Characterization of matrix metalloproteinases produced by rat alveolar macrophages.

Evidence presented in the accompanying article (Gibbs, D. F., T. P. Shanley, R. L. Warner, H. S. Murphy, J. Varani, and K. J. Johnson. 1999. Role of matrix metalloproteinases in models of macrophage-dependent acute lung injury: evidence for alveolar macrophage as source of proteinases. Am. J. Respir. Cell Mol. Biol. 20:1145-1154) implicates alveolar macrophage matrix metalloproteinases (MMPs) in two models of acute lung inflammation in the rat. As a prerequisite to understanding which specific MMPs might be involved in the injury and how they might function, it was necessary to know the spectrum of enzymes present. To this end, alveolar macrophages were obtained from normal rat lungs by bronchoalveolar lavage, placed in culture with and without various agonists, and assessed by a variety of techniques for MMPs. The identification process involved characterization by gelatin, beta-casein, and kappa-elastin zymography, with confirmation of identity by Western blot/immunoprecipitation. Message levels of detected MMPs were assessed by Northern blot. Rat alveolar macrophages were found to produce a low constitutive level of MMP-2 (72-kD gelatinase A) that was only modestly upregulated following stimulation with phorbol myristate acetate, bacterial lipopolysaccharide, or immunoglobulin A-containing immune complexes. Although control cells were found to produce little or no MMP-9 (92-kD gelatinase B) or MMP-12 (metalloelastase), both enzymes were markedly upregulated upon stimulation. In the same stimulated macrophages there was little activity against type I collagen (associated with MMP-13 [collagenase-3] on the basis of Western blotting), no activity suggestive of stromelysin or matrilysin, and no measurable secretion of the serine proteinases, elastase and cathepsin G. These data demonstrate the ability of rat alveolar macrophages to elaborate certain MMPs under proinflammatory conditions, consistent with their possible involvement in the progression of acute inflammation.     

中性粒细胞弹性蛋白酶、髓过氧化物酶在大鼠哮喘中的变化及意义

目的：观察中性粒细胞（PMN）、中性粒细胞弹性蛋白酶（NE）和髓过氧化物酶（MPO）在大鼠哮喘中的表达水平变化及意义。方法:18只大鼠被随机平均分成2组：哮喘组、正常对照组，以卵清白蛋白（OVA）致敏激发法复制大鼠哮喘模型，对血PMN进行分离纯化，免疫组化和比色法检测MPO的表达水平，ELISA法测定NE的蛋白浓度。 结果:（1）免疫组化法显示哮喘组血PMN和支气管壁中MPO的表达水平均显著高于对照组（P<0.01），比色法显示哮喘组支气管肺泡灌洗液（BALF）和肺组织中MPO的活性显著高于对照组（P<0.05，P<0.01）；（2）哮喘组PMN和BALF中NE蛋白浓度显著高于对照组（P<0.01）；（3）哮喘组BALF、支气管壁、肺组织中PMN的计数均显著高于对照组（P<0.01）。 结论:PMN 计数、NE和MPO的表达水平在此实验性哮喘中增加，PMN可能通过分泌NE、MPO参与哮喘炎症过程。     

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.     

Contrasting roles for tumor necrosis factor in the pathogeneses of IgA and IgG immune complex lung injury.

