Dietary advanced glycation end-products, its pulmonary receptor, and high mobility group box 1 in aspiration lung injury.

BACKGROUND: Gastric aspiration is a significant cause of acute lung injury and acute respiratory distress syndrome. Environmental risk factors, such as a diet high in proinflammatory advanced glycation end-products (AGEs), may render some patients more susceptible to lung injury after aspiration. We hypothesized that high dietary AGEs increase its pulmonary receptor, RAGE, producing an amplified pulmonary inflammatory response in the presence of high mobility group box 1 (HMGB1), a RAGE ligand and an endogenous signal of epithelial cell injury after aspiration. MATERIALS AND METHODS: CD-1 mice were fed either a low AGE or high AGE diet for 4 wk. After aspiration injury with acidified small gastric particles, bronchoalveolar lavage and whole-lung tissue samples were collected at 5 min, 1 h, 5 h, and 24 h after injury. RAGE, soluble RAGE (sRAGE), HMGB1, cytokine and chemokine concentrations, albumin levels, neutrophil influx, and lung myeloperoxidase activity were measured. RESULTS: We observed that high AGE-fed mice exhibited greater pulmonary RAGE levels before aspiration and increased bronchoalveolar lavage sRAGE levels after aspiration compared with low AGE-fed mice. Lavage HMGB1 levels rose immediately after aspiration, peaking at 1 h, and strongly correlated with sRAGE levels in both dietary groups. High AGE-fed mice demonstrated higher cytokine and chemokine levels with increased pulmonary myeloperoxidase activity over 24 h versus low AGE-fed mice. CONCLUSIONS: This study indicates that high dietary AGEs can increase pulmonary RAGE, augmenting the inflammatory response to aspiration in the presence of endogenous damage signals such as HMGB1.

Low pH environmental stress inhibits LPS and LTA-stimulated proinflammatory cytokine production in rat alveolar macrophages.

Gastric aspiration increases the risks for developing secondary bacterial pneumonia. Cytokine elaboration through pathogen recognition receptors (PRRs) is an important mechanism in initiating innate immune host response. Effects of low pH stress, a critical component of aspiration pathogenesis, on the PRR pathways were examined, specifically toll-like receptor-2 (TLR2) and TLR4, using isolated rat alveolar macrophages (aMØs). We assessed the ability of aMØs after brief exposure to acidified saline to elaborate proinflammatory cytokines in response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) stimulation, known ligands of TLR4 and TLR2, respectively. Low pH stress reduced LPS- and LTA-mediated cytokine release (CINC-1, MIP-2, TNF-α, MCP-1, and IFN-ß). LPS and LTA increased intracellular Ca²âº concentrations while Ca²âº chelation by BAPTA decreased LPS- and LTA-mediated cytokine responses. BAPTA blocked the effects of low pH stress on most of LPS-stimulated cytokines but not of LTA-stimulated responses. In vivo mouse model demonstrates suppressed E. coli and S. pneumoniae clearance following acid aspiration. In conclusion, low pH stress inhibits antibacterial cytokine response of aMØs due to impaired TLR2 (MyD88 pathway) and TLR4 signaling (MyD88 and TRIF pathways). The role of Ca²âº in low pH stress-induced signaling is complex but appears to be distinct between LPS- and LTA-mediated responses.

Role of pulmonary artery reactivity and nitric oxide in injury and inflammation following lung contusion.

The mechanisms contributing to hypoxia in lung contusion (LC) remain unclear and not temporally associated with the peak onset of acute inflammation. We investigated the role of oxidative stress in alteration of pulmonary arterial (PA) reactivity following LC. In addition, the role of antioxidants in reversing this process was examined. PaO2 and PA reactivity were measured in rats subjected to bilateral LC. Rings were pretreated with a nitric oxide synthase (NOS) inhibitor, L-nitro arginine (10(-3) M), or PEG-superoxide dismutase (SOD) and PEG-catalase (CAT), or both (L-nitro arginine + SOD/CAT). Rings were constricted with norepinephrine and relaxed with an NOS agonist (A23187) or NO donor (SNAP [S-nitrosyl amino penicillamine]). Immunochemical and mass spectrometric quantification for nitrotyrosine was performed. Rats were hypoxemic at 4 h after contusion compared with controls, but recovered by 24 h (PaO(2)/FIO(2) ratio: baseline, 443 ± 28; 4 h, 288 ± 46; and 24 h, 417 ± 23). Pulmonary arterial constriction to NOS inhibition and relaxation to A23187 were impaired 4 h after LC. Pulmonary arterial relaxation to SNAP was decreased at 4 and 24 h after LC. These alterations in PA reactivity were reversed by SOD/CAT pretreatment. SOD1 and 2 mRNA were upregulated, and soluble guanylyl cyclase mRNA was downregulated 24 h after LC. Immunohistochemistry and mass spectrometry revealed that levels of 3-nitrotyrosine were increased markedly at 4 h following LC consistent with superoxide generation and formation of peroxynitrite. Collectively, these data suggest that consumption of NO due to excess superoxide resulting in peroxynitrite formation leads to diminished vascular reactivity following LC.

