Pulmonary aspiration: new therapeutic approaches in the experimental model

BACKGROUND: Acute lung injury caused by gastric aspiration is a frequent occurrence in unconscious patients. Acute respiratory distress syndrome in association with gastric aspiration carries a mortality of up to 30% and accounts for up to 20% of deaths associated with anesthesia. Although the clinical condition is well known, knowledge about the exact inflammatory mechanisms is still incomplete. This study was performed to define the role of alveolar macrophages in this inflammatory response. In addition, potentially modifying effects of intratracheally applied nuclear factor kappaB inhibitor pyrrolidine dithiocarbamate were investigated. METHODS: Rat alveolar macrophages were depleted by intratracheal administration of clodronate liposomes, and lung injury was evaluated 6 h after instillation of 0.1N hydrochloric acid. In a second set of experiments, pyrrolidine dithiocarbamate was intratracheally instilled 3 h after hydrochloric acid application, and injury parameters were determined. RESULTS: Depletion of alveolar macrophages resulted in decreased production of inflammatory mediators in acid aspiration (23-80% reduction of messenger RNA or protein of inflammatory mediators; P < 0.05) and consequently also in diminished neutrophil recruitment (36% fewer neutrophils; P < 0.01). Treatment with pyrrolidine dithiocarbamate was highly effective in decreasing neutrophil recruitment (66%; P < 0.01) and vascular permeability (80%; P < 0.001). CONCLUSIONS: These data suggest that alveolar macrophages play an essential role in the inflammatory response of acid-induced lung injury. For the first time, attenuation of acid-induced lung injury with an inhibitor, applied after the onset of injury, is shown.     

Pulmonary Aspiration: New Therapeutic Approaches in the Experimental Model

Background: Acute lung injury caused by gastric aspiration is a frequent occurrence in unconscious patients. Acute respiratory distress syndrome in association with gastric aspiration carries a mortality of up to 30% and accounts for up to 20% of deaths associated with anesthesia. Although the clinical condition is well known, knowledge about the exact inflammatory mechanisms is still incomplete. This study was performed to define the role of alveolar macrophages in this inflammatory response. In addition, potentially modifying effects of intratracheally applied nuclear factor [kappa]B inhibitor pyrrolidine dithiocarbamate were investigated.

Methods: Rat alveolar macrophages were depleted by intratracheal administration of clodronate liposomes, and lung injury was evaluated 6 h after instillation of 0.1N hydrochloric acid. In a second set of experiments, pyrrolidine dithiocarbamate was intratracheally instilled 3 h after hydrochloric acid application, and injury parameters were determined.

Results: Depletion of alveolar macrophages resulted in decreased production of inflammatory mediators in acid aspiration (23-80% reduction of messenger RNA or protein of inflammatory mediators; P < 0.05) and consequently also in diminished neutrophil recruitment (36% fewer neutrophils; P < 0.01). Treatment with pyrrolidine dithiocarbamate was highly effective in decreasing neutrophil recruitment (66%; P < 0.01) and vascular permeability (80%; P < 0.001).     

Alpha chemokines regulate direct lung ischemia-reperfusion injury.

BACKGROUND: Alpha chemokines function predominantly to recruit and activate neutrophils, which are important effectors of acute lung injury. This study evaluated whether blockade of 2 potent alpha chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC), is protective against lung ischemia-reperfusion injury in a warm in situ hilar clamp model. METHODS: Left lungs of Long-Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received antibodies to MIP-2 or CINC immediately prior to reperfusion. Lung injury was quantitated by vascular permeability to (125)I-radiolabeled bovine serum albumin, lung tissue neutrophil sequestration (myeloperoxidase [MPO] content), and alveolar leukocyte content in bronchoalveolar lavage (BAL) fluid. CINC and MIP-2 mRNA expression were assessed by northern blot, while ribonuclease protection assays were performed to evaluate mRNA expression for a number of early response cytokines. MIP-2 and CINC protein expression in injured lungs was determined by immunoblotting. RESULTS: Treatment with antibodies to CINC or MIP-2 was associated with significant protection against increases in vascular permeability, MPO content and alveolar leukocyte sequestration in injured lungs. Expression of CINC and MIP-2 mRNA peaked after 2 hours of reperfusion in injured lungs, and protein levels were evident on immunoblotting after 3 hours of reperfusion. Neither CINC nor MIP-2 blockade appeared to modulate cytokine mRNA expression. CONCLUSIONS: CINC and MIP-2 are important mediators involved in direct lung ischemia-reperfusion injury. They appear to function by modulating neutrophil recruitment, but not inflammatory cytokine release.     