Recent studies suggest that development of acute gamma G immunoglobulin (IgG) immune complex lung injury is partially dependent on a tumor necrosis factor (TNF)-dependent mechanisms of neutrophil (PMN) recruitment. The authors have sought to further define the role of intrapulmonary TNF in IgG alveolitis and to examine its role in IgA immune complex alveolitis, a neutrophil-independent model of acute lung injury. IgG immune complex lung injury resulted in a marked rise in intrapulmonary TNF activity accompanied by progressive pulmonary PMN accumulation. Intratracheal instillation of neutralizing concentrations of anti-TNF markedly reduced PMN influx measured at 4 hours but had no effect on PMN recruitment quantitated at 2 hours. IgA immune complex deposition resulted in acute lung injury accompanied by increased numbers of intrapulmonary mononuclear phagocytes but few neutrophils. Lung lavage fluids obtained from IgA immune complex-injured rats contained both neutrophil and monocyte chemotactic activities, albeit at twofold to fourfold lower concentrations than observed in IgG-mediated alveolitis. In contrast to IgG complex-mediated alveolitis, lung lavage fluids from IgA-injured rats contained no TNF activity. Intratracheal administration of anti-TNF antibodies had no effect on the development of IgA lung injury as assessed by morphology and measurements of vascular permeability. In vitro exposure of isolated alveolar macrophages to performed IgG immune complexes resulted in dose-dependent TNF secretion, while exposure to IgA complexes resulted in very low levels of TNF secretion. These data suggest that TNF-mediated pulmonary neutrophil recruitment (in IgG lung injury) is manifest chiefly in the late phase (approximately 4 hours) of developing alveolitis. The virtual absence of intrapulmonary TNF activity in evolving IgA immune complex alveolitis may in part account for the limited PMN recruitment observed in this model.     

外源性一氧化氮抗内毒素致肺微血管高通透性的作用机制初探

目的和方法:应用气管内滴注脂多糖(LPS)致SD大鼠急性肺损伤(ALI)模型和体外培养人血中性粒细胞(PMN),观察一氧化氮(NO)供体硝普钠(SNP)对LPS所致肺内PMN聚集、微血管通透性增高及PMN凋亡的影响。结果:①整体实验中LPS组(气管滴注LPS100μg/只)的支气管肺泡灌洗液(BALF)中蛋白含量、PMN数量、肺组织中伊文思蓝(EB)含量和染料单星蓝(MB)颗粒标记的肺微血管数目均明显高于假手术组,而LPS+SNP组(气管同时滴注LPS和SNP5μg/只)上述指标均明显低于LPS组;②体外培养人血PMN,SNP(5×10^-3mol/L)组的PMN凋亡百分率明显高于对照组,SNP+LPS组明显高于LPS(10μg/L)组。结论:气管内滴注SNP能够减少PMN在肺内的聚集,在一定程度上起到抗LPS所致的以肺微血管高通透性为特征的ALI的作用。促进PMN凋亡可能是SNP减轻PMN在肺内聚集的机制之一。     

Effects of Granulocyte Colony-Stimulating Factor (G-CSF) and Neutrophil Elastase Inhibitor (ONO-5046) on Acid-Induced Lung Injury in Rats

It has been suggested that neutrophils play an important role in acid-aspirated lung injury. We examined the effects of the high dose of granulocyte-colony stimulating factor (G-CSF), which is capable of increasing peripheral neutrophils, and a specific neutrophil elastase inhibitor (ONO-5046) on acid lung injury in rats. Animals were anesthetized and normal saline (NS, 2 mL kg⁻¹) or hydrochloric acid (HCl, 0.1 N 2 mL kg⁻¹) was then instilled into trachea. Thirty minutes before HCl instillation, G-CSF (150 μg kg⁻¹) was injected subcutaneously or ONO-5046 (10 mg kg⁻¹ h⁻¹) was infused continuously into the right jugular vein. Animals were ventilated during the experiments. Five hours after HCl or NS instillation, bronchoalveolar lavage fluid (BALF) and lung tissue samples were obtained. Total nuclear cell count, absorbance, albumin, tumor necrosis factor (TNF)-α, interleukin (IL)-6, cytokine-induced neutrophil chemoattractant (CINC), neutrophil elastase in BALF, wet-to-dry (W/D) ratio were measured. HCl aspiration markedly increased these values in BALF and W/D ratio. Both ONO-5046 and G-CSF attenuated the parameters increased by acid-induced lung injury in rats. The data suggests that neutrophils play an important role in acid-induced lung injury. However, high-dose G-CSF does not exacerbate acid-aspirated lung injury in rats, although this agent causes an increase in peripheral neutrophils.     