Role of macrophage chemoattractant protein-1 in acute inflammation after lung contusion.

Lung contusion (LC), commonly observed in patients with thoracic trauma is a leading risk factor for development of acute lung injury/acute respiratory distress syndrome. Previously, we have shown that CC chemokine ligand (CCL)-2, a monotactic chemokine abundant in the lungs, is significantly elevated in LC. This study investigated the nature of protection afforded by CCL-2 in acute lung injury/acute respiratory distress syndrome during LC, using rats and CC chemokine receptor (CCR) 2 knockout (CCR2(-/-)) mice. Rats injected with a polyclonal antibody to CCL-2 showed higher levels of albumin and IL-6 in the bronchoalveolar lavage and myeloperoxidase in the lung tissue after LC. Closed-chest bilateral LC demonstrated CCL-2 localization in alveolar macrophages (AMs) and epithelial cells. Subsequent experiments performed using a murine model of LC showed that the extent of injury, assessed by pulmonary compliance and albumin levels in the bronchoalveolar lavage, was higher in the CCR2(-/-) mice when compared with the wild-type (WT) mice. We also found increased release of IL-1ß, IL-6, macrophage inflammatory protein-1, and keratinocyte chemoattractant, lower recruitment of AMs, and higher neutrophil infiltration and phagocytic activity in CCR2(-/-) mice at 24 hours. However, impaired phagocytic activity was observed at 48 hours compared with the WT. Production of CCL-2 and macrophage chemoattractant protein-5 was increased in the absence of CCR2, thus suggesting a negative feedback mechanism of regulation. Isolated AMs in the CCR2(-/-) mice showed a predominant M1 phenotype compared with the predominant M2 phenotype in WT mice. Taken together, the above results show that CCL-2 is functionally important in the down-modulation of injury and inflammation in LC.

Doxorubicin-conjugated quantum dots to target alveolar macrophages and inflammation.

The ability to provide targeted therapeutic delivery in the lung would be a major advancement in pharmacological treatments for many pulmonary diseases. Critical issues for such successful delivery would require the ability to target specific cell types, minimize toxicity (e.g., inflammatory response), and deliver therapeutic levels of drugs. We report here on the ability of nanoconjugates of CdSe/CdS/ZnS quantum dots (QDs) and doxorubicin (Dox) to target alveolar macrophages (aMØs), cells that play a critical role in the pathogenesis of inflammatory lung injuries. Confocal imaging showed the release of Dox from the QD-Dox nanoconjugate, as was evident by its accumulation in the cell nucleus and induction of apoptosis, implying that the drug retains its bioactivity after coupling to the nanoparticle. Inflammatory injury parameters (albumin leakage, proinflammatory cytokines, and neutrophil infiltration) were recorded after in vivo administration of QD-Dox and Dox, observing no significant effect after QD-Dox treatment compared with Dox. These results demonstrate that nanoparticle platforms can provide targeted macrophage-selective therapy for the treatment of pulmonary disease. FROM THE CLINICAL EDITOR: Pulmonary inflammatory diseases still often remain challenging to treat, despite decades of advances and several available agents. In this study, a quantum dot-based alveolar delivery system is presented, targeting macrophages with doxorubicin.

Predictive modeling and inflammatory biomarkers in rats with lung contusion and gastric aspiration.