Inhibition of nuclear factor-kappaB activation by pyrrolidine dithiocarbamate prevents chronic FK506 nephropathy

BACKGROUND: Chronic tacrolimus (FK506) nephrotoxicity is characterized by renal fibrosis with interstitial inflammation. Since nuclear factor-kappaB (NF-kappaB) plays a key role in chronic inflammatory diseases including renal disease, the present study was conducted to elucidate the role of NF-kappaB in the pathogenesis of chronic FK506-induced nephropathy. METHODS: FK506 (1 mg/kg/day, SC) was administered daily to rats maintained on low sodium diet for 42 days. Some rats were treated with a putative NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC; 100, 200 mg/kg/day, by gavage). The renal function, renal histology, renal NF-kappaB-DNA binding activity and gene expression profile were examined. RESULTS: FK506 caused a decline in glomerular filtration and induced characteristic renal morphologic changes including arteriolopathy, tubular atrophy and interstitial fibrosis. FK506 markedly activated renal cortical NF-kappaB-DNA binding. PDTC administration inhibited NF-kappaB-DNA binding activity in a dose dependent manner. With higher dose, NF-kappaB-DNA binding activity was decreased to a control level. PDTC had little effect on FK506-induced renal dysfunction. Renal cortical monocyte/macrophage infiltration observed in FK506-treated rats was dramatically suppressed by PDTC. FK506 up-regulated renal cortical gene expression of chemoattractant proteins, monocyte chemoattractant protein-1 (MCP-1) and osteopontin. PDTC significantly blocked MCP-1 gene expression but had no effect on osteopontin gene expression. Tubular atrophy and tubulointerstitial fibrosis, but not arteriolopathy, were significantly attenuated by PDTC. FK506 increased renal mRNA expression of fibrogenic molecules and extracellular matrices that also were attenuated by PDTC treatment. CONCLUSIONS: NF-kappaB plays an important role in mediating cortical monocyte/macrophage infiltration and in the pathogenesis of tubular injury and interstitial fibrosis in experimental FK506-induced chronic nephropathy.     

The role of neutrophils, oxidants, and proteases in the pathogenesis of acid pulmonary injury.

We recently reported a biphasic injury pattern of nonlethal acid aspiration pneumonitis in rats. The first phase consisted of the immediate effects of the direct tissue injury, and the second phase was associated with a neutrophilic inflammatory response. Using this model, the present report examines the possible role of neutrophils, oxidants, and proteases in the pathogenesis of the second phase of this lung injury. Acid aspiration injury was induced by instillation of saline/HCl, pH = 1.25, into the trachea of rats. Lung injury was assessed by measuring the degree of alveolar capillary permeability to 125I-labeled albumin (permeability index [PI]). Rats made neutropenic with polyclonal antineutrophil antibody had a lower PI (0.44 +/- 0.07, P less than 0.05) 6 h after acid aspiration than similarly injured animals with normal whole blood neutrophil counts (PI = 0.85 +/- 0.03). Even though neutrophils appeared necessary for the full development of the lung injury in this model, the administration of different intravenous and/or intratracheal concentrations of either deferoxamine or catalase offered no protection against injury. This suggests that neutrophil oxidants were minimally involved in the injury. Large increases in leukocyte-free serine protease activity (1,477 +/- 438 u/ml, P less than 0.05) were detected in the bronchoalveolar lavage fluid from the saline/HCl, pH = 1.25, injured rats at 6 h postinjury, as compared to saline/HCl, pH = 5.3, treated control animals (2.7 +/- 0.2 u/ml). This study supports the hypothesis that neutrophils are necessary for the full expression of acid-induced lung injury and that the generation of leukocyte-derived oxidants does not appear to be the primary mechanism involved in this injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel broad-spectrum chemokine inhibitor protects against lung ischemia-reperfusion injury.

