family, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) cleave type IV collagen, which is an important constituent of basement membrane. They are posttranslationally regulated by TIMP-1 and -2, which make complexes with active form of either MMP-2 or -9, and with latent form of MMP-9 and -2, respectively (9).
Tumor cells penetrate basement membrane by making holes with secreted MMP-2 and -9 (18). Recently, MMP-2 and -9 were reported in in vitro system to play a crucial role also in the transmigration of lymphocytes (13), and neutrophils (14) through basement membrane components. Furthermore, we demonstrated that MMP-9 was also important in the in vitro transmigration of basement membrane components by eosinophils (19), and that MMP-9 was overexpressed by eosinophils accumulating in airway walls of asthmatics (20). However, despite the results that MMP-2 and -9 are crucial for the in vitro transmigration of inflammatory cells and that MMPs are produced by inflammatory cells at the site of inflammation, it has not been determined yet whether they are also crucial for the in vivo accumulation of inflammatory cells to the airways in bronchial asthma. Thus, we hypothesized that MMP-2 and/or -9 play important roles in the in vivo migration of inflammatory cells to the airways in which the cells have to transverse endothelial and epithelial basement membrane. To examine the hypothesis, using a murine model of allergic asthma, we studied first the production of MMP-2 and -9 in airways after Ag challenge, and secondly the effect of MMP inhibitors, recombinant human (rh) TIMP-1 and -2, and a synthetic inhibitor of MMPs, on inflammatory cell accumulation to airways and on airway hyperresponsiveness.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free female BALB/c mice, 6 to 8 wk old, purchased from our animal facility (Institute for Experimental Animals, Tohoku University School of Medicine, Sendai, Japan), were sensitized and challenged, as previously described (21). Briefly, mice were sensitized by i.p. injections of OVA (Sigma, St. Louis, MO) (8 µg/mouse) adsorbed with aluminum hydroxide (Wako Pure Chemical Industries, Osaka, Japan) on days 0 and 5. Ten to fourteen days after the last injection, mice were challenged with aerosolized OVA or saline as a control. At various time points, mice were anesthetized with diethylether (Wako), and blood samples were obtained by retro-orbital bleeding. Blood smears were stained with Diff-Quick solution (International Reagents, Kobe, Japan) for differential cell counting. Total white cell number in peripheral blood was counted with a hemocytometer after RBC lysis using RBC lysis buffer. Bronchoalveolar lavage (BAL) was performed and lung tissues were collected, as previously described (21). Lungs were washed twice by the injection of HBSS (Life Technologies, Grand Island, NY) (0.25 ml and 0.20 ml, respectively) through the trachea, and approximately 0.4 ml of the instilled fluid was consistently recovered from each mouse. Total cell numbers were counted with a hemocytometer. Smears of BAL cells prepared with a Cytospin II (Shandon, Runcorn, U.K.) were stained with Diff-Quick solution for differential cell counting or fixed with PLP solution (10 mM sodium m-periodate (Sigma) to 75 mM L-lysine monohydrochloride (Wako)-2% paraformaldehyde (Nacalai Tesque, Kyoto, Japan) in 37.5 mM PBS) for immunocytochemistry. Counts of cells in peripheral blood and BAL fluids were performed by a person unaware of the experiment. The percentages of cell differentials were determined by counting at least 300 cells under a light microscopy. After centrifugation of BAL fluid, supernatants were stored at -20°C until for use. Tracheal tissues were fixed with PLP solution and embedded in OCT compound (Miles Laboratories, Naperville, IL) with liquid nitrogen. Five-micrometer-thick sections were used for the counting of tissue eosinophils after staining with Diff-Quick solution and for that of CD3-positive cells after immunohistochemical staining. The numbers of eosinophils and CD3-positive cells were counted in whole areas of submucosae and expressed as a number/mm of basement membrane of epithelium. The length of the basement membrane was measured with MCID image analyzer (Imaging Research, St. Catherines, Ontario, Canada). An average of data in three sections was used as the result for each mouse. These studies had been approved by the Ethics Committee for Animal Experiments of the Tohoku University School of Medicine.
Administration of TIMPs and a MMP inhibitor
rhTIMP-1 (Fuji Chemical Industries, Toyama, Japan) (25 µg in 25 µl of saline/mouse), rhTIMP-2 (Fuji) (25 µg in 25 µl of saline/mouse), or saline alone (25 µl/mouse), each of which contained <0.005 endotoxin units (EU) of endotoxin, was instilled intranasally six times, in some experiments fourteen times, with 12-h interval, beginning at 1 h before OVA inhalation. A synthetic inhibitor of MMPs, R-94138 (IC50 on MMP-9; 1.2 nM, MMP-2; 38 nM, MMP-3; 120 nM, MMP-7; 23 µM, MMP-13; 38 nM) (compound 31f in 22) (kindly provided by Dr. K. Tanzawa, Sankyo, Tokyo, Japan), was injected i.p. (30 mg/kg body weight) three times with 24-h interval, beginning at 30 min before OVA inhalation. Three days after the OVA inhalation, mice were sacrificed and samples were collected, as mentioned above.
