Intratracheal Instillation Of LPS Research Articles

Macrolides attenuate mucus hypersecretion in rat airways through inactivation of NF‐κB

To examine the effect of a 14-membered ring macrolide on airway mucus hypersecretion in rats treated with LPS. Mucus hypersecretion in rat airways was induced by intratracheal instillation of LPS. Rats treated with or without LPS were administered roxithromycin (1-10 mg/kg), josamycin (10 mg/kg) or amoxicillin (40 mg/kg), orally for 4 days. Expression of Muc5ac, nuclear factor (NF)-kappaB, and the mitogen-activated protein (MAP) kinases p38 and ERK1/2 in bronchial epithelium were detected by RT-PCR, immunohistochemistry or western blotting. Mucins, IL-1beta, IL-8 and tumour necrosis factor (TNF)-alpha in BAL fluid were assayed by enzyme-linked lectin assay and ELISA. LPS significantly induced the expression of Muc5ac mRNA and protein in bronchial epithelium, increased the release of mucins, IL-1beta, IL-8 and TNF-alpha, and increased neutrophil numbers in BAL. Moreover, LPS increased staining for NF-kappaB in the cytoplasm as well as nuclear translocation of NF-kappaB in airway epithelial cells. Upregulated expression of Muc5ac mRNA correlated positively with NF-kappaB activation and the levels of cytokines (P < 0.05). Roxithromycin (5 and 10 mg/kg) significantly attenuated bronchial Muc5ac expression and NF-kappaB nuclear translocation stimulated by LPS, and reduced neutrophil numbers, mucins and inflammatory cytokines in BAL (P < 0.05). However, LPS-stimulated expression of p38 and ERK1/2 in airway epithelium was not affected by roxithromycin. Josamycin and amoxicillin had no effects on Muc5ac expression, NF-kappaB activation or cytokine release. Roxithromycin inhibits the pulmonary inflammatory response and airway mucus hypersecretion induced by LPS. The inhibitory effect of roxithromycin on airway mucus hypersecretion may be mediated through reduction of NF-kappaB activation, neutrophil infiltration and release of inflammatory cytokines in the lung.

Activation of Toll‐like Receptors is a Critical Determinant of Lung Neutrophil Sequestration and Injury Induced by High Tidal Volume Mechanical Ventilation

The infiltration of polymorphonuclear neutrophils (PMNs) into lungs is an important feature of ventilator-induced lung injury (VILI) associated with pneumonia, but the mechanisms that recruit PMNs are poorly understood. Using Toll-like receptor 4 knockout mice (TLR4−/−), we addressed the role of TLR4 signaling in PMN recruitment. Wild type (WT) or TLR4−/− mice were challenged by intratracheal instillation of LPS (3.0 mg/kg) for 2 hr and then subjected to high tidal volume mechanical ventilation (28 ml/kg) for 2 hr. Ventilated WT mice exhibited significant increases in PMN sequestration (5-fold), bronchoalveolar lavage PMN count (3.6-fold), airway fluid albumin concentration (2.4-fold), and edema formation (1.6-fold). Quantitative lung histopathology demonstrated PMN infiltration, alveolar hemorrhage, edema, and alveolar wall thickening in lungs of ventilated, LPS-challenged WT mice. However, TLR4−/− mice showed negligible PMN sequestration, microvascular barrier breakdown, and edema formation. Thus, high tidal volume ventilation during pneumonia induces PMN sequestration and lung injury via TLR4-dependent signaling pathways. The results suggest the important role of lung innate immunity regulated by TLRs in the mechanism of PMN sequestration and injury resulting from mechanical ventilation at high tidal volumes. Supported by NIH R01 HL 071626, P01 HL 60678, and P01 HL 077806.

Polar head groups are important for barrier-protective effects of oxidized phospholipids on pulmonary endothelium