BACKGROUND: Functional roles for chemokines have been demonstrated in several models of ischemia-reperfusion injury. The redundancy inherent to chemokine pathways makes administration of neutralizing antibodies to any single chemokine ineffective in ameliorating injury. This study was undertaken to define the pattern of chemokine expression in lung ischemia-reperfusion injury (LIRI), and to determine whether a broad-spectrum chemokine inhibitor, NR58-3.14.3, could confer significant protection against LIRI. METHODS: Left lungs of rats were rendered ischemic for 90 minutes and then reperfused for 4 hours. Chemokine secretion into the alveolar space was quantified by enzyme-linked immunoassay. Treated animals received NR58-3.14.3 prior to reperfusion. Vascular injury was measured by lung permeability index, neutrophil accumulation in lung parenchyma was determined by myeloperoxidase (MPO) activity, and alveolar leukocyte counts were quantified in bronchoalveolar lavage (BAL) effluent. A ribonuclease protection assay evaluated mRNA expression of various chemokines. RESULTS: Lavage effluent in untreated animals demonstrated significant increases in the secretion of cytokine-induced neutrophil chemoattractant (CINC), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2), MIP-1alpha and monocyte chemoattractant protein-1 (MCP-1). Animals receiving NR58-3.14.3 demonstrated a 37% (p < 0.001) reduction in vascular injury and a marked reduction in lung MPO activity (p < 0.001) and alveolar cell counts (p = 0.005). Chemokine inhibition decreased mRNA expression of a number of early response cytokines when compared with positive control animals, and caused a significant decrease (p < 0.04) in the secretion of TNF-alpha. CONCLUSIONS: These studies demonstrate that chemokines are expressed after lung ischemia and reperfusion, and that broad-spectrum chemokine inhibition ameliorates reperfusion injury. mRNA expression of early response cytokines was modulated, and the secretion of TNF-alpha was decreased.     

内毒素休克大鼠核因子κB活化规律及其在生物蝶呤诱生中的作用

目的观察内毒素休克大鼠血浆及主要脏器核因子(NF)κB活化规律及其对生物蝶呤(BH4)和一氧化氮(NO)表达水平的影响,探讨内毒素休克时NF-κB信号通路对BH4诱生NO的分子调控机制及其与多器官功能损害的关系。方法将47只大鼠按表格随机法分为正常组(8只)、内毒素／脂多糖(LPS)组(24只,每观察时相点8只,均同时注射LPS制成休克模型)和拮抗组[15只,每观察时相点5只,均同时注射LPS并以吡咯烷二硫代氨基甲酸盐(PDTC)拮抗]。休克及拮抗组于注射LPS后2、6、12 h观察,并与正常组同法处死,无菌留取大鼠血标本及肝、肺、肾组织,测定组织中NF-κB活性和三磷酸鸟苷环水解酶Ⅰ(GTP-CHⅠ)和诱导型一氧化氮合酶(iNOS)mRNA表达水平、血浆和组织中的BH4含量及NO水平、肝脏和肾脏功能指标、肺组织髓过氧化物酶活性。结果与正常组(例如肺组织中NF-κB活性为26±6)比较,LPS组大鼠组织中NF-κB迅速活化(P<0．01),并于注射后2 h达峰值(肺组织中为291±44);LPS组各组织中GTP-CHⅠ和iNOS mRNA表达、BH4和NO水平也较正常组明显升高(P<0．05或0．01),至伤后12 h仍持续较高水平。此外,该组相应器官功能均受到不同程度的损害。应用PDTC的拮抗组大鼠各组织中NF-κB活性均较LPS组有所降低,GTP-CHⅠ、iNOS mRNA表达及BH4、NO水平显著受抑,肝、肺、肾功能明显改善。结论内毒素休克时机体内NF-κB通路高度活化,并对BH4／NO系统具有明显调节效应;可通过下调BH4介导的iNOS的过度活化抑制NF-κB信号途径,从而减轻组织炎性反应,对机体脏器功能起到保护作用。     

Interactions between angiotensin II and NF-kappaB-dependent pathways in modulating macrophage infiltration in experimental diabetic nephropathy