Zymography
Gelatin zymography was performed as described previously (23). A total of 10 µl of BAL fluid underwent electrophoresis in 7.5% polyacrylamide gels containing 2.8 mg/ml gelatin (Sigma), in the presence of SDS (Wako) under nonreducing conditions. After electrophoresis, gels were washed three times each for 15 min in 50 mM Tris-HCl (Sigma) (pH 7.5) containing 2.5% Triton X-100 (Sigma), 10 mM CaCl2 (Wako), and 1 µM ZnCl2 (Wako), and then incubated in 1% Triton X-100 at 37°C for 24 h in the presence or absence of EDTA (Dojin Chemical Laboratories, Kumamoto, Japan) (20 mM), 1,10-phenanthroline (Sigma) (5 mM), or PMSF (Sigma) (2 mM). Following incubation, the gels were stained with Coomassie brilliant blue R 250 (Fluka Chemie AG, Buchs, Switzerland) and destained in a solution of 1% formic acid with 30% methanol. Gelatinolytic activity was detected as clear bands against a blue background. The intensity of the bands in inverted image of zymogram was estimated using densitometry with National Institute of Health image 1.61. hMMP-2 and hMMP-9 from HT1080 cells were used as positive controls in gelatin zymography.
Immunoprecipitation
To determine types of MMPs to which intranasally administered rhTIMP-2 bound, BAL fluids from rhTIMP-2-administered mice after OVA inhalation were immunoprecipitated with a mouse monoclonal anti-hTIMP-2 IgG1, 68-6H4 (Fuji), which recognizes free TIMP-2, TIMP-2 binding to active MMP-2 and -9, and pro-MMP-2 (24). A total of 300 µl of BAL fluids was incubated with anti-hTIMP-2 Ab at the concentration of 70 µg/ml in a 1.5-ml Eppendorf tube. After overnight incubation at 4°C, samples were added with 30 µl of packed protein G-Sepharose (4FF; Amersham, Buckinghamshire, U.K.), and further incubated for 90 min. The immunoprecipitates were collected as pellets after centrifugation. After the supernatants were removed, the pellets were washed and suspended with 300 µl of electrophoresis sample buffer. Then the immunoprecipitates and the supernatants were subjected to gelatin zymography, as mentioned above.
Western blot analysis of BAL fluids
Immunoblotting was performed to certify the gelatinolytic activity as MMP-9. BAL fluids concentrated five times by Microcon 10 (Amicon, Beverly, MA) and purified hMMP-9 were loaded to SDS-PAGE under nonreducing conditions, and were transferred to a membrane (Immobilon PVDF; Millipore, Bedford, MA). After blocking with 22% skim milk, the blots were incubated overnight with fibronectin-absorbed polyclonal anti-hMMP-9 sheep IgG (25). After several washes and blocking again, rabbit IgG anti-sheep IgG conjugated with horseradish peroxidase (ICN Pharmaceuticals, Costa Mesa, CA) were applied to the blots. After several washes, blots were soaked in enhanced chemoluminescence (ECL) solution (Amersham Pharmacia Biotech, Uppsala, Sweden) and were exposed to x-ray films. Western blotting was performed also with fibronectin-absorbed polyclonal anti-hMMP-2 rabbit IgG and anti-hMMP-1 and -3 sheep serum (25).
Immunostaining
CD3-positive cells in sections of tracheal tissues were investigated with anti-mouse CD3e hamster IgG (PharMingen, San Diego, CA) or normal hamster IgG (PharMingen) as a control Ab, using biotin-labeled anti-hamster IgG (PharMingen) and alkaline phosphatase-conjugated avidin-biotin complex (Strept ABComplex/AP; Dako, Carpenteria, CA), and visualized with a Fast Red Substrate System (Dako), followed by counterstaining with hematoxylin. Immunoreactivity of MMP-9 in BAL cells was examined with fibronectin-absorbed polyclonal anti-hMMP-9 sheep IgG or normal sheep IgG (ICN) as a control Ab, and rabbit IgG anti-sheep IgG conjugated with horseradish peroxidase, and was visualized with diaminobenzidine, followed by counterstaining with methyl green.
Measurement of IL-5 in BAL fluids
The content of IL-5 in BAL fluids was measured with a specific mouse (m) IL-5 ELISA kit (Amersham). The detection limit of the IL-5 assay was 5 pg/ml, and the assay is said by the manufacturer to be specific for IL-5.
Airway hyperresponsiveness
Airway responsiveness of mice 3 days after OVA inhalation, followed by intranasal administration of rhTIMP-2 or saline as mentioned above, was assessed. As a control, sensitized mice were inhaled with saline, and then intranasally administered with saline in the same way. Airway responsiveness was assessed as a change in pulmonary resistance (Rl) after injections of increasing dose of methacholine (MCh, acetyl-ß-methylcholine chloride) (Wako) (0.1–30 mg/kg) in 50-µl volumes. Mice were anesthetized by i.p. injection of pentobarbital sodium (Wako) (50 mg/kg), and were tracheostomized. Air flow rate at airway opening during spontaneous breathing was monitored by a pneumotachogram (8430B; Hans Rudolph, Kansas City, MO) combined with a differential pressure transducer (LCVR, 0–2 cm H2O; Celesco, Canoga Park, CA). Esophageal pressure monitored by a water-filled tube and a pressure transducer (Ohmeda, Singapore) was used as transpulmonary pressure, because the pressure difference generated by the pneumotachogram connected to tracheal tube was very small (more than 100 times) compared with the amplitude of esophageal pressure. Rl was calculated by the subtraction method of Mead and Whittenberger (26). An average Rl of three breaths at 3 min after each injection of MCh was calculated, and expressed as a percentage of baseline Rl that was measured and calculated in the same way after the injection of saline used as a diluent of MCh.
