Matrix metalloproteinase-2 and -9 expression increases in Mycoplasma-infected airways but is not required for microvascular remodeling

Peter Baluk,1,2,6 Wilfred W. Raymond,1,3 Erin Ator,1,2 Lisa M. Coussens,4,5 Donald M. McDonald,1,2,6 and George H. Caughey1,3,6

1Cardiovascular Research Institute and Departments of 2Anatomy and 3Medicine, 4Cancer Research Institute; and 5Department of Pathology and 6Comprehensive Cancer Center, University of California, San Francisco, California 94143-0130

Submitted 21 November 2003 ; accepted in final form 6 April 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Murine Mycoplasma pulmonis infection induces chronic lung and airway inflammation accompanied by profound and persistent microvascular remodeling in tracheobronchial mucosa. Because matrix metalloproteinase (MMP)-2 and -9 are important for angiogenesis associated with placental and long bone development and skin cancer, we hypothesized that they contribute to microvascular remodeling in airways infected with M. pulmonis. To test this hypothesis, we compared microvascular changes in airways after M. pulmonis infection of wild-type FVB/N mice with those of MMP-9–/– and MMP-2–/–/MMP-9–/– double-null mice and mice treated with the broad-spectrum MMP inhibitor AG3340 (Prinomastat). Using zymography and immunohistochemistry, we find that MMP-2 and MMP-9 rise strikingly in lungs and airways of infected wild-type FVB/N and C57BL/6 mice, with no zymographic activity or immunoreactivity in MMP-2–/–/MMP-9–/– animals. However, microvascular remodeling as assessed by Lycopersicon esculentum lectin staining of whole-mounted tracheae is as severe in infected MMP-9–/–, MMP-2–/–/MMP-9–/– and AG3340-treated mice as in wild-type mice. Furthermore, all groups of infected mice develop similar inflammatory infiltrates and exhibit similar overall disease severity as indicated by decrease in body weight and increase in lung weight. Uninfected wild-type tracheae show negligible MMP-2 immunoreactivity, with scant MMP-9 immunoreactivity in and around growing cartilage. By contrast, MMP-2 appears in epithelial cells of infected, wild-type tracheae, and MMP-9 localizes to a large population of infiltrating leukocytes. We conclude that despite major increases in expression, MMP-2 and MMP-9 are not essential for microvascular remodeling in M. pulmonis-induced chronic airway inflammation.

angiogenesis; Mycoplasma pulmonis; matrix metalloproteinase; MMP-2; MMP-9; AG3340; Prinomastat


ANGIOGENESIS, remodeling of blood vessels, and extravasation of leukocytes are features of many chronic inflammatory diseases, including asthma. Angiogenesis is also required for tumor growth; indeed, there are similarities between stromal responses to tumors and the healing of wounds (10, 11). Much effort has been made to target drugs to angiogenic vessels in the hope of developing novel forms of treatment for these conditions. One approach is to inhibit matrix metalloproteinases (MMPs), which are believed to initiate the remodeling of the extracellular matrix and basement membrane that surrounds established vessels (18). MMPs are a family of enzymes currently comprising at least 23 members (38, 41). Of these, MMP-2 and MMP-9 (also known as gelatinases A and B) are thought to be particularly important in microvascular remodeling because of their involvement in several types of angiogenesis, including that associated with placental and long bone growth and progression of skin cancer (8–10, 20, 42). Furthermore, MMPs (and MMP-2 and -9 especially) have been implicated in control of inflammatory cell infiltration in various murine models of lung and airway inflammation (7, 23, 26).

In the present study, we examined the respiratory tract of mice infected with Mycoplasma pulmonis as a model of chronic inflammation. Some strains of mice with this airway infection, e.g., C57BL/6 mice, respond by sprouting angiogenesis of the airway microvasculature, whereas in other strains, e.g., C3H mice, the dominant response appears to be microvascular remodeling in which microvessels enlarge, such that capillaries become venules (40). Both types of response are accompanied by an influx of large numbers of inflammatory cells. Several inflammatory cell types, e.g., neutrophils, which are abundant in the airways in the early stages of M. pulmonis infection, synthesize and secrete MMPs and other proteolytic enzymes. Therefore, we reasoned that MMP-2 and -9 might contribute to microvascular remodeling and inflammation in airways of infected mice. To test this hypothesis, we compared microvascular changes in the airways after M. pulmonis infection of wild-type mice with those of MMP-9–/– mice, MMP-2–/–/MMP-9–/– mice and mice treated with the broad-spectrum MMP inhibitor AG3340.