Change in the concentration of neutrophil elastase in bronchoalveolar lavage fluid during anesthesia and its inhibition by cholesterol sulfate

Cholesterol sulfate (CS) in the gastrointestinal tract exhibits a mucosal protective activity in mouse ulcer model. To clarify the possible role of CS for protection from the epithelial injury due to neutrophil elastase in the tracheobronchi, the authors determined the concentrations of CS and neutrophil elastase in bronchoalveolar lavage fluid (BALF) from patients under anesthesia, and they examined the inhibitory activity of CS toward neutrophil elastase. The concentrations of CS and neutrophil elastase were determined by thin-layer chromatography and enzyme-linked immunosorbent assaying, respectively, and the effect of CS on the activity of elastase was determined with a chromogenic substrate. CS was found to be present in human lung, tracheal mucosa, and BALF, and a high synthesis of it was detected in the tracheal mucosa, in which cellular cholesterol sulfotransferase was induced depending on the density of tracheal cells. Among lipids in the tracheal mucosa, only CS was demonstrated to exhibit inhibitory activity toward neutrophil elastase, a powerful erosive agent in inflammation. The secretion of elastase from neutrophils into BALF was stimulated during the course of general anesthesia. In contrast, the amount of CS in BALF gradually decreased during anesthesia. On immune-precipitation of neutrophil elastase in BALF, CS was detected in the immune precipitate, which indicates a possible association of CS with neutrophil elastase in BALF. Conclusion: CS, which is a major acidic lipid in the tracheobronchial epithelium, might function as an epithelial inhibitor toward neutrophil elastase secreted in response to several stimuli such as anesthesia.     

Intrapulmonary tumor necrosis factor triggers local platelet-activating factor production in rat immune complex alveolitis

Recent studies suggest that intrapulmonary tumor necrosis factor (TNF) participates in the pathogenesis of acute IgG immune complex alveolitis through a mechanism involving neutrophil polymorphonuclear leukocyte (PMN) recruitment. There are few in vivo studies that address mechanisms of TNF-dependent PMN recruitment and tissue injury. We have examined the relationship between intrapulmonary TNF and locally generated platelet-activating factor (PAF) in the development of acute alveolitis. Intratracheal instillation of IgG anti-bovine serum albumin followed by intravenous bovine serum albumin results in acute neutrophil-mediated alveolitis. Induction of IgG immune complex lung injury resulted in a marked increase in the whole lung and bronchoalveolar lavage PAF levels. Intratracheal instillation of the PAF antagonists, L-652,731 (Merck, Sharpe, and Dohme, Rahway, New Jersey) or WEB-2086 (Boehringer), attenuated pulmonary vascular leakage and PMN recruitment into the alveolar compartment. Neutralization of intrapulmonary TNF with anti-TNF antibodies reduced pulmonary vascular permeability, PMN recruitment, and whole lung PAF levels. Incubation of isolated mouse alveolar macrophages with recombinant murine TNF resulted in rapid (30 to 60 minutes), cell concentration-dependent PAF release. The presence of high concentrations of PAF in whole lungs obtained from PMN-depleted rats after immune complex deposition suggest that recruited PMN are not the predominant source of intrapulmonary PAF in this model. These data suggest that acute IgG immune complex alveolitis is in part mediated by TNF-triggered PAF production and that locally produced PAF promotes recruitment of PMN into the alveolar compartment.     