Data analysis
All data are presented as means ± SEM. Significant differences in counts of cells in BAL fluids and in tracheal walls during the time course after OVA or saline inhalation were determined using one-way analysis of variance with post hoc analysis of Fisher’s Protected Least Significance. Significant differences between groups in the others were determined using unpaired Student’s t test. These analyses were performed using StatView 4.11 (Abacus Concepts, Berkeley, CA) for Macintosh. A p value of less than 0.05 was taken as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Total cell numbers in BAL fluids were significantly increased at 3
to 14 days after OVA inhalation compared with either those before the
inhalation or those after saline inhalation (Fig. 1
, A and B). The
increase of total cell numbers was associated with significant increase
of eosinophils and lymphocytes at 3 to 7 days, that of macrophages at 3
days, and that of neutrophils at 5 days after OVA inhalation. In
contrast, no significant changes in cell numbers were observed after
saline inhalation (Fig. 1B).
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Eosinophils in tracheal walls significantly increased at 12 h
after OVA inhalation and remained increased until day 5 compared with
those before the inhalation (Fig. 1
C). In contrast, only a
few increases of eosinophils were observed after saline inhalation
(data not shown).
Gelatinolytic activities in BAL fluids
Gelatin zymography of BAL fluids showed the constitutive
expression of 65- and 60-kDa gelatinase activities and the induction of
105-kDa gelatinase activity after OVA inhalation (Fig. 2, A and B). The
gelatinolytic activities in BAL fluids at 3 days after OVA inhalation,
as well as those of purified hMMP-2 and -9, were inhibited by EDTA or
by 1,10-phenanthroline, but not by PMSF (data not shown). The
gelatinolytic activity with a molecular mass of 105 kDa is consistent
with being the zymogen of mMMP-9 (27). The identity of pro-MMP-9 was
further confirmed by Western blotting analysis (Fig. 3). Western analysis of concentrated BAL
fluids from OVA-inhaled mice with anti-hMMP-9 Ab also showed the
presence of a 95-kDa band (Fig. 3), which corresponds to the active
mMMP-9 (27). The 65- and 60-kDa gelatinolytic activities correspond to
a pro- and active forms of mMMP-2, respectively (28). Anti-hMMP-2 could
not detect any band in blots of BAL fluids. We also performed Western
blot analysis of BAL fluids with anti-hMMP-1 and -3 Abs, but these
MMPs were not detected. Semiquantitative analysis of gelatinolytic
activities revealed that pro-MMP-9 activities were significantly higher
on days 3 and 5 in OVA-inhaled mice than in saline-inhaled mice (Fig. 2C), and that pro-MMP-2 and active MMP-2 activities were
significantly higher on day 3 (Fig. 2, D and E).
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Immunocytochemistry with anti-hMMP-9 Ab used in Western
blotting analysis showed the localization of immunoreactive MMP-9 on
BAL cells, including macrophage-, lymphocyte-, and eosinophil-like
cells, from sensitized mice at 3 days after OVA inhalation (Fig. 4B). In contrast, no
immunoreactivity was detected on BAL cells from those at 3 days after
saline inhalation (Fig. 4A). No positive staining was
detected with control Ab in BAL cells from either group of mice (data
not shown).
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Intranasal administration of rhTIMP-2 (n = 6)
significantly inhibited the enhanced leukocyte accumulation in the
airway lumen at 3 days after OVA inhalation, compared with intranasal
administration of saline (n = 4), as shown by total
cell counts (3.35 ± 0.37 versus 7.50 ± 0.70 x
105/ml, p < 0.05) and differential cell
counts including macrophages (2.21 ± 0.19 versus 3.34 ±
0.36, p < 0.05), eosinophils (0.80 ± 0.14 versus
3.37 ± 0.43, p < 0.01), and lymphocytes
(0.19 ± 0.03 versus 0.61 ± 0.05, p < 0.05)
(Fig. 5A). The inhibitory
effect of rhTIMP-2 was observed also at 7 days after OVA inhalation
(data not shown). There was no significant difference in both total and
differential cell counts between rhTIMP-2- and saline-administered mice
after saline inhalation (data not shown). Similarly, either rhTIMP-1
(Fig. 5B) or R-94138, a synthetic MMP inhibitor (Fig. 5C), inhibited leukocyte infiltration after OVA inhalation.
The MMP inhibitors used in this study had no effect on neutrophil
number, which was not significantly increased after OVA inhalation.
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The concentration of IL-5 in BAL fluids was increased at 3 days after OVA inhalation (173.8 ± 47.1 pg/ml, n = 5) compared with saline inhalation (6.4 ± 1.1 pg/ml, n = 5). However, the increase of IL-5 concentration was not significantly affected by the administration of rhTIMP-2 (144.9 ± 36.2 pg/ml, n = 5).
