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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Matrix metalloproteinases (MMPs) are upregulated locally in sites of inflammation, including the lung. Several MMP activities are upregulated in acute lung injury models but the exact role that these MMPs play in the development of the lung injury is unclear due to the absence of specific inhibitors. To determine the involvement of individual MMPs in the development of lung injury, mice genetically deficient in gelatinase B (MMP-9) and stromelysin 1 (MMP-3) were acutely injured with immunoglobulin G immune complexes and the intensity of the lung injury was compared with genetically identical wild-type (WT) mice with normal MMP activities. In the WT mice there was upregulation of gelatinase B and stromelysin 1 in the injured lungs which, as expected, was absent in the genetically deficient gelatinase B- and stromelysin 1-deficient mice, respectively. In the deficient mice there was little in the way of compensatory upregulation of other MMPs. The gelatinase B- and the stromelysin 1-deficient mice had less severe lung injury than did the WT controls, suggesting that both MMPs are involved in the pathogenesis of the lung injury. Further, the mechanism of their involvement in the lung injury appears to be different, with the stromelysin 1-deficient mice having a reduction in the numbers of neutrophils recruited into the lung whereas the gelatinase B-deficient mice had the same numbers of lung neutrophils as did the injured WT controls. These studies indicate, first, that both gelatinase B and stromelysin 1 are involved in the development of experimental acute lung injury, and second, that the mechanisms by which these individual MMPs function appear to differ.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Matrix metalloproteinases (MMPs) are a group of neutral proteinases that share common structural domains, contain zinc, and are produced as inactive proenzymes (reviewed in Refs. 1-3). Members of this family include collagenases; gelatinases, stromelysins, matrilysin, and membrane-bound MMPs. Most are secreted in the inactive proenzyme form with the exception of the membrane-bound MMPs and stromelysin 3, which are secreted in active form (3). The secreted proenzyme MMPs can be activated by several proinflammatory agents such as oxidants, elastase, and other MMPs, and they can degrade all components of the extracellular matrix (3, 4). They are felt to play important roles in embryonic development and postnatal growth (2). In addition, MMPs mediate normal tissue remodeling such as wound healing, hormonally dependent involution, and organ morphogenesis (reviewed in Ref. 5). However, in pathologic conditions such as inflammation, high levels of MMPs are produced that can facilitate tissue injury. Neutrophils and macrophages produce high levels of MMPs and they are felt to be the primary source of these activities in inflammation (6, 7). In inflammation associated with rheumatoid and osteoarthritis, increased MMP collagenolytic and stromelysin activities are present in the joints, which is associated with degradation of type II collagen and proteoglycans (8, 9). MMPs have also been implicated in many other inflammatory conditions, such as periodontitis and skin diseases (reviewed in Ref. 2).
There is also evidence that MMPs are involved in pulmonary inflammation. In adult respiratory distress syndrome (ARDS) associated with neutrophil infiltration, increased levels of gelatinase B are present in the bronchoalveolar lavage fluid (BALF) (10). In chronic lung injury with pulmonary fibrosis, where macrophage infiltration is present, there is increased gelatinase B and collagenase (MMP-1) expression in the lung, and increased gelatinase B expression has also been described in the lungs of patients with asthma (11, 12).
Experimental models of lung injury have suggested an important role for MMPs. Models of acute lung injury induced by immune complexes, hyperoxia, ozone, or lipopolysaccharide have shown increased gelatinolytic and collagenolytic activities in the lung (13). The evidence is particularly strong for gelatinase B, where high levels of this MMP are present in BALF and tissues. In models of acute and chronic lung injury, we and others have found evidence of macrophage-derived metalloelastase (MMP-12) and gelatinase B activities in the lung (17, 18). The use of MMP inhibitors such as the naturally occurring tissue inhibitors of metalloproteinases (TIMPs), which inhibit all of the MMPs, reduces the degree of lung injury in these models (19). Stromelysin 1 activities in the lung have not been described but in other sites of inflammation, in particular arthritis, there are increased stromelysin activities in the inflamed tissues (8, 9).