槲皮素对重症急性胰腺炎肺损伤大鼠肺泡中性粒细胞凋亡的影响

 目的：探讨槲皮素对重症急性胰腺炎（SAP）大鼠肺泡中性粒细胞（polymorphonuclear neutrophil, PMN）凋亡的影响及机制。方法：48只成年SD大鼠随机分为4组,每组12只,即假手术（sham组）、SAP组、槲皮素低剂量（50 mg/kg）组及槲皮素高剂量（100 mg/kg）组。逆行性胆胰管注射5%牛磺胆酸钠建立SAP大鼠模型。槲皮素组在胰腺炎模型基础上予槲皮素干预,sham组和SAP组给予等量生理盐水。经支气管肺泡灌洗分离收集PMN。荧光显微镜观察细胞形态学变化;流式细胞术检测PMN凋亡指数;Western blotting检测Bax和Bcl-2蛋白表达;荧光分光光度计法检测caspase-3活性。结果：荧光显微镜下观察发现SAP组PMN固缩,核染色质凝聚;与sham组比较,SAP组PMN凋亡指数降低,Bax蛋白表达减弱,Bcl-2蛋白表达增强,caspase-3活性减弱（P<0.01）;而槲皮素处理后PMN凋亡指数升高,Bax蛋白表达增强,Bcl-2蛋白表达减弱,caspase-3活性增强（P<0.05）,呈现浓度依赖性,而且,槲皮素能减轻SAP引起的肺损伤。结论：SAP时存在肺泡PMN凋亡延迟,槲皮素通过上调PMN Bax蛋白表达和抑制Bcl-2蛋白表达,诱导PMN凋亡。槲皮素减轻SAP引起的肺损伤可能与其诱导PMN凋亡有关。     

白藜芦醇通过激活SGK1上调急性肺损伤小鼠肺泡上皮钠离子通道的表达

目的探讨白藜芦醇(resveratrol)对急性肺损伤(ALI)小鼠肺泡上皮钠离子通道(ENaC)的作用及可能机制。方法将小鼠随机分为对照(control)组、LPS组、RES组和PP242组,每组6只。苏木精-伊红(HE)染色观察肺组织病理;BCA法测肺泡灌洗液(BALF)中蛋白含量,酶联免疫吸附实验(ELISA)检测炎性因子水平;流式细胞计量术检测BALF中性粒细胞比例;Western blot检测肺组织α-ENaC蛋白表达和SGK1磷酸化水平,实时荧光定量PCR(qPCR)检测肺组织α-ENaC mRNA转录水平。结果 1)与对照组相比,LPS组肺组织损伤明显,BALF中性粒细胞比例、蛋白含量和炎性因子水平明显升高(P0.05),肺组织α-ENaC表达和SGK1磷酸化水平显著下调(P0.05);2)与LPS组相比,RES组肺损伤明显减轻,BALF中性粒细胞比例、蛋白含量和炎性因子水平明显降低(P0.05),伴α-ENaC表达和SGK1磷酸化水平显著上调(P0.05);3)与RES组相比,PP242组肺损伤明显加重,BALF中性粒细胞比例、蛋白含量和炎性因子水平明显升高(P0.05),同时伴α-ENaC表达和SGK1磷酸化水平显著下调(P0.05)。结论 SGK1介导的α-ENaC上调机制参与了RES对ALI的保护作用。     

Prolonged monocyte accumulation in the lung during bleomycin-induced pulmonary fibrosis. A noninvasive assessment of monocyte kinetics by scintigraphy.