The effect of TIMP-2 on the accumulation of eosinophils and CD3-positive cells in tracheal walls
The increased numbers of eosinophils and CD3-positive cells in
tracheal walls of sensitized mice at 3 days after OVA inhalation were
significantly reduced (eosinophils; 38.5 ± 6.4 (n
= 6) versus 18.7 ± 2.8 (n = 6) cells/mm,
p < 0.05, CD3-positive cells; 11 ± 2.1
(n = 4) versus 4.3 ± 1.3 (n = 4)
cells/mm, p < 0.05) by the intranasal administration
of rhTIMP-2 (Fig. 6).
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In contrast to the reduction of cell counts in airway lumen and
tracheal walls, total leukocyte, eosinophil, and lymphocyte counts in
peripheral blood were significantly increased in mice administered with
rhTIMP-2 (n = 6) compared with those administered with
saline (n = 4) after OVA inhalation (17,080 ±
1,336.6 versus 10,920 ± 1,555, p < 0.05;
1,076.3 ± 163.2 versus 516.1 ± 95, p <
0.05; and 13,655.6 ± 1,112.2 versus 8,942.1 ± 1,323.7
103/ml, p < 0.05, respectively) (Fig. 7).
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Baseline Rl of saline-challenged and
saline-administered, OVA-challenged and saline-administered, and
OVA-challenged and rhTIMP-2-administered groups were 0.29 ± 0.11
(n = 4), 0.26 ± 0.06 (n = 6), and
0.33 ± 0.11 (n = 4)
cmH2O · ml-1·sec, respectively,
and there was no significant difference between three groups. Airway
responsiveness assessed as percentage of increase in Rl in
response to increasing dose of MCh was increased after the OVA
challenge (Fig. 8). Compared with the
saline-challenged group, dose-response curve of percentage of
Rl shifted to left side, and percentage of Rl
significantly increased at 10 mg/kg of MCh in the OVA-challenged group
(717 ± 153%, p < 0.05). Administration of
rhTIMP-2 reduced airway hyperresponsiveness induced by OVA challenge.
In the OVA-challenged group with rhTIMP-2 administration, percentage of
Rl at 10 mg/kg of MCh was significantly reduced (212 ±
94%, p < 0.05) compared with the OVA-challenged group
with saline administration, and was almost as same as the
saline-challenged group (207 ± 31%). The inhibitory effect of
rhTIMP-2 on airway hyperresponsiveness was observed also at 7 days
after OVA inhalation (data not shown).
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There was no significant difference in gelatinolytic activities
detected by zymography in BAL fluids between rhTIMP-2- and
saline-administered mice after OVA inhalation (data not shown). Gelatin
zymography of immunoprecipitates with anti-hTIMP-2 Ab showed that
rhTIMP-2 intranasally administered had made complexes with, at least,
active and pro-MMP-2 in mice with OVA inhalation, while not with
pro-MMP-9, as expected (Fig. 9,
lane 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The up-regulation of MMP-9 release observed in this study is consistent with the previous reports that MMP-9 content in BAL fluids from untreated asthmatics was increased compared with that from control subjects including steroid-treated asthmatics (12), and that Ag challenge increased enzyme activity mainly due to MMP-9 in BAL fluids from asthmatics (29). In this study, we could not detect MMP-2 in BAL fluids by Western blotting analysis using anti-hMMP-2 antiserum that cross-reacts with mouse MMP-2 (25). A reason for this failure is most likely due to low sensitivity of the assay. MMP-1 and -3 were also not detected either by zymography or Western blotting. However, because of the same reason as in the case of MMP-2, i.e., the limitation of the assay sensitivity, the possibility of the presence of these and other types of MMPs could not be ruled out.
Sources of MMP-2 and -9 detected in BAL fluids after OVA inhalation
were not determined in this study. However, the result of
immunostaining of BAL cells with anti-MMP-9 Ab suggests that
macrophages, lymphocytes, and eosinophils in airways are potential
sources of the MMPs, and this is supported by the studies that have
been done on human leukocytes (12, 13, 20). It has also been
demonstrated that bronchial and alveolar epithelial cells
constitutively express MMP-2 and -9, and that the expression is
increased by the stimulation with TNF-
and IL-1ß (11), which are
increased in asthmatic inflammation (30, 31). The release of MMP-2 and
-9 from bovine airway mucosa is enhanced when cocultured with
eosinophils (32). These reports suggest that the release of MMP-2 and
-9 from structural cells including epithelial cells during the
activation by proinflammatory cytokines and eosinophils might occur in
our model. In our study, the peak of MMP-9 activity was at 5 days after
OVA challenge, while MMP-2 activity was maximal at 3 days. These data
suggest that the expressions of MMP-2 and MMP-9 after Ag challenge are
differentially regulated. More importantly, the levels of MMPs in BAL
fluids were not reduced by rhTIMP-2 in OVA-challenged mice, but the
cell infiltration to airway lumen was reduced. Thus, a variety of cell
types and inductive mechanisms are likely to be involved in the
production of MMPs during asthmatic reaction.
