AJP - Lung AJP citation statistics
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 293: L779-L789, 2007. First published June 22, 2007; doi:10.1152/ajplung.00442.2006
1040-0605/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/3/L779    most recent
00442.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martin, E. L.
Right arrow Articles by Veldhuizen, R. A. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martin, E. L.
Right arrow Articles by Veldhuizen, R. A. W.

Contribution of alveolar macrophages to the response of the TIMP-3 null lung during a septic insult

Erica L. Martin,1 Tanya A. Sheikh,2 Kevin J. Leco,1 James F. Lewis,1,2 and Ruud A. W. Veldhuizen1,2

Departments of 1Physiology and Pharmacology and 2Medicine, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada

Submitted 8 November 2006 ; accepted in final form 16 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice deficient in tissue inhibitor of metalloproteinase-3 (TIMP-3) develop an emphysema-like phenotype involving increased pulmonary compliance, tissue degradation, and matrix metalloproteinase (MMP) activity. After a septic insult, they develop a further increase in compliance that is thought to be a result of heightened metalloproteinase activity produced by the alveolar macrophage, potentially modeling an emphysemic exacerbation. Therefore, we hypothesized that TIMP-3 null mice lacking alveolar macrophages would not be susceptible to the altered lung function associated with a septic insult. TIMP-3 null and wild-type (WT) mice were depleted of alveolar macrophages before the induction of a septic insult and assessed for alteration in lung mechanics, alveolar structure, metalloproteinase levels, and inflammation. The results showed that TIMP-3 null mice lacking alveolar macrophages were protected from sepsis-induced alterations in lung mechanics, particularly pulmonary compliance, a finding that was supported by changes in alveolar structure. Additionally, changes in lung mechanics involved primarily peripheral tissue vs. central airways as determined using the flexiVent system. From investigation into possible molecules that could cause these alterations, it was found that although several proteases and inflammatory mediators were increased during the septic response, only MMP-7 was attenuated after macrophage depletion. In conclusion, the alveolar macrophage is essential for the TIMP-3 null sepsis-induced compliance alterations. This response may be mediated in part by MMP-7 activity but occurs independently of inflammatory cytokine and/or chemokine concentrations.

compliance; chronic obstructive pulmonary disease; metalloproteinase; inflammation

MATRIX METALLOPROTEINASES (MMPs) are degradative enzymes that are regulated, in part, by their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Both are found throughout the body, where their primary function is tissue turnover and remodeling (3). MMPs degrade the extracellular matrix, whereas TIMPs regulate MMP activity (51). A shift in the balance between MMP and TIMP activity can lead to excessive or insufficient tissue breakdown and, consequently, altered tissue function. An MMP-TIMP imbalance is associated with many lung diseases, including asthma (35), idiopathic pulmonary fibrosis (60), acute respiratory distress syndrome (5, 6), and pulmonary emphysema/chronic obstructive pulmonary disease (COPD) (48).

Previous characterization of the TIMP-3 knockout (KO) lung phenotype found chronic air space enlargement, decreased lung function, no compensation of the other TIMPs, and an MMP-TIMP imbalance, thereby modeling the disease of pulmonary emphysema (39). To better understand the role of TIMP-3 in the development and progression of injury and disease, TIMP-3 null mice were exposed to various lung insults (44, 45). After indirect septic insults, TIMP-3 KO lungs developed a rapid increase in lung compliance and altered tissue structure (44, 45). However, when the TIMP-3 KO mice were exposed to direct lung insults (mechanical ventilation or hyperoxia), lung compliance was not altered, suggesting that this response was specific to some component of the septic insult (44). Although it was unclear which cell types and molecules were involved in the TIMP-3 null lung's susceptibility to sepsis, MMPs were believed to be involved in this response to sepsis, since they were increased after septic insults but remained unchanged after direct lung insults (44, 45).

Interestingly, this TIMP-3 null lung response to sepsis displays clinical similarities to exacerbations of COPD, which involve destructive structural alterations and permanently worsened airflow in response to a lung insult (57). Although respiratory infection is the leading cause of COPD exacerbations (57), sepsis is known to activate similar signaling pathways (10, 49, 56) and, therefore, may also be a risk factor for this condition. Since COPD patients experiencing frequent exacerbations deteriorate more rapidly (12), an understanding of the mechanisms underlying this condition has therapeutic value.

Given that TIMP-3 null lungs develop increased compliance selectively after septic insults, this response is thought to involve activation of the inflammatory cascade (40). Several inflammatory cells secrete MMPs during a septic insult (58, 67); however, the inflammatory cell population in the TIMP-3 null lung 6 h after a septic insult consists of >99% macrophages. Furthermore, alveolar macrophages are known to be pivotal to the pathophysiology of COPD, since they are correlated with disease severity and associated with increased compliance (11, 33, 52, 61). This pathophysiology may occur via secretion of various MMPs, cytokines, chemokines, and other proteases from the macrophage (61, 63), many of which can alter lung structure and increase compliance (9, 36, 37, 43, 73).

On the basis of this information, it was hypothesized that the alveolar macrophage is responsible for the increased compliance in the TIMP-3 null lung after a septic insult. In the present study we will test this hypothesis by depleting TIMP-3 null mice of alveolar macrophages and, subsequently, subjecting them to a septic insult.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal breeding and preparation. A full description of the generation and breeding of the TIMP-3 null mice was previously reported (39, 45). Young adult (9–12 wk of age) male C57/BL6 mice were group housed and had free access to standard rodent chow and water in an automatically controlled facility providing a 12:12-h light-dark cycle. All procedures were approved by the Animal Use Sub-Committee of the University of Western Ontario.

Depletion of alveolar macrophages via liposome administration. Phosphatidylcholine and cholesterol, in a ratio of 43 mg:4 mg and dissolved in chloroform, were used to form liposomes containing dichloromethylene-diphosphonic acid (DMDP) or PBS, as previously described by van Rooijen and Sanders (68, 69). TIMP-3 wild-type (WT) and KO animals were anesthetized with a 0.45-ml intraperitoneal injection of 7.5 mg of ketamine (Sandoz, Quebec, PQ, Canada) and 0.75 mg of xylazine (Bayer, Toronto, ON, Canada). Mice were randomized to receive 100 µl of 5 mg/ml of DMDP- or PBS-encapsulated liposomes, which were intratracheally instilled using a 30-gauge needle. Researchers were blinded with respect to the experimental groups throughout the experimentation. Animals were then sutured and allowed to recover while being monitored every 12 h. During this time, animals were assigned health scores on the basis of respiration and stress as indicated by appearance, activity, and behavior.

