Model Of Acute Lung Inflammation Research Articles

Protective Effects of Anthocyanins from Blackberry in a Rat Model of Acute Lung Inflammation

Anthocyanins are a group of naturally occuring phenolic compounds related to the coloring of plants, flowers and fruits. These pigments are important as quality indicators, as chemotaxonomic markers and for their antioxidant activities. Here, we have investigated the therapeutic efficacy of anthocyanins contained in blackberry extract (cyanidin-3-O-glucoside represents about 80% of the total anthocyanin contents) in an experimental model of lung inflammation induced by carrageenan in rats. Injection of carrageenan into the pleural cavity elicited an acute inflammatory response characterized by fluid accumulation which contained a large number of neutrophils as well as an infiltration of polymorphonuclear leukocytes in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate (NOx) and prostaglandin E2 (PGE2). All parameters of inflammation were attenuated in a dose-dependent manner by anthocyanins (10, 30 mg kg-1 30 min before carrageenan). Furthermore, carrageenan induced an upregulation of the adhesion molecule ICAM-1, nitrotyrosine and poly (ADP-ribose) synthetase (PARS) as determined by immunohistochemical analysis of lung tissues. The degree of staining was lowered by anthocyanins treatment. Thus, the anthocyanins contained in the blackberry extract exert multiple protective effects in carrageenan-induced pleurisy.

Cytoplasmic deposition of NFkappaB decoy oligonucleotides is insufficient to inhibit bleomycin-induced pulmonary inflammation.

Lung inflammation leads to severe tissue destruction and ultimately organ failure in a number of diseases, including cystic fibrosis (CF). The transcription factor nuclear factor kappa B (NFkappaB) regulates expression of many pro-inflammatory mediators. We have assessed the effect of topical administration of NFkappaB decoys in a bleomycin model of acute lung inflammation. Using fluorescein-labelled decoy oligonucleotides (ODN) (80 microg/mouse) we have shown that lipid-complexed and 'naked' ODN transfect conducting airway epithelium in a comparable manner (approximately 65% of cells). However, the ODN were detectable in the cytoplasm, but not in the nucleus of transfected cells. An increase of ODN dose to 500 microg/mouse did not increase nuclear transfection significantly. We determined the effect of cytoplasmic NFkappaB decoys on bleomycin-induced inflammation. We transfected mice with 'naked' decoy and scrambled ODN (500 microg) 1 h before intratracheal administration of bleomycin. We measured IL6 secretion in BALF and lung homogenates and total and differential cell counts in BALF 5 days after bleomycin administration. We did not detect a difference between NFkappaB decoy and scrambled ODN-treated animals in any of the parameters tested. We suggest that access of ODN to the nucleus of airway epithelial cells is a key problem, limiting the efficacy of such decoy strategies, as well as attempts at gene repair.

Pharmacological profile of PKF242-484 and PKF241-466, novel dual inhibitors of TNF-alpha converting enzyme and matrix metalloproteinases, in models of airway inflammation.

1. TNF-alpha converting enzyme (TACE) and matrix metalloproteinases (MMPs) are believed to play a role in various airway inflammatory disorders. Therefore we have tested the effect of two new inhibitors of TACE/MMPs (PKF242-484, PKF241-466) in models of airway inflammation. 2. PKF242-484 and PKF241-466 inhibited purified MMP-1, -2, -3, -9, -13 and rat collagenase at low nanomolar range. Both compounds inhibited the TNF-alpha release from activated human peripheral blood mononuclear cells with IC(50) values of 56+/-28 and 141+/-100 nM, respectively and had no significant effect on the activation of other human leukocytes, as neither neutrophils and eosinophils oxidative burst nor proliferation or cytokines production by T cells were inhibited in vitro. 3. PKF242-484 and PKF241-466 had beneficial effects in two different murine models of acute lung inflammation in vivo. The influx of neutrophils and lymphocytes into the airways was reduced 3 and 24 h after intranasal LPS challenge. This was accompanied by reduced levels of myeloperoxidase and elastase activities in the bronchoalveolar lavage. Furthermore, a complete inhibition of TNF-alpha release into the airways was observed. In addition, PKF242-484 effectively reduced the influx of neutrophils, eosinophils and lymphocytes in a model of acute allergic lung inflammation. 4. PKF242-484 and PKF241-466 are two novel and potent dual inhibitors of TACE and MMPs, which show activity in in vivo models of lung inflammation. Such compounds could have beneficial effects in airway inflammatory conditions such as asthma and chronic obstructive pulmonary disease.