Even though there is good evidence for MMP involvement in lung injury it is not clear which of the upregulated MMP activities are responsible for the tissue injury. This is largely due to lack of selective MMP inhibitors such as gelatinase inhibitors. In an attempt to directly address the role of specific MMPs, in particular gelatinase B and stromelysin 1, in acute lung injury, we have used mice made deficient in gelatinase B and stromelysin 1 by targeted mutagenesis and compared the degrees of acute lung injury in these animals with that seen in their wild-type (WT) control animals having normal levels of the MMPs. The use of these selected MMP knockout (KO) animals allows for definitive analysis of the roles of the individual MMPs in the development of the lung injury.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Reagents and Materials
All materials were reagent grade or better and, except where noted, were purchased from Sigma Chemical Company (St. Louis, MO).
Laboratory Animals
The gelatinase B-deficient mice and their WT control animals were obtained from the laboratory of Dr. Robert Senior (Washington University, St. Louis, MO). As described in recent publications, the gelatinase B-deficient animals are bred on a 129SvEv background and are genetically identical to the WT 129SvEv animals (20). The stromelysin 1-deficient mice and their genetically identical WT littermates were obtained commercially from Taconic Laboratories (Germantown, NY). These animals were originally developed in the laboratory of Dr. John Mudgett (Merck Research Laboratories, Rahway, NJ) and the donor strain is 129/SvEV ES cell implanted into C57BL/6J mice (21). For the present studies, the animals were bred and maintained under pathogen-free conditions. All husbandry and animal procedures were done under the jurisdiction of the Unit for Laboratory Animal Medicine at the University of Michigan.
Immunoglobulin G Immune Complex-Mediated Acute Alveolitis
WT and KO mice 8 to 12 wk of age were used for the studies. The animal care committee at the University of Michigan approved these studies. The animals were anesthetized by intraperitoneal injection of a mixture of Ketaset (Fort Dodge Animal Health, Fort Dodge, IA) and Rompun (Bayer Corp., Shawnee Mission, KS) at doses of 1.66 and 0.033 mg/g body weight, respectively. Immunoglobulin (Ig) G immune complex lung injury was induced by the intratracheal instillation of 0.5 mg of IgG antibody to bovine serum albumin (BSA). The BSA antigen was instilled intravenously. This model of acute lung injury is mostly neutrophil-dependent as established by specific neutrophil depletion studies, although alveolar macrophages play an important role in leukocyte recruitment as well as directly producing oxidants and proteinases (22, 23). The animals were killed after 4 h and the degree of lung injury quantified by histopathology as well as assessment of BALF protein levels, which we have found to correlate with lung permeability changes (24). Leukocyte emigration into the alveoli was assessed by analysis of the cell pellets in the BALF.
Morphologic Studies
For histopathologic analysis, at least eight animals per group were evaluated. The lungs were inflated with buffered formalin at low pressure (10 mm H2O). At the time of the animal's death and the tissue was embedded in paraffin and routinely processed for light microscopy analysis using hematoxylin and eosin (H&E)-stained sections.
Quantitative Morphometric Evaluation
To quantify more precisely the extent of the lung injury, analysis was carried out using quantitative morphometry. A minimum of 60 random ×100 oil immersion fields were analyzed from three animals per group using an Olympus BX40 microscope with a video camera attached to a digital video imaging analysis system. The system uses software obtained from IP Labs Spectrum (Signal Analytics, Vienna, VA) and NIH Image (Scion Corp., Walkerville, MD) to precisely quantify such parameters as intraovular hemorrhage, leukocyte influx, and the surface area of edema around blood vessels. This analysis has been used in the past in our laboratories (25).
BAL
Animals were killed 4 h after injury by lethal injection of Ketamine. The animals were exsanguinated and their thoracic cavities opened to reveal the lungs and trachea. The lungs were lavaged in situ, initially with 0.7 ml of sterile phosphate-buffered saline. After centrifugation the recovered supernatant was used for MMP and protein determinations and the cell pellet was combined with lavage fluid from subsequent lavages (three lavages of 1 ml each). Cell numbers were determined using a hemocytometer, and morphologic comparisons of cell pellets were made using modified Wright's stain. The fluids were evaluated for protein levels using trichloroacetic acid precipitation followed by folin assay, using a standard curve for comparison. The fluids were also assayed for MMP activities by techniques described later.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Substrate-Embedded Enzymography
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) substrate-embedded zymography was carried out by a modification of the method of Heusen and Dowdle to characterize the gelatinase B activity in the BALF (26). Briefly, SDS-PAGE gels were prepared for minigels from 30:1 acrylamide/bis with the addition of 1 mg/ml gelatin. The gelatin gels were 7.5% acrylamide. BALF samples were electrophoresed into the gels at a constant voltage of 150 V in an ice bath under nonreducing conditions. After electrophoresis, the gels were washed with Tris buffer containing Triton S-100 and stained with Coomassie Brilliant Blue 250-R. After destaining, zones of enzymatic activity were stained as regions of negative staining. These zones were scanned by densitometry for quantification. This technique allows for the assessment of active and total enzyme activity.