Induction of the cecal ligation-perforation insult. At 42 h after instillation of liposomes, which previous studies determined to be a sufficient time for significant depletion of macrophages (2, 20), TIMP-3 WT and KO mice were reanesthetized with ketamine and xylazine (see above). Animals were randomized to cecal ligation and perforation-induced sepsis (CLP) or a laparotomy (sham) surgery. The CLP procedure, as previously described (45), involved a laparotomy and ligation of the cecum distal to the ileocecal valve. The cecum was then punctured twice with an 18-gauge needle, once slightly distal to the ligature and once at the distal end of the cecum, and a small amount of fecal matter was extruded from each puncture. Sham animals were subjected to laparotomy with no manipulation of the cecum. The abdomen was then sutured, and the animals were allowed to recover for 6 h. During this time, animals were assigned health scores by an investigator blinded to the experimental groups (see above) (44, 45). The combination of these procedures created four WT and four KO experimental groups, PBS sham, PBS CLP, DMDP sham, and DMDP CLP, resulting in a total of eight groups.

Assessment of lung mechanics. Mice were euthanized 6 h after the CLP or sham insult with an injection of pentobarbital sodium (110 mg/kg ip); then the descending aorta was transected, and the animal was exsanguinated. A tracheotomy was performed, and mice were connected to the flexiVent ventilator (Scireq, Montreal, PQ, Canada) for ~5 min for measurement of lung mechanics. During this time, the ventilation parameters were set as follows: 150 breaths/min, 10 ml/kg body wt tidal volume, and zero end-expiratory pressure. The respiratory mechanics were not measured during regular ventilation but, rather, during brief interruptions in normal ventilation. During these interruptions, the valves connecting the ventilator to the atmosphere were closed and a volume signal was applied to the lungs. Whole lung dynamic respiratory mechanics included pulmonary compliance, resistance, and elastance, which were determined by fitting the linear single-compartment model using a multiple linear regression (31, 53), for which the first-order linear equation of motion was used (38, 72)

Formula
where compliance is the inverse of elastance (compliance = 1/elastance). To obtain this measurement, normal ventilation is interrupted for 1.1 s; during this interruption, a sinusoidal flow at 256 Hz is used to mimic a standardized breath. Therefore, the dependent variable is pressure, and flow and volume are independent variables; however, flow is calculated from the derivative of the volume signal, and resistance and elastance are the two coefficients for which we are solving. After the measurements of whole lung mechanics, the flexiVent was also utilized to measure central (airway) and peripheral (tissue) lung mechanics: central airway resistance (Rn), inertance (I), tissue damping (G), and tissue elastance (H), which were obtained using the forced oscillation technique that fits the constant-phase model to input impedence (31, 53). Briefly, this perturbation involves an 8-s signal, which utilizes a range of frequencies, including 0.5, 0.75, 1.25, 1.75, 2.75, 3.25, 4.25, 4.75, 5.75, 7.25, 9.25, 10.25, 11.75, 14.75, 16.75, 18.25, and 19.75 Hz. These frequencies were used because 1) they range from being well below to well above the normal breathing frequency and 2) they are mutually prime, so that the harmonics of one frequency do not interfere with the output of another. The values of respiratory mechanics were obtained as follows (26, 27, 64)

Formula
where {alpha} = (2/{pi}) x atan(H/G) and {omega} is the angular frequency. This technique has been established and evaluated by Schuessler and Bates (59). Data were rejected if coherence values were <0.95.

All the assessments of lung mechanics were performed identically for all experimental groups with regard to the specific settings for the perturbations as well as the order of the measurements. After assessment of lung mechanics, mice were split into two cohorts: 1) lavage, inflammatory cell assessment, cytokine/chemokine measurement, and zymography and 2) histology.

Inflammatory cell assessment. After the assessment of lung mechanics, two separate 1-ml volumes of 0.15 M NaCl were used to lavage the lungs through the endotracheal tube (1). The saline was instilled and withdrawn from the lung three times, the recovered lavage was combined, and the total volume was recorded. The remaining lung tissue was snap frozen in liquid nitrogen for zymographic analysis. The lavage was centrifuged for 10 min at 400 g at 4°C for isolation of inflammatory cells within the lavage as previously described (30). The supernatant from this spin of the lavage was snap frozen in liquid nitrogen for cytokine analysis. The pellet, which contained the inflammatory cells, was resuspended in 200 µl of Plasmalyte, and from this resuspended solution, 50 µl were spun onto a cytoslide and stained by HemaColor (EM Science, Gibbstown, NJ) for differential cell analysis. In addition, the resuspended solution was combined with an equal volume of trypan blue stain, and total live cells were counted using a hemocytometer and light microscope.

Histology and morphometric analysis. Lungs were removed from the thoracic cavity, inflated with air to a pressure of 25 cmH2O, and immediately fixed at this pressure in 10% phosphate-buffered formalin for 24 h. Lungs were rinsed in PBS for 1 h and then stored in 70% ethanol. Lungs were embedded in paraffin and sectioned at 7 µm. For structural analysis, the sections were deparaffinized to water and stained with hematoxylin and eosin. Ten random digital images of the peripheral alveolar structure of each animal were captured using a transmitted light microscope (Carl Zeiss, New York, NY) at a magnification of x20. Automatic image analysis of lung sections for calculation of surface area, alveolar diameter, and alveolar wall thickness was performed with Northern Eclipse (version 7.0) morphometric analysis software (Empix Imaging, Mississauga, ON, Canada). Briefly, for acquisition of the surface area and alveolar diameter measurements, this software system first identified all tissue through threshold intensity, and then the exterior lung surface, which was present along one side of each photograph, and significant blood vessels were blocked out, resulting in identification of the remaining alveolar air spaces. The software then calculated the average diameter of each air space and the total perimeter (or surface area) of these alveoli. For alveolar wall thickness, a grid of three horizontal lines was overlaid on the same photographs, and each time the grid crossed an alveolar wall, the thickness was measured. All measurements were performed by investigators blinded to the experimental group, and since each of the 10 photographs normally contain >100 alveoli, >1,000 alveoli were assessed for each animal.

Zymography. Zymography was used to analyze MMPs, as previously described (45). Briefly, frozen, lavaged lungs were homogenized in extraction buffer [50 mM Tris·Cl (pH 7.5), 150 M NaCl, 1% SDS, and 1 EDTA-free Protease Inhibitor Cocktail Pill (Roche Diagnostics, Laval, QC, Canada) for every 10 ml of buffer], and protein content of the homogenate was determined using a Micro BCA protein assay reagent kit (Pierce Chemical, Biolynx, Brockville, ON, Canada). Subsequently, 50 µg of lung homogenate protein were separated by electrophoresis in 10% SDS-polyacrylamide gels containing 1 mg/ml gelatin (for analysis of MMP-2 and MMP-9) or 1 mg/ml casein (for analysis of MMP-8 and MMP-7). Gels were washed in 2.5% Triton X-100 solution, rinsed in double-distilled H2O, and then incubated at 37°C in incubation buffer [50 mM Tris·Cl (pH 7.5), 5 mM CaCl2, and 5 µM ZnCl2] for 18–45 h. Gels were stained in Coomassie brilliant blue dye for 60 min (Bio-Rad Laboratories, Hercules, CA) and destained in 30% methanol and 10% acetic acid for 15 min. Gels were analyzed by spot densitometry using an Alpha Innotech Imager 2200 and the AlphaEase image analysis software (Alpha Innotech, San Leandro, CA). Clear resolution of the gelatin zymography allowed for individual quantification of latent and active forms of MMP-2 and MMP-9. However, the lower resolution of casein zymography (a limitation of this analysis) prevented accurate quantitative separation of the latent and active bands, resulting in combined analysis for MMP-8 and MMP-7. Replicate gels containing the same samples were incubated in incubation buffer containing 5 mM EDTA to confirm that the proteolytic activity was due to metalloproteinase activity.