This study had several goals: 1) to determine whether MMP gelatinases increase in airways of mice infected with M. pulmonis, 2) to determine the cellular sources of MMP-2 and -9 in normal vs. infected airways, and 3) to determine whether genetic or pharmacological elimination of MMP-9 or MMP-2 and -9 prevents remodeling of airway vessels and inflammation in infected animals. The results, as detailed below, reveal a dramatic rise in infected animals of MMP-2 and MMP-9 expression in epithelium and infiltrating leukocytes, respectively, and suggest that these MMPs do not play major roles in Mycoplasma-induced microvascular remodeling and inflammation.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Animals. The generation of MMP-2–/– and MMP-9–/– homozygous-null mice has been reported (19, 42). Before MMP-2–/– and MMP-9–/– mice were generated, MMP-2+/– and MMP-9+/– heterozygous animals were individually backcrossed into the FVB/N background for five generations, then intercrossed to generate MMP-2–/–/MMP-9–/– double-null mice. Wild-type FVB/N and C57BL/6 mice were purchased from Charles River (Hollister, CA) or bred in-house. All mice were housed under specific pathogen-free barrier conditions, and sentinel mice were routinely tested serologically for common murine pathogens. Uninfected mice were maintained in the same environmental conditions as the infected mice. Mice were given food and water ad libitum and were used from 8 to 12 wk of age. Before experimental procedures, animals were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) injected intramuscularly. All procedures were approved by the Institutional Animal Care and Use Committee of the University of California at San Francisco.

M. pulmonis inoculation. Specific pathogen-free male or female mice were inoculated intranasally on day 0 with 50 µl of M. pulmonis (strain UAB CT7, University of Alabama at Birmingham) broth (25 µl per nostril). FVB/N and C57BL/6 mice received doses of 105 and 106 colony-forming units of M. pulmonis, respectively, and were studied 1 wk (FVB/N) or 2 and 4 wk later (C57BL/6).

Gelatin zymography. Gelatin substrate zymography was performed essentially as described previously (13). In brief, the vasculature of anesthetized mice was cleared of blood by aortic perfusion with phosphate-buffered saline (PBS). Tracheae and lungs were rapidly removed, weighed, and frozen. Protein was extracted from lung and tracheal homogenates into 10 mM bis-Tris·HCl, pH 6.1. Electrophoresis of aliquots of extracts was performed under nonreducing conditions on SDS-PAGE gels containing gelatin. Gels were then incubated overnight (16 h) and stained with Coomassie blue. Extracts from wild-type or single-mutant tissues served as positive controls for MMP-2 and MMP-9 expression. The detection limit for the assays was ~10 pg of MMP-2 and MMP-9.

Lectin staining of blood vessels and morphometry. In preparation for lectin staining of tracheal vessels, 100 µg of biotinylated Lycopersicon esculentum lectin were injected intravenously and allowed to circulate for 2 min. The vasculature of the anesthetized mice was then perfused with fixative (1% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M PBS, pH 7.4). Tracheae were removed and stained as whole mount preparations. Lectin-labeled blood vessels were detected by avidin-biotin complex histochemistry (39), and their diameters and area densities were measured by morphometric methods, as described previously (40). In brief, for each trachea, the diameter of 10 vessels crossing the cartilage rings was measured at a final magnification of 736. We assessed the overall microvascular density by overlaying a rectangular graticule of 126 points on 10 regions of mucosa and counting the points of intersection with blood vessels. These regions, each 1.4 mm2 in area observed at a final magnification of 184, included regions between and over the cartilage rings.

Treatment with MMP inhibitor. To assess effects of a broad-spectrum MMP inhibitor, we treated some mice with AG3340 (200 mg/kg ip, twice daily; Pfizer Agouron Pharmaceuticals) or with vehicle (acidified water, pH 2.3) starting on day 0 for 7 days. This drug has antitumor and antiangiogenic effects in other mouse models at doses considerably below the one used here (36, 37).

Lung histology and pathology. Wet weights of lungs and bronchial lymph nodes were determined as indexes of the severity of inflammation (12). Organ weights were normalized to body weight. Lungs were embedded in wax, and 3-µm sections were stained with hematoxylin and eosin.

