内毒素诱导大鼠急性肺损伤模型的动态研究

目的:建立内毒素诱导大鼠急性肺损伤的模型并筛选出敏感检测指标。方法:90只wistar大鼠随机分为10组,其中9组以气管内滴注内毒素(Lipopolysaccharide,LPS)建立大鼠急性肺损伤(Acute lung injury,ALI)模型,另一组作为空白对照组。观察造模8、12、24、36、48、72、96、120 h后的肺泡灌洗液(BALF)中肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)水平、肺组织的病理形态学变化、12h造模组与空白组的BALF中的多形核白细胞(PMN)百分比和蛋白浓度。结果:造模后BALF中的TNF-α、IL-6的浓度随时间延长显著升高且均在24 h达到峰值(P0.01);肺组织病理损伤也逐渐加重,12 h已出现明显的肺泡损伤、肺水肿、炎性细胞浸润等病变;12 h模型组BALF中PMN百分比和蛋白浓度较空白对照组显著增加(P0.01)。结论:在该实验条件下,气管内滴注LPS 8 mg/kg,12 h后即可建立ALI模型,可通过检测BALF中的TNF-α、IL-6浓度及肺组织的病变程度等指标进行模型评价。     

The effect of suramin on bleomycin-induced lung injury

Since transforming growth factor beta (TGF-beta) is presumed to play a role in lung fibrosis, we evaluated the effect of suramin (Sur), a substance with an anti-TGF-beta effect, in vivo on bleomycin (Bleo)-induced pulmonary injury in mice and in vitro on human lung fibroblasts. Four groups of C57BL/6 mice each received one of four treatments: (1) intratracheal (i.t.) instillation of Bleo and intraperitoneal (i.p.) injections of Sur, every other day, starting one day before i.t. instillation of Bleo (Bleo-Sur); (2) i.t. Bleo and i.p. injections of saline (Bleo-Sal); (3) i.t. saline and i.p. Sur (Sal-Sur); and (4) i.t. and i.p. saline (Sal-Sal). Animals were sacrificed 14 days after i.t. treatment. Lung injury was evaluated by analysis of bronchoalveolar lavage (BAL) fluid, histologically by the semiquantitative morphological index, and biochemically by analysis of lung hydroxyproline content. In vitro, Sur did not affect TGF-beta induced increase of alpha1 (I) collagen mRNA in human lung fibroblasts. In vivo treatment of mice with Sur did not affect Bleo-induced lung injury. These results indicate that despite its potential anti TGF-beta and lymphocytotoxic effects, Sur is not a therapeutic candidate drug for rescue of lung fibrosis.     

Sustained lung activity of a novel chimeric protein, SOD2/3, after intratracheal administration

Delivery of recombinant superoxide dismutase to the lung is limited by its short half-life and poor tissue penetration. We hypothesized that a chimeric protein, SOD2/3, containing the enzymatic domain of manganese superoxide dismutase (SOD2) and the heparan-binding domain of extracellular superoxide dismutase (SOD3), would allow for the delivery of more sustained lung and pulmonary vascular antioxidant activity compared to SOD2. We administered SOD2/3 to rats by intratracheal (i.t.), intraperitoneal (i.p.), or intravenous (i.v.) routes and evaluated the presence, localization, and activity of lung SOD2/3 1 day later using Western blot, immunohistochemistry, and SOD activity gels. The effect of i.t. SOD2/3 on the pulmonary and systemic circulation was studied in vivo in chronically catheterized rats exposed to acute hypoxia. Active SOD2/3 was detected in lung 1 day after i.t. administration but not detected after i.p. or i.v. SOD2/3 administration or i.t. SOD2. The physiologic response to acute hypoxia, vasoconstriction in the pulmonary circulation and vasodilation in the systemic circulation, was enhanced in rats treated 1 day earlier with i.t. SOD2/3. These findings indicate that i.t. administration of SOD2/3 effectively delivers sustained enzyme activity to the lung as well as pulmonary circulation and has a longer tissue half-life compared to native SOD2. Further testing in models of chronic lung or pulmonary vascular diseases mediated by excess superoxide should consider the longer tissue half-life of SOD2/3 as well as its potential systemic vascular effects.     

