LPS-treated Lungs Research Articles

Enhanced clearance of surfactant protein D during LPS-induced acute inflammation in rat lung.

Pulmonary surfactant participates in the regulation of alveolar compliance and lung host defense. Surfactant homeostasis is regulated through a combination of synthesis, secretion, clearance, recycling, and degradation of surfactant components. The extracellular pool size of surfactant protein (SP) D fluctuates significantly during acute inflammation. We hypothesized that changes in SP-D levels are due, in part, to altered clearance of SP-D. Clearance pathways in rats were assessed with fluorescently labeled SP-D that was instilled into control lungs or lungs that had been treated with lipopolysaccharide (LPS) 16 h earlier. SP-D clearance from lavage into lung tissue was time dependent from 5 min to 1 h and 1.7-fold greater in LPS-treated lungs than in control lungs. Analysis of cells isolated by enzymatic digestion of lung tissue revealed differences in the SP-D-positive cell population between groups. LPS-treated lungs had 28.1-fold more SP-D-positive tissue-associated neutrophils and 193.6-fold greater SP-D association with those neutrophils compared with control lungs. These data suggest that clearance of SP-D into lung tissue is increased during inflammation and that tissue-associated neutrophils significantly contribute to this process.

Mechanisms of endotoxin-induced airway and pulmonary vascular hyperreactivity in mice.

Endotoxin is thought to contribute to pulmonary hyperresponsiveness in byssinosis, asthma, and the acute respiratory distress syndrome (ARDS). The aim of this study was to elucidate the mechanism of this phenomenon in the isolated, blood-free perfused mouse lung. Perfusion with lipopolysaccharide (LPS) had no effect on pulmonary resistance or pulmonary artery pressure, but induced airway hyperreactivity (AHR) to methacholine (MCh) and pulmonary vascular hyperreactivity (VHR) to platelet-activating factor (PAF). Blockade of the thromboxane/endoperoxide (TP) receptor with SQ29.548 completely protected against LPS-induced AHR and VHR. Blockade of cyclooxygenase-2 (COX-2) abolished LPS-induced VHR but suppressed LPS-induced AHR only marginally. COX-2 messenger RNA was upregulated in LPS-treated lungs, and inhibition of transcription with actinomycin D or of protein biosynthesis with cycloheximide protected against LPS-induced VHR but not AHR. Pretreatment with the radical scavenger N-acetylcysteine partly protected against LPS-induced AHR. In addition, perfusion of mouse lungs with the isoprostane 8-epiprostaglandin F(2alpha) (8-epi-PGF(2alpha)), which may be formed as a consequence of oxidative stress in the lung, elicited AHR, which was completely blocked by SQ29.548. Enzyme immunoassay did not detect either 8-epi-PGF(2alpha )or thromboxane B(2) in perfusate samples. Our findings show that LPS induces AHR and VHR in mouse lungs via activation of the TP receptor. Although induction of VHR depends on COX-2 activity, AHR is largely mediated by a non-COX-derived TP agonist, which might be a product of radical-induced lipid peroxidation.

Differential expression of CCAAT enhancer binding protein family in rat alveolar epithelial cell proliferation and in acute lung injury

Although alveolar reorganization after acute lung injury depends on regeneration of alveolar epithelial cells, there is little knowledge of regulation of pulmonary healing process. Transcription factors may play key roles in this regulation. To investigate whether the CCAAT enhancer binding protein (C/EBP) family, alpha, beta, and delta, were involved in alveolar reorganization after injury, we examined expression of C/EBP proteins and mRNAs in lung injuries induced by lipopolysaccharide (LPS) or bleomycin (Bleo) and in cell proliferation by keratinocyte growth factor (KGF). By immunohistochemistry, we demonstrated that C/EBP alpha and C/EBP beta were expressed in alveolar type II cells and alveolar macrophages, but C/EBP delta was expressed restrictedly in some of alveolar type II cells in a spatial pattern in the control lungs. Further, these three C/EBP family members were differentially expressed in alveolar cell proliferation and in acute lung injury, in which, interestingly, C/EBP alpha and C/EBP delta were reciprocally expressed in alveolar type II cell proliferation and in pulmonary fibrosis. However, expressions of their mRNAs by in situ hybridization were dramatically increased in the affected lesions of the lungs by LPS and Bleo, and Northern blot analysis showed an increased abundance of the mRNA for C/EBP beta in LPS-treated lungs and for C/EBP delta in Bleo-treated lungs, compared with those in the control lungs. Thus, differential expression of the C/EBP family may be required to maintain and reorganize the basic integrity of alveolar structure during pathological states, which suggests an important role for the C/EBP family in maintaining normal alveolar architecture and function and in repairing the damaged epithelium after injury.

Early alterations in intracellular and alveolar surfactant of the rat lung in response to endotoxin.