Stromelysin 1 Assay
Stromelysin 1 activity was assessed using a kit obtained from Chemicon International (Temecula, CA). The assay is based upon the fluorescent measurement of peptide substrate fragments released upon cleavage of the substrate by stromelysin. The assay was run in the presence and absence of trypsin to evaluate total versus active stromelysin activity.
Statistical Analysis
Experimental data were tested for normality using the Kolmogorov-Smirnov test and equal variance was tested using the Levene Median test. Data sets that passed both normality and equal variance tests were considered parametric data, and those that failed either test were considered nonparametric data. For parametric analyses, comparisons of two variables were made using Student's t test, and comparisons of more than two variables were by a one-way analysis of variance, with subsequent multiple comparisons made using a Tukey test. For nonparametric analyses, comparisons of two variables were by means of a Mann-Whitney rank sum test and comparisons of more than two variables were made using a Kruskal-Wallis analysis of variance on ranks with subsequent multiple analyses using a Tukey test. For all statistical tests, differences were considered significant when P < 0.05. All of the analyses were performed using Sigma Stat 2.0 (Jandel Corp., San Rafael, CA).
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Results |
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Abstract
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Materials and Methods
Results
Discussion
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Gelatinolytic Activities in the Injured Lungs
Because elevated levels of gelatinase A as well as gelatinase B have been described in some models of lung injury, we wanted to determine the levels of gelatinase A and B as well as stromelysin 1 in the injured lungs of the WT versus the MMP KO animals. BALF was evaluated in noninjured and injured animals for MMP activity. As shown in Figure 1, when gelatinase B activity was evaluated in the BALF of the stromelysin 1 WT animals there was little activity in the negative control animals given saline instead of the IgG immune complexes. This was also true for the gelatinase B WT animals. When the animals were injured with IgG immune complexes there was a dramatic increase in gelatinase B activity in the BALF of both WT strains as compared with the saline control animals. In the gelatinase B KO animals, as expected, no gelatinase B activity could be detected. In the stromelysin 1 KO animals, increased gelatinase B activity could be detected in the BALF of the injured animals but the levels were about the same as in the injured WT controls. Also illustrated in Figure 1 are gelatinase A activities in the BALF. As compared with gelatinase B there was only a modest increase in MMP-2 activity in injured lungs, and no significant differences were noted between WT and KO animals. Thus, gelatinolytic activities, in particular gelatinase B, were increased in this model of acute lung injury and gelatinase B KO animals showed the expected lack of gelatinolytic activity. In addition, the deletion of another MMP activity stromelysin 1 did not induce a compensatory increase in gelatinase B activity.
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Stromelysin Activity in the Injured Lungs
Evaluation of the BALF for stromelysin 1 activity in the WT animals as compared with KO animals is illustrated in Figure 2. In the WT controls for the stromelysin 1 KO animals there was some intrinsic stromelysin 1 activity in the BALF of uninjured animals, which was increased in the injured animals. As expected, in the stromelysin 1 KO animals little or no stromelysin 1 activity could be detected. In the gelatinase B WT animals there was virtually no intrinsic stromelysin 1 activity in the BALF in the injured as well as noninjured animals. In the gelatinase B KO animals there was an increased amount of stromelysin 1 activity in the uninjured as well as injured animals, but these levels were lower than in the WT stromelysin 1 animals. Thus, stromelysin 1 activity appears to be intrinsically present in the lungs of the C57BL/6 stromelysin 1 WT but not the 129sv gelatinase B WT animals, suggesting strain variations in the levels of this MMP. In the injured lungs, the levels of stromelysin 1 were increased in the stromelysin 1 WT animals and only marginally in the gelatinase B KO animals as compared with the uninjured controls. Thus, the C57BL/6 strain of mice appears to have higher constitutive expression of stromelysin 1, which is increased during injury, as compared with the 129SvEv strain. In addition, there appears to be an increase in stromelysin 1 activity in the gelatinase B-deficient mice as compared with that in the WT gelatinase B animals, which is also slightly increased during injury.