Adrenomedullin ameliorates lipopolysaccharide-induced acute lung injury in rats

Adrenomedullin (AM), an endogenous peptide, has been shown to have a variety of protective effects on the cardiovascular system. However, the effect of AM on acute lung injury remains unknown. Accordingly, we investigated whether AM infusion ameliorates lipopolysaccharide (LPS)-induced acute lung injury in rats. Rats were randomized to receive continuous intravenous infusion of AM (0.1 microg x kg(-1) x min(-1)) or vehicle through a microosmotic pump. The animals were intratracheally injected with either LPS (1 mg/kg) or saline. At 6 and 18 h after intratracheal instillation, we performed histological examination and bronchoalveolar lavage and assessed the lung wet/dry weight ratio as an index of acute lung injury. Then we measured the numbers of total cells and neutrophils and the levels of tumor necrosis factor (TNF)-alpha and cytokine-induced neutrophil chemoattractant (CINC) in bronchoalveolar lavage fluid (BALF). In addition, we evaluated BALF total protein and albumin levels as indexes of lung permeability. LPS instillation caused severe acute lung injury, as indicated by the histological findings and the lung wet/dry weight ratio. However, AM infusion attenuated these LPS-induced abnormalities. AM decreased the numbers of total cells and neutrophils and the levels of TNF-alpha and CINC in BALF. AM also reduced BALF total protein and albumin levels. In addition, AM significantly suppressed apoptosis of alveolar wall cells as indicated by cleaved caspase-3 staining. In conclusion, continuous infusion of AM ameliorated LPS-induced acute lung injury in rats. This beneficial effect of AM on acute lung injury may be mediated by inhibition of inflammation, hyperpermeability, and alveolar wall cell apoptosis.     

Acute lung injury augments hypoxic ventilatory response in the absence of systemic hypoxemia.

The objective of the present study was to examine the impact of early stages of lung injury on ventilatory control by hypoxia and hypercapnia. Lung injury was induced with intratracheal instillation of bleomycin (BM; 1 unit) in adult, male Sprague-Dawley rats. Control animals underwent sham surgery with saline instillation. Five days after the injections, lung injury was present in BM-treated animals as evidenced by increased neutrophils and protein levels in bronchoalveolar lavage fluid, as well as by changes in lung histology and computed tomography images. There was no evidence of pulmonary fibrosis, as indicated by lung collagen content. Basal core body temperature, arterial Po(2), and arterial Pco(2) were comparable between both groups of animals. Ventilatory responses to hypoxia (12% O(2)) and hypercapnia (7% CO(2)) were measured by whole body plethysmography in unanesthetized animals. Baseline respiratory rate and the hypoxic ventilatory response were significantly higher in BM-injected compared with control animals (P = 0.003), whereas hypercapnic ventilatory response was not statistically different. In anesthetized, spontaneously breathing animals, response to brief hyperoxia (Dejours' test, an index of peripheral chemoreceptor sensitivity) and neural hypoxic ventilatory response were augmented in BM-exposed relative to control animals, as measured by diaphragmatic electromyelograms. The enhanced hypoxic sensitivity persisted following bilateral vagotomy, but was abolished by bilateral carotid sinus nerve transection. These data demonstrate that afferent sensory input from the carotid body contributes to a selective enhancement of hypoxic ventilatory drive in early lung injury in the absence of pulmonary fibrosis and arterial hypoxemia.     

Modulation of endoperoxide product levels and cyclophosphamide-induced injury by glutathione repletion