Our studies suggest the relationship between the release of MMP-2 and
-9 and the recruitment of leukocytes to the site of inflammation. To
investigate further the role of the MMPs in allergic airway
inflammation, we applied two types of natural inhibitors of MMPs,
TIMP-1 and -2, and a synthetic MMP inhibitor to this model. rhTIMP-2
reduced the cellular infiltrates in BAL fluids and tracheal walls that
were increased after OVA inhalation. It is unlikely that rhTIMP-2 was
cytotoxic to cellular infiltrates, because leukocyte counts in
peripheral blood were increased in contrast to those in BAL fluids and
tracheal walls. In addition, there was no change in cell counts in BAL
fluids from sensitized mice treated with rhTIMP-2 after saline
challenge. Therefore, the reduction of the number of inflammatory cells
in BAL fluids and tracheal walls by the administration of rhTIMP-2 is
likely to be due to the inhibition of the movement of leukocytes from
circulation to the site of inflammation, resulting in an increase of
blood leukocytes. As demonstrated in Fig. 9, rhTIMP-2 was bound to
active and pro-MMP-2 in vivo. rhTIMP-1 and a synthetic MMP inhibitor
that inhibit enzyme activity of MMP-2 and MMP-9 (9, 10, 22) also
prevented the cellular infiltration in BAL fluids. Together with these
results, the inhibitory effect of rhTIMP-2 on the cell infiltration was
attributed to the inhibition of enzymatic activities of MMPs,
including, at least, MMP-2 and -9 at airway lumen and submucosae, where
the presence of rhTIMP-2 was demonstrated by reverse zymography of BAL
fluids and by immunohistochemistry of tracheal tissues (data not
shown). Alternatively, these results indicate the participation of
MMPs, at least MMP-2 and -9, to the cellular infiltration in allergic
airway responses.
In the processes of cellular infiltration from circulation to inflammatory sites, MMP-2 and -9 could be involved in the migration of inflammatory cells through endothelial and epithelial basement membrane, as suggested by in vitro studies demonstrating that lymphocytes, neutrophils, and eosinophils passed through the basement membrane by degrading its components with their own MMP-2 and MMP-9 (13, 14, 19). In this study, we observed the delay in eosinophil accumulation in BAL fluids compared with that in the airway walls. Previously, we observed that eosinophils purified from peripheral blood passed through the basement membrane in vitro within 30 min upon stimulation (19), and that tissue eosinophils in airway mucosa of asthmatics synthesized MMP-9 (20). These results led us to assume that eosinophils utilize stored MMP-9 for the transmigration through the subendothelial basement membrane, and, after extravasation, start to newly synthesize MMP-9 for the transmigration through the subepithelial basement membrane in response to microenvironmental stimulation such as cytokines, LTs, and PAF.
Cytokines and chemokines are believed to play a central role in the induction of allergic airway inflammation in bronchial asthma (3, 6, 7). Among them, IL-5, whose expression is increased in BAL fluids and bronchial biopsy tissues from asthmatics (33), is considered as one of the key cytokines promoting eosinophil accumulation (34, 35). In our model, the level of IL-5 in BAL fluids increases at 3 days after OVA inhalation (21). Interestingly, the level of IL-5 in rhTIMP-2-treated mice, in which the eosinophil number in airways was significantly reduced, was similar to that in saline-treated mice after OVA inhalation. These results indicate that the inhibitory effect of rhTIMP-2 on eosinophil infiltration was not, at least, due to the down-regulation of IL-5. Although the accumulation of lymphocytes, which are main sources of IL-5 in airways (33), was reduced significantly by the administration of rhTIMP-2, the IL-5 content in BAL fluids of the rhTIMP-2-treated mice was not changed. One possible interpretation of this observation was that a small number of lymphocyte, eosinophil (36), and/or mast cells (37) residing in or infiltrated to airways might be enough to secrete the same level of IL-5 as in OVA-challenged and saline-administered mice.
The administration of rhTIMP-2 also reduced airway hyperresponsiveness, which is a pathophysiologic feature of bronchial asthma (1). Increasing pieces of evidence have demonstrated that eosinophilic airway inflammation plays a crucial role in the induction of airway hyperresponsiveness. During the activation at the site of inflammation, eosinophils release toxic granule protein such as eosinophilic cationic protein, and major basic protein, LTC4, and PAF. These products can cause the physiologic manifestation of airway hyperresponsiveness (38). T lymphocytes are also thought to play a role in the development of airway hyperresponsiveness (39), although the mechanism is not clear. Nonetheless, our studies have demonstrated that inhibition of cell infiltration by rhTIMP-2 abolished airway hyperresponsiveness, indicating that infiltrated cells after Ag challenge, but not resident cells, are important in the development of airway hyperresponsiveness.
Although neutrophil infiltration to airways is not so prominent as eosinophils in our model, the number of neutrophils in BAL fluids was not different between rhTIMP-2-treated mice and saline-treated mice after OVA inhalation. In a separate experiment, we examined the effect of rhTIMP-2 on neutrophil accumulation into airway lumen in mice inhaled with LPS (50 µg/ml, 30 min). In this case, the accumulation of neutrophils to BAL fluids was not inhibited by rhTIMP-2 (data not shown). It is suggested that neutrophils utilize both MMP-9 and elastase in their trans-basement membrane migration in vitro (14), and neutrophil infiltration to airways in an animal model with LPS inhalation is inhibited by an elastase inhibitor (40). Thus, infiltration of neutrophils appears to be more dependent on elastase than MMPs, and this may be one of the reasons that neutrophils and eosinophils accumulate to the site of inflammation differently.