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Lung Injury Is Decreased in the Gelatinase B KO Mice
Because gelatinase B activities are increased in the injured lungs of these animals we wanted to determine whether the absence of this activity in the gelatinase B KO mice was associated with a reduction in the intensity of the lung injury. In fact, this appears to be the case. As shown in Figure 3, when the levels of BALF protein leakage were quantified there was an expected large increase in BALF protein in the injured gelatinase B WT animals as compared with negative control animals, reflecting increased vascular permeability. By comparison, in the lung injury induced in the gelatinase B-deficient animals there was a marked reduction in the amount of BALF protein present, with a 64% reduction in protein levels (P = 0.001) as compared with the WT mice. Thus, it appears that selective depletion of gelatinase B is protective against the development of acute lung injury.
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To determine whether this protective effect of gelatinase B deletion was associated with a reduction in inflammatory cell infiltration into the lung, BALF leukocyte counts were compared in the WT versus the gelatinase B KO animals. These studies are summarized in Figure 4. As expected, the uninjured animals showed very low levels of neutrophils in the BALF with normal levels of alveolar macrophages. In the injured animals there was a marked increase in the numbers of neutrophils in the BALF in both the WT as well as the gelatinase B KO animals. The numbers of macrophages were not increased. Thus, the protective effect seen in the permeability studies in the gelatinase B KO animals does not appear to be due to a reduction in the number of infiltrating neutrophils. This argues for a direct effect of gelatinase B on the lung extracellular matrix rather than indirect modulation of the leukocyte influx.
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Lung Injury Is Decreased in the Stromelysin KO Mice
Similar studies were done to determine the role of stromelysin 1 in the development of acute lung injury. These studies are summarized in Figures 5 and 6. As shown in Figure
5, when lung permeability was assessed in the WT mice as
compared with the stromelysin 1-deficient animals there
was a marked reduction in the amount of protein present
in the BALF of the KO animals as compared with the WT
animals, with a 57% reduction in protein noted (P = 0.0001). Thus, similar to what was seen with the gelatinase B-deficient animals, a loss of a selective MMP activity
in
this case, stromelysin 1is associated with a reduction in
the degree of lung injury. To determine whether this reduction in lung injury was associated with a reduction in
infiltrating neutrophils, BALF leukocytes were quantified
in these animals. As shown in Figure 6, the stromelysin 1-
deficient animals had a 64% reduction (P = 0.001) in the
numbers of BALF neutrophils as compared with the WT
controls (P < 0.05). Thus, it appears that the reduced lung injury seen in the stromelysin 1-deficient animals is associated with a reduction in leukocyte recruitment, which is
different from the effect seen in the gelatinase B-deficient
animals.
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Histologic Appearance of Lung Injury in MMP KO versus WT Animals
As illustrated in Figure 7, histologic analysis of the lung injury in these studies reveals that both protease-deficient animals showed less severe lung injury. However, correlating with the BALF leukocyte counts, there were differences in the numbers of neutrophils in the two groups. In the gelatinase B-deficient animals there was a reduction in the intensity of the lung injury as compared with the WT animals, but no apparent differences in neutrophil accumulation in the lung were observed. By comparison, in the stromelysin 1-deficient mice the degree of lung injury was also reduced as compared with the WT controls, but in these animals there was also a reduction in the number of neutrophils in the lung.
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Morphometric studies were done to more precisely quantify the intensity of the lung injury. As shown in Table 1, the gelatinase B KO mice had less injury than did the WT controls, as manifested by a reduction in hemorrhage and edema. However, the numbers of infiltrating neutrophils and macrophages in the gelatinase B KO mice were the same as for the WT controls. By comparison, the stromelysin 1 KO animals also had a reduction in injury as compared with the WT controls, with a marked reduction in edema and hemorrhage. However, unlike the gelatinase B animals, the stromelysin 1 KO animals had a significant reduction in the number of infiltrating leukocytes, with 96% less neutrophils and 84% less macrophages than the WT controls.