Immunofluorescence histochemistry. To determine the cells of origin of airway MMPs, we stained whole mount preparations of trachea for MMP-2, MMP-9, and CD31 platelet endothelial cell adhesion molecule immunoreactivity and imaged them by confocal fluorescence microscopy. The chest of each anesthetized mouse was opened rapidly, and the vasculature was perfused for 2 min with 1% paraformaldehyde in PBS, pH 7.4. Tracheae were removed and immersed in the same fixative for 1 h. After several rinses with PBS, tracheal whole mounts were pinned on silicone rubber slabs and were incubated in 5% normal goat or donkey serum (Jackson ImmunoResearch, West Grove, PA) in PBS containing 0.3% Triton X-100 (Sigma), 0.2% bovine serum albumin (Sigma), and 0.01% thimerosal (Sigma) for 1 h at room temperature to block nonspecific antibody binding. Next, whole mounts were incubated overnight at room temperature in combinations of primary antibodies diluted in the medium described above. MMP-2 was identified with a rabbit polyclonal antibody (AB809, 1:500; Chemicon, Temecula, CA), and MMP-9 was identified with a goat polyclonal antibody (AF909, 1:1,000; R&D Systems, Minneapolis, MN). Endothelial cells were identified with a monoclonal antibody to CD31 that stains endothelial cell borders (hamster clone 2H8, 1:500; Chemicon). After rinses with PBS, specimens were incubated for overnight at room temperature with fluorescent (FITC or Cy3) secondary antibodies (goat anti-rat, anti-hamster, or anti-rabbit, or donkey anti-goat; Jackson ImmunoResearch) diluted 1:400 in PBS. Finally, we rinsed specimens with PBS, mounted them in Vectashield medium (Vector Laboratories, Burlingame, CA), and examined them with a Zeiss LSM 510 confocal microscope, scanning through the full thickness of the tracheal mucosa (~60 µm).

Statistics. Values are presented as means ± SE. Groups consisted of four to eight mice per group. The significance of differences between means was assessed by analysis of variance followed by the Dunn-Bonferroni test, with the level of significance set at P < 0.05.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Increase in MMP gelatinases in infected airways. MMP gelatinases are detected in lungs of C57BL/6 mice and in lungs and tracheae of wild-type, MMP-9–/–, and MMP-2–/–/MMP-9–/– FVB/N mice by gelatin substrate zymography (Figs. 1 and 2). Lungs of uninfected wild-type C57BL/6 mice show little MMP gelatinolytic activity, but upon infection with M. pulmonis, electrophoretic bands appear at ~92 and 84 kDa, corresponding to proenzyme and proteolytically processed forms of MMP-9, respectively. Band intensity increases with duration of infection (Fig. 1). In infected MMP-9–/– mice, no bands were observed at ~92 and 84 kDa. However, in tracheae and lungs of infected MMP-9–/– mice, bands became apparent at ~70 and 56 kDa, corresponding to proenzyme and processed forms of MMP-2, respectively (Fig. 1). This observation, which indicates a potential increase of MMP-2 in infected MMP-9–/– mice compared with wild-type mice, led us to examine gelatinolytic activity in MMP-2–/–/MMP-9–/– animals. Extensive destaining of the gel shown in Fig. 2 allowed us to detect trace amounts of MMP-2 and MMP-9 in tracheal and lung extracts, respectively, of uninfected, wild-type FVB/N mice. In extracts of MMP-2–/–/MMP-9–/– mice, as expected, no bands are seen. As noted C57BL/6 mice, MMP-2 and MMP-9 are present in extracts of tracheae and lungs of infected, wild-type FVB/N mice, where MMP-2 is expressed more strongly in the trachea and MMP-9 more strongly in the lung (Fig. 2).



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Fig. 1. Zymography of lung and tracheal extracts of wild-type (WT) and matrix metalloproteinase (MMP)-9-deficient mice. In these electrophoretograms, 1 µg of protein in each lane was subjected to SDS-PAGE under nonreducing conditions in the presence of gelatin and then stained with Coomassie blue. Clear bands reveal MMP gelatinolytic activity. Left panel: results from lung (Lu) extracts of C57BL/6 mice with or without infection with Mycoplasma pulmonis. Extracts in lanes 2 and 3 were obtained from mice 2 and 4 wk after infection, respectively. Trace pro-MMP-2 activity is apparent at the 4-wk time point. This panel reveals that proenzyme and proteolytically processed forms of lung MMP-9 activity increase with duration of infection. Right panel: results obtained from lung and tracheal (Tr) extracts of MMP-9–/– FVB/N mice with or without infection with M. pulmonis for 1 wk. No bands corresponding to MMP-9 are seen in any of the extracts. However, strong bands corresponding to proenzyme and processed forms of MMP-2 appear in tracheal as well as lung extracts of infected mice.