In summary, we have demonstrated that inhibitors of MMPs, TIMP-1, TIMP-2, and a synthetic inhibitor reduced the development of allergic airway inflammation, possibly by inhibiting MMP-2 and MMP-9, and that the administration of rhTIMP-2 itself does not affect the physiologic cell infiltration. Our studies suggest that the inhibition of MMPs is a new therapeutic strategy for bronchial asthma, although further investigation is necessary for our clear understanding of the regulatory mechanisms of MMP expression and the exact role of different MMPs in asthmatic airways.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: LT, leukotriene; BAL, bronchoalveolar lavage; h, human; m, mouse; MCh, methacholine; MMP, matrix metalloproteinase; PAF, platelet-activating factor; Rl, pulmonary resistance; TIMP, tissue inhibitor of metalloproteinase.
Received for publication September 28, 1998. Accepted for publication December 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Am. J. Physiol. 273:L866.: a potential mediator of asthma. Clin. Exp. Allergy 25:1038.[Medline]
) cytokines in bronchoalveolar lavage and bronchial biopsies from atopic asthmatic and normal control subjects. Am. J. Respir. Cell Mol. Biol. 12:477.[Abstract]
in normal and asthmatic airways: evidence for the human mast cell as a source of these cytokines. Am. J. Respir. Cell Mol. Biol. 10:471.[Abstract]
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 M. Abel and H. Vliagoftis Mast Cell-Fibroblast Interactions Induce Matrix Metalloproteinase-9 Release from Fibroblasts: Role for IgE-Mediated Mast Cell Activation J. Immunol., March 1, 2008; 180(5): 3543 - 3550. [Abstract] [Full Text] [PDF]
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E. L. Martin, T. A. Sheikh, K. J. Leco, J. F. Lewis, and R. A. W. Veldhuizen Contribution of alveolar macrophages to the response of the TIMP-3 null lung during a septic insult Am J Physiol Lung Cell Mol Physiol, September 1, 2007; 293(3): L779 - L789. [Abstract] [Full Text] [PDF]
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 C. M. Swaisgood, M. A. Aronica, S. Swaidani, and E. F. Plow Plasminogen Is an Important Regulator in the Pathogenesis of a Murine Model of Asthma Am. J. Respir. Crit. Care Med., August 15, 2007; 176(4): 333 - 342. [Abstract] [Full Text] [PDF]
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G. Candiano, M. Bruschi, N. Pedemonte, L. Musante, R. Ravazzolo, S. Liberatori, L. Bini, L. J. V. Galietta, and O. Zegarra-Moran Proteomic analysis of the airway surface liquid: modulation by proinflammatory cytokines Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L185 - L198. [Abstract] [Full Text] [PDF]
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N. Di Girolamo, I. Indoh, N. Jackson, D. Wakefield, H. P. McNeil, W. Yan, C. Geczy, J. P. Arm, and N. Tedla Human Mast Cell-Derived Gelatinase B (Matrix Metalloproteinase-9) Is Regulated by Inflammatory Cytokines: Role in Cell Migration J. Immunol., August 15, 2006; 177(4): 2638 - 2650. [Abstract] [Full Text] [PDF]
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 M. A. Birrell, S. Wong, A. Dekkak, J. De Alba, S. Haj-Yahia, and M. G. Belvisi Role of Matrix Metalloproteinases in the Inflammatory Response in Human Airway Cell-Based Assays and in Rodent Models of Airway Disease J. Pharmacol. Exp. Ther., August 1, 2006; 318(2): 741 - 750. [Abstract] [Full Text] [PDF]
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D. H. Lim, J. Y. Cho, M. Miller, K. McElwain, S. McElwain, and D. H. Broide Reduced peribronchial fibrosis in allergen-challenged MMP-9-deficient mice. Am J Physiol Lung Cell Mol Physiol, August 1, 2006; 291(2): L265 - L271. [Abstract] [Full Text] [PDF]
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J. J. Atkinson and S. D. Shapiro Neutrophil-mediated eosinophil transmigration: parting the red sea or leaving bread crumbs. Am. J. Respir. Cell Mol. Biol., June 1, 2006; 34(6): 645 - 646. [Full Text] [PDF]
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 R O'Donnell, D Breen, S Wilson, and R Djukanovic Inflammatory cells in the airways in COPD Thorax, May 1, 2006; 61(5): 448 - 454. [Abstract] [Full Text] [PDF]
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S. J. Park, M. T. Wiekowski, S. A. Lira, and B. Mehrad Neutrophils Regulate Airway Responses in a Model of Fungal Allergic Airways Disease J. Immunol., February 15, 2006; 176(4): 2538 - 2545. [Abstract] [Full Text] [PDF]
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S. Chakrabarti, J. M. Zee, and K. D. Patel Regulation of matrix metalloproteinase-9 (MMP-9) in TNF-stimulated neutrophils: novel pathways for tertiary granule release J. Leukoc. Biol., January 1, 2006; 79(1): 214 - 222. [Abstract] [Full Text] [PDF]