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Thus, the histology data correlates with the BAL studies and suggests that although both stromelysin 1 and gelatinase B are involved in the pathogenesis of lung injury, their mechanism(s) of action may differ.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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As described earlier, there are many observations in humans and experimental animals that MMPs are upregulated in a variety of disease processes including inflammatory lung injury. These observations, coupled with the fact that TIMP activities are upregulated in vivo in lung injury and that exogenous TIMP administration inhibits lung injury, provide definitive evidence of the important role that MMPs play in the development of acute lung injury. However, because several MMP activities are upregulated in the inflammatory models and there are no selective MMP inhibitors, it has not been possible to determine by inhibition studies the contributions of individual MMPs to the lung injury. For that reason, MMP-targeted mutagenesis (KO) mice lacking a specific MMP activity hold great promise as a way to precisely characterize the role of specific MMPs in these complex biologic processes. Using these MMP-deficient mice we have been able to provide definitive evidence that stromelysin 1 and gelatinase B are involved in the pathogenesis of experimental macrophage- and neutrophil-mediated acute lung injury. There is a considerable degree of evidence for gelatinase B involvement in lung injury, with several human as well as experimental models of lung injury showing increased expression of gelatinase B. However, this work represents the first study in which a direct role for gelatinase B in the development of acute lung injury was shown using the gelatinase B-deficient animals. The use of these animals provides a powerful tool to ascertain the role of the MMPs and is the only way to directly assess a role for the MMPs, inasmuch as selective gelatinolytic inhibitors are not currently available.
Our studies also suggest a role for stromelysin in the development of the lung injury model. Unlike the data available for gelatinase B there are no previous studies on stromelysin involvement in acute lung injury, although stromelysin is upregulated in the joint in inflammatory arthritis, and elevated circulating levels of this MMP are present in arthritis as well as other inflammatory diseases, such as lupus (8, 9). However, these circulating levels do not correlate with disease activity, and the stromelysin 1 KO animals have not shown decreased severity of joint injury in the model of collagen-induced arthritis (21). Our studies are the first to show not only that stromelysin activity is upregulated in acute lung injury but also that it plays a role in the development of the lung injury induced by acute inflammation, as evidenced by a reduction in the degree of lung injury in the stromelysin 1-deficient animals.
These studies also show that there does not appear to be much in the way of compensatory increases in other MMP activities in the lungs of the mice with selective MMP deletions. In the stromelysin 1 KO mice there was no increase in gelatinase B activity in the injured lungs. There was some increase in stromelysin 1 activity in the gelatinase B KO animals as compared with the WT, but this increased activity was seen in the uninjured animals as well. Thus, it appears that the selective depletion of these MMPs with the subsequent reduction in lung injury cannot be compensated for by alterations in the levels of other MMPs.
These studies also indicate that there are differences in the mechanisms of action of these MMPs. For gelatinase B, the diminution in lung injury in the gelatinase B-deficient animals was not associated with a reduction in the intensity of leukocyte recruitment, arguing for a direct effect of gelatinase B on the breakdown of the extracellular matrix. In fact, studies with the gelatinase B mice in a model of dermal inflammation also suggest a direct effect on the intensity of the inflammatory response without a reduction in neutrophil recruitment (20). By comparison, the studies with the stromelysin 1-deficient mice clearly show that the protective effect in the lung is associated with a reduction in leukocyte recruitment, suggesting that stromelysin 1 is involved in neutrophil recruitment into sites of inflammation as well as perhaps a direct effect on the extracellular matrix. Similar findings were seen in a recent study by Wang and colleagues (27), using an experimental model of delayed type hypersensitivity (DTH) in the mouse, where stromelysin 1-deficient mice had marked inhibition of the inflammation-induced edema as compared with the WT mice with normal levels of stromelysin 1. This inhibitory effect was overcome by local injection of stromelysin 1 (25). Thus, these studies using a model of chronic rather than acute inflammation are consistent with our studies on the important role of stromelysin 1 in inflammation. The DTH response in mice is characterized by extensive neutrophil as well as mononuclear inflammatory cell infiltration, and it would be of interest to determine whether the inhibition of the inflammation observed in the stromelysin 1-deficient mice in this model is also associated with a reduction in neutrophil influx.