Cytokine/chemokine analysis. The lavage concentrations of 16 cytokines or chemokines [IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, TNF-{alpha}, IFN-{gamma}, keratinocyte-derived chemokine (KC), monocyte chemotactic protein-1, granulocyte-macrophage colony stimulating factor, macrophage inflammatory protein-1{alpha}, and regulated on activation, normal T cell expressed and secreted] were measured using the Luminex mouse inflammatory cytokine kit (Cedarlane Laboratories, Hornby, ON, Canada) according to the manufacturer's instructions. Briefly, lavage samples (25 µl) were diluted in assay buffer and plated onto a 96-well filter plate preequilibrated with assay buffer. The samples were incubated with 25 µl of the antibody-coupled beads selected for the assay on a plate shaker overnight at 4°C. Detection antibody cocktail was added to the wells, and the samples were incubated on a plate shaker for 2 h at room temperature. Streptavidin-phycoerythrin was added to the wells, and the samples were incubated at room temperature for 30 min. The vacuum manifold was used to filter the unbound fraction out of the wells, and the bound beads were washed twice with wash buffer (200 µl/well). Finally, 100 µl of sheath fluid were added to each well, and the plate was placed on a plate shaker for 5 min. The samples were analyzed on the Luminex100 according to the manufacturer's instructions (RCYTO, Linco Research) (13, 65, 70).

Statistical analysis. Values are means ± SE. Differences were considered statistically significant at P < 0.05. Significance was determined by a three-way ANOVA using genotype, liposome administration, and insult as grouping variables. This analysis determines main effects of each individual variable, as well as interactions between variables. Statistical analysis was performed using the SPSS statistical software package for Windows version 11.0 (SPSS, Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Eighty-eight male mice were used in this study; there were no significant differences in age (9–12 wk) or body weight (22–24 g) among the groups. Results from health monitoring showed no significant differences after intratracheal instillation of DMDP or PBS liposomes, whereas mice further randomized to CLP had significantly poorer health scores than sham animals; however, this response was not significantly different between WT and KO mice (data not shown).

Inflammatory cells in the lavage. To determine the inflammatory cell populations within the lung and confirm our method of macrophage depletion, we performed total and differential cell counts. Analysis of the total number of macrophage cells (Fig. 1A) in the lavage revealed a significant decrease in DMDP groups compared with PBS controls. Figure 1B shows the number of polymorphonuclear neutrophils, which was significantly decreased in TIMP-3 KO vs. WT mice.


Figure 1
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 1. Inflammatory cell counts and differential analysis. Wild-type (WT) and TIMP-3 null [knockout (KO)] mice were treated with PBS or dichloromethylene-diphosphonic acid (DMDP) liposomes before sham surgery or cecal ligation and perforation-induced sepsis (CLP), creating 4 WT groups [PBS sham (n = 6), PBS CLP (n = 6), DMDP sham (n = 7), and DMDP CLP (n = 6)] and 4 KO groups [PBS sham (n = 6), PBS CLP (n = 6), DMDP sham (n = 6), and DMDP CLP (n = 6)]. Statistical significance (P < 0.05) is shown as main effect of genotype [KO vs. WT (a)] and main effect of liposome administration [DMDP vs. PBS (b)].

 
Respiratory mechanics. Results from the analysis of dynamic lung compliance and resistance are shown in Fig. 2. TIMP-3 KO lungs developed increased dynamic compliance (Fig. 2A) after sepsis; however, after DMDP liposome administration, TIMP-3 KO lungs were protected from this sepsis-induced compliance alteration. This effect on whole lung compliance was confirmed by three-way ANOVA, which found significant main effects of all three variables (genotype, liposome administration, and insult). There were also significant interactions between genotype and liposomes, genotype and insult, and liposomes and insult. Additionally, there was a significant three-way interaction between genotype, liposome administration, and insult. Whole lung resistance was not significantly different among experimental groups (Fig. 2B).


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 2. Whole lung dynamic compliance (A) and resistance (B), indicating distensibility and level of constriction, respectively. Values are means ± SE of flexiVent measurements of WT groups [PBS sham (n = 10), PBS CLP (n = 13), DMDP sham (n = 12), and DMDP CLP (n = 10)] and KO groups [PBS sham (n = 12), PBS CLP (n = 10), DMDP sham (n = 11), and DMDP CLP (n = 10)]. Statistical significance (P < 0.05) is shown as main effect of genotype (a), main effect of liposome administration (b), main effect of insult [CLP vs. sham (c)], interaction of genotype and liposome administration (d), interaction of genotype and surgery (e), interaction of liposome administration and surgery (f), and interaction of genotype, liposome administration, and surgery (g).

 
From analysis of the central airway and peripheral tissue contributions to whole lung mechanics, it was determined that central airway resistance (Fig. 3A) was significantly altered between genotypes. Specifically, central airway resistance was higher in TIMP-3 KO lungs than in WT controls. Tissue damping (Fig. 3B), reflective of tissue resistance, also had a significant effect of genotype: tissue damping was lower in TIMP-3 KO lungs than in WT controls. Tissue elastance (Fig. 3C) showed significant main effects of all variables (genotype, liposome administration, and insult). In addition, a significant three-way interaction between genotype, liposome administration, and surgery revealed decreased tissue elastance after CLP in TIMP-3 KO lungs; however, DMDP liposome administration may be protective in regard to this septic alteration.


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 3. Central and peripheral lung mechanics: central airway resistance, tissue damping (or tissue resistance), and tissue elastance. Values are means ± SE; see Fig. 2 legend for number of animals in each group. For explanation of statistical significance (P < 0.05), see Fig. 2 legend.

 
Histology and morphometric analysis. Typical alveolar structure is shown in Fig. 4, with corresponding morphometric analysis in Table 1. All measurements had a main effect of genotype, where alveolar diameter was significantly greater in TIMP-3 KO mice than in WT controls, and alveolar surface area and alveolar wall thickness were significantly lower in TIMP-3 KO mice than in WT controls. Additionally, alveolar wall thickness also had a main effect of liposome administration: values were significantly higher in DMDP groups than PBS controls.