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 R. L. Warner, N. W. Lukacs, S. D. Shapiro, N. Bhagarvathula, K. C. Nerusu, J. Varani, and K. J. Johnson Role of Metalloelastase in a Model of Allergic Lung Responses Induced by Cockroach Allergen Am. J. Pathol., December 1, 2004; 165(6): 1921 - 1930. [Abstract] [Full Text] [PDF]
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I. Fajardo, L. Svensson, A. Bucht, and G. Pejler Increased Levels of Hypoxia-sensitive Proteins in Allergic Airway Inflammation Am. J. Respir. Crit. Care Med., September 1, 2004; 170(5): 477 - 484. [Abstract] [Full Text] [PDF]
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P. Baluk, W. W. Raymond, E. Ator, L. M. Coussens, D. M. McDonald, and G. H. Caughey Matrix metalloproteinase-2 and -9 expression increases in Mycoplasma-infected airways but is not required for microvascular remodeling Am J Physiol Lung Cell Mol Physiol, August 1, 2004; 287(2): L307 - L317. [Abstract] [Full Text] [PDF]
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 I. I. Ekekezie, D. W. Thibeault, S. D. Simon, M. Norberg, J. D. Merrill, R. A. Ballard, P. L. Ballard, and W. E. Truog Low Levels of Tissue Inhibitors of Metalloproteinases With a High Matrix Metalloproteinase-9/Tissue Inhibitor of Metalloproteinase-1 Ratio Are Present in Tracheal Aspirate Fluids of Infants Who Develop Chronic Lung Disease Pediatrics, June 1, 2004; 113(6): 1709 - 1714. [Abstract] [Full Text] [PDF]
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S. J. McMillan, J. Kearley, J. D. Campbell, X.-W. Zhu, K. Y. Larbi, J. M. Shipley, R. M. Senior, S. Nourshargh, and C. M. Lloyd Matrix Metalloproteinase-9 Deficiency Results in Enhanced Allergen-Induced Airway Inflammation J. Immunol., February 15, 2004; 172(4): 2586 - 2594. [Abstract] [Full Text] [PDF]
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M. A. Pouladi, C. S. Robbins, F. K. Swirski, M. Cundall, A. N.J. McKenzie, M. Jordana, S. D. Shapiro, and M. R. Stampfli Interleukin-13-Dependent Expression of Matrix Metalloproteinase-12 Is Required for the Development of Airway Eosinophilia in Mice Am. J. Respir. Cell Mol. Biol., January 1, 2004; 30(1): 84 - 90. [Abstract] [Full Text] [PDF]
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E. L. Martin, B. Z. Moyer, M. C. Pape, B. Starcher, K. J. Leco, and R. A. W. Veldhuizen Negative impact of tissue inhibitor of metalloproteinase-3 null mutation on lung structure and function in response to sepsis Am J Physiol Lung Cell Mol Physiol, December 1, 2003; 285(6): L1222 - L1232. [Abstract] [Full Text] [PDF]
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D. Wingett and C. P. Nielson Divergence in NK cell and cyclic AMP regulation of T cell CD40L expression in asthmatic subjects J. Leukoc. Biol., October 1, 2003; 74(4): 531 - 541. [Abstract] [Full Text] [PDF]
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Y Oshita, T Koga, T Kamimura, K Matsuo, T Rikimaru, and H Aizawa Increased circulating 92 kDa matrix metalloproteinase (MMP-9) activity in exacerbations of asthma Thorax, September 1, 2003; 58(9): 757 - 760. [Abstract] [Full Text] [PDF]
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K. Y. Vermaelen, D. Cataldo, K. Tournoy, T. Maes, A. Dhulst, R. Louis, J.-M. Foidart, A. Noel, and R. Pauwels Matrix Metalloproteinase-9-Mediated Dendritic Cell Recruitment into the Airways Is a Critical Step in a Mouse Model of Asthma J. Immunol., July 15, 2003; 171(2): 1016 - 1022. [Abstract] [Full Text] [PDF]
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 M. Selman, J. Cisneros-Lira, M. Gaxiola, R. Ramirez, E. M. Kudlacz, P. G. Mitchell, and A. Pardo Matrix Metalloproteinases Inhibition Attenuates Tobacco Smoke-Induced Emphysema in Guinea Pigs Chest, May 1, 2003; 123(5): 1633 - 1641. [Abstract] [Full Text] [PDF]
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J. J. Atkinson and R. M. Senior Matrix Metalloproteinase-9 in Lung Remodeling Am. J. Respir. Cell Mol. Biol., January 1, 2003; 28(1): 12 - 24. [Abstract] [Full Text] [PDF]
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D. D. Cataldo, J. Bettiol, A. Noel, P. Bartsch, J.-M. Foidart, and R. Louis Matrix Metalloproteinase-9, but Not Tissue Inhibitor of Matrix Metalloproteinase-1, Increases in the Sputum From Allergic Asthmatic Patients After Allergen Challenge Chest, November 1, 2002; 122(5): 1553 - 1559. [Abstract] [Full Text] [PDF]