We have previously described protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cell (EC) barrier function and demonstrated the critical role of cyclopentenone-containing modifications of arachidonoyl moiety in OxPAPC protective effects. In this study we used oxidized phosphocholine (OxPAPC), phosphoserine (OxPAPS), and glycerophosphate (OxPAPA) to investigate the role of polar head groups in EC barrier-protective responses to oxidized phospholipids (OxPLs). OxPAPC and OxPAPS induced sustained barrier enhancement in pulmonary EC, whereas OxPAPA caused a transient protective response as judged by measurements of transendothelial electrical resistance (TER). Non-OxPLs showed no effects on TER levels. All three OxPLs caused enhancement of peripheral EC actin cytoskeleton. OxPAPC and OxPAPS completely abolished LPS-induced EC hyperpermeability in vitro, whereas OxPAPA showed only a partial protective effect. In vivo, intravenous injection of OxPAPS or OxPAPC (1.5 mg/kg) markedly attenuated increases in the protein content, cell counts, and myeloperoxidase activities detected in bronchoalveolar lavage fluid upon intratracheal LPS instillation in mice, although OxPAPC showed less potency. All three OxPLs partially attenuated EC barrier dysfunction induced by IL-6 and thrombin. Their protective effects against thrombin-induced EC barrier dysfunction were linked to the attenuation of the thrombin-induced Rho pathway of EC hyperpermeability and stimulation of Rac-mediated mechanisms of EC barrier recovery. These results demonstrate for the first time the essential role of polar OxPL groups in blunting the LPS-induced EC dysfunction in vitro and in vivo and suggest the mechanism of agonist-induced hyperpermeability attenuation by OxPLs via reduction of Rho and stimulation of Rac signaling.

Gastrin-releasing Peptide Receptor Antagonist Effects on an Animal Model of Sepsis

Several new therapeutic strategies have been described for the treatment of sepsis, but to date none are related to alterations in the bombesin/gastrin-releasing peptide (GRP) receptor pathways. To determine the effects of a selective GRP receptor antagonist, RC-3095, on cytokine release from macrophages and its in vivo effects in the cecal ligation and puncture (CLP) model of sepsis and in acute lung injury induced by intratracheal instillation of LPS. We determined the effects of RC-3095 in the CLP model of sepsis and in acute lung injury induced by intratracheal instillation of LPS. In addition, we determined the effects of RC-3095 on tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-10, and nitric oxide release from activated macrophages. The GRP antagonist attenuated LPS- or CLP-induced TNF-alpha, IL-1beta, and nitric oxide release in cultured macrophages and decreased the mRNA levels of inducible nitric oxide synthase. The administration of RC-3095 (0.3 mg/kg) 6 h after sepsis induction improved survival in the CLP model, and diminished lung damage after intratracheal instillation of LPS. These effects were associated with attenuation on the circulating TNF-alpha and IL-1beta levels and decreased myeloperoxidase activity in several organs. We report that a selective GRP receptor antagonist attenuates the release of proinflammatory cytokines in vitro and in vivo and improves survival in "established" sepsis. These are consistent with the involvement of a new inflammatory pathway relevant to the development of sepsis.

Effect of CD14 blockade on endotoxin-induced acute lung injury in mice.

CD14 functions as a cell surface receptor for endotoxin (lipopolysaccharide [LPS]) and is thought to have an essential role in innate immune responses to infection. Previous studies have revealed attenuation of the systemic response after sepsis by blocking CD14. In this study, we tested the hypothesis that CD14 blockade protects against inflammatory responses associated with LPS pneumonia. We examined the effect of an anti-murine CD14 monoclonal antibody (4C1) on the development of acute lung injury induced by intratracheal LPS in mice. We also measured the production of cytokines (tumor necrosis factor-alpha, interleukin-6, and macrophage inflammatory protein-2) and nitric oxide by murine peritoneal macrophages exposed to LPS in vitro. Nuclear factor (NF)-kappa B translocation was evaluated in nuclear extracts from lung homogenates. 4C1 significantly attenuated pulmonary edema and neutrophil emigration after LPS administration. The production of cytokines and nitric oxide by LPS-stimulated macrophages was significantly decreased by 4C1 treatment. NF-kappa B translocation induced by LPS instillation was also suppressed by 4C1. These results suggest that blockade of CD14 might attenuate acute lung injury after intratracheal instillation of LPS through the suppression of NF-kappa B translocation. The inhibitory effect of CD14 blockade on cytokine production and nitric oxide release of macrophages might contribute to the attenuation of lung injury.