Figure 4
View larger version (76K):
[in this window]
[in a new window]

  		Fig. 4. Histological changes in overall gross lung alveolar structure in the 8 experimental groups described in Fig. 1 legend (n = 4). Images were obtained from fixed paraffin-embedded 7-µm lung sections stained with hematoxylin and eosin. Representative images show increased air spaces in KO mice compared with WT animals. This air space enlargement is enhanced by CLP in PBS-liposome-treated, but not in DMDP-treated, KO animals. All images were obtained from the lung periphery without knowledge of experimental group at x20 magnification. Scale bar, 250 µm.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Morphometric analysis of alveolar structure

 
MMP analysis. MMPs were assessed by gelatin and casein zymography, and band intensities were quantified by densitometry (Table 2). On the basis of a limitation of the number of samples that can be analyzed on one gel, TIMP-3 WT and KO samples were run on separate gels; however, all samples were standardized to the PBS sham control, and previous studies showed no significant difference in MMPs between TIMP-3 WT and KO control lungs (44, 45). Typical zymograms of WT mice are displayed in Fig. 5; results from zymograms of TIMP-3 KO mice were identical.


View this table:
[in this window]
[in a new window]

  		Table 2. MMP levels as analyzed by gelatin and casein zymography

 

Figure 5
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 5. Typical zymogram of samples from WT mice quantified in Table 2. MMP, matrix metalloproteinase. Molecular sizes are as follows: 72 kDa (MMP-2), 28 kDa (MMP-7), 75 kDa (MMP-8), and 105 kDa (MMP-9).

 
After gelatin zymography analysis, we observed that the abundance of both forms of MMP-2 (72 kDa) and the latent form of MMP-9 (105 kDa) were not significantly different between groups. The active form of MMP-9, however, was significantly increased in CLP groups compared with sham groups. Results from casein zymography analysis showed that MMP-7 (28 kDa) and MMP-8 (75 kDa) abundance was significantly increased in CLP groups compared with sham groups. Furthermore, there was a significant interaction for MMP-7 between liposome administration and surgery: MMP-7 was increased to a lesser extent after DMDP than after PBS liposome administration.

Cytokine/chemokine analysis. The lavage concentration of 16 inflammatory cytokines and chemokines was analyzed. To determine whether any of these molecules are mechanistically involved in the TIMP-3 KO lung's altered response to sepsis, the concentrations of these molecules were examined in relation to the whole lung compliance data. This was accomplished by a linear regression correlation analysis of the cytokine/chemokine concentrations compared with compliance in each of the 24 TIMP-3 KO lungs that were lavaged, producing r2 values to quantify the degree of correlation (Fig. 6). From this analysis, it was determined that none of the 16 cytokines/chemokines showed any correlation to changes in compliance of the TIMP-3 KO lung, since all r2 values were <0.4. Figure 7 displays the lavage concentration values for three of these molecules, IL-6, KC, and TNF-{alpha}, which had a similar expression pattern. Statistically, IL-6 and KC had significant main effects of liposome administration and insult, both resulting in increased cytokine concentration, as well as a significant interaction between liposome administration and insult, showing a synergistic increase in these cytokines. Although TNF-{alpha} concentrations showed a similar synergistic pattern (Fig. 6C), it was not statistically significant.


Figure 6
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 6. Correlation analysis of tissue inhibitor of metalloproteinase-3 (TIMP-3) KO lungs. Dynamic lung compliance and cytokine/chemokine concentrations in lavage in 24 TIMP-3 KO mice described in Fig. 1 legend were compared using linear regression analysis. KC, keratinocyte-derived chemokine; GMCSF, granulocyte-macrophage colony stimulating factor; MIP-1{alpha}, macrophage inflammatory protein-1{alpha}; MCP-1, macrophage chemoattractant protein-1; RANTES, regulated on activation, normal T cell expressed and secreted.

 

Figure 7
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 7. Cytokine concentrations in whole lung lavage assessed by a Luminex100 multiplex assay for experimental groups and number of animals described in Fig. 1 legend. For explanation of statistical significance (P < 0.05) see Fig. 2 legend.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The TIMP-3 KO mouse is an established in vivo model of MMP-TIMP imbalance that leads to progressive air space enlargement similar to the pathology of emphysema (39, 45). TIMP-3 null mice developed a rapid increase in compliance after a septic insult (44, 45), which may reflect aspects of the irreversible alterations to lung structure and function that patients with emphysema can develop in response to various lung insults (57). The increase in compliance was accompanied by significantly decreased collagen and fibronectin levels but no changes to the pulmonary surfactant system, indicating that a rapid degradation of extracellular matrix components due to MMP activity was, in part, responsible for the effect (44, 45). To further examine this phenomenon and since 1) the altered response of the TIMP-3 KO lung was specific to a septic insult, 2) alveolar macrophages are activated by the inflammatory cascade during sepsis (30, 34, 62), and 3) the main inflammatory cell found in the lungs of this model are alveolar macrophages, the main focus of this study was to determine whether depletion of alveolar macrophages in the TIMP-3 KO lung before the septic insult could prevent the alterations in pulmonary compliance.

The technique used to deplete macrophages was established by van Rooijen and Sanders (68, 69) and is highly specific to macrophages due to 1) the high phagocytic action of macrophages toward liposomes, 2) the short (~15-min) half-life of DMDP, and 3) the inability of free DMDP to cross cell membranes, limiting its effect on other cell types and quarantining it within the lung.

Examination of respiratory mechanics showed that depletion of alveolar macrophages completely eliminated the sepsis-induced compliance increase in the TIMP-3 KO lungs, thereby supporting our original hypothesis that the alveolar macrophage is required for this response. In addition to compliance, this study explored other respiratory mechanics to elucidate the mechanisms responsible for the TIMP-3 KO response to sepsis. Although central airway resistance and peripheral tissue damping may influence the initial null phenotype, they were not affected by sepsis. The peripheral tissue elastance measured using the constant-phase model was consistent with compliance measured using the single-compartment model, since this was decreased in the lungs of TIMP-3 KO mice after sepsis, yet it was maintained if lungs were depleted of alveolar macrophages. It is acknowledged that there are some limitations when the constant-phase model is used to assess central and peripheral lung mechanics, including a tendency to underestimate central airway resistance and/or overestimate peripheral tissue damping (64). However, since our results show an increase in central airway resistance (which may be underestimated) and a decrease in peripheral tissue damping (which may be overestimated), this limitation of the constant-phase model would reduce the magnitude of the changes observed in our experiments. Furthermore, Thamrin et al. (64) reported that an alteration in tissue damping can be clouded by an alteration in central airway resistance; however, they also showed that a corresponding change in tissue elastance, as seen in our data, supports the validity of the change in tissue damping. The alterations in alveolar morphometry, including increased alveolar diameter and decreased alveolar surface area and wall thickness, showed trends similar to the lung mechanics, which supports the more sensitive findings of lung compliance and elastance. These histologically based measurements reflect the static properties of the lung and suggest that the dynamic alteration of compliance is the result of structural modifications. However, although fixation of lung tissue at a set pressure was utilized to histologically visualize alterations in lung compliance and that all tissues appeared adequately fixed during sectioning, a limitation of this external fixation technique is the variation of wall thickness, which could result in uneven fixative penetration and, thereby, uneven fixation.