It has become more evident that monocytes, macrophages, and their products interact in a complex manner with various cell types in the lung, and may under the proper set of conditions contribute to the pathogenesis of pulmonary fibrosis. Current methods used to assess the lung content of mononuclear cells, which include tissue immunohistochemistry and bronchoalveolar lavage fluid analysis, sample the lung at one point in time and therefore provide only a "snapshot" of dynamic process. We utilized external imaging (scintigraphy) to provide a dynamic assessment of the trafficking patterns of radiolabeled monocytes in the lungs of rabbits in conjunction with lung tissue morphometry and bronchoalveolar lavage fluid analysis to determine the kinetics of neutrophil and monocyte accumulation in the alveolar walls and alveolar spaces of the lung during bleomycin-induced pulmonary fibrosis. We found that scintigraphy accurately reflected the accumulation of monocyte-associated radioactivity in the alveolar walls over time as well as the subsequent migration of these cells into alveolar spaces during the acute phase of bleomycin-induced lung injury (days 0 to 14) when compared with lung tissue morphometry. The scintigraphy, lavage, and morphometry data together showed that neutrophil influx into both of these lung compartments preceded that of monocytes by days, and that the influx of monocytes accounted for a major proportion of mononuclear cells found in the alveolar walls and alveolar spaces of the lung during this acute phase of inflammation. The increased numbers of neutrophils and mononuclear cells in alveolar spaces normalized by days 14 and 28 respectively, but in contrast to the normalization of neutrophil content in alveolar walls by day 10, increased numbers of mononuclear cells persisted in alveolar walls for up to 56 days, a time when there was a significant increase in the hydroxyproline content of these lungs. These data also show that the increased number of mononuclear cells present in the alveolar walls on days 28 and 56 was not due to a persistent influx of blood monocytes. These data suggest: (a) that differential pathways of efflux existed for alveolar wall versus alveolar space mononuclear cells, (b) that a delay in efflux from the alveolar walls occurred and/or that an increase in the local proliferation of mononuclear cells in this compartment may have been occurring during the later phases of bleomycin-induced lung injury, and (c) that this prolonged residence of mononuclear cells in the alveolar walls occurred concurrently with the development of pulmonary fibrosis.     

Collagenase and gelatinase activities in bronchoalveolar lavage fluids during bleomycin-induced lung injury

Basement membrane degradation can be indicative of tissue injury, but the process may also release matrix-bound cytokines to stimulate cell regeneration. To investigate this process, acute lung injury was induced in rats by intratracheal bleomycin and animals were killed from 3 days to 8 weeks later. The lungs were lavaged with saline to collect bronchoalveolar lavage (BAL) fluid and cell proliferation was assessed by pulse incorporation of tritiated thymidine. Bleomycin induced rapid inflammation with increased cell numbers and protein levels in BAL. Collagen degradation products were also increased in BAL fluid from 3 days to 4 weeks. Incubating samples of BAL fluid with radiolabelled collagens I and IV showed that high levels of activity, particularly for the degradation of type IV collagen, were present as early as 3 days post-bleomycin and persisted over the 8-week period. Zymograms demonstrated the highest level of gelatinase A (MMP-2) activity in BAL fluid in the first 2 weeks after bleomycin. Coincident with peak basement membrane degradative activity was the onset of a phase of epithelial cell proliferation, as measured by labelled nuclei in autoradiographs. The results show that enzymes capable of degrading the alveolar basement membrane are secreted early in the lung injury phase and that their presence in BAL fluid can be used as a measure of alveolar wall damage. It is possible that this enzyme action may release bound cytokines from the basement membrane, since maximal gelatinase activity correlates with alveolar epithelial cell proliferation. © 1998 John Wiley & Sons, Ltd.     