Glutathione is a tripeptide important in a number of diverse cellular functions including enzymatic reactions involved in prostaglandin endoperoxide metabolism. We have previously reported that cyclophosphamide administration to rats results in acute lung injury manifested by increased bronchoalveolar lavage albumin concentrations. In the current study we examine whether cyclophosphamide treatment affects pulmonary glutathione stores or bronchoalveolar endoperoxide metabolic product levels and whether these effects may be related to acute lung injury caused by the drug. We show that cyclophosphamide treatment causes a dose-dependent reduction in pulmonary glutathione stores 4 h after drug administration. In addition, acute lung injury as the result of cyclophosphamide can be abrogated by coadministration of oxothiazolidine carboxylate, an intracellular cysteine delivery system that also reverses pulmonary glutathione depletion induced by cyclophosphamide in our study. Finally, cyclophosphamide treatment reduces prostaglandin E2 concentrations in bronchoalveolar lavage and alveolar macrophage culture supernatant in a dose-dependent fashion and increases bronchoalveolar thromboxane concentrations in low dose-treated animals. These effects are reversed to a variable degree by coadministration of oxothiazolidine carboxylate. Our study suggests in vivo pulmonary arachidonic acid metabolism and cyclophosphamide-induced acute lung injury are modulated by cellular glutathione stores. These findings may have important implications for the treatment of acute lung injury.     

Halofuginone does not reduce fibrosis in bleomycin-induced lung injury

Halofuginone, a coccidiostatic alkaloid, has anti-fibrotic properties, and may be useful as a therapeutic agent in lung fibrosis. To test this hypothesis we investigated the effect of halofuginone on bleomycin-induced lung fibrosis in Sprague-Dawley rats. Treatment groups included: (1) a single intratracheal (IT) instillation of 1.2U bleomycin, and intraperitoneal (IP) injection of halofuginone (0.5 mg/dose), every other day; (2) IT 1.2U bleomycin and IP distilled water (D.W.), every other day; (3) IT 0.8U bleomycin and daily IP halofuginone (0.5 mg/dose); (4) IT 0.8U bleomycin and daily IP D.W.; (5) IT saline and IP halofuginone, every other day; (6) IT saline and daily IP D.W.; (7) IT 0.625U bleomycin and oral halofuginone (10 mg/kg rodent lab chow); (8) IT 0.625U bleomycin and standard lab chow. Animals were studied 14 days after IT instillation. Lung injury was evaluated by total and differential cell count in bronchoalveolar lavage fluid, by a semi-quantitative morphological index of lung injury, and by biochemical analysis of lung hydroxyproline content. Overt signs of lung injury were apparent in bleomycin-treated rats by all measures. These changes were not affected by treatment with halofuginone, irrespective of the treatment regimen used. This study does not support the use of halofuginone to prevent or ameliorate lung fibrosis.     

Effect of surfactant on pulmonary expression of type IIA PLA(2) in an animal model of acute lung injury

We previously showed that the seminatural surfactant Curosurf inhibits the in vitro synthesis of secretory type IIA phospholipase A(2) (sPLA(2)-IIA) in alveolar macrophages (AM). These cells are the main source of sPLA(2)-IIA in a guinea pig model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Here, we investigate the effect of Curosurf on the pulmonary synthesis of sPLA(2)-IIA in this ALI model. Our results showed that intratracheal administration of LPS (330 microg/kg) induced an increase in pulmonary expression of sPLA(2)-IIA, which was inhibited when animals received Curosurf (16 mg/guinea pig) 30 min or 8 h after LPS instillation. When AM were isolated from LPS-treated animals and cultured in conditioned medium, they expressed higher levels of sPLA(2)-IIA than AM from saline-treated animals. This ex vivo sPLA(2)-IIA expression was significantly reduced when guinea pigs received Curosurf 30 min after LPS instillation. Finally, we examined the effect of Curosurf on pulmonary inflammation measured 8 or 24 h after LPS administration. Curosurf instillation 30 min or 8 h after LPS reversed the increase in tumor necrosis factor-alpha expression, polymorphonuclear cell extravasation, and protein concentration in bronchoalveolar lavage fluids. Curosurf also decreased the bronchial reactivity induced by LPS. We conclude that Curosurf inhibits the pulmonary expression of sPLA(2)-IIA and exhibits palliative anti-inflammatory effects in an animal model of ALI.     