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D. D. Cataldo, K. G. Tournoy, K. Vermaelen, C. Munaut, J.-M. Foidart, R. Louis, A. Noel, and R. A. Pauwels Matrix Metalloproteinase-9 Deficiency Impairs Cellular Infiltration and Bronchial Hyperresponsiveness during Allergen-Induced Airway Inflammation Am. J. Pathol., August 1, 2002; 161(2): 491 - 498. [Abstract] [Full Text] [PDF]
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J. D. Moffatt, K. L. Jeffrey, and T. M. Cocks Protease-Activated Receptor-2 Activating Peptide SLIGRL Inhibits Bacterial Lipopolysaccharide-Induced Recruitment of Polymorphonuclear Leukocytes into the Airways of Mice Am. J. Respir. Cell Mol. Biol., June 1, 2002; 26(6): 680 - 684. [Abstract] [Full Text] [PDF]
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 L. Joos, J.-Q. He, M. B. Shepherdson, J. E. Connett, N. R. Anthonisen, P. D. Pare, and A. J. Sandford The role of matrix metalloproteinase polymorphisms in the rate of decline in lung function Hum. Mol. Genet., March 1, 2002; 11(5): 569 - 576. [Abstract] [Full Text] [PDF]
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H. Hakonarson, E. Halapi, R. Whelan, J. Gulcher, K. Stefansson, and M. M. Grunstein Association Between IL-1beta /TNF-alpha -Induced Glucocorticoid-Sensitive Changes in Multiple Gene Expression and Altered Responsiveness in Airway Smooth Muscle Am. J. Respir. Cell Mol. Biol., December 1, 2001; 25(6): 761 - 771. [Abstract] [Full Text] [PDF]
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 A. M. Abu El-Asrar, I. Van Aelst, S. Al-Mansouri, L. Missotten, G. Opdenakker, and K. Geboes Gelatinase B in Vernal Keratoconjunctivitis Arch Ophthalmol, October 1, 2001; 119(10): 1505 - 1511. [Abstract] [Full Text] [PDF]
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K. Cederqvist, T. Sorsa, T. Tervahartiala, P. Maisi, K. Reunanen, P. Lassus, and S. Andersson Matrix Metalloproteinases-2, -8, and -9 and TIMP-2 in Tracheal Aspirates From Preterm Infants With Respiratory Distress Pediatrics, September 1, 2001; 108(3): 686 - 692. [Abstract] [Full Text] [PDF]
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 C. Faveeuw, G. Preece, and A. Ager Transendothelial migration of lymphocytes across high endothelial venules into lymph nodes is affected by metalloproteinases Blood, August 1, 2001; 98(3): 688 - 695. [Abstract] [Full Text] [PDF]
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 M. Girard, P. Cléroux, P. Tremblay, S. Dea, and Y. St-Pierre Increased proteolytic activity and matrix metalloprotease expression in lungs during infection by porcine reproductive and respiratory syndrome virus J. Gen. Virol., June 1, 2001; 82(6): 1253 - 1261. [Abstract] [Full Text]
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C. Daniel, J. Duffield, T. Brunner, K. Steinmann-Niggli, N. Lods, and H.-P. Marti Matrix Metalloproteinase Inhibitors Cause Cell Cycle Arrest and Apoptosis in Glomerular Mesangial Cells J. Pharmacol. Exp. Ther., April 1, 2001; 297(1): 57 - 68. [Abstract] [Full Text]
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 S. Nagai, S.-i. Hashimoto, T. Yamashita, N. Toyoda, T. Satoh, T. Suzuki, and K. Matsushima Comprehensive gene expression profile of human activated Th1- and Th2-polarized cells Int. Immunol., March 1, 2001; 13(3): 367 - 376. [Abstract] [Full Text] [PDF]
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S. L. F. Pender, M. T. Salmela, G. Monteleone, D. Schnapp, C. McKenzie, J. Spencer, S. Fong, U. Saarialho-Kere, and T. T. MacDonald Ligation of {{alpha}}4{beta}1 Integrin on Human Intestinal Mucosal Mesenchymal Cells Selectively Up-Regulates Membrane Type-1 Matrix Metalloproteinase and Confers a Migratory Phenotype Am. J. Pathol., December 1, 2000; 157(6): 1955 - 1962. [Abstract] [Full Text] [PDF]
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E. A. BECKY KELLY, W. W. BUSSE, and N. N. JARJOUR Increased Matrix Metalloproteinase-9 in the Airway after Allergen Challenge Am. J. Respir. Crit. Care Med., September 1, 2000; 162(3): 1157 - 1161. [Abstract] [Full Text]
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W.-Y. Su, R. H. Jaskot, J. Richards, S. R. Abramson, J. F. Woessner Jr., W.-H. Yu, and K. L. Dreher Induction of pulmonary matrilysin expression by combustion and ambient air particles Am J Physiol Lung Cell Mol Physiol, July 1, 2000; 279(1): L152 - L160. [Abstract] [Full Text] [PDF]
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H. Shirota, K. Sano, T. Kikuchi, G. Tamura, and K. Shirato Regulation of T-helper Type 2 Cell and Airway Eosinophilia by Transmucosal Coadministration of Antigen and Oligodeoxynucleotides Containing CpG Motifs Am. J. Respir. Cell Mol. Biol., February 1, 2000; 22(2): 176 - 182. [Abstract] [Full Text]
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