内源性CO在CCK-8减轻脂多糖所致的急性肺损伤中的作用

目的：探讨内源性一氧化碳（CO）在八肽胆囊收缩素（CCK-8）减轻LPS所致急性肺损伤（ALI）中的作用。 方法： 将56只大鼠随机分为正常对照组、LPS组、LPS+ZnPP（HO-1特异性阻断剂）组、LPS+Hm（CO供体）组、CCK-8+LPS组、CCK-8+LPS+ZnPP组、CCK-8组7组，每组8只。各组给药后2 h，6 h，12 h行支气管肺泡灌洗、检测支气管肺泡灌洗液（BALF）中中性粒细胞（PMN）数目，并进行肺组织的形态学观察；计算大鼠死亡率；测定肺组织中MDA、CO含量。 结果： 给药2 h和6 h后，各组大鼠死亡率均为0，LPS 注入12 h后大鼠死亡率高于相应对照组，LPS+Hm和CCK-8+LPS组大鼠死亡率均低于LPS组，LPS+ZnPP和CCK-8+LPS+ZnPP组大鼠死亡率分别高于LPS和CCK-8+LPS组；LPS组肺组织均出现损伤变化，同时BALF中PMN数目和肺组织中MDA和CO含量高于相应对照组；LPS+Hm和CCK-8+LPS组肺组织损伤程度、BALF中PMN数目和肺组织中MDA含量低于相应LPS组，但肺组织中CO含量高于相应LPS组；LPS+ZnPP和CCK-8+LPS+ZnPP组肺组织损伤程度、BALF中PMN数目和肺组织中MDA含量分别高于相应LPS和CCK-8+LPS组，而肺组织中CO含量分别低于相应LPS组和CCK-8+LPS组。 结论： CCK-8可通过内源性CO介导的抗氧化、抑制PMN聚集等效应来发挥改善LPS所致的肺损伤作用。     

Mice lacking neutrophil elastase are resistant to bleomycin-induced pulmonary fibrosis

Neutrophil elastase is a serine protease stored in the azurophilic granules of leukocytes. It has been implicated in the pathology of several lung diseases and is generally presumed to contribute to the tissue destruction and extracellular matrix damage associated with these conditions. To delineate the role of neutrophil elastase in pulmonary inflammation and fibrosis, neutrophil elastase-null mice were intratracheally instilled with bleomycin. In neutrophil elastase-null mice, biochemical and morphological characteristics of pulmonary fibrosis were attenuated for at least 60 days after bleomycin administration despite a typical response to bleomycin as evidenced by assessment of indices of DNA and cell damage. Neutrophil burden of bleomycin-treated wild-type and neutrophil elastase-null mice was comparable, and marked neutrophilic alveolitis was manifest in bleomycin-treated neutrophil elastase-null mice. An absence of immunostaining for active transforming growth factor (TGF)-beta in lung tissue from bleomycin-treated neutrophil elastase-null mice suggested a defect in TGF-beta activation, which was confirmed by biochemical assessment of TGF-beta levels in bronchoalveolar lavage fluid and lung tissue. These data point to novel and unexpected fibrogenic consequences of neutrophil elastase activity in the inflamed lung.     

Iloprost inhibits neutrophil-induced lung injury and neutrophil adherence to endothelial monolayers.

We hypothesized that Iloprost, a long-acting prostacyclin analog, would inhibit neutrophil (PMN)-induced lung injury and decrease PMN adherence to vascular endothelium. Human PMNs infused into isolated buffer-perfused rat lungs subsequently stimulated with phorbol myristate acetate (PMA) resulted in lung injury as assessed by the accumulation of [125I]bovine serum albumin (125I-BSA) in lung parenchyma and alveolar lavage fluid. Addition of Iloprost to the lung perfusate, prior to activation of the PMNs, reduced lung injury as assessed by a decrease in the accumulation of 125I-BSA in the lung. This protective effect was not due to the vasodilatory effect of Iloprost. Protection by Iloprost was not linked to a reduction in PMA-induced PMN superoxide production since Iloprost did not reduce the amount of superoxide released into lung perfusate. In vitro, Iloprost caused a dose-dependent inhibition of PMA-stimulated PMN adherence to endothelial cells. Iloprost did not affect the number of Mo1 adhesion molecules constitutively expressed or the number of receptors expressed on the PMNs following PMA. Addition of cAMP or dibutyryl cAMP to the endothelial cells mimicked the effects of Iloprost, diminishing PMA-stimulated PMN adhesion. In separate experiments, addition of the phosphodiesterase inhibitor IBMX to Iloprost resulted in a greater inhibition of PMA-stimulated PMN adherence, while addition of an adenylate cyclase inhibitor, SQ 22,536, or cAMP antibodies with the Iloprost abolished Iloprost's inhibitory effect on PMN adhesion. Thus, Iloprost inhibits PMA-activated PMN-induced lung injury despite continued superoxide production. Iloprost inhibition of PMN adhesion is dependent on cAMP.     