NF-kappaB-dependent pathways play an important role in macrophage infiltration and kidney injury. NF-kappaB is regulated by angiotensin II (AII). However, the role of this pathway in diabetic nephropathy has not been clearly delineated. First, the activation of NF-kappaB, monocyte chemoattractant protein-1 (MCP-1), and macrophage infiltration in the diabetic kidney were explored, in a temporal manner. The active subunit of NF-kappaB, p65, was elevated in the diabetic animals in association with increased MCP-1 gene expression and macrophage infiltration. Second, the effects of treatment for 4 wk with the AII type 1 receptor antagonist valsartan, the AII type 2 receptor antagonist PD123319, or pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB and on these parameters were assessed. These treatments were associated with a reduction in p65 activation, MCP-1 gene expression, and macrophage infiltration. These findings demonstrate a role for activation of NF-kappaB, in particular the p65 subunit, in the pathogenesis of early renal macrophage infiltration in experimental diabetes. In the context of the known proinflammatory effects of AII, it is postulated that the renoprotection conferred by angiotensin II receptor antagonism is at least partly related to the inhibition of NF-kappaB-dependent pathways.     

Fish-oil emulsion (omega-3 polyunsaturated fatty acids) attenuates acute lung injury induced by intestinal ischemia–reperfusion through Adenosine 5′-monophosphate-activated protein kinase–sirtuin1 pathway

Background

Activated macrophage infiltration into the lungs is paramount in the pathogenesis of acute lung injury (ALI) induced by intestinal ischemia–reperfusion (I/R). Omega-3 polyunsaturated fatty acid (ω-3 PUFA) is a potent activator of the Adenosine 5′-monophosphate-activated protein kinase–sirtuin1 (AMPK/SIRT1) pathway against macrophage inflammation. We aimed to evaluate whether ω-3 PUFAs may protect against ALI induced by intestinal I/R via the AMPK/SIRT1 pathway.

Methods

Ischemia in male Wistar rats was induced by superior mesenteric artery occlusion for 60 min and reperfusion for 240 min. One milliliter per day of fish-oil emulsion (FO emulsion, containing major ingredients as ω-3 PUFAs) or normal saline (control) was administered by intraperitoneal injection for three consecutive days to each animal. All animals were sacrificed at the end of reperfusion. Blood and tissue samples were collected for analysis.

Results

Intestinal I/R caused intestinal and lung injury, evidenced by severe lung tissue edema and macrophage infiltration. Pretreatment with FO emulsion improved the integrity of microscopic structures in the intestine and lungs. Intestinal I/R induced the expression of macrophage-derived mediators (macrophage migration inhibitory factor and macrophage chemoattractant protein-1), inflammatory factors (nuclear factor κB, tumor necrosis factor α, interleukin 6, and interleukin 1β), and proapoptosis factor p66shc. There was a decrease in the expression of AMPK, SIRT1, and claudin 5. FO emulsion significantly inhibited macrophage infiltration into the lungs, inflammatory factor expression, and p66shc phosphorylation. Importantly, FO emulsion restored AMPK, SIRT1, and claudin 5 in the lungs.

Conclusions

Pretreatment with ω-3 PUFAs effectively protects intestinal and lung injury induced by intestinal I/R, reduces macrophage infiltration, suppresses inflammation, inhibits lung apoptosis, and improves the lung endothelial barrier after intestinal I/R in a manner dependent on AMPK/SIRT1. Thus, there is a potential for developing AMPK/SIRT1 as a novel target for patients with intestinal I/R–induced ALI.     

TNF receptor I mediates chemokine production and neutrophil accumulation in the lung following systemic lipopolysaccharide.