BACKGROUND: This study uses statistical predictive modeling and hierarchical cluster analyses to examine inflammatory mediators and cells in bronchoalveolar lavage (BAL) as putative biomarkers in rats with blunt trauma lung contusion (LC), gastric aspiration (combined acid and small gastric food particles, CASP), or a combination of the two. METHODS: Specific parameters assessed in the innate pulmonary inflammatory response were leukocytes, macrophages, and polymorphonuclear neutrophils (PMNs) in BAL; whole lung myeloperoxidase activity; and a series of cytokines or chemokines present in BAL at 5 or 24 hours after injury: tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, interferon-gamma, IL-10, macrophage inflammatory protein-2, cytokine-induced neutrophil chemoattractant-1, and monocyte chemoattractant protein-1. RESULTS: Rats with LC, CASP, LC + CASP all had severe lung injury compared with uninjured controls based on decreased arterial oxygenation or increased BAL albumin at 5 or 24 hours postinsult. However, the injury groups had distinct overall patterns of inflammation that allowed them to be discriminated accurately by hierarchical cluster analysis (29 of 30 and 35 of 37 rats were correctly classified in hierarchical clusters at 5 and 24 hours, respectively). Moreover, predictive analyses based on an extension of standard receiver-operator characteristic methodology discriminated individual animals and groups with similar high accuracy based on a maximum of two inflammatory parameters per group (29 of 30 and 36 of 37 rats were correctly classified at 5 hours and 24 hours, respectively). CONCLUSIONS: These results support the possibility that inflammatory biomarker profiles could be developed in the future to improve the diagnosis and management of trauma patients with unwitnessed (occult) gastric aspiration who have an increased risk of clinical acute lung injury or the acute respiratory distress syndrome.

Superimposed gastric aspiration increases the severity of inflammation and permeability injury in a rat model of lung contusion.

INTRODUCTION: Lung contusion (LC) from blunt thoracic trauma is a clinically-prevalent condition that can progress to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Patients with LC are at risk for gastric aspiration at the time of trauma, but the combined insults have not been well-studied in animal models. This study tests the hypothesis that concurrent gastric aspiration (combined acid and small gastric particles, CASP) at the time of trauma significantly increases permeability injury and inflammation compared with LC alone, and also modifies the inflammatory response to include distinct features compared with the aspiration component of injury. MATERIALS AND METHODS: Four groups of adult male Long-Evans rats were studied (LC, CASP, LC+CASP, uninjured controls). LC was induced in anesthetized rats at a fixed impact energy of 2.0 J, and CASP (1.2 mL/kg body weight, 40 mg particles/mL, pH=1.25) was instilled through an endotracheal tube. Lung injury and inflammation were assessed by arterial blood gases and levels of albumin, cells, and cytokines/chemokines in bronchoalveolar lavage (BAL) at 5 and 24 h. RESULTS: Rats with LC+CASP had lower mean PaO(2)/FiO(2) ratios compared with LC alone at 24 h, and higher BAL albumin concentrations compared with either LC or CASP alone. Rats with LC+CASP versus LC had more severe inflammation based on higher levels of PMN in BAL at 5 h, increased whole lung myeloperoxidase (MPO) activity at 5 and 24 h, and increased levels of inflammatory mediators in BAL (TNFalpha, IL-1beta, and MCP-1 at 5 and 24 h; IL-10, MIP-2, and CINC-1 at 5 h). Rats with LC+CASP also had distinct aspects of inflammation compared with CASP alone, i.e., significantly higher levels of IL-10 (5 and 24 h), IL-1beta (24 h), CINC-1 (24 h), and MCP-1 (24 h), and significantly lower levels of MPO (5 h), MIP-2 (5 h), and CINC-1 (5 h). CONCLUSIONS: Concurrent gastric aspiration can exacerbate permeability lung injury and inflammation associated with LC, and also generates a modified inflammatory response compared with aspiration alone. Unwitnessed gastric aspiration has the potential to contribute to more severe forms of LC injury associated with progression to ALI/ARDS and pneumonia in patients with thoracic trauma.

Lung contusion: inflammatory mechanisms and interaction with other injuries.

This article reviews current animal models and laboratory studies investigating the pathophysiology of lung contusion (LC), a common and severe condition in patients with blunt thoracic trauma. Emphasis is on studies elucidating cells, mediators, receptors, and processes important in the innate pulmonary inflammatory response that contribute to LC injury. Surfactant dysfunction in the pathogenesis of LC is also discussed, as is the potential role of epithelial cell or neutrophil apoptosis. Studies examining combination injuries where LC is exacerbated by secondary insults such as gastric aspiration in trauma patients are also noted. The need for continuing mechanism-based research to further clarify the pathophysiology of LC injury, and to define and test potential therapeutic interventions targeting specific aspects of inflammation or surfactant dysfunction to improve clinical outcomes in patients with LC, is also emphasized.