Functions of IκB Proteins in Inflammatory Responses toEscherichia coliLPS in Mouse Lungs

Acute inflammation induced by intrapulmonary LPS requires nuclear factor (NF)-kappaB RelA. This study elucidates the effects of intrapulmonary LPS on IkappaB proteins, endogenous inhibitors of RelA, and the effects of deficiency of IkappaB-beta. IkappaB-alpha, IkappaB-beta, and IkappaB-epsilon each complexed with RelA in uninfected murine lungs. Intratracheal instillation of LPS induced the degradation of IkappaB-alpha and IkappaB-beta, as measured by the loss of immunoreactive proteins in non-nuclear fractions. Degradation was apparent by 2 h and sustained through 6 h. In contrast, net IkappaB-epsilon content increased over this period. The small amounts of IkappaB-alpha and IkappaB-beta that were detected in nuclear fractions from the lungs also decreased over this time frame, whereas intranuclear NF-kappaB content (including both RelA and p50) increased. The hypophosphorylated form of IkappaB-beta, which facilitates transcription induced by NF-kappaB, was not detected. Neutrophil recruitment and edema accumulation did not differ between wild type mice and gene-targeted mice deficient in IkappaB-beta, suggesting that IkappaB-beta is not specifically required for these responses. Altogether, these data suggest that RelA is liberated during LPS-induced pulmonary inflammation by the regulated degradation of both IkappaB-alpha and IkappaB-beta. In the absence of IkappaB-beta, IkappaB-alpha or other inhibitory proteins can regulate NF-kappaB functions essential to acute neutrophil emigration in the lungs.

The Effects of Surfactant on Neutrophil Apoptosis in Lipopolysaccharide Induced Acute Lung Injury in Rat

Background : The therapeutic effects of surfactant on acute lung injury derive not only from its recruiting action on collapsed alveoli but also from its anti-inflammatory effects. Pro-apoptotic action on alveolar neutrophils represents one of the important anti-inflammatory mechanisms of surfactant. In the present study, we evaluated the effects of sufactant on the apoptosis of human peripheral and rat alveolar neutrophils. Methods : In the (Ed- the article is not definitely needed but it helps to separate the two prepositions 'in') in vitro study, human neutrophils were collected from healthy volunteers. An equal number of neutrophils () (Ed-confirm) was treated with LPS (10, 100, 1000ng/ml), surfactant (10, 100, ), or a combination of LPS (1000ng/ml) and surfactant (10, 100, ). After incubation for 24 hours, the apoptosis of neutrophils was evaluated by Annexin V method. In the in vivo study, induction of acute lung injury in SD rats by intra-tracheal instillation of LPS (5mg/kg) was followed by intra-tracheal administration of either surfactant (30mg/kg) or normal saline (5ml/kg). Tenty-four hours after LPS instillation, alveolar neutrophils were collected and the apoptotic rate was evaluated by Annexin V method. In addition, changes of the respiratory mechanics of rats (respiratory rate, tidal volume, and airway resistance) were evaluated with one chamber body plethysmography before, and 23 hours after, LPS instillation. Results : in the in vitro study, LPS treatment decreased the apoptosis of human peripheral blood neutrophils (control: , LPS 10ng/ml; , LPS 100ng/ml; , LPS 1000ng/ml; ). The combination of low to moderate doses of surfactant with LPS promoted apoptosis (LPS 1000ng/ml + Surf ; , LPS 1000ng/ml +Surf ; ). The high dose of surfactant () decreased apoptosis () and augmented the anti-apoptotic effect of LPS (LPS 1000ng/ml + Surf ; ). In the in vivo study, the apoptotic rate of alveolar neutrophils of surfactant-treated rats was higher than that of normal saline-treated rats ( vs. ). The airway resistance (represented by Penh) of surfactant-treated rats was lower than that of normal saline-treated rats at 23 hours after LPS injury ( vs. , p

The Relationship Between the NF-γ B Activity and Anti-inflammatory Action of Surfactant in the Acute Lung Injury of Rats

Background : The therapeutic effects of surfactants on acute lung injury derive not only from their recruiting action on collapsed alveoli but also from their anti-inflammatory action in the alveolar sapce. This study evaluated the anti-inflammatory action of a surfactant in an acute lung injury model of rats by neutrophils were recollected from the BAL fluid and the NF- activity of the neutrophilic nuclear protein was evaluated. Methods : Male Sprague-Dawley rats weighing approximately 300 gram were divided into 3 groups, which consisted of 6 rats respectively. In the control group, normal saline(3ml/kg) was instilled into the trachea twice with 30 minute interval. In two other groups, acute lung injury was induced by the intra-tracheal instillation of LPS(5mg/kg). Thirty minutes later, either a surfactant(ST group; 30mg/kg) or normal saline(NT group: 3ml/kg) was instilled via the trachea. Twenty-four hours after the LPS instillation, the BAL fluid was retrieved to measure the WBC count and cytokine(IL- and IL-6) levels. The neutrophils were isolated from the BAL fluid and the nuclear protein was extracted to evaluate the NF- activity using a eletrophoretic mobility shift assay(EMSA). Results : The WBC count of the BAL fluid of the ST group() was higher than that of the control group()(p)(p from the NT group() was higher than those of the ST group()(p)(p activity of the neutrophilic nuclear protein in the ST group and NT group was similar. Conclusion : The surfactant, attenuates the alveolar inflammation in the acute lung injury of rats model. However, its anti-inflammatory action does no't appear to be mediated by the inhibition of NF- activity.