 


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Fig. 2. Zymography of lung and tracheal extracts of WT and MMP-2–/–/MMP-9–/– mice. In each lane, 1 µg protein was electrophoresed under nonreducing conditions on an SDS-PAGE gel containing gelatin, then stained with Coomassie blue. Clear bands reveal pro- and processed forms of MMP-2 and MMP-9. Note that low-level pro-MMP activity is seen in extracts of uninfected WT mice. These levels increase in WT mice infected for 1 wk, along with activated forms of MMPs. No MMP activity is detected in extracts of MMP-2–/–/MMP-9–/– null mice. This zymogram was more extensively destained than that in Fig. 1, which may have increased the visibility of MMP-2-associated bands.

 
Effect of MMP-2 and MMP-9 deficiency on vessel remodeling. The effect of M. pulmonis infection on tracheal vessel morphology was studied in wild-type C57BL/6 and FVB/N mice and in MMP-9- or MMP-2/MMP-9-deficient FVB/N mice. Tracheae of uninfected mice have well-ordered microvascular beds arranged in neat arcades, with most collecting venules located between the cartilage rings and capillaries running across the cartilage (Fig. 3A). Upon infection, vessels in the tracheae of wild-type C57BL/6 mice show microvascular enlargement and growth of many new capillaries by angiogenesis. Collecting venules enlarge. Adherent leukocytes are evident in postcapillary and collecting venules (Fig. 3B). The tracheal vasculature of uninfected FVB/N mice resembles that of C57BL/6 mice in overall features (Fig. 3C). Upon infection, vessels in wild-type FVB/N tracheae mainly show enlargement, rather than growth of new vessels by traditional angiogenesis (Fig. 3D). The change is particularly noticeable in the capillaries that formerly overlay the cartilages. These vessels widen into venules and become laden with numerous adherent leukocytes (Fig. 3, E and F). Some blunt vascular sprouts appear on enlarged vessels (Fig. 3F). This enlargement is not simply due to vasodilatation, but to proliferation of endothelial cells, as is evident in CD31-stained preparations in which endothelial cell borders are revealed (Fig. 3, G and H).



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Fig. 3. Blood vessels in tracheae of uninfected and infected WT mice. Uninfected C57BL/6 mouse (A) and mouse infected for 28 days (B). Arrows: Lycopersicon esculentum lectin-stained capillaries overlying cartilages in A and profusion of new capillaries in B. Uninfected WT FVB/N mouse (C) and mouse infected for 1 wk (D). Arrows indicate capillaries, which are enlarged in D. E and F: enlargement of regions in C and D. Arrows: capillaries in E and blunt vascular sprouts in F. Many adherent leukocytes (brown circles) are apparent within the enlarged vessels in F. G and H: confocal fluorescence micrographs of uninfected (G) and 1-wk M. pulmonis-infected (H) FVB/N mouse tracheal whole mounts stained with a hamster antibody to CD31, a marker of endothelial cell borders. Arrows indicate blood vessels overlying the cartilages that are enlarged and composed of multiple endothelial cells in H. Scale bar: 300 µm for A–D and 100 µm for E–H.

 
The tracheal vasculature of uninfected MMP-9–/– and MMP-2–/–/MMP-9–/– mice resembles that of wild-type littermates (Fig. 4A). Vessels are enlarged, and leukocytes are abundant in infected mice. Vessels in infected tracheae of MMP-9–/– and MMP-2–/–/MMP-9–/– mice widen and appear similar to those in infected wild-type littermates, with abundant adherent leukocytes (Fig. 4B). No statistical differences were found in measurements of capillary diameter or total vessel area density between infected wild-type and MMP-2–/–/MMP-9–/– mice (Fig. 5, A and B).



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Fig. 4. Blood vessels and lung sections of WT and MMP-2–/–/MMP-9–/– mice. A and B: L. esculentum lectin-stained tracheal blood vessels in uninfected (A) and 1-wk M. pulmonis infected MMP-2 and MMP-9 double-deficient mouse (B). Vessels are enlarged in B, and adherent leukocytes are abundant. The appearance is similar to that of an infected WT mouse (Fig. 3D). C–F: hematoxylin and eosin-stained sections of lung from uninfected (C) and infected WT mouse (D) and uninfected (E) and infected MMP-2–/–/MMP-9–/– mouse (F). Leukocytes are abundant in the infected mice, mostly neutrophils in the airway lumen and lymphocytes in the lung parenchyma. G and H: lectin-stained tracheal blood vessels in 1-wk M. pulmonis infected WT FVB/N mouse concurrently treated for 7 days with vehicle (G) or with the broad-spectrum MMP inhibitor AG3340 (H). The degree of microvascular enlargement is similar in the 2 conditions. Scale bar: 200 µm for A, B, G, and H and 150 µm for C–F.