There are several potential mechanisms by which stromelysin 1 could be regulating leukocyte emigration in sites of
inflammation. One potential mechanism is a reduction in
chemotactic activity. In models of acute lung injury, several neutrophil chemotactic peptides are upregulated, including the complement chemotactic peptide C5a as well
the CXC family of 
chemokines, including macrophage
inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (26). In addition, the B-chemokine MIP-1 has also been shown to be involved in neutrophil recruitment as well. These chemotactic agents are
generated primarily by the alveolar macrophage, as are
many of the MMP activities; and alterations in the levels of
the MMPs by the use of MMP inhibitors such as TIMP
have been associated with reductions in chemotactic activities in the BALF (19). There is also evidence in a model
of chronic lung injury associated with smoking that macrophage elastase (MMP-12) regulates monocyte recruitment
into the lung via the generation of elastin fragments chemotactic for these cells (18). Finally, a direct role for
stromelysin 1 in the generation of chemotactic activity for
leukocytes has been shown in a recent publication (27)
where macrophage-dependent vertebral disc resorption was assessed in the stromelysin 1 KO mice as compared with
the WT. They found that chondrocyte-derived stromelysin
1 was required for the generation of a macrophage chemoattractant and the subsequent infiltration of the disc tissue by
the macrophages (27). Whether or not a similar stromelysin 1-dependent chemotactic activity for leukocytes is
generated during lung injury remains to be determined.
Another possible mechanism for the reduction in neutrophil infiltration in the stromelysin 1 KO animals is decreased neutrophil migration into the lung parenchyma, inasmuch as MMP-induced matrix degradation has been implicated in cell migration during tissue regeneration, wound healing, and inflammation (2). In vitro, decreased neutrophil migration through Matrigel-coated filters has been reported in the presence of MMP inhibitors (28). However, because neutrophils do not produce stromelysin it is not likely that stromelysin would be directly responsible for neutrophil migration into the tissues. The potential roles of other neutrophil MMPs in neutrophil infiltration in the WT as compared with the KO mice are currently being evaluated in the laboratory.
The critical role for MMPs in the development of the
acute lung injury may also involve alterations in the levels
of biologically active inflammatory mediators. In the DTH
study, gelatinase B-deficient mice had no reduction in the
leukocyte influx but there were alterations in the levels of
interleukin (IL)-10, suggesting that gelatinase B is involved in the upregulation of this anti-inflammatory cytokine at least in chronic inflammation (25). Also, MMPs
and the closely related ADAMs have the ability to shed
biologically active molecules from cell membranes including tumor necrosis factor- and ILs such as IL-6 which are
known to play important roles in inflammation, including
lung injury (28). Thus, it is possible that MMPs are involved in the pathogenesis of lung injury by the regulation of pro- and anti-inflammatory cytokines.
Finally, the findings with the stromelysin 1-deficient mice where gelatinase B levels in the injured lungs were similar to the WT controls in spite of reductions in the numbers of infiltrating neutrophils is supportive of our previous studies that suggest that much of the MMP activity generated in these acute lung injury models is from alveolar macrophages. In our previous studies using this model and others, macrophages were increased in the injured lungs and lavaged macrophages contained high levels of MMP activities (17, 29). Thus, it appears that macrophages rather than neutrophils may be the primary source of MMPs in acute lung injury. These studies therefore provide additional evidence of the critical role that macrophages play in lung injury not only by modulating inflammation by cytokine and chemokine elaboration but also by the direct production of proteinases and oxidants.
In summary, these studies provide definitive evidence for a role for stromelysin 1 and gelatinase B in the pathogenesis of acute lung injury. Further, the inhibition of neutrophil infiltration in the stromelysin 1-deficient animals suggests that stromelysin 1 is somehow involved in neutrophil recruitment. Future studies will need to be done to determine the precise mechanism of this effect as well as the other possible biologic effects of these MMPs in inflammation. In addition, other MMPs, such as macrophage metalloelastase (MMP-12), may also play important roles in the development of acute lung injury. Studies using the MMP-12-deficient mice should address this issue.
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Footnotes |
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Address correspondence to: Kent J. Johnson, M.D., Dept. of Pathology, University of Michigan Medical School, M7520 Medical Science Research I, Box 0602, 1301 Catherine Rd., Ann Arbor, MI 48109-0602. E-mail: kjjkjj{at}umich.edu
(Received in original form March 7, 2000 and in revised form November 28, 2000).