Introduction of the interleukin-10 gene into mice inhibited bleomycin-induced lung injury in vivo

Interleukin (IL)-10 has been shown to reduce many inflammatory reactions. We investigated the in vivo effects of IL-10 on a bleomycin-induced lung injury model. Hemagglutinating virus of Japan (HVJ)-liposomes containing a human IL-10 expression vector (hIL10-HVJ) or a balanced salt solution as a control (Cont-HVJ) was intraperitoneally injected into mice on day -3. This was followed by intratracheal instillation of bleomycin (0.8 mg/kg) on day 0. Myeloperoxidase activity of bronchoalveolar lavage fluid and tumor necrosis factor-alpha mRNA expression in bronchoalveolar lavage fluid cells on day 7 and hydroxyproline content of the whole lung on day 21 were inhibited significantly by hIL10-HVJ treatment. However, Cont-HVJ treatment could not suppress any of these parameters. We also examined the in vitro effects of IL-10 on the human lung fibroblast cell line WI-38. IL-10 significantly reduced constitutive and transforming growth factor-beta-stimulated type I collagen mRNA expression. However, IL-10 did not affect the proliferation of WI-38 cells induced by platelet-derived growth factor. These data suggested that exogenous IL-10 may be useful in the treatment of pulmonary fibrosis.     

Influence of DMT1 and iron status on inflammatory responses in the lung

Divalent metal transporter 1 (DMT1) is the major iron transporter responsible for duodenal dietary iron absorption and is required for erythropoiesis. Recent studies suggest that loss of DMT1 activity could be involved in metal-related lung injury, but little is known about the effects of iron status and DMT1 function on pulmonary inflammation. To better define the role of DMT1 and iron status in pulmonary inflammatory responses, we performed bronchoalveolar lavage (BAL) following intratracheal instillation of lipopolysaccharide (LPS) to the Belgrade rat, an animal model deficient in DMT1 function. In the basal state, the BAL fluid of Belgrade rats had more macrophages and higher lactate dehydrogenase, myeloperoxidase, albumin, and hemoglobin levels compared with heterozygote control rats. Following LPS instillation, the macrophage fraction relative to total BAL cell content and levels of albumin and IgM were increased in Belgrade rats compared with controls. In contrast, heterozygote Belgrade rats made anemic by diet-induced iron deficiency exhibited attenuated inflammatory responses to LPS. These combined results show that pulmonary inflammation can be modified by both DMT1 and iron status. Loss of DMT1 alters pulmonary responses necessary for lung homeostasis in the basal state and enhances LPS-induced inflammation and therefore would contribute to progression of lung injury.     

Inducible lung-specific urokinase expression reduces fibrosis and mortality after lung injury in mice

Plasminogen activator inhibitor-1 (PAI-1)-deficient transgenic mice have improved survival and less fibrosis after intratracheal bleomycin instillation. We hypothesize that PAI-1 deficiency limits scarring through unopposed plasminogen activation. If this is indeed true, then we would expect increased urokinase-type plasminogen activator (uPA) expression to result in a similar reduction in scarring and improvement in mortality. To test our hypothesis, using the tetracycline gene regulatory system, we have generated a transgenic mouse model with the features of inducible, lung-specific uPA production. After doxycycline administration, these transgenic animals expressed increased levels of uPA in their bronchoalveolar lavage (BAL) fluid that accelerated intrapulmonary fibrin clearance. Importantly, this increased plasminogen activator production led to a reduction in both lung collagen accumulation and mortality after bleomycin-induced injury. These results suggest that PAI-1 deficiency does protect against the effects of bleomycin-induced lung injury through unopposed plasmin generation. By allowing the manipulation of plasminogen activation at different phases of the fibrotic process, this model will serve as a powerful tool in further investigations into the pathogenesis of pulmonary fibrosis.     

Abatement of bleomycin-induced pulmonary injury by cell-impermeable inhibitor of phospholipase A2