 


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Fig. 5. Vessel size and inflammation in uninfected and infected WT and MMP-deficient mice. Capillary diameter (A) and total vessel area (B) measured in whole mounts of uninfected and infected WT and MMP-2–/–/MMP-9–/– mice. Lung (C) and lymph node (D) weights in uninfected and infected WT and MMP-2–/–/MMP-9–/– mice. Lung (E) and lymph node (F) weights in uninfected and infected WT FVB/N mice treated concurrently with vehicle or with MMP-inhibitor AG3340. *Significantly different from uninfected control group; {dagger}significantly different from infected WT group; P < 0.05. Group size of 4 mice per group for A and B and 6–8 mice per group for C–F.

 
Infection with M. pulmonis induced lethargy in all mice, with a loss of appetite and body weight. Thus over the first week of infection, wild-type mice lost ~2 g in body weight, from 25.5 ± 0.4 to 23.2 ± 0.7 g. However, MMP-2–/–/MMP-9–/– mice lost significantly more weight, ~6 g, from 23.6 ± 0.9 to 17.3 ± 0.6 g. In all infected mice, the lungs increased in wet weight. This was most likely the result of the influx of inflammatory cells and edema, but the increase was significantly more in MMP-deficient mice. Thus in wild-type mice the weight of the lungs increased from 206 ± 6 to 302 ± 31 mg; however, in MMP-2–/–/MMP-9–/– mice, the lungs increased in weight from 178 ± 24 to 371 ± 20 mg. In all infected mice there was also an increase in the weight of the bronchial lymph node that drains the lungs. To compensate for the different body weights of mice, we show the relative weights of lungs and lymph nodes normalized to body weight in pathogen-free and 1-wk infected wild-type, MMP-9–/–, and MMP-2–/–/MMP-9–/– mice in Fig. 5, C and D.

Histologically, the lungs of uninfected and infected MMP-9–/– and MMP-2–/–/MMP-9–/– mice resemble those of the corresponding wild-type littermates (Fig. 4, C–F). Lungs of infected mice show massive accumulations of leukocytes: mainly lymphocytes in lung parenchyma and neutrophils in airway lumen (Fig. 4, F and H).

Effect of MMP inhibition on vessel and airway remodeling. Tracheal blood vessels in infected FVB/N mice treated for 1 wk with vehicle or with AG3340 appear similar. Treatment with AG3340 does not block leukocyte adhesion or microvascular enlargement (Fig. 4, G and H). No statistical differences are found in measurements of vessel diameter or area density between wild-type mice infected for 1 wk and treated concomitantly with vehicle or AG3340 (vessel are density 42.5 ± 5.6 vs. 43.9 ± 5.2%, P > 0.05). Similarly, relative lung and lymph node weights do not differ between mice treated with vehicle and those treated with AG3340 (Fig. 5, E and F).

Immunohistochemical localization of MMP-2 and MMP-9 in infected airways. Immunohistochemical staining for MMP-2 and MMP-9 was done in tracheal whole mounts of uninfected and infected FVB/N wild-type mice and MMP-2–/–/MMP-9–/– mice and in wild-type C57BL/6 mice. Immunoreactivity for MMP-2 was weak or absent in tracheae of wild-type uninfected FVB/N mice (Fig. 6A) but markedly increased in a population of epithelial cells (Fig. 6D) after infection with M. pulmonis. Leukocytes appeared to be unstained. In wild-type mice, moderately strong MMP-9 immunoreactivity was found in clusters of chondrocytes within the tracheal cartilage rings and also in individual cells at the edges of the cartilage rings (Fig. 6B). In uninfected and infected MMP-2–/–/MMP-9–/– mice no immunoreactivity was detected for MMP-2 (not shown) or MMP-9 (Fig. 6, C and F).