On the basis of the lung mechanics measurements, it was concluded that the alveolar macrophage is essential for the sepsis-induced increased compliance in the TIMP-3 KO lung and that this phenomenon occurs through a mechanism that specifically alters peripheral tissue elastance. This implied role of the macrophage is based on the significant decrease in macrophage cell number in the lung when the macrophage depletion model is used. Depletion was confirmed in our study, which showed an 88% decrease in alveolar macrophages in lung lavage of TIMP-3 KO and WT mice 48 h after DMDP administration, which is comparable to other studies in the literature (2, 14). A limitation of this model, however, is that alterations in lung homeostasis due to side effects of macrophage depletion may contribute to these findings. For example, this technique has recently been observed to increase levels of pulmonary surfactant in the lung (18), which could potentially alter lung function and inflammation (15, 24, 41). However, because 1) DMDP treatment alone does not alter any of our measured lung mechanics, histology, or inflammatory markers and 2) increased surfactant levels by surfactant administration does not increase compliance above normal values (55, 74), it is unlikely that this side effect of DMDP administration plays a primary role in the TIMP-3 KO response to sepsis.

The secondary focus of this study was to assess MMPs, cytokines, and chemokines to potentially further our understanding of how the macrophage induces physiological changes in compliance. Various MMPs were investigated on the basis of the knowledge that 1) macrophages secrete various MMPs during an inflammatory insult (17, 22, 50), 2) the increased compliance in the TIMP-3 KO lung after sepsis is believed to result from altered lung structure (45), 3) excessive MMP activity can alter lung structure, resulting in an emphysema-like phenotype (8, 9, 17, 19), and 4) MMPs are less inhibited in the TIMP-3 KO lung, resulting in an MMP-TIMP imbalance (39, 45). MMP-2, MMP-7, MMP-8, and MMP-9 were analyzed using zymography, and consistent with previous findings, MMP-7, MMP-8, and MMP-9 were increased in CLP groups compared with sham groups (44, 45). Because of the absence of regulation by TIMP-3, it was previously thought that the increased abundance of these MMPs was the major cause of the sepsis-induced compliance alteration in the TIMP-3 KO lung (45). Nevertheless, the results of this study indicate that MMP-2, MMP-8, and MMP-9 are not involved in the sepsis-induced compliance alterations of the TIMP-3 KO lung. Among the MMPs measured, only the increase in MMP-7 during sepsis was attenuated after macrophage depletion, which suggests that MMP-7 may be involved in the development of altered compliance of the TIMP-3 KO lung after sepsis. This potential role of MMP-7 is supported by other studies. It has been shown that macrophage cells directly produce MMP-7 (16, 21, 25) and that this expression can be stimulated after LPS exposure (4). Macrophages may also activate lung epithelial cells, leading to increased MMP-7 production in response to bacterial exposure (42). Furthermore, MMP-7 produced from macrophages has previously been implicated in the development of acquired pulmonary emphysema, which is characterized by altered lung structure and increased compliance (7). Despite this evidence, our results in the TIMP-3 KO animals showed that macrophage depletion caused only an attenuation of MMP-7 levels after CLP, not a complete reduction to baseline values. In contrast, the increase in compliance observed in the KO animals after CLP was completely eliminated by alveolar macrophage depletion, suggesting that it is unlikely that MMP-7 is solely responsible for the increased compliance after sepsis. In this regard, it is important to realize that only 4 of 26 members of the MMP family were assessed in our study, and it remains likely that other MMPs contribute to an MMP-TIMP imbalance that is involved in this process. For example, MMP-12, which is produced primarily by mouse macrophages (32) and is a known contributor to emphysema phenotypes (28, 29, 36, 46, 47), may also contribute to the septic response and, therefore, should be investigated in future studies.

During an inflammatory injury such as sepsis, macrophages secrete many cytokines and chemokines that are involved in the propagation of the inflammatory cascade (23, 34). Overexpression of several of these molecules, including IL-1, IL-13, TNF-{alpha}, and IFN-{gamma}, has been shown to induce an increase in lung compliance (36, 37, 43, 73). Therefore, a large panel of inflammatory cytokines/chemokines was analyzed in lung lavage in an attempt to identify any molecules that may contribute to the mechanism involved in the CLP-induced compliance alterations of the TIMP-3 KO lung. As expected, many of these cytokines were increased in response to the CLP-induced septic insult, which confirmed our inflammatory model (only data for IL-6 and KC are shown). However, none of the lavage concentrations of these molecules correlated with the alteration of TIMP-3 KO lung compliance; therefore, they do not appear to have a causative role in this response. Interestingly, a few cytokines, namely, IL-6, KC, and TNF-{alpha}, appeared to be reduced in lavage concentrations after CLP-induced sepsis in TIMP-3 KO vs. WT mice. Although these changes did not reach statistical significance, this difference between genotypes may be related to the fact that TIMP-3 KO mice had significantly fewer neutrophils than WT controls. Furthermore, previous studies have shown that macrophages are involved in the downregulation of neutrophil infiltration and inflammation (54, 66). Specifically, Elder et al. (14) found that alveolar macrophages play an important suppressive role in the pulmonary inflammatory response to an LPS injury. Similarly, in this study, WT mice have a significantly greater lung inflammatory response to a CLP-induced septic insult in the absence of alveolar macrophages; however, it is believed that since the TIMP-3 KO lung has significantly fewer neutrophils, they are protected from this response.

The clinical implication of this study is that since the TIMP-3 KO mice have a lung phenotype similar to that of emphysema, emphysemic patients may be susceptible to a sepsis-induced injury possibly mimicking the symptoms of an exacerbation. Furthermore, since our results show that the macrophage is essential for the susceptibility of TIMP-3 KO mice to sepsis and in COPD patients macrophage numbers correlate with severity of disease (11), the macrophage may also play a significant role in the response of COPD patients to insults and their resulting exacerbations. This concept is supported by a recent review by Vlahos et al. (71) that implicated a role for granulocyte-macrophage colony stimulating factor, a major regulator of macrophages, in the development and treatment of COPD exacerbations.

In conclusion, the present study demonstrates that the alveolar macrophage is required for the increased compliance observed in the TIMP-3 KO lung after a septic insult. Furthermore, we have shown that although the initial TIMP-3 KO phenotype involves alterations to central and peripheral lung mechanics, the altered response of these mice to sepsis only involves alterations to peripheral tissue. Although the specific enzymes and mediators through which the alveolar macrophage influences pulmonary compliance in TIMP-3 KO mice during sepsis remains unclear, the results of this study indicate that MMP-2, MMP-8, and MMP-9 and the major inflammatory cytokines/chemokines are not involved, whereas MMP-7 may play a partial role in this process.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by Ontario Thoracic Society Grant RO361A10 (to R. A. W. Veldhuizen) and Canadian Institutes of Health Research Grant MOP-42556 (to R. A. W. Veldhuizen). Salary support for E. L. Martin was provided in part by an Ontario Graduate Scholarship.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Li-Juan Yao, Lynda McCaig, Cynthia Pape, and Pamela Joseph for technical assistance, Dr. Martin Post's laboratory for assistance with the Luminex cytokine assay, Kalamo Farley for teaching the DMDP liposome technique, and Dr. Yves Bureau for statistical consultation.