The mechanism of bleomycin (Bleo)-induced pulmonary injury is not fully understood. Elevated levels of lung phospholipase A₂ (PLA₂) have been previously reported following intratracheal (IT) instillation of Bleo, but the role of this enzyme in the pathogenesis of lung injury is not clear.In this pilot study, we have evaluated the effect of a cell impermeable inhibitor of PLA₂ (CME) on Bleo-induced pulmonary inflammation in hamsters. Pulmonary injury was induced by a single IT instillation of Bleo (1 unit/0.5 ml saline). Three groups of male Syrian hamsters were evaluated: 1) BLEO-CME animals received IT Bleo and daily intraperitoneal (IP) injections of CME (1 μmole/kg), starting 1 day before IT instillation; 2) BLEO-SAL animals-received IT Bleo and IP injections of saline and 3) SAL-SAL animals — treated with IT and IP administrations of saline. Animals were sacrificed 14 days after IT treatment and lung injury was evaluated histologically by a semiquantitative morphologic index and by a differential cell count of bronchoalveolar lavage fluid. CME treatment significantly ameliorated Bleo-induced lung injury compared to BLEO-SAL animals (P < 0.05). The percentage of neutrophiles in bronchoalveolar lavage fluid was reduced from 17.7 ± 3.2% (mean ± S.E.) in BLEO-SAL group to 7.3 ± 1.7% in BLEO-CME group (P < 0.05), achieving levels comparable to SAL-SAL control animals. These results suggest that treatment with an extracellular PLA₂ inhibitor-CME abates Bleo-induced pulmonary injury. This may indicate an active role of PLA₂ in the pathogenesis of interstitial pulmonary fibrosis.     

Body temperature control in sepsis-induced acute lung injury

Body temperature is precisely regulated to maintain homeostasis in homeothermic animals. Although it remains unproved whether change of body temperature constitutes a beneficial or a detrimental component of the septic response, temperature control should be an important entity in septic experiments. We investigated the effect of body temperature control on the lipopolysaccharide (LPS)-induced lung injury. Acute lung injury in rats was induced by intratracheal spray of LPS and body temperature was either clamped at 37 degrees C for 5 hours or not controlled. The severity of lung injury was evaluated at the end of the experiment. Intratracheal administration of aerosolized LPS caused a persistent decline in body temperature and a significant lung injury as indicated by an elevation of protein-concentration and LDH activity in the bronchoalveolar lavage (BAL) fluid and wet/dry weight (W/D) ratio of lungs. Administration of LPS also caused neutrophil sequestration and lipid peroxidation in the lung tissue as indicated by increase in myeloperoxidase (MPO) activity and malondialdehyde (MDA) production, respectively. Control of body temperature at 37 degrees C after LPS (LPS/BT37, n = 11) significantly reduced acute lung injury as evidenced by decreases in BAL fluid protein concentration (983 +/- 189 vs. 1403 +/- 155 mg/L) and LDH activity (56 +/- 10 vs. 123 +/- 17 deltamAbs/min) compared with the LPS group (n = 11). Although the W/D ratio of lung and MDA level were lower in the rats received temperature control compared with those received LPS only, the differences were not statistically significant. Our results demonstrated that intratracheal administration of aerosolized LPS induced a hypothermic response and acute lung injury in rats and controlling body temperature at a normal range may alleviate the LPS-induced lung injury.     

Human insulin-like growth factor-IA expression in transgenic mice promotes adenomatous hyperplasia but not pulmonary fibrosis

Insulin-like growth factor-I (IGF-I) has been implicated in postnatal alveolar development, pulmonary fibrosis, and non-small cell lung cancer. To further investigate the role of IGF-I, we created a line of transgenic mice in which alveolar type II epithelial cells express human IGF-IA under the control of the surfactant protein C promoter. We determined the effect of pulmonary overexpression of human IGF-IA on 1) pulmonary inflammation and fibrosis in response to intratracheal instillation of bleomycin, 2) premalignant pulmonary adenomatous hyperplasia, and 3) adenoma formation. Transgenic expression of human IGF-IA had no effect on baseline gross lung pathology, cellularity of bronchoalveolar lavage, or total lung collagen content. In addition, there were no significant differences between transgenic mice and nontransgenic littermate controls in the development of pulmonary inflammation or pulmonary fibrosis in response to intratracheal bleomycin instillation. However, pulmonary expression of human IGF-IA in older mice (>12 mo) significantly increased the incidence of premalignant adenomatous hyperplastic lesions compared with littermate controls without affecting adenoma formation. These findings suggest that increased expression of human IGF-IA in alveolar air spaces does not affect the development of pulmonary fibrosis but promotes premalignant changes in the alveolar epithelium.     