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Fig. 6. MMP immunoreactivity in tracheae of uninfected and infected WT and MMP-2–/–/MMP-9–/– mice. Confocal micrographs of tracheal whole mounts from uninfected (A–C) and infected (D–F and G–I) WT and MMP-2–/–/MMP-9–/– mice. A: MMP-2 immunoreactivity is weak or absent in uninfected mice. B: some MMP-9 immunoreactivity is found within (arrows) and around (arrowheads) the cartilage rings in uninfected mice. C: MMP-9 immunoreactivity is absent from tracheae of uninfected MMP-2–/–/MMP-9–/– mice. D: MMP-2 immunoreactivity is increased in some epithelial cells of 7-day M. pulmonis-infected WT mouse. Inset: enlarged region of D showing polygonal epithelial cells (E) MMP-9 immunoreactivity in numerous infiltrating leukocytes in infected WT mouse. F: MMP-9 immunoreactivity is absent from tracheae of infected MMP-2–/–/MMP-9–/– mice. G–I: infected WT FVB/N mouse. G: MMP-9 immunoreactivity is expressed strongly in leukocytes (arrowheads) and weakly in blood vessel basement membrane (arrows). H: CD31 immunoreactivity is expressed strongly in blood vessels (arrows) and less so in leukocytes (arrowheads). I: merged image. J: infected C57BL/6 mouse trachea. Endothelial cells labeled for CD31 (green). MMP-9 immunoreactivity (reddish orange) is found mainly in leukocytes, both intravascular and extravascular. Regions overlying cartilages (arrows). Inset: region of J enlarged. Intravascular (arrows) and extravascular (arrowheads) leukocytes, some of which appear to be degranulating (arrowheads). Scale bar: 300 µm for A–F, 150 µm for G–I, and 100 µm for J.

 
Upon infection of wild-type mice, MMP-9 immunoreactivity greatly increases and localizes to swarms of infiltrating leukocytes that obscure underlying structures (Fig. 6E). From their size, polymorphic nuclei, and ameboid shape, most immunoreactive leukocytes appear to be neutrophils. Epithelial cells and mast cells are not stained. At higher magnifications, weaker MMP-9 immunoreactivity was found in the basement membrane of some blood vessels and also on nerves and other cells in the tracheal mucosa of infected mice (Fig. 6, G–I). MMP-9-immunoreactive leukocytes are more abundant in infected FVB/N mice (Fig. 6, E and G–I) than in infected C57BL/6 mice (Fig. 6J). The leukocytes are mostly extravascular, but double staining for CD31 shows that some immunoreactive leukocytes are intravascular (Fig. 6J). CD31 stains endothelial cells strongly and leukocytes to a lesser extent. Clouds of MMP-9 immunoreactivity surround some leukocytes, suggesting degranulation.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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In this study we examined the contribution of two MMP gelatinases to microvascular remodeling in airways and lungs of mice infected with M. pulmonis. We reasoned that if these MMPs were important to microvascular remodeling in this chronic disease, they should be detectable in increased amounts in infected airways compared with uninfected airways. Gelatin zymography suggests that MMP-2 and MMP-9 increase in airways of infected mice. Accordingly, we sought to determine whether MMPs of the gelatinase class were essential for the remodeling by comparing the changes in the airways after M. pulmonis infection of wild-type mice with those of mice deficient for one or both MMPs. Our initial experiments showed that MMP-9 was not essential and suggested that in its absence there was a potentially offsetting increase in infected airways of MMP-2, which is similar to MMP-9 in some of its substrate preferences. We therefore extended the experiments to MMP-2–/–/MMP-9–/– mice to determine the functional significance of increased MMP-2. Somewhat to our surprise, we found that neither MMP is essential, as microvascular remodeling occurs in all groups of infected mice. Likewise, our attempts to block gelatinase-induced remodeling by inhibiting MMP activity with a broad-spectrum inhibitor does not alter the characteristics of infection. Our immunohistochemical results support the findings from zymography. Both show a dramatic increase in MMP protein in airways of infected mice. MMP-2 immunoreactivity appears to be endogenous and predominantly epithelial in origin, whereas most MMP-9 is exogenous, from infiltrating neutrophils. However, a small amount of MMP-9 immunoreactivity also appears to be in the basement membrane of some blood vessels and other cells, perhaps deposited there by migrating leukocytes.

Role of MMPs in microvascular remodeling. It is generally believed that for angiogenesis to proceed, the endothelial basement membrane must be remodeled before endothelial sprout extension (2, 33). Several lines of evidence suggest that MMPs are involved in matrix and basement membrane penetration by migrating tumor, vascular smooth muscle, and inflammatory cells (15, 24, 27, 35). MMP-2 and MMP-9 are of particular interest because some of their preferred substrates include type IV collagen, laminin, and fibronectin (1, 38, 43), all of which are prominent constituents of basement membranes (21). Furthermore, MMP mRNAs are upregulated in many pathological conditions associated with angiogenesis and inflammation, including allergic inflammation of the airways (7, 23). These features and the reported effectiveness of inhibitors of MMPs in inhibiting the growth of some tumors, presumably by inhibiting angiogenesis, prompted us to examine the role of MMP-2 and MMP-9 in angiogenesis and microvascular remodeling in response to chronic inflammation in the airways.