Abbreviations: bronchoalveolar lavage fluid, BALF; immunoglobulin, Ig; knockout, KO; matrix metalloprotease, MMP; tissue inhibitor of metalloproteinase, TIMP; wild-type, WT.Acknowledgments: This study was supported in part by National Institute Heart, Lung grant #HL-42607 and by National Cancer Institute grant #CA60958. The authors thank Dr. Robert Senior for providing the gelatinase B-deficient mice.
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References |
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Materials and Methods
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Discussion
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1. Woessnner, J. F. Jr.. 1994. The family of matrix metalloproteinases. Ann. NY Acad. Sci. 732: 11-21 [Medline].
2.
Birkedal-Hansen, H.,
W. G. I. Moore,
M. K. Bodden,
L. J. Windsor,
B. Birkedal-Hansen,
A. DeCarolo, and
J. A. Engler.
1993.
Matrix metalloproteinases: a review.
Crit. Rev. Oral Biol. Med.
4:
197-250
3. Cawston, T. E.. 1996. Metalloproteinase inhibitors and the prevention of connective tissue breakdown. Pharmacol. Ther. 3: 163-182 .
4. Alexander, C. M., and Z. Werb. 1992. Extracellular matrix degradation. In Cell Biology of the Extracellular Matrix. E. D. Hay, editor. Plenum Press, New York. 255-302.
5.
Rudolph-Owen, L. A.,
D. L. Hulboy,
C. L. Wilson,
J. Mudgett, and
L. M. Matrisian.
1997.
Coordinate expression of matrix metalloproteinase family
member in the uterus of normal, matrilysin-deficient, stromelysin 1-deficient mice.
Endocrinology
138:
4902-4911
6.
Shipley, J. M.,
R. L. Wesselschmidt,
D. K. Kobayshi,
T. J. Ley, and
S. D. Shapiro.
1996.
Metalloelastase is required for macrophage-mediated porteolysis and matrix invasion in mice.
Proc. Natl. Acad. Sci. USA
93:
3942-3946
7. Palmgren, M. S., R. D. deShazo, R. M. Carter, M. L. Zimny, and S. V. Shah. 1992. Mechanisms of neutrophil damage to human alveolar extracellular matrix: the role of serine and metalloproteinases. J. Allergy Clin. Immunol. 89: 905-915 [Medline].
8. Case, J. P., H. Sana, R. Lafyatis, E. F. Remmers, G. K. Kukkumian, and R. L. Wilder. 1989. Stromelysin gene expression in the synovium of rats with experimental erosive arthritis. J. Clin. Invest. 84: 1731-1740 .
9. Cawston, T. E. 1995. Proteinases and connective tissue breakdown. In Mechanisms and Models in Rheumatoid Arthritis. B. Henderson, J. C. W. Edwards, and E. R. Pettipher, editors. Academic Press, London. 333-362.
10. Ricou, B., L. Nicod, S. Lacraz, H. G. Welgus, P. M. Suter, and J. Dayer. 1996. Matrix metalloproteinases and TIMP in acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 154: 346-352 [Abstract].
11. Hayashi, T., W. G. Stetler-Stevenson, and M. Fleming. 1996. Immunohistochemical study of metalloproteinases and their tissue inhibitor in the lungs of patients with diffuse alveolar damage and idiopathic pulmonary fibrosis. Am. J. Pathol. 149: 531-538 [Abstract].
12.
Mautino, G.,
N. Oliver,
P. Chanez,
J. Bousquest, and
J. Capony.
1997.
Increased release of matrix metalloproteinase-9 in bronchoalveolar lavage
fluid and by alveolar macrophages of asthmatics.
Am. J. Respir. Cell Mol.
Biol.
17:
583-591
13.
Mulligan, M. S.,
P. E. Desrochers,
A. M. Chinnaiyan,
D. F. Gibbs,
J. Varani,
K. J. Johnson, and
P. A. Ward.
1993.
In vivo suppression of immune complex-induced alveolitis by secretory leukoproteinase inhibitor and tissue inhibitor of metalloproteinases 2.
Proc. Natl. Acad. Sci. USA
90:
11523-11527
14.
Kleeberger, S. R.,
J. P. Bassett,
G. J. Jakab, and
R. C. Levitt.
1990.
A genetic model for evaluation of susceptibility to ozone-induced inflammation.
Am. J. Physiol.
258:
L313-L320
15.