Intratracheal gene transfer of tissue factor pathway inhibitor attenuates pulmonary fibrosis

Activation of the coagulation system and increased expression of tissue factor (TF) in pulmonary fibrosis associated with acute and chronic lung injury have been previously documented. In the present study, we evaluated the effect of TF inhibition with intratracheal gene transfer of tissue factor pathway inhibitor (TFPI), a potent and highly specific endogenous inhibitor of TF-dependent coagulation activation, in a rat model of bleomycin-induced lung fibrosis. Significant lung fibrotic changes as assessed by histologic findings and hydroxyproline content, and increased procoagulant activity and thrombin generation in bronchoalveolar lavage fluid were detected in rats after intratracheal injection of bleomycin. Intratracheal administration of an adenovirus vector expressing TFPI significantly decreased bleomycin-induced procoagulant and thrombin generation resulting in a strong inhibition of pulmonary fibrosis. TFPI-overexpression in the lung was associated with a significant reduction in gene expression of the connective tissue growth factor, a potent profibrotic growth factor. This is the first report showing that direct inhibition of TF-mediated coagulation activation abrogates bleomycin-induced pulmonary fibrosis.     

Neutrophil defensins mediate acute inflammatory response and lung dysfunction in dose-related fashion

High concentrations of neutrophil defensins from airway and blood have been reported in patients with inflammatory lung diseases, but their exact role is unclear. We investigated the direct effect of defensins on the lungs of mice. Intratracheal instillation of purified defensins (5-30 mg/kg) induced a progressive reduction in peripheral arterial O(2) saturation, increased lung permeability, and enhanced the lung cytochrome c content. These indexes of acute lung dysfunction were associated with an increased total cell number and a significant neutrophil influx into the lung [5.1 +/- 0.04% in control vs. 48.6 +/- 12.7% in the defensin (30 mg/kg) group, P < 0.05]. Elastase concentrations in the bronchoalveolar lavage (BAL) fluids increased from 38 +/- 11 ng/ml (control) to 80 +/- 4 ng/ml (defensins, P < 0.05). Five hours after defensin instillation, concentrations of tumor necrosis factor-alpha and macrophage inflammatory protein-2 in BAL fluid were significantly increased. High levels of monocyte chemoattractant protein-1 in BAL fluid and plasma were also found after defensin stimulation. We conclude that intratracheal instillation of defensins causes acute lung inflammation and dysfunction, suggesting that high concentrations of defensins in the airways may play an important role in the pathogenesis of inflammatory lung diseases.     

Role of interferon-gamma in the evolution of murine bleomycin lung fibrosis

IFN-gamma production is upregulated in lung cells (LC) of bleomycin-treated C57BL/6 mice. The present study characterizes the time course, cellular source, and regulation of IFN-gamma expression in bleomycin-induced lung injury. IFN-gamma mRNA in LC from bleomycin-treated mice peaked 3 days after intratracheal instillation. IFN-gamma protein levels were increased at 6 days, as was the percentage of LC expressing IFN-gamma. CD4+, CD8+, and natural killer cells each contributed significantly to IFN-gamma production. IL-12 mRNA levels were increased at 1 day in LC of bleomycin-treated mice. Anti-IL-12 and anti-IL-18 antibodies decreased IFN-gamma production by these cells. To define the role of endogenous IFN-gamma in the evolution of bleomycin lung injury, we compared the effect of bleomycin in mice with a targeted knockout mutation of the IFN-gamma gene (IFN-gamma knockout) and wild-type mice. At 14 days after intratracheal bleomycin, total bronchoalveolar lavage cell counts and lung hydroxyproline were decreased in IFN-gamma knockouts compared with wild-type animals. There was no difference in morphometric parameters of fibrosis. Our data show that enhanced IFN-gamma production in the lungs of bleomycin-treated mice is at least partly IL-12 and IL-18 dependent. Absence of IFN-gamma in IFN-gamma knockout mice does not increase pulmonary fibrosis. Endogenous IFN-gamma may play a proinflammatory or profibrotic role in bleomycin-induced lung fibrosis.     