A rat model for isolated bilateral lung contusion from blunt chest trauma.

Lung contusion affects 17%-25% of adult blunt trauma patients, and is the leading cause of death from blunt thoracic injury. A small animal model for isolated bilateral lung contusion has not been developed. We induced lung contusion in anesthetized rats by dropping a 0.3-kg weight onto a precordial protective shield to direct the impact force away from the heart and toward the lungs. Lung injury was characterized as a function of chest impact energy (1.8-2.7 J) by measurements of arterial oxygenation, bronchoalveolar lavage (BAL) albumin and cytology, pressure-volume mechanics, and histopathology. Histology confirmed bilateral lung contusion without substantial cardiac muscle trauma. Rats receiving 2.7 J of chest impact energy had 33% mortality that exceeded prospectively defined limits for sublethal injury. Hypoxemia in rats with maximal sublethal injury (2.45 J) met criteria for acute lung injury at < or =24 h, improving by 48 h. BAL albumin levels were highest at < or =24 h, and remained elevated along with increased BAL leukocytes and decreased lung volumes at 48 h. We concluded that an impact energy of 2.45 J induces isolated, bilateral lung contusion and provides a useful model for future mechanistic pathophysiological assessments.

The evolution of isolated bilateral lung contusion from blunt chest trauma in rats: cellular and cytokine responses.

Lung contusion is the leading cause of death from blunt thoracic trauma in adults, but its mechanistic pathophysiology remains unclear. This study uses a recently developed rat model to investigate the evolution of inflammation and injury in isolated lung contusion. Bilateral lung contusion with minimal cardiac trauma was induced in 54 anesthetized rats by dropping a 0.3-kg hollow cylindrical weight onto a precordial shield (impact energy, 2.45 Joules). Arterial oxygenation, pressure-volume (P-V) mechanics, histology, and levels of erythrocytes, leukocytes, albumin, and inflammatory mediators in bronchoalveolar lavage (BAL) were assessed at 8 min, at 4, 12, 24, and 48 h, and at 7 days after injury. The role of neutrophils in the evolution of inflammatory injury was also specifically studied by depleting these cells with intravenous vinblastine before lung contusion. Arterial oxygenation was severely reduced at 8 min to 24 h postcontusion, but became almost normal by 48 h. Levels of erythrocytes, leukocytes, and albumin in BAL were increased at

Progressive, severe lung injury secondary to the interaction of insults in gastric aspiration.

This study examines lung injury and inflammation over 24 hours following intratracheal instillation of hydrochloric acid (acid), small nonacidic gastric particles (SNAP), or combined acid and small particles (CASP) in adult rats. The severity and duration of injury was significantly greater for CASP compared to acid or SNAP based on PaO2/FiO2, bronchoalveolar lavage (BAL) albumin, and BAL cell numbers. The inflammatory response associated with aspiration injury from CASP was distinct in several respects. Tumor necrosis factor (TNF)-alpha was greatly reduced in CASP compared to SNAP or acid, whereas interleukin (IL)-1beta was increased. Levels of cytokine-induced neutrophil chemoattractant (CINC)-1, monocyte chemoattractant protein (MCP)-1, and IL-10 in lavage were also significantly increased in animals injured with CASP compared to other forms of aspiration. Statistical analysis showed that BAL levels of IL-10 correlated most strongly with albumin leakage in aspiration-injured animals at 6 and 24 hours, followed by BAL levels of MCP-1. Additional cytokine cluster analyses indicated that levels of MCP-1 and CINC-1 in BAL from all injured animals were strongly correlated with inflammatory neutrophil numbers at 6 and 24 hours post aspiration, and that IL-10 levels in BAL were strongly correlated with inflammatory cell numbers at 24 hours. Preliminary blocking experiments showed that administration of anti-IL-10 antibody increased the albumin permeability index at 6 hours in SNAP and CASP animals, but anti-MCP-1 antibody did not affect the severity of injury. The results of this study support the possibility that different forms of aspiration are associated with identifiable cytokine profiles, and that specific cytokines, including IL-10 and MCP-1, may have utility as diagnostic or prognostic markers in clinical applications.

Gastric acid and particulate aspiration injury inhibits pulmonary bacterial clearance.