BACKGROUND: Tumor necrosis factor (TNF)-alpha is a critical effector of lipopolysaccharide (LPS)-induced acute lung injury, and its effects are mediated by two structurally related receptors, RI and RII. Cellular adhesion molecules and C-X-C chemokines (Keratinocyte chemoattractant (KC) and macrophage inflammatory protein [MIP]-2) regulate tissue neutrophil polymorphonuclear neutrophil (PMN) accumulation in a multitude of inflammatory states. We hypothesized that TNFRI signaling dictates PMN accumulation in the lung via regulation of chemokine molecule production. Therefore, the purposes of this study were to (1) delineate LPS-induced lung TNF-alpha production and (2) characterize the contribution of both TNF receptors to lung chemokine production and neutrophil influx following systemic LPS. METHODS: Wild-type or TNFRI and TNFRII knockout (KO) mice were injected with vehicle (saline) or LPS (Escherichia coli 0.5 mg/kg intraperitoneally). After 2, 4, 6, or 24 h, lungs were analyzed for TNF-alpha and chemokine (KC and MIP-2) protein expression (enzyme-linked immunosorbent assay) and PMN accumulation (myeloperoxidase assay). RESULTS: There was an increase in total lung TNF-alpha (vehicle, 5.0 +/- 1.2 pg/mg total protein vs LPS, 950 +/- 318; P < 0.05) after LPS. Lung chemokine production and PMN accumulation were also increased compared to vehicle-injected mice. Lung chemokine production and PMN accumulation were significantly lower in TNFRI KO, but not TNFRII KO, mice, despite no difference in TNF-alpha production (TNFRI KO, 925 +/- 301 vs TNFRII KO, 837 +/- 267, P = 0.82). CONCLUSIONS: Acute lung injury following systemic LPS administration is characterized by increased lung (1) TNF-alpha production, (2) C-X-C chemokine production, and (3) neutrophil accumulation. The maximal effect of LPS-induced lung neutrophil accumulation appears to be dependent upon the TNFRI receptor but not the TNFRII receptor. .     

The cytokine TWEAK modulates renal tubulointerstitial inflammation

TNF-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily of cytokines. In addition to binding and activating the fibroblast growth factor-inducible 14 receptor, TWEAK may regulate apoptosis, proliferation, and inflammation; however, the role of this system in kidney injury is unknown. In vitro, it was found that TWEAK induced the sustained activation of NF-kappaB in a murine tubular epithelial cell line (MCT). NF-kappaB activation was associated with degradation of IkappaB-alpha; translocation of RelA to the nucleus; and increased mRNA and protein expression of monocyte chemoattractant protein-1, RANTES, and IL-6. Similarly, in vivo, the systemic administration of TWEAK induced renal NF-kappaB activation, chemokine and IL-6 expression, and interstitial inflammation in mice. Parthenolide, which prevents IkappaB-alpha degradation, inhibited TWEAK-induced NF-kappaB activation and prevented the aforementioned changes in vitro and in vivo. After folic acid-induced acute kidney injury, fibroblast growth factor-inducible 14 expression increased in mouse tubular epithelium. Neutralization of TWEAK decreased the expression of chemokines in tubular cells and reduced interstitial inflammation. In conclusion, TWEAK has NF-kappaB-dependent proinflammatory effects on tubular epithelial cells in vitro and in vivo. Moreover, blockade of TWEAK reduces tubular chemokine expression and macrophage infiltration, suggesting that TWEAK modulates acute kidney injury by regulating the inflammatory response.     

Protective effect of a nuclear factor-kappaB inhibitor on ischemia-reperfusion injury in a rat epigastric flap model

We examined whether nuclear factor-kappa B (NF-kappaB) activation was involved in the ischemia-reperfusion (I/R) injury in a rat skin flap model and whether administration of pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor, could improve flap viability. Eighty-four Sprague-Dawley rats were divided into control group (n = 28), I/R group (n = 28), and PDTC-treated group (n = 28). An abdominal skin flap (4 x 5 cm) was elevated and subjected to 10 hours of ischemia in both the I/R group and the PDTC-treated group. A bolus of PDTC (300 mg/kg) was infused 5 minutes before reperfusion, followed by a second dose during the first 30 minutes of reperfusion in the PDTC-treated group. Flap tissues were assessed by electrophoretic mobility shift assay at 1, 2, 3, and 6 hours of reperfusion, and myeloperoxidase activity and neutrophil infiltration were assessed at 12 hours of reperfusion. The viability of flaps was assessed 7 days postoperatively. NF-kappaB was activated after reperfusion in the I/R group and displayed peak activity at 1 and 3 hours of reperfusion. In the PDTC-treated group, NF-kappaB activity was significantly reduced at 1, 2, and 6 hours of reperfusion. Myeloperoxidase activity was significantly decreased, and little neutrophil infiltration could be observed. In the PDTC-treated group, the survival of flaps was 86.88 +/- 13.63%, which was significantly greater than the I/R group, in which only 19.20 +/- 7.52% of the flap survived. NF-kappaB is activated during reperfusion in a rat skin flap I/R model. Administration of PDTC can significantly improve flap survival by regulating the early activation of NF-kappaB and suppressing neutrophil infiltration within the flap.     