Role of insulin on PGE2 generation during LPS-induced lung inflammation in rats

Alterations in arachidonic acid (AA) metabolism have been reported to occur in diabetes mellitus. The present study was carried out to verify if these alterations are due to the relative lack of insulin or to high levels of blood glucose. Male Wistar rats were rendered diabetic by alloxan injection (42 mg/kg, i.v.), 10 or 30 days before the experiments. Some diabetic rats received a single dose (4 IU, s.c.) of NPH insulin 2 h before an intratracheal instillation of lipopolysaccharide (LPS, 750 microg) or saline. Six hours after LPS challenge, the following parameters were analysed: blood glucose levels, total and differential leukocyte counts in bronchoalveolar lavage (BAL) fluid; linoleic acid and AA content in blood neutrophils (HPLC), and levels of prostaglandin (PG)E(2) in BAL (ELISA). Relative to controls, a reduced number of neutrophils (18%) and decreased amounts of PGE(2) (40%) were observed in the BAL fluid of diabetic rats in response to LPS. A single dose of insulin was not able to reduce blood sugar levels to normal values, but instead resulted in the normalization of both leukocyte migration to the lungs and levels of PGE(2). Accordingly, these abnormalities might be primarily linked to a continuing insulin deficiency rather than to secondary hyperglycaemia occurring in the diabetic rat. In conclusion, data presented suggest that insulin might regulate neutrophil migration and generation of PGE(2) during the course of acute lung injury induced by LPS.     

Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice

Activation of the Fas/Fas ligand (FasL) system in the lungs results in a form of injury characterized by alveolar epithelial apoptosis and neutrophilic inflammation. Studies in vitro show that Fas activation induces apoptosis in alveolar epithelial cells and cytokine production in alveolar macrophages. The main goal of this study was to determine the contribution of alveolar macrophages to Fas-induced lung inflammation in mice, by depleting alveolar macrophages using clodronate-containing liposomes. Liposomes containing clodronate or PBS were instilled by intratracheal instillation. After 24 h, the mice received intratracheal instillations of the Fas-activating monoclonal antibody Jo2 or an isotype control antibody and were studied 18 h later. The Jo2 MAb induced increases in bronchoalveolar lavage fluid (BALF) total neutrophils, lung caspase-3 activity, and BALF total protein and worsened histological lung injury in the macrophage-depleted mice. Studies in vitro showed that Fas activation induced the release of the cytokine KC in a mouse lung epithelial cell line, MLE-12. These results suggest that the lung inflammatory response to Fas activation is not primarily dependent on resident alveolar macrophages and may instead depend on cytokine release by alveolar epithelial cells.     

Role of complement in endotoxin/platelet-activating factor-induced lung injury.

C receptor-1 is a protein involved in the regulation of C3 and C5-convertases. Recombinant human soluble C receptor-1 has recently been produced and shown to reduce infarct size in a rat model of myocardial ischemia/reperfusion injury. The present study aimed to investigate whether recombinant human soluble C receptor-1 exerts any protective effect on pulmonary injury produced in a rodent model of adult respiratory distress syndrome. In this model, Escherichia coli endotoxin (LPS, 0.1 microgram/kg) combined with platelet-activating factor (1 pmol/kg/min over 60 min, n = 10) caused microvascular lung injury characterized by elevation of myeloperoxidase activity, deposition of C3 and C5b-9 on the endothelium of pulmonary vessels, and pulmonary edema. Furthermore, bronchoalveolar lavage revealed increased neutrophil count and elevated protein concentration. These pulmonary responses were associated with elevated serum TNF-alpha. Pretreatment (10 min, i.v.) with recombinant human soluble C receptor-1 at 10 mg/kg (n = 13), but not at 1 mg/kg, prevented the LPS/platelet-activating factor-induced pulmonary edema (p less than 0.01) and changes in the bronchoalveolar lavage fluid cell count (p less than 0.01) and protein concentration (p less than 0.05), and attenuated the deposition of C3 and C5b-9 to lung vessels. There was no effect on lung myeloperoxidase activity and serum TNF-alpha. Also, C depletion by cobra venom factor (500 U/kg, i.v.) eliminated the pulmonary edema and elevated leukocyte count in bronchoalveolar lavage fluid, but had no effect on lung myeloperoxidase activity and serum TNF-alpha. These data suggest that C factors may play an important role in the pathophysiology of adult respiratory distress syndrome.