Role of tumor necrosis factor-alpha in endotoxin-induced lung parenchymal hyporesponsiveness in mice

Although changes in airway responsiveness in pulmonary inflammation are commonly related to the action of infiltrated leukocytes, our previous report suggested a direct role of inflammatory cytokines in LPS-induced lung hyporesponsiveness. The aim of this study was to define if cytokines detected in the BALF (bronchoalveolar lavage fluid) of intratracheal LPS-treated mice could be, at least in part, responsible for 5-HT (5-hydroxytryptamine) lung hyporeactivity. Our results show that intratracheal instillation of LPS induced a time-dependent increase in IL-(interleukin-)1β, IL-6, and TNF (tumor necrosis factor)α in the BALF. Cytokine production was paralleled by 5-HT lung hyporesponsiveness, and intratracheal administration of TNFα proved to be very efficient in inhibiting 5-HT responsiveness. In addition, systemic treatment with rolipram, an inhibitor of TNFα production, was paralleled by a significant recovery of lung responsiveness. On the contrary, IL-1β and IL-6 were not demonstrated to play a relevant role in 5-HT hyporesponsiveness. It is concluded that TNFα could be a crucial mediator of LPS-induced lung hyporesponsiveness.

Neutrophil emigration in the lungs, peritoneum, and skin does not require gelatinase B.

Polymorphonuclear leukocytes (PMN) release gelatinase B in response to variable stimuli. Gelatinase B degrades basement membrane components in vitro, and inhibition of matrix metalloproteinase activity blunts PMN migration through a prototype basement membrane (Matrigel) and amnionic membranes. Accordingly, it has been speculated that gelatinase B is necessary for PMN emigration. To test this hypothesis we induced acute inflammation in the lungs, peritoneum, and skin in mice with a null mutation of the gelatinase B gene (gelatinase B-/-) and littermate controls (gelatinase B+/+). At 3, 6, 12, and 24 h after intratracheal instillation of LPS, the emigration of PMN in the lung, as determined by PMN in bronchoalveolar lavage fluid, was similar in gelatinase B-/- and gelatinase B+/+ mice. The number of PMN in the peritoneal cavity 4 h after thioglycollate-induced peritonitis was also comparable in gelatinase B-/- and gelatinase B+/+ mice. At 4 h after an intradermal injection of interleukin-8, numerous PMN were present extravascularly in the dermis in both gelatinase B-/- and gelatinase B+/+ mice and the myeloperoxidase activities of the skin at the injection sites were indistinguishable between the two types of mice. PMN from gelatinase B-/- mice migrated through Matrigel in response to zymosan-activated serum with the same efficiency as did PMN from gelatinase B+/+ mice. In vitro, gelatinase B-/- PMN killed Staphylococcus aureus and Klebsiella pneumoniae as effectively as did PMN from gelatinase B+/+ mice. These findings indicate that gelatinase B is not required for PMN emigration, and suggest that the antibacterial function of PMN is preserved despite gelatinase B deficiency.

The Role of Leukocyte Emigration and IL-8 on the Development of Lipopolysaccharide-Induced Lung Injury in Rabbits

Leukocyte emigration and alveolar macrophage-derived cytokines may contribute to lung microvascular injury associated with adult respiratory distress syndrome. We have used mAbs against cell adhesion molecules on leukocytes (anti-CD18 and anti-CD49d) or against IL-8 to investigate these contributions. Intratracheal (i.t.) instillation of LPS (50 microg/kg) caused a significant increase in bronchoalveolar lavage polymorphonuclear leukocytes (PMNs) without an increase in mononuclear cells (MNCs) or an increase in lung permeability. Injection of LPS (10 microg/kg) i.v. at 24 h after i.t. LPS caused significant increases in bronchoalveolar lavage PMNs, MNCs, IL-8, and monocyte chemotactic protein-1, as well as increases in lung permeability. Rabbits that were administered i.t. LPS followed by i.v. LPS and treated with anti-CD18 mAb had a significantly lower lung permeability index and emigration of fewer PMNs but no change in MNC emigration compared with saline treatment. Anti-IL-8 mAb treatment resulted in a significantly lower lung permeability index with no change in PMN emigration compared with no treatment. These results suggest that PMN emigration is necessary but not sufficient for the development of LPS-induced lung injury, and that IL-8 plays a significant role in PMN-dependent lung injury, independent of PMN emigration.