Present address of E. L. Martin: Univeristà di Torino, Dipartimento di Discipline Medico Chirurgiche, Sezione di Anestesiologia e Rianimazione, Ospedale S. Giovanni Battista, Turin 10126, Italy.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. W. Veldhuizen, Lawson Health Research Institute F4-117, 268 Grosvenor St., London, ON, Canada N6A 4V2 (e-mail: rveldhui{at}uwo.ca)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bailey TC, Martin EL, Zhao L, Veldhuizen RA. High oxygen concentrations predispose mouse lungs to the deleterious effects of high stretch ventilation. J Appl Physiol 94: 975–982, 2003.[Abstract/Free Full Text]
  2. Berg JT, Lee ST, Thepen T, Lee CY, Tsan MF. Depletion of alveolar macrophages by liposome-encapsulated dichloromethylene diphosphonate. J Appl Physiol 74: 2812–2819, 1993.[Abstract/Free Full Text]
  3. Birkedal-Hansen H, Moore WG, Bodden MK, Windsor LJ, Birkedal-Hansen B, DeCarlo A, Engler JA. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med 4: 197–250, 1993.[Abstract/Free Full Text]
  4. Busiek DF, Baragi V, Nehring LC, Parks WC, Welgus HG. Matrilysin expression by human mononuclear phagocytes and its regulation by cytokines and hormones. J Immunol 154: 6484–6491, 1995.[Abstract]
  5. Carney DE, McCann UG, Schiller HJ, Gatto LA, Steinberg J, Picone AL, Nieman GF. Metalloproteinase inhibition prevents acute respiratory distress syndrome. J Surg Res 99: 245–252, 2001.[CrossRef][ISI][Medline]
  6. Corbel M, Boichot E, Lagente V. Role of gelatinases MMP-2 and MMP-9 in tissue remodeling following acute lung injury. Braz J Med Biol Res 33: 749–754, 2000.[ISI][Medline]
  7. Cuvelier A, Kuntz C, Sesboue R, Muir JF, Martin JP. Metalloproteinases in the extracellular matrix: structure and activity. Rev Mal Respir 14: 1–10, 1997.[ISI][Medline]
  8. Dalal S, Imai K, Mercer B, Okada Y, Chada K, D'Armiento JM. A role for collagenase (matrix metalloproteinase-1) in pulmonary emphysema. Chest 117: 227S–228S, 2000.[CrossRef][ISI][Medline]
  9. D'Armiento J, Dalal SS, Okada Y, Berg RA, Chada K. Collagenase expression in the lungs of transgenic mice causes pulmonary emphysema. Cell 71: 955–961, 1992.[CrossRef][ISI][Medline]
  10. Delclaux C, Rezaiguia-Delclaux S, Delacourt C, Brun-Buisson C, Lafuma C, Harf A. Alveolar neutrophils in endotoxin-induced and bacteria-induced acute lung injury in rats. Am J Physiol Lung Cell Mol Physiol 273: L104–L112, 1997.[Abstract/Free Full Text]
  11. Di Stefano A, Capelli A, Lusuardi M, Balbo P, Vecchio C, Maestrelli P, Mapp CE, Fabbri LM, Donner CF, Saetta M. Severity of airflow limitation is associated with severity of airway inflammation in smokers. Am J Respir Crit Care Med 158: 1277–1285, 1998.[Abstract/Free Full Text]
  12. Donaldson GC, Seemungal TA, Bhowmik A, Wedzicha JA. Relationship between exacerbation frequency and lung function decline in chronic obstructive pulmonary disease. Thorax 57: 847–852, 2002.[Abstract/Free Full Text]
  13. Dupont NC, Wang K, Wadhwa PD, Culhane JF, Nelson EL. Validation and comparison of Luminex multiplex cytokine analysis kits with ELISA: determinations of a panel of nine cytokines in clinical sample culture supernatants. J Reprod Immunol 66: 175–191, 2005.[CrossRef][ISI][Medline]
  14. Elder A, Johnston C, Gelein R, Finkelstein J, Wang Z, Notter R, Oberdorster G. Lung inflammation induced by endotoxin is enhanced in rats depleted of alveolar macrophages with aerosolized clodronate. Exp Lung Res 31: 527–546, 2005.[CrossRef][ISI][Medline]
  15. Epaud R, Ikegami M, Whitsett JA, Jobe AH, Weaver TE, Akinbi HT. Surfactant protein B inhibits endotoxin-induced lung inflammation. Am J Respir Cell Mol Biol 28: 373–378, 2003.[Abstract/Free Full Text]
  16. Filippov S, Caras I, Murray R, Matrisian LM, Chapman HA Jr, Shapiro S, Weiss SJ. Matrilysin-dependent elastolysis by human macrophages. J Exp Med 198: 925–935, 2003.[Abstract/Free Full Text]
  17. Finlay GA, O'Driscoll LR, Russell KJ, D'Arcy EM, Masterson JB, FitzGerald MX, O'Connor CM. Matrix metalloproteinase expression and production by alveolar macrophages in emphysema. Am J Respir Crit Care Med 156: 240–247, 1997.[Abstract/Free Full Text]
  18. Forbes A, Pickell M, Foroughian M, Yao LJ, Lewis JF, Veldhuizen RA. Alveolar macrophage depletion results in increased surfactant pool sizes in adult rats. J Appl Physiol. doi:10.1152/japplphysiol.00995.2006.
  19. Foronjy RF, Okada Y, Cole R, D'Armiento J. Progressive adult-onset emphysema in transgenic mice expressing human MMP-1 in the lung. Am J Physiol Lung Cell Mol Physiol 284: L727–L737, 2003.[Abstract/Free Full Text]
  20. Fujimoto J, Wiener-Kronish JP, Hashimoto S, Sawa T. Effects of Cl2MDP-encapsulating liposomes in a murine model of Pseudomonas aeruginosa-induced sepsis. J Liposome Res 12: 239–257, 2002.[CrossRef][ISI][Medline]
  21. Furman C, Copin C, Kandoussi M, Davidson R, Moreau M, McTaggiart F, Chapman MJ, Fruchart JC, Rouis M. Rosuvastatin reduces MMP-7 secretion by human monocyte-derived macrophages: potential relevance to atherosclerotic plaque stability. Atherosclerosis 174: 93–98, 2004.[CrossRef][ISI][Medline]
  22. Gibbs DF, Shanley TP, Warner RL, Murphy HS, Varani J, Johnson KJ. Role of matrix metalloproteinases in models of macrophage-dependent acute lung injury. Evidence for alveolar macrophage as source of proteinases. Am J Respir Cell Mol Biol 20: 1145–1154, 1999.[Abstract/Free Full Text]
  23. Gouwy M, Struyf S, Proost P, Van Damme J. Synergy in cytokine and chemokine networks amplifies the inflammatory response. Cytokine Growth Factor Rev 16: 561–580, 2005.[CrossRef][ISI][Medline]
