Attenuation of pulmonary injury by an inhaled MMP inhibitor in the endotoxin lung injury model

Abstract

Acute respiratory distress syndrome (ARDS) is characterized by pulmonary edema and poor gas exchange resulting from severe inflammatory lung injury. Neutrophilic infiltration and increased pulmonary vascular permeability are hallmarks of early ARDS and precipitate a self-perpetuating cascade of inflammatory signaling. The biochemical processes initiating these events remain unclear. Typically associated with extracellular matrix degradation, recent data suggest matrix metalloproteinases (MMPs) are regulators of pulmonary inflammation. To demonstrate that inhalation of a broad MMP inhibitor attenuates LPS induced pulmonary inflammation. Nebulized CGS27023AM (CGS) was administered to LPS-injured mice. Pulmonary CGS levels were examined by mass spectroscopy. Inflammatory scoring of hematoxylin-eosin sections, examination of vascular integrity via lung wet/dry and bronchoalveolar lvage/serum FITC-albumin ratios were performed. Cleaved caspase-3 levels were also assessed. Differential cell counts and pulse-chase labeling were utilized to determine the effects of CGS on neutrophil migration. The effects of CGS on human neutrophil migration and viability were examined using Boyden chambers and MTT assays. Nebulization successfully delivered CGS to the lungs. Treatment decreased pulmonary inflammatory scores, edema, and apoptosis in LPS treated animals. Neutrophil chemotaxis was reduced by CGS treatment, with inhalation causing significant reductions in both the total number and newly produced bromodeoxyuridine-positive cells infiltrating the lung. Mechanistic studies on cells isolated from humans demonstrate that CGS-treated neutrophils exhibit decreased chemotaxis. The protective effect observed following treatment with a nonspecific MMP inhibitor indicates that one or more MMPs mediate the development of pulmonary edema and neutrophil infiltration in response to LPS injury. In accordance with this, inhaled MMP inhibitors warrant further study as a potential new therapeutic avenue for treatment of acute lung injury.

Keywords: inflammation; lung; matrix metalloproteinase; neutrophil.

Fig. 1.

Inhaled CGS27023AM (CGS) is absorbed by the lungs. A: C57/BL6 mice received nebulized CGS at high dose (10 mg/kg), medium dose (5 mg/kg), or low dose (2 mg/kg). Animals were euthanized at 1, 3, and 5 h (n = 3 mice per time point), followed by collection of lung homogenates. Homogenates were sent to the Columbia Proteomics Core for mass spectroscopy to determine CGS levels in the lung. Dose size had no significant effect on CGS concentration at 1, 3, or 5 h, and subsequently dosing groups were pooled for analysis at each time point. At 1 h postinhalation, CGS levels were significantly increased compared with the 3- or 5-h time points, whereas no difference was found between the 3- and 5-h groups (n = 6; **P < 0.01). B: mass spectroscopy was performed on serum samples from mice receiving inhaled CGS. Again, no significant difference in concentration was observed between dosing groups. Pooling the dose groups for each timepoint revealed a significant difference in serum concentration at 1 h compared with 3 or 5 h (n = 3; *P < 0.05). Importantly, serum concentration was lower than that observed in the lungs for each time point examined. C: treatment paradigm for mice receiving inhaled CGS. Mice were injured with lipopolysaccharide (LPS) and permitted to rest for 24 h following injury. CGS was delivered via nebulizer (12 mg/kg) every 6 h for 4 doses. PBS was administered as a control.

Fig. 2.

Treatment with inhaled CGS27023AM (CGS) attenuates lipopolysaccharide (LPS)-induced pulmonary inflammation. A: representative hematoxylin-eosin (H&E)-stained paraffin-embedded sections. Increased inflammatory infiltrate is apparent in the animal receiving LPS followed by PBS. Inflammation was visibly reduced by treatment with CGS. B: lung injury scoring system as adapted from Matute-Bello et al. (2008). C: lung inflammatory score (LIS) from experimental mice. As shown, LPS significantly increased the LIS compared with both PBS control animals and those receiving CGS (n = 5; ***P < 0.001). Treatment with CGS significantly reduced the LPS-mediated increase in the LIS (n = 5; ###P < 0.001). A total of 5 fields per section were analyzed for each mouse.

Fig. 3.

CGS27023AM (CGS) treatment attenuated the development of lipopolysaccharide (LPS)-induced pulmonary edema and increase in vascular permeability. LPS-injured mice were treated with CGS every 6 h for 4 doses. A: to determine the extent of pulmonary edema, lung wet/dry ratios were determined by weighing the right lung after removal followed by desiccation for 72 h to determine dry weights. LPS treatment significantly increased wet/dry ratios compared with both PBS controls and CGS-treated animals (n = 5; **P < 0.01). Importantly, CGS treatment a significantly attenuated the LPS-induced increase in edema (n = 5; ##P < 0.01). B: 1 h before the 4th dose, 3 mg of FITC-albumin was administered by tail vein injection to assess pulmonary vascular permeability. Bronchoalvelolar lavage fluid (BALF) and serum were collected from each animal to determine the BALF/serum ratio of FITC-albumin. As shown, LPS treatment significantly increased pulmonary vascular permeability as measured by FITC-albumin ratio compared with PBS controls and animals treated with CGS (n = 5; *P < 0.05). Treatment with inhaled CGS resulted in significant attenuation of LPS-induced vascular permeability (n = 5; #P < 0.05).