Lung Injury Indices Depending on Tumor Necrosis Factor-alpha Level and Novel 35 kDa Protein Synthesis in Lipopolysaccharide-Treated Rat

Background : TNF- appears to be a central mediator of the host response to sepsis. While TNF- is mainly considered a proinflammatory cytokine, it can also act as a direct cytotoxic cytokine. However, there are not so many studies about the relationship bet ween TNF- level and lung injury severity in ALI, particularly regarding the case of ALI caused by direct lung injury such as diffuse pulmonary infection. Recently, a natural defense mechanism, known as the stress response or the heat shock response, has been reported in cellular or tissue injury reaction. There are a number of reports examining the protective role of pre-induced heat stress proteins on subsequent LPS-induced TNF- release from monocyte or macrophage and also on subsequent LPS-induced ALI in animals. However it is not well established whether the stress protein synthesis such as HSP can be induced from rat alveolar macrophages by in vitro or in vivo LPS stimulation. Methods : We measured the level of TNF-, the percentage of inflammatory cells in bronchoalveolar lavage fluid, protein synthesis in alveolar macrophages isolated from rats at 1, 2, 3, 4, 6, 12, and 24 hours after intratracheal LPS instillation. We performed histologic examination and also obtained histologic lung injury index score in lungs from other rats at 1, 2, 3, 4, 6, 12, 24 h after intratracheal LPS instillation. Isolated non-stimulated macrophages were incubated for 2 h with different concentration of LPS (0, 1, 10, 100 ng/ml, 1, or 10 ). Other non-stimulated macrophages were exposed at for 15 min, then returned to at in 5% CO2-95% for 1 hour, and then incubated for 2 h with LPS (0, 1, 10, 100ng/ml, 1, or 10 ). Results : TNF- levels began to increase significantly at 1 h, reached a peak at 3 h (P levels in supernatants of LPS-stimulated macro phages were significantly higher than those of non-stimulated macrophages(p levels in supernatants were significantly lower after heat treatment than in those without heat treatment (p. Conclusion : TNF- has a direct or indirect close relationship with lung injury severity in acute lung injury or acute respiratory distress syndrome. In vivo and in vitro LPS stimulation dose not induce heat stress protein 72 in alveolar macrophages. It is likely that 35 kDa protein, synthesized by alveolar macrophage after LPS instillation, does not have a defense role in acute lung injury.

Nitric oxide synthase in human and rat lung: immunocytochemical and histochemical localization.

Nitric oxide synthase (NOS) produces nitric oxide, a mediator of potential importance in numerous physiologic and inflammatory processes in the lung. We localized constitutive NOS (c-NOS) and inducible NOS (i-NOS) within lung tissue by immunoperoxidase labeling with specific antibodies or by histochemical demonstration of the characteristic NADPH diaphorase activity of NOS. We analyzed human airway (n = 4) or parenchyma (n = 10) specimens obtained from uninvolved areas of surgical tumor resections. We also studied human fetal lung samples (n = 6) and normal or inflamed (16 h after intratracheal LPS instillation) rat lung tissue. Immunostaining with anti-c-NOS identified c-NOS antigen in rat lung nerves, endothelium, and airway epithelium. Normal or inflamed rat macrophages were not stained. Human nerve elements and large-vessel endothelium showed immunostaining with the anti-c-NOS, but no labeling of the airway or alveolar epithelium was seen. Immunostaining with anti-i-NOS showed strong labeling of rat macrophages after LPS treatment, in vivo or in vitro, while normals were negative. Human alveolar macrophages were occasionally positive for i-NOS, especially in areas of chronic inflammation, which also showed focal immunolabeling of endothelium. Uniform labeling of epithelium in large, cartilaginous airways was found with anti-i-NOS in both human bronchi and normal rat trachea samples, suggesting a constitutive role for a NOS that shares epitope(s) with or is highly homologous to the inducible, macrophage type of NOS. Histochemical staining for NADPH diaphorase activity was consistent with immunolocalization of NOS antigens.(ABSTRACT TRUNCATED AT 250 WORDS)