Adenoviral gene transfer of macrophage inflammatory protein-2 in rat lung.

Replication-defective adenoviral vectors are capable of localized transfer and expression of incorporated gene product in lung tissue. We have constructed an adenoviral vector that expresses rat macrophage inflammatory protein (MIP)-2, a C-X-C chemokine specifically chemotactic for neutrophils, Supernatants from 293 cells, infected with the adenoviral MIP-2 (ADMIP-2) construct, showed potent chemotactic activity and the ability of the ADMIP-2 vector to transcribe and make functional protein was confirmed. In vivo analysis of bronchoalveolar lavage fluid from rats after intratracheal instillation of ADMIP-2 (10(9) plaque-forming units) showed a 10-fold increase in the absolute number of neutrophils in bronchoalveolar lavage fluid as opposed to rats treated with an equal titer of an E1-disabled control virus expressing firefly luciferase (ADCA-18). Neutrophils constituted 65% of total BAL cells with alveolar macrophages being the other major cell type recovered. Rat MIP-2 protein was increased (nanograms per milliliter) in bronchoalveolar lavage fluid over a period of 7 days in ADMIP-2-treated animals. MIP-2 mRNA was demonstrated by Northern blot analysis in lung tissue, and histological analysis confirmed the presence of massive localized tissue neutrophilia. Evidence of chronic tissue injury and repair (ie, fibrosis) was not detected up to 2 weeks after the neutrophil infiltrate had resolved, subsequent to decreased chemokine presence. Adenoviral gene transfer proved an effective tool for the assessment of lung tissue expression of this chemokine in vivo and is useful in developing rodent models of tissue neutrophilia.     

Early Increase in Alveolar Macrophage Prostaglandin 15d-PGJ2 Precedes Neutrophil Recruitment into Lungs of Cytokine-Insufflated Rats

Early detection and prevention is an important goal in acute respiratory distress syndrome research. We determined the concentration of the anti-inflammatory 15-deoxy-Δ^12,14-prostaglandin-J2 (15d-PGJ2) and other components of the cyclopentenone prostaglandin cascade in relation to lung inflammation in cytokine (IL-1/LPS)-insufflated rats. We found that 15d-PGJ2 levels increase in the bronchoalveolar lavage (BAL) fluid of rats insufflated with cytokines 2 h before. BAL 15d-PGJ2 increases preceded neutrophil recruitment, lung injury, and oxidative stress in the lungs of cytokine-insufflated rats. 15d-PGJ2 was localized in alveolar macrophages that decreased following cytokine insufflation. 15d-PGJ2 may constitute an early biomarker of lung inflammation and may reflect an endogenous attempt to regulate ongoing inflammation in macrophages and elsewhere after cytokine insufflation.     

Role of alveolar macrophages in Candida-induced acute lung injury.