  24. Haagsman HP, Diemel RV. Surfactant-associated proteins: functions and structural variation. Comp Biochem Physiol A 129: 91–108, 2001.[CrossRef][Medline]
  25. Hagemann T, Robinson SC, Schulz M, Trumper L, Balkwill FR, Binder C. Enhanced invasiveness of breast cancer cell lines upon co-cultivation with macrophages is due to TNF-{alpha} dependent up-regulation of matrix metalloproteases. Carcinogenesis 25: 1543–1549, 2004.[Abstract/Free Full Text]
  26. Hantos Z, Collins RA, Turner DJ, Janosi TZ, Sly PD. Tracking of airway and tissue mechanics during TLC maneuvers in mice. J Appl Physiol 95: 1695–1705, 2003.[Abstract/Free Full Text]
  27. Hantos Z, Daroczy B, Suki B, Nagy S, Fredberg JJ. Input impedance and peripheral inhomogeneity of dog lungs. J Appl Physiol 72: 168–178, 1992.[Abstract/Free Full Text]
  28. Hautamaki RD, Kobayashi DK, Senior RM, Shapiro SD. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 277: 2002–2004, 1997.[Abstract/Free Full Text]
  29. Houghton AM, Quintero PA, Perkins DL, Kobayashi DK, Kelley DG, Marconcini LA, Mecham RP, Senior RM, Shapiro SD. Elastin fragments drive disease progression in a murine model of emphysema. J Clin Invest 116: 753–759, 2006.[CrossRef][ISI][Medline]
  30. Huang W, McCaig LA, Veldhuizen RA, Yao LJ, Lewis JF. Mechanisms responsible for surfactant changes in sepsis-induced lung injury. Eur Respir J 26: 1074–1079, 2005.[Abstract/Free Full Text]
  31. Ito S, Ingenito EP, Brewer KK, Black LD, Parameswaran H, Lutchen KR, Suki B. Mechanics, nonlinearity, and failure strength of lung tissue in a mouse model of emphysema: possible role of collagen remodeling. J Appl Physiol 98: 503–511, 2005.[Abstract/Free Full Text]
  32. Kassim SY, Fu X, Liles WC, Shapiro SD, Parks WC, Heinecke JW. NADPH oxidase restrains the matrix metalloproteinase activity of macrophages. J Biol Chem 280: 30201–30205, 2005.[Abstract/Free Full Text]
  33. Keatings VM, Collins PD, Scott DM, Barnes PJ. Differences in interleukin-8 and tumor necrosis factor-{alpha} in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 153: 530–534, 1996.[Abstract]
  34. Kopydlowski KM, Salkowski CA, Cody MJ, Van Rooijen N, Major J, Hamilton TA, Vogel SN. Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo. J Immunol 163: 1537–1544, 1999.[Abstract/Free Full Text]
  35. Kumagai K, Ohno I, Okada S, Ohkawara Y, Suzuki K, Shinya T, Nagase H, Iwata K, Shirato K. Inhibition of matrix metalloproteinases prevents allergen-induced airway inflammation in a murine model of asthma. J Immunol 162: 4212–4219, 1999.[Abstract/Free Full Text]
  36. Lanone S, Zheng T, Zhu Z, Liu W, Lee CG, Ma B, Chen Q, Homer RJ, Wang J, Rabach LA, Rabach ME, Shipley JM, Shapiro SD, Senior RM, Elias JA. Overlapping and enzyme-specific contributions of matrix metalloproteinases-9 and -12 in IL-13-induced inflammation and remodeling. J Clin Invest 110: 463–474, 2002.[CrossRef][ISI][Medline]
  37. Lappalainen U, Whitsett JA, Wert SE, Tichelaar JW, Bry K. Interleukin-1beta causes pulmonary inflammation, emphysema, and airway remodeling in the adult murine lung. Am J Respir Cell Mol Biol 32: 311–318, 2005.[Abstract/Free Full Text]
  38. Lauzon AM, Bates JH. Estimation of time-varying respiratory mechanical parameters by recursive least squares. J Appl Physiol 71: 1159–1165, 1991.[Abstract/Free Full Text]
  39. Leco KJ, Waterhouse P, Sanchez OH, Gowing KL, Poole AR, Wakeham A, Mak TW, Khokha R. Spontaneous air space enlargement in the lungs of mice lacking tissue inhibitor of metalloproteinases-3 (TIMP-3). J Clin Invest 108: 817–829, 2001.[CrossRef][ISI][Medline]
  40. Lei MG, Gao JJ, Morrison DC, Qureshi N. Pathogenesis of sepsis: current concepts and emerging therapies. Mol Med 100: 524–529, 2003.
  41. Lewis JF, Veldhuizen R. The role of exogenous surfactant in the treatment of acute lung injury. Annu Rev Physiol 65: 613–642, 2003.[CrossRef][ISI][Medline]
  42. Lopez-Boado YS, Wilson CL, Hooper LV, Gordon JI, Hultgren SJ, Parks WC. Bacterial exposure induces and activates matrilysin in mucosal epithelial cells. J Cell Biol 148: 1305–1315, 2000.[Abstract/Free Full Text]
  43. Lundblad LK, Thompson-Figueroa J, Leclair T, Sullivan MJ, Poynter ME, Irvin CG, Bates JH. Tumor necrosis factor-{alpha} overexpression in lung disease: a single cause behind a complex phenotype. Am J Respir Crit Care Med 171: 1363–1370, 2005.[Abstract/Free Full Text]
  44. Martin EL, McCaig LA, Moyer BZ, Pape MC, Leco KJ, Lewis JF, Veldhuizen RA. Differential response of TIMP-3 null mice to the lung insults of sepsis, mechanical ventilation, and hyperoxia. Am J Physiol Lung Cell Mol Physiol 289: L244–L251, 2005.[Abstract/Free Full Text]
  45. Martin EL, Moyer BZ, Pape MC, Starcher B, Leco KJ, Veldhuizen RA. 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 285: L1222–L1232, 2003.[Abstract/Free Full Text]
  46. Molet S, Belleguic C, Lena H, Germain N, Bertrand CP, Shapiro SD, Planquois JM, Delaval P, Lagente V. Increase in macrophage elastase (MMP-12) in lungs from patients with chronic obstructive pulmonary disease. Inflamm Res 54: 31–36, 2005.[CrossRef][ISI][Medline]
  47. Morris DG, Huang X, Kaminski N, Wang Y, Shapiro SD, Dolganov G, Glick A, Sheppard D. Loss of integrin {alpha}vbeta6-mediated TGF-beta activation causes MMP12-dependent emphysema. Nature 422: 169–173, 2003.[CrossRef][Medline]
  48. Ohnishi K, Takagi M, Kurokawa Y, Satomi S, Konttinen YT. Matrix metalloproteinase-mediated extracellular matrix protein degradation in human pulmonary emphysema. Lab Invest 78: 1077–1087, 1998.[ISI][Medline]