D'Ortho, M. P.,
P. H. Janeau,
C. Delacourt,
J. MacQuin-Mavier,
M. LeVane,
S. Pezet,
A. Harf, and
C. Lafuma.
1994.
Matrix metalloproteinases
and elastase activities in LPS-induced acute lung injury in guinea pigs.
Am.
J. Physiol.
266:
L209-L216
16. Pardo, A., M. Selman, K. Ridge, R. Barrios, and I. Sznajder. 1996. Increased expression of gelatinases and collagenase in rat lungs exposed to 100% oxygen. Am. J. Respir. Crit. Care Med. 154: 1067-1075 [Abstract].
17.
Gibbs, D. F.,
T. P. Shanley,
R. L. Warner,
J. Varani, and
K. J. Johnson.
1999.
The role of matrix metalloproteinases in models of acute lung injury: evidence for the alveolar macrophage as the source of the proteinases.
Am. J. Respir. Cell Mol. Biol.
20:
1145-1154
18.
Hautamaki, R. D.,
D. K. Koyayashi,
R. M. Senior, and
S. D. Shapiro.
1997.
Requirement for macrophage elastase from cigarette smoke-induced emphysema in mice.
Science
277:
2002-2004
19.
Gipson, T. S.,
D. F. Gibbs,
N. M. Bless,
T. P. Shanley,
L. Crouch,
K. J. Johnson, and
P. A. Ward.
1999.
Regulatory effect of endogenous protease
inhibitors in acute lung inflammatory injury.
J. Immunol.
162:
3653-3662
20.
Liu, Z.,
J. M. Shipley,
T. Vu,
X. Zhou,
L. A. Diaz,
Z. Werb, and
R. M. Senior.
1998.
Gelatinase B deficient mice are resistant to experimental
bullous pemphigoid.
J. Exp. Med.
188:
475-482
21. Mudgett, J. S., N. I. Hutchinson, N. M. A. Chartrain, A. J. Forsyth, J. McDonnel, I. I. Singel, E. K. Bayne, J. Flanagan, D. Kawka, C. F. Shen, K. Stevens, H. Chen, M. Trumbauer, and D. M. Visco. 1998. Susceptibility of stromelysin 1-deficient mice to collagen-induced arthritis and cartilage destruction. Arthritis Rheum. 41: 110-121 [Medline].
22. Johnson, K. J., and P. A. Ward. 1974. Acute immunologic pulmonary alveolitis. J. Clin. Invest. 54: 349-352 .
23. Warren, J. S., S. L. Kunkel, T. W. Cunningham, K. J. Johnson, and P. A. Ward. 1987. In vitro activation of rat neutrophils and alveolar macrophages with IgA and IgG immune complexes: implications for immune complex induced lung injury. Am. J. Pathol. 129: 578-585 [Abstract].
24. Kubo, H., D. Morgenstern, W. M. Quinian, P. A. Ward, M. C. Dinauer, and C. M. Doerschuk. 1996. Preservation of complement-induced lung injury in mice with deficiency of NADPH oxidase. J. Clin. Invest. 97: 2680-2684 [Medline].
25. Guice, K. S., K. Oldham, K. L. Johnson, R. G. Kunkel, M. L. Morganroth, and P. A. Ward. 1987. Pancreatitis-induced acute lung injury. Ann. Surg. 208: 71-77 .
26. Heussen, C., and E. B. Dowdle. 1980. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. 102: 196-202 [Medline].
27.
Wang, M. X.,
X. Qin,
J. S. Mudgett,
T. A. Ferguson,
R. M. Senior, and
H. G. Welgus.
1999.
Matrix metalloproteinase deficiencies affect contact hypersensitivity: stromelysin 1 deficiency prevents the response and gelatinase B
deficiency prolongs the response.
Proc. Natl. Acad. Sci. USA
96:
6885-6889
28.
Czermak, B. J.,
V. Sarma,
N. M. Bless,
H. Schmal,
H. P. Frield, and
P. A. Ward.
1999.
In vitro and in vivo dependency of chemokine generation on
C5a and TNFa.
J. Immunol.
162:
2321-2325
29. Declaux, C., C. Delacourt, M. P. d'Ortho, V. Boyer, C. Lafuma, and A. Harf. 1996. Role of gelatinase B and elastase in human polymorphonuclear neutrophil migration across basement membrane. Am. J. Respir. Cell Mol. Biol. 14: 288-295 [Abstract].
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