Recent studies have shown that alveolar macrophages (AMs) not only act as phagocytes but also play a central role as potent secretory cells in various lung diseases, including pneumonia and acute respiratory distress syndrome. The behavior of AMs during disseminated candidiasis, however, is insufficiently elucidated. This study is the first to report disseminated candidiasis in AM-depleted mice and to analyze the effect of AMs on Candida-induced acute lung injury. While all AM-sufficient mice died by day 2 after infection with Candida albicans, no mortality was observed among AM-depleted mice. Unexpectedly, the CFU numbers of C. albicans isolated from the lungs of AM-depleted mice were significantly higher than those for C. albicans isolated from AM-sufficient mice. The lung wet-to-dry weight ratio was lower for AM-depleted mice than for AM-sufficient mice, although this difference was not significant. We found that bronchoalveolar lavage fluid (BALF) from AM-depleted mice in candidemia contained fewer neutrophils than BALF from AM-sufficient mice. In addition, myeloperoxidase activities in lung homogenates of AM-depleted mice were significantly lower than those in homogenates of AM-sufficient mice. A significant decrease in levels of murine macrophage inflammatory protein 2 (MIP-2), a potent chemoattractant for neutrophils, was noted in lung homogenates from AM-depleted mice compared with levels in homogenates from AM-sufficient mice. Immunohistochemical studies using anti-MIP-2 antibodies revealed that AMs were the cellular source of MIP-2 within the lung during candidemia. We observed that AM depletion decreased levels of AM-derived neutrophil chemoattractant, alleviated acute lung injury during candidemia, and prolonged the survival of mice in candidemia, even though clearance of C. albicans from the lungs was reduced.     

Oncostatin M production by blood and alveolar neutrophils during acute lung injury

Polymorphonuclear neutrophils (PMN) are involved in the pathogenesis of acute lung injury (ALI), secreting numerous mediators such as proteases, reactive oxygen species, and cytokines. Because we had recently observed the ability of normal human PMN to degranulate and synthesize oncostatin M (OSM), an IL-6-family cytokine, we quantified OSM production ex vivo by highly purified blood and alveolar PMN from 24 ventilated patients with ALI, including some patients with severe pneumonia. Most of the patients had no detectable OSM in plasma, and OSM production by cultured blood PMN was similar to that of healthy controls. However, OSM was present in bronchoalveolar lavage (BAL) fluid supernatant, with significantly higher levels during pneumonia. In addition, alveolar OSM levels correlated with the number of PMN obtained by BAL, suggesting that PMN are an important source of OSM within the alveoli. Indeed, purified alveolar PMN from all of the patients, especially those with pneumonia, strongly produced OSM. Interestingly, in the latter patients, alveolar PMN always produced more OSM than autologous blood PMN. These results document the functional duality of PMN in ALI by showing the participation of PMN in the modulation of lung inflammation.     

HO-1在CCK-8减轻脂多糖所致的急性肺损伤中的作用

目的： 探讨血红素氧合酶（HO）-1在八肽胆囊收缩素（CCK-8）减轻脂多糖（LPS）所致急性肺损伤（ALI）中的作用。方法: 将大鼠随机分为5组：正常对照组、LPS组、CCK-8+LPS组、LPS+Hm（氯血红素，CO供体）组、LPS+ZnPP（锌原卟啉，HO-1特异性阻断剂）组。各组给药后2 h、6 h、12 h行支气管肺泡灌洗、检测支气管肺泡灌洗液（BALF）中中性粒细胞（PMN）数目；进行肺组织的形态学观察；测定肺组织中丙二醛（MDA）含量和HO-1蛋白活性；应用RT-PCR和Western blotting技术检测给药后6h肺组织中HO-1 mRNA和蛋白的表达情况。结果: LPS组肺组织出现损伤性变化，同时BALF中PMN数目、肺组织中MDA含量、HO-1蛋白活性、HO-1 mRNA和蛋白的表达均高于相应对照组（均P<0.05）；CCK-8+LPS和LPS+Hm组肺组织损伤程度、BALF中PMN数目和肺组织中MDA含量低于相应LPS组，而肺组织中HO-1蛋白活性、HO-1 mRNA和蛋白的表达均高于相应LPS组（均P<0.05）；LPS+ZnPP组肺组织损伤程度、BALF中PMN数目和肺组织中MDA含量分别高于相应LPS组，而肺组织中HO-1蛋白活性、HO-1 mRNA和蛋白的表达分别低于相应LPS组（均P<0.05）。结论: CCK-8可部分通过HO-1介导的抗氧化、抑制PMN聚集等效应来发挥减轻LPS所致的肺损伤作用。