  49. Okamoto T, Akuta T, Tamura F, Van D, V, Akaike T. Molecular mechanism for activation and regulation of matrix metalloproteinases during bacterial infections and respiratory inflammation. Biol Chem 385: 997–1006, 2004.[CrossRef][ISI][Medline]
  50. Pagenstecher A, Stalder AK, Kincaid CL, Volk B, Campbell IL. Regulation of matrix metalloproteinases and their inhibitor genes in lipopolysaccharide-induced endotoxemia in mice. Am J Pathol 157: 197–210, 2000.[Abstract/Free Full Text]
  51. Parks WC, Wilson CL, Lopez-Boado YS. Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat Rev Immunol 4: 617–629, 2004.[CrossRef][ISI][Medline]
  52. Pesci A, Balbi B, Majori M, Cacciani G, Bertacco S, Alciato P, Donner CF. Inflammatory cells and mediators in bronchial lavage of patients with chronic obstructive pulmonary disease. Eur Respir J 12: 380–386, 1998.[Abstract]
  53. Pillow JJ, Korfhagen TR, Ikegami M, Sly PD. Overexpression of TGF-{alpha} increases lung tissue hysteresivity in transgenic mice. J Appl Physiol 91: 2730–2734, 2001.[Abstract/Free Full Text]
  54. Qualls JE, Kaplan AM, Van Rooijen N, Cohen DA. Suppression of experimental colitis by intestinal mononuclear phagocytes. J Leukoc Biol 80: 802–815, 2006.[Abstract/Free Full Text]
  55. Rasaiah VP, Malloy JL, Lewis JF, Veldhuizen RA. Early surfactant administration protects against lung dysfunction in a mouse model of ARDS. Am J Physiol Lung Cell Mol Physiol 284: L783–L790, 2003.[Abstract/Free Full Text]
  56. Remick DG. Applied molecular biology of sepsis. J Crit Care 10: 198–212, 1995.[CrossRef][ISI][Medline]
  57. Rennard SI, Farmer SG. Exacerbations and progression of disease in asthma and chronic obstructive pulmonary disease. Proc Am Thorac Soc 1: 88–92, 2004.[Abstract/Free Full Text]
  58. Russell RE, Thorley A, Culpitt SV, Dodd S, Donnelly LE, Demattos C, Fitzgerald M, Barnes PJ. Alveolar macrophage-mediated elastolysis: roles of matrix metalloproteinases, cysteine, and serine proteases. Am J Physiol Lung Cell Mol Physiol 283: L867–L873, 2002.[Abstract/Free Full Text]
  59. Schuessler TF, Bates JH. A computer-controlled research ventilator for small animals: design and evaluation. IEEE Trans Biomed Eng 42: 860–866, 1995.[CrossRef][ISI][Medline]
  60. Selman M, Ruiz V, Cabrera S, Segura L, Ramirez R, Barrios R, Pardo A. TIMP-1, -2, -3, and -4 in idiopathic pulmonary fibrosis. A prevailing nondegradative lung microenvironment? Am J Physiol Lung Cell Mol Physiol 279: L562–L574, 2000.[Abstract/Free Full Text]
  61. Shapiro SD. The macrophage in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 160: S29–S32, 1999.[Abstract/Free Full Text]
  62. Simovart HE, Poldoja E, Kokk K, Tapfer H, Liigant A, Talvik R, Roosaar P. Changes of activated macrophages and apoptotic cell count in the organs of rats during experimental sepsis. Medicina (B Aires) 39: 932–939, 2003.
  63. Tetley TD. Macrophages and the pathogenesis of COPD. Chest 121: 156S–159S, 2002.[CrossRef][ISI][Medline]
  64. Thamrin C, Janosi TZ, Collins RA, Sly PD, Hantos Z. Sensitivity analysis of respiratory parameter estimates in the constant-phase model. Ann Biomed Eng 32: 815–822, 2004.[CrossRef][ISI][Medline]
  65. Tsuchida S, Engelberts D, Roth M, McKerlie C, Post M, Kavanagh BP. Continuous positive airway pressure causes lung injury in a model of sepsis. Am J Physiol Lung Cell Mol Physiol 289: L554–L564, 2005.[Abstract/Free Full Text]
  66. Valstar DL, Schijf MA, Arts JH, Kuper CF, Nijkamp FP, Storm G, Bloksma N, Henricks PA. Alveolar macrophages suppress non-specific inflammation caused by inhalation challenge with trimellitic anhydride conjugated to albumin. Arch Toxicol 80: 561–571, 2006.[CrossRef][ISI][Medline]
  67. Van Den Steen PE, Proost P, Grillet B, Brand DD, Kang AH, Van Damme J, Opdenakker G. Cleavage of denatured natural collagen type II by neutrophil gelatinase B reveals enzyme specificity, post-translational modifications in the substrate, and the formation of remnant epitopes in rheumatoid arthritis. FASEB J 16: 379–389, 2002.[Abstract/Free Full Text]
  68. Van Rooijen N. The liposome-mediated macrophage "suicide" technique. J Immunol Methods 124: 1–6, 1989.[CrossRef][ISI][Medline]
  69. Van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Methods 174: 83–93, 1994.[CrossRef][ISI][Medline]
  70. Vignali DA. Multiplexed particle-based flow cytometric assays. J Immunol Methods 243: 243–255, 2000.[CrossRef][ISI][Medline]
  71. Vlahos R, Bozinovski S, Hamilton JA, Anderson GP. Therapeutic potential of treating chronic obstructive pulmonary disease (COPD) by neutralising granulocyte macrophage-colony stimulating factor (GM-CSF). Pharmacol Ther 112: 106–115, 2006.[CrossRef][ISI][Medline]
  72. Wagers S, Lundblad L, Moriya HT, Bates JH, Irvin CG. Nonlinearity of respiratory mechanics during bronchoconstriction in mice with airway inflammation. J Appl Physiol 92: 1802–1807, 2002.[Abstract/Free Full Text]
  73. Wang Z, Zheng T, Zhu Z, Homer RJ, Riese RJ, Chapman HA Jr, Shapiro SD, Elias JA. Interferon-{gamma} induction of pulmonary emphysema in the adult murine lung. J Exp Med 192: 1587–1600, 2000.[Abstract/Free Full Text]
  74. Welk B, Malloy JL, Joseph M, Yao LJ, Veldhuizen AW. Surfactant treatment for ventilation-induced lung injury in rats: effects on lung compliance and cytokines. Exp Lung Res 27: 505–520, 2001.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
Y.-C. T. Huang
Lung compliance measurement in mice
Am J Physiol Lung Cell Mol Physiol, April 1, 2008; 294(4): L815 - L815.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
E. L. Martin and R. A. W. Veldhuizen
Reply to Huang
Am J Physiol Lung Cell Mol Physiol, April 1, 2008; 294(4): L816 - L816.
[Full Text] [PDF]

This Article