Fig. 4.

CGS27023AM (CGS) treatment attenuates apoptosis in lipopolysaccharide (LPS)-treated lungs. Mice received nasally instilled LPS followed by inhaled CGS every 6 h for 4 doses. Animals were euthanized, and assessment of apoptosis was performed. A: representative Western blot for whole and cleaved caspase-3. Each lane contains lung homogenate (40 μg) from an individual animal. B: relative quantification of Western blots for caspase-3. Whole and cleaved caspase-3 were normalized to actin, and the ratio of cleaved to whole caspase-3 was determined. As shown, LPS treatment increased the cleaved/whole caspase-3 ratio compared with control animals (n ≥ 5; ##P < 0.01, ###P < 0.001). CGS treatment attenuated the LPS-induced increase in cleaved caspase-3 (n ≥ 5; **P < 0.01). C: lung homogenates were also analyzed using a caspase-3/7 activity assay. In agreement with Western blot analysis, LPS treatment increased caspase activity significantly compared with PBS controls (n ≥ 4; #P < 0.05). This increase was attenuated by treatment with CGS (n ≥ 4; *P < 0.05). D: ×20 images of representative sections of lung tissue fluorescently stained for cleaved caspase-3 (red, Alexa 647) and cell nuclei (blue, DAPI) (n = 3).

Fig. 5.

Treatment with inhaled CGS27023AM (CGS) decreases pulmonary neutrophil counts in response to lipopolysaccharide (LPS). Mice were nasally instilled with LPS, followed by treatment with CGS every 6 h for 4 doses. Bronchoalveolar lavage (BAL) was performed, and lungs were collected for analysis. A: paraffin embedded sections (5 μM) were stained for myeloperoxidase (MPO), a neutrophil marker, followed by detection using DAB. As shown, significantly more MPO-positive cells (brown; black arrow) were present in LPS-PBS animals compared with PBS controls or those receiving CGS (×10 images). B: representative DifQuik stains of cytospins from BAL fluid (BALF) showing a decrease in neutrophils (red arrows) following treatment of LPS-injured mice with inhaled CGS (green arrows are macrophages). All images are ×20 magnification. C: differential cell counts were taken from each animal’s cytosine, with at least 300 cells counted per animal. LPS treatment significantly increased the total inflammatory cell count compared with PBS controls and mice receiving inhaled CGS (n ≥ 4; **P < 0.01, ***P < 0.001). Inhaled CGS treatment significantly decreased the number of neutrophils present in the BALF (n ≥ 5; ###P < 0.001). MPO ELISAs were performed on BALF and lung homogenate from experimental animals. D: CGS attenuated the LPS induced increased in MPO in BALF, consistent with a decrease in neutrophils (n ≥ 5; **P < 0.01). E: CGS-treated animals were similarly characterized by decreased levels of MPO in response to LPS in lung tissue homogenate (n ≥ 5; ***P < 0.001). F: inhibition of matrix metalloproteinase (MMP)-9 by inhaled CGS was assessed using a Quantikine assay. LPS significantly increased MMP-9 activity compared with PBS-only controls, as determined by MMP-9 Quantikine ELISA kit (n = 6; ###P < 0.001). Treatment with CGS significantly reduced the LPS-mediated increase in MMP-9 activity (n = 6 per group; ***P < 0.001). This decrease would be expected with a substantial fall in neutrophil count.

Fig. 6.

CGS27023AM (CGS) inhibits lipopolysaccharide (LPS)-induced influx of newly synthesized neutrophils from the vascular pool. LPS-treated mice received ip injections of bromodeoxyuridine (BrdU) and were treated with CGS 2.5 h postinjection. Bronchoalveolar lavage fluid (BALF) was collected 15 and 45 min following CGS treatment. Confocal images were taken of cytospins stained for BrdU and myeloperoxidase (MPO). The number of BrdU(+) and BrdU(−) neutrophils was quantified. A: CGS treatment at 15 and 45 min significantly decreased the percentage of BrdU(+) neutrophils in LPS-treated mice compared with LPS alone (300 cells counted/cytospin, n = 3 mice; ***P < 0.001). B: representative immunofluorescent images for stained cytospins showing BrdU-stained neutrophils (red arrow) and macrophages (yellow arrow) with BrdU localized in the cytoplasm likely from phagocytized neutrophils. All images are ×20 magnification.

Fig. 7.

CGS27023AM (CGS) prevents migration of human neutrophils in response to N-formylmethionyl-leucyl-phenylalanine (FMLP) but does not affect cell viability in vitro. A: schema for migration experiments in a modified Boyden chamber. Briefly, the basal chamber was filled with medium containing the chemoattractant FMLP or vehicle. The apical chamber was seeded with ∼100,000 neutrophils with the addition of CGS or vehicle. Neutrophils were permitted to migrate for 1 h, followed by fixation and staining of the basal chamber with DAPI. Five fields per well were imaged, and the number of neutrophils counted using NIH ImageJ. B: FMLP treatment resulted in a significant increase in neutrophil migration into the basal chamber compared with (51) CGS-treated neutrophils and vehicle controls (n = 4; **P < 0.01). Treatment with 20 nM CGS significantly reduced neutrophil migration in response to FMLP (n = 4; ##P < 0.01). C: to determine whether CGS influenced neutrophil viability, cells were exposed to CGS for 1 h, followed by MTT reduction assay. As shown, CGS had no significant effect on neutrophil viability in vitro.