Zymography and enzyme activity. Our zymography experiments establish that both pro- and processed forms of MMP-2 and MMP-9 increase strongly in wild-type infected mice. It is possible, however, that some proteolytically "activated" gelatinase is inactive in vivo because of binding to endogenous inhibitors, such as tissue inhibitor of metalloproteinase-1 (TIMP-1), which dissociate from MMPs in SDS-PAGE gels. On the other hand, recent evidence suggests that the proenzyme form of MMPs can become active without propeptide removal in some circumstances (3). Finally, the zymography results show that the genetically null mice are truly deficient in gelatinolytic enzymatic activity and that the bands attributed to MMP-2 and MMP-9 are accurately identified.

Characteristics of the M. pulmonis model. In mice, M. pulmonis causes lifelong infection of the respiratory tract accompanied by microvascular remodeling, inflammatory cell influx, and fibrosis and epithelial cell remodeling in the airways. The microvascular remodeling phenotype is strain dependent, with strains such as C57BL/6 showing gradual sprouting of new capillaries in addition to widening of existing vessels and thickening of tracheal mucosa in bronchus-associated lymphoid tissue, which is normally absent from the trachea (40). Other strains, such as C3H, show more rapid enlargement of vessels, such that capillaries are converted into venules and support leukocyte adhesion and transmigration (40). In this first detailed description of microvascular remodeling in FVB/N airways, we show that FVB/N mice resemble C3H mice in their response to infection. Immunohistochemical staining for CD31, which marks the borders of endothelial cells, shows that the increased diameter of blood vessels is due to proliferation of endothelial cells composing the vessel wall, and not to simple vasodilatation.

Comparison to other models. In M. pulmonis-infected airways, there was a massive influx of inflammatory cells into the airway mucosa and lumen. This feature was shared in common with ovalbumin-induced allergic models of inflammation in mouse lungs (7, 23). However, the number and types of cells differ somewhat, with lung weight in M. pulmonis-infected mice increasing due to influx of lymphocytes in the mucosa and neutrophils in the lumen, whereas in the allergen models, lymphocytes and eosinophils predominate. Both cited allergic models note increased MMP-2 and MMP-9 in allergen-inflamed lung, and both report that eliminating MMP-2 activity, either by the use of inhibitors or MMP-2–/– mice, reduces influx of inflammatory cells into the airway lumen (7, 23). One of these studies notes that although egress of inflammatory cells into the lumen is inhibited, influx into mucosa is not and is actually greater in MMP-2–/– mice (7). An additional study reports that MMP-9 deficiency impairs cellular infiltration in murine allergic inflammation (6). In the present study of M. pulmonis-induced lung inflammation, it is evident that inhibiting MMP-2 and MMP-9 in combination, either by chemical inhibition or genetic deletion, does not prevent leukocyte influx into the airway lumen and lung mucosa. Lung weights are actually greater in MMP-9–/– and MMP-2–/–/MMP-9–/– mice than in wild-type mice. This is consistent with the recent observation that lavage recovery of neutrophils from inflamed lungs of mice expressing an IL-13 transgene is greater in MMP-9-deficient animals (26). In the present study, leukocyte trafficking was also reflected in the weights of the sentinel bronchial lymph nodes that drain the lungs and which have been used as an index of host defense by us in Mycoplasma-infected mice (12) and by others in mice with intradermal bacterial infections (29). In infected MMP-deficient mice or in wild-type mice treated with MMP inhibitor, these lymph nodes were as large as, or larger than, the corresponding nodes in groups of infected wild-type mice or infected mice treated with the vehicle of the MMP inhibitor.

Inhibiting or eliminating MMP-2 or MMP-9 inhibits several other types of microvascular remodeling. In MMP-2–/– mice, ocular angiogenesis is inhibited in cornea and in choroid (5, 22) and also in experimentally induced tumors (20). Elimination of MMP-9 inhibits angiogenesis in endochondral bone and slows bone growth. In the eye, lack of MMP-9 reduces, but does not eliminate, the choroidal neovascularization associated with age-related macular degeneration (25). Inhibition of MMPs with AG3340, administered at doses considerably lower than those used here, inhibits hypoxia-induced retinal neovascularization in newborn mice (14). Furthermore, inhibitors of MMP-9 and other MMPs reduce ocular angiogenesis induced by herpes simplex virus (28). Several studies report that tumors grow more slowly in MMP-9-deficient mice (4, 8).