OBJECTIVE: To establish a model of secondary bacterial pneumonia following gastric aspiration and to identify possible mechanisms involved in the suppressed antibacterial defenses following the initial pulmonary insult. DESIGN: A controlled, in vivo laboratory study. SETTING: Research laboratory of a health sciences university. SUBJECTS: Ninety-five Long-Evans rats. INTERVENTIONS: Animals were anesthetized for neck dissection and placement of a 14-gauge catheter in the trachea. Gastric aspirate (1.2 mL/kg of saline, pH 1.25, and 40 mg/mL sterile rat gastric particles) or an equal amount of normal saline (pH 5.3) was instilled intratracheally. One minute after this insult, animals received an intratracheal instillation of either 5.6 x 10 colony-forming units of Escherichia coli or an equal volume of normal saline. The animals remained in room air until kill at 4 hrs or 24 hrs after the intratracheal instillation. The lungs were homogenized for quantitative bacterial cultures. Bronchoalveolar lavage fluid was obtained for cell counts and measurements of albumin, tumor necrosis factor-alpha, interleukin-1 beta, cytokine-induced neutrophil chemoattractant-1, macrophage inflammatory protein-2, monocyte chemoattractant protein-1, and interleukin 10. MEASUREMENTS AND MAIN RESULTS: Animals that received gastric aspirate (followed by normal saline or E. coli) had increased injury as assessed by significant reductions in oxygenation and elevations in bronchoalveolar lavage albumin. At 24 hrs, animals that received gastric aspirate inoculation followed by E. coli had significantly higher pulmonary bacterial counts compared with animals that received E. coli alone. Gastric aspiration injury followed by bacterial inoculation also resulted in acute, but transient, increases in tumor necrosis factor-alpha, interleukin-1 beta, cytokine-induced neutrophil chemoattractant-1, and macrophage inflammatory protein-2 and more sustained elevations of monocyte chemoattractant protein-1 and interleukin-10. CONCLUSIONS: Lung injury increases and bacterial clearance decreases in this experimental model of E. coli pneumonia following gastric aspiration. Cytokine profiles suggest possible mechanisms for the impaired antibacterial host defense.

The effects of Escherichia coli capsule, O-antigen, host neutrophils, and complement in a rat model of Gram-negative pneumonia.

Gram-negative enteric bacilli are agents of life-threatening pneumonia. The role of the bacterial capsule and O-antigen moiety of lipopolysaccharide in the pathogenesis of Gram-negative pneumonia was assessed. In a rat model of pneumonia the LD(50) of a wild-type extraintestinal pathogenic Escherichia coli strain (CP9) was significantly less than its isogenic derivatives deficient in capsule (CP9.137), O-antigen (CP921) or both capsule and O-antigen (CP923) (P< or =0.003). Studies using complement depleted or neutropenic animals established that both neutrophils and complement are important for the pulmonary clearance of E. coli. Data from these studies also support that capsule and O-antigen serve, at least in part, to counter the complement and neutrophil components of the pulmonary host defense response. Lastly, the contribution of E. coli versus neutrophils in causing lung injury was examined. Findings suggest that E. coli virulence factors and/or non-neutrophil host factors are more important mediators of lung injury than neutrophils. These findings extend our understanding of Gram-negative pneumonia and have treatment implications.

Total extracellular surfactant is increased but abnormal in a rat model of gram-negative bacterial pneumonia.

An in vivo rat model was used to evaluate the effects of Escherichia coli pneumonia on lung function and surfactant in bronchoalveolar lavage (BAL). Total extracellular surfactant was increased in infected rats compared with controls. BAL phospholipid content in infected rats correlated with the severity of alveolar-capillary leak as reflected in lavage protein levels (R(2) = 0.908, P < 0.0001). Western blotting showed that levels of surfactant protein (SP)-A and SP-D in BAL were significantly increased in both large and small aggregate fractions at 2 and 6 h postinstillation of E. coli. SP-B was also increased at these times in the large aggregate fraction of BAL, whereas SP-C levels were increased at 2 h and decreased at 6 h relative to controls. The small-to-large (S/L) aggregate ratio (a marker inversely proportional to surfactant function) was increased in infected rats with >50 mg total BAL protein. There was a significant correlation (R(2) = 0.885, P < 0.0001) between increasing S/L ratio in BAL and pulmonary damage assessed by total protein. Pulmonary volumes, compliance, and oxygen exchange were significantly decreased in infected rats with >50 mg of total BAL protein, consistent with surfactant dysfunction. In vitro surface cycling studies with calf lung surfactant extract suggested that bacterially derived factors may have contributed in part to the surfactant alterations seen in vivo.