The aim of this study was to characterize early ultrastructural, biochemical, and functional alterations of the pulmonary surfactant system induced by Salmonella minnesota lipopolysaccharide (LPS) in rat lungs. Experimental groups were: (1) control in vitro, 150 min perfusion; (2) LPS in vitro, 150 min perfusion, infusion of 50 microg/ml LPS after 40 min; (3) control ex vivo, 10 min perfusion; (4) LPS ex vivo, lungs perfused for 10 min from rats treated for 110 min with 20 mg/kg LPS intraperitoneally. Morphometry of type II pneumocytes showed that LPS increased stored surfactant. Lamellar bodies were increased in size, but decreased in numerical density, suggesting that giant lamellar bodies observed in LPS-treated lungs may result from fusion of normal bodies. Structural analysis of alveolar surfactant composition showed that LPS elicited an increase in lamellar body-like and multilamellar forms. Bronchoalveolar lavage (BAL) material from LPS-treated lungs was decreased in phospholipids. BAL bubble surfactometer analysis showed a reduction in hysteresis area caused by LPS. We conclude that LPS leads to alterations of intracellular and alveolar surfactant within 2 h: fusion of lamellar bodies, reduction in surfactant secretion, and changes in alveolar surfactant transformation, composition, and function, which may contribute to the development of respiratory distress.

Lipopolysaccharide-induced alveolar epithelial permeability: the role of nitric oxide.

Intratracheal instillation of lipopolysaccharide (LPS) in the rat has been used as a model of acute lung inflammation. Among the early events in this process is a transient increase in airspace epithelial permeability which peaks 4 h after intratracheal instillation of LPS. The increased epithelial permeability is concomitant with the influx of neutrophils into the airspaces, peaking 8 h postinstillation. We have investigated the mechanism of this LPS-induced increase in epithelial permeability. The role of the neutrophil in LPS-induced epithelial permeability was assessed by pretreatment with neutrophil antibody to abolish neutrophil influx, which did not affect the increase in epithelial permeability. Because LPS instillation also induced increased tumor necrosis factor alpha (TNF-alpha) activity in bronchoalveolar lavage (BAL) fluid, and its release by cultured BAL leukocytes from treated animals, TNF-alpha antibody was coinstilled intratracheally with LPS in rats. TNF-alpha antibody eliminated TNF-alpha activity in BAL fluid, but had no effect on LPS-induced increased epithelial permeability. Increased levels of nitric oxide (NO), measured as nitrite, were also present in BAL fluid from LPS-treated rat lungs and LPS-elicited BAL leukocytes produced increased NO in culture. Treatment of rats with the specific NO synthase inhibitor L-NMMA significantly diminished the LPS-induced increased epithelial permeability. These data suggest that NO is involved in LPS-induced changes in epithelial integrity. However, other mechanisms should be evoked in addition to NO to explain completely the increased epithelial permeability produced by LPS.

Endotoxin alters hypoxic pulmonary vasoconstriction in isolated rat lungs.

Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism for maintaining oxygenation, which may be altered by endotoxin. We determined that acute endotoxemia alters the HPV response secondary to changes in endothelium-derived vasoactive products. Rats were treated with Salmonella typhimurium lipopolysaccharide (LPS; 15 mg/kg i.p.) either 1 to 6 h before lung isolation and compared with control rats (no LPS). Additional 6-h LPS-treated and control rats were pretreated with either indomethacin (15 mg/kg i.p.), a cyclooxygenase inhibitor, or bosentan (10 mg/kg po), a nonselective endothelin-receptor antagonist. The rats lungs were isolated and challenged with 3% O2 for 10 min to elicit HPV responses before and after nitric oxide (NO) synthase inhibition with N omega-nitro-L-arginine methyl ester (L-NAME; 100 microM). LPS (6 h) significantly increased the peak HPV responses by 108%. L-NAME had no significant effect in LPS-treated lungs but increased the peak HPV response in control lungs to levels equal to LPS-treated lungs. Bosentan increased the peak HPV response in all lungs, and indomethacin increased the peak HPV in LPS-treated lungs. The HPV response was sustained in control lungs at 10 min and in additional 20-min studies. In contrast, in LPS-treated lungs the HPV response faded after 10 min to levels equal to control, and in 20-min studies it faded by 82% to levels significantly less than in control lungs. The 10-min fade in LPS-treated lungs was attenuated by indomethacin (51%) and bosentan (80%) but not by L-NAME. In conclusion, acute endotoxemia with LPS increased the peak HPV response, but this effect was not sustained and by 20 min was nearly abolished. Inhibition of endogenous NO by LPS may explain the increased peak HPV response, but NO is not involved in the fade. The fade is at least partially due to increased vasodilating cyclooxygenase products and endothelins.