In view of this abundant evidence that MMP gelatinases are upregulated in inflammatory situations and that inhibiting them can reduce angiogenesis, why was no effect observed in the present study? The unrestrained microvascular remodeling in MMP-9–/– mice and the upregulation of MMP-9 in these circumstances prompted us to repeat the studies in MMP-2–/–/MMP-9–/– mice. We observe essentially the same lack of effect on microvascular remodeling and inflammatory cell influx. It is possible that there are offsetting increases in the activity of other MMPs, since there appears to be considerable overlap and potential for complex regulatory interactions among the many MMPs (26). However, the lack of effect of a broad-spectrum MMP inhibitor in the present study makes a major contribution of closely related MMPs unlikely. Another possible explanation is suggested by recent studies of angiogenesis in skeletal muscle (16, 34). Two distinct patterns of remodeling of the same vessels occur in response to muscle stretch or increased shear stress induced by vasodilatation. Although VEGF expression and endothelial cell proliferation increased in both models, MMP-2 was increased by muscle stretch alone and not by shear stress. Thus it appears that different microvascular remodeling pathways may be used in response to different stimuli.

A further consideration in interpreting the present results is the realization that MMPs can have multiple and sometimes opposing pro- and antiangiogenic effects (18). Thus MMP-2 can degrade plasminogen to produce angiostatin, and MMP-9 can degrade type IV collagen alpha3 chains to produce tumstatin; both products are well-established antiangiogenic molecules (17, 30). Nevertheless, the data in the present study establish that the profound alterations in airway mucosal vessels provoked by M. pulmonis infection are largely independent of MMP-2 and MMP-9, in contrast to findings in several other varieties of microvascular remodeling.

Immunohistochemical localization of MMPs. Numerous cell types have been reported as expressing either constitutive or inducible MMP activity. Taken by itself, our immunohistochemical approach to the localization of MMPs must be cautiously interpreted for several reasons. First, MMPs can be secreted from the true cells of origin and then bind to other cells and extracellular matrix. The presence of the protein does not directly identify the cell of origin. Thus in the present study, the weak MMP-9 immunoreactivity observed on the basement membrane of some blood vessels may not necessarily originate from endothelial cells but could be secreted by other cells, such as migrating neutrophils (32). Inactive and active forms of MMP-2 and MMP-2 can bind to collagen IV, a component of the basement membrane (1). Second, even though active forms of MMPs are identified by zymography, the resultant activity in tissue can be suppressed by TIMPs. Even so, the inhibition by TIMP is reversible, and the activity of TIMP-bound MMP can be rescued by destruction of TIMP by proteases such as chymase (13). Irrespective of these considerations, our results suggest that the major MMP-2- and MMP-9-expressing cell types in Mycoplasma-inflamed airways are epithelial cells and leukocytes, respectively. Neutrophils have long been known to be a rich source of MMP-9 (31). The clouds of MMP-9 immunoreactivity found around some leukocytes are also consistent with reports of inflammatory cytokines that can degranulate neutrophils and release secreted MMP-9 (31).

In summary, we find that MMP-2 and MMP-9 protein levels increase strikingly in the lungs and airways of wild-type mice infected with M. pulmonis. MMP-2 localizes to epithelial cells and MMP-9 localizes to a large of infiltrating leukocytes in infected wild-type mice but not in infected MMP-2–/–/MMP-9–/– double-null mice. However, microvascular remodeling, inflammatory cell influx, and changes in lymph node weight are not reduced in infected MMP-2–/–/MMP-9–/– mice compared with infected wild-type animals. Therefore, we conclude that MMP-2 and MMP-9 are not essential for microvascular remodeling in M. pulmonis-induced chronic airway inflammation.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported in part by National Institutes of Health Grants HL-24136 and HL-59157 (to D. M. McDonald), HL-24136 (to G. H. Caughey), and CA-72006 and CA-98075 (to L. M. Coussens).


    ACKNOWLEDGMENTS
 
We thank Dr. David Shalinsky of Pfizer Agouron Pharmaceuticals (San Diego, CA) for the kind gift of AG3340 and Dr. Kenneth Fang of University of California-San Francisco for assistance with the zymography experiments. We also thank Lidiya Korets and Carolyn Woo for help in care of the mice.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. Baluk, Cardiovascular Research Institute, Univ. of California, 513 Parnassus Ave., San Francisco, CA 94143-0130 (E-mail: pbaluk{at}itsa.ucsf.edu)

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.


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 MATERIALS AND METHODS
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 DISCUSSION
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