Pressure-Time Curve Predicts Minimally Injurious Ventilatory Strategy in an Isolated Rat Lung Model

Background: We tested the hypothesis that the pressure-time (P-t) curve during constant flow ventilation can be used to set a noninjurious ventilatory strategy.

Methods: In an isolated, nonperfused, lavaged model of acute lung injury, tidal volume and positive end-expiratory pressure were set to obtain: (1) a straight P-t curve (constant compliance, minimal stress); (2) a downward concavity in the P-t curve (increasing compliance, low volume stress); and (3) an upward concavity in the P-t curve (decreasing compliance, high volume stress). The P-t curve was fitted to: P =a [middle dot] t b +c, where b describes the shape of the curve, b = 1 describes a straight P-t curve, b < 1 describes a downward concavity, and b > 1 describes an upward concavity. After 3 h, lungs were analyzed for histologic evidence of pulmonary damage and lavage concentration of inflammatory mediators. Ventilator-induced lung injury occurred when injury score and cytokine concentrations in the ventilated lungs were higher than those in 10 isolated lavaged rats kept statically inflated for 3 h with an airway pressure of 4 cm H2O.

Results: The threshold value for coefficient b that discriminated best between lungs with and without histologic and inflammatory evidence of ventilator-induced lung injury (receiver-operating characteristic curve) ranged between 0.90-1.10. For such threshold values, the sensitivity of coefficient b to identify noninjurious ventilatory strategy was 1.00. A significant relation (P < 0.001) between values of coefficient b and injury score, interleukin-6, and macrophage inflammatory protein-2 was found.     

MCP-1 induces inflammatory activation of human tubular epithelial cells: involvement of the transcription factors,nuclear factor-kappaB and activating protein-1

Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine synthesized by several cell types, e.g., inflammatory cells, such as monocytes, and resident renal cells, such as human tubular epithelial cells (TECs). Besides induction of monocyte recruitment, MCP-1 has been suggested to induce non-leukocytes to produce cytokines and adhesion molecules. Inflammation of the tubulointerstitium is a hallmark of many renal diseases and contributes to progression of renal failure; the purpose therefore of this study was to investigate the influence of MCP-1 on markers of inflammatory activation in human TECs. MCP-1 stimulated interleukin-6 (IL-6) secretion and intercellular adhesion molecule-1 (ICAM-1) synthesis in a time- and dose-dependent manner. In parallel, MCP-1 increased IL-6 and ICAM-1 mRNA expression in human TECs. Pretreatment with pertussis toxin, GF109203X, BAPTA-AM, and pyrrolidine dithiocarbamate inhibited MCP-1-dependent IL-6 and ICAM-1 synthesis, suggesting the involvement of Gi-proteins, protein kinase C, intracellular Ca(2+), and nuclear factor-kappaB (NF-kappaB) in MCP-1 signaling. Using electrophoretic gel mobility shift assay, we observed that MCP-1 stimulated binding activity of NF-kappaB. Binding activity of the activator protein-1 (AP-1), which has been implicated to regulate induction of the IL-6 gene together with NF-kappaB, was also stimulated by MCP-1. In the present experiments, NF-kappaB and AP-1 were involved in the MCP-1-mediated induction of IL-6, as demonstrated by cis element double-stranded (decoy) oligonucleotides (ODN). In contrast to IL-6 release, MCP-1-induced ICAM-1 expression was predominantly dependent on NF-kappaB activation. These results document for the first time that MCP-1 induces an inflammatory response in human TECs. This may be an important new mechanism in the pathogenesis of tubulointerstitial inflammation.