Hyperbaric Oxygen Increases the Lung's Susceptibility to Inhaled Lipopolysaccharide in Mice

Hyperbaric oxygen (HBO2) has been shown to inhibit the adhesion function of beta(2)-integrin, which is important in mediating cell-to-cell adhesion and extravasation of inflammatory cells. In the present study, we examined the effects of HBO2 exposure on neutrophil infiltration and tissue injury in a model of acute lung inflammation induced by lipopolysaccharide (LPS) inhalation. Male C57BL/6 mice of 8 weeks old were exposed to 3 atmosphere absolute (ATA) 100% HBO2, 3 ATA hyperbaric air (HBA), or room air for 90 min. After exposure, they were exposed to aerosolized LPS solution (1 mg/ml) or saline in a plexiglass chamber for 10 min. Four hours after inhalation, bronchoalveolar lavage (BAL) was performed to determine protein concentration, LDH activity, total cells, and differential cell counts in the lavage fluid (BALF). Myeloperoxidase (MPO) content, lung histopathology, and plasma nitric oxide (NO) metabolite concentrations were also determined in separate sets of animals. We observed that LPS inhalation increased neutrophil number in the BALF, which was significantly inhibited by HBO2 but not HBA pre-exposure. However, MPO content in the lung was prominently increased by HBO2 pre-exposure, which correlated with increased PMN infiltration in lung tissues. Further, HBO2 plus LPS, but not saline inhalation caused a significant increase in the BALF protein level and LDH activity compared with that of LPS inhalation alone. LPS exposure induced significant increase in plasma NO metabolites, which was not potentiated by HBO2 pre-exposure. The inducible nitric oxide synthase inhibitor, aminoguanidine, significantly attenuated the increases in plasma NO metabolites and tissue MPO content as well as lung injuries. In summary, our data suggest that HBO2 pre-exposure increases the lung's susceptibility to inhaled LPS, which may be related to increased tissue neutrophil infiltration and dependent on interaction(s) between HBO2 exposure with LPS-induced nitric oxide production.

Role of IL-18 in acute lung inflammation.

We have examined the role of IL-18 after acute lung inflammation in rats caused by intrapulmonary deposition of IgG immune complexes. Constitutive IL-18 mRNA and protein expression (precursor form, 26 kDa) were found in normal rat lung, whereas in inflamed lungs, IL-18 mRNA was up-regulated; in bronchoalveolar (BAL) fluids, the 26-kDa protein form of IL-18 was increased at 2-4 h in inflamed lungs and remained elevated at 24 h, and the "mature" protein form of IL-18 (18 kDa) appeared in BAL fluids 1-8 h after onset of inflammation. ELISA studies confirmed induction of IL-18 in inflamed lungs (in lung homogenates and in BAL fluids). Prominent immunostaining for IL-18 was found in alveolar macrophages from inflamed lungs. When rat lung macrophages, fibroblasts, type II cells, and endothelial cells were cultured in vitro with LPS, only the first two produced IL-18. Intratracheal administration of rat recombinant IL-18 in the lung model caused significant increases in lung vascular permeability and in BAL content of neutrophils and in BAL content of TNF-alpha, IL-1beta, and cytokine-induced neutrophil chemoattractant, whereas intratracheal instillation of anti-IL-18 greatly reduced these changes and prevented increases in BAL content of IFN-gamma. Intratracheal administration of the natural antagonist of IL-18, IL-18 binding protein, resulted in suppressed lung vascular permeability and decreased BAL content of neutrophils, cytokines, and chemokines. These findings suggest that endogenous IL-18 functions as a proinflammatory cytokine in this model of acute lung inflammation, serving as an autocrine activator to bring about expression of other inflammatory mediators.

REGULATION OF CHEMOKINE EXPRESSION BY IL-10 IN LUNG INFLAMMATION

We have been interested in understanding the mechanisms regulating the inflammatory process underlying acute lung injury. The current studies have employed a model of acute lung inflammation in mice triggered by bacterial lipopolysaccharide. The development of this injury was associated with increased expression of the chemokines, MIP-1α and MIP-2, that coordinate recruitment of neutrophils to the lung. IL-10 is a potent, endogenous anti-inflammatory molecule that has been shown to decrease lung inflammation partly on the basis of TNF-α and IL-1β inhibition. In these studies we tested the hypothesis that endogenous IL-10 modulates chemokine expression using the IL-10 knock-out mouse, and then explored the molecular mechanisms by which IL-10 might do so. The results demonstrate that significant elevations in both chemokines were observed in the absence of IL-10 and that these findings were associated with significant increases in lung neutrophil accumulation. In vitro studies defined two, gene-specific, mechanisms by which IL-10 regulated chemokine expression: mRNA destabilization and NF-κB inhibition. These results suggested that IL-10 is an important, endogenous regulator of chemokine expression in acute lung inflammation.

Regulation of chemokine mRNA expression in a rat model of vanadium-induced pulmonary inflammation.

Environmental and occupational exposure to vanadium dusts results in toxic effects mainly confined to the respiratory system. Using a rat model of acute lung inflammation induced by intratracheal instillation of sodium metavanadate (NaVO3) at the dose of 200 microg V/kg, we investigated the relationship between the cytologic characterization of pulmonary inflammation and the expression of chemokine mRNA. Significant polymorphonuclear leukocyte (PMN) influx (P < 0.01) into the lung was noted 4 h after NaVO3 instillation, whereas alveolar macrophages (AMs) in bronchoalveolar lavage (BAL) cells appeared to decrease significantly. In contrast, neither PMNs nor AMs changed substantially 1 h after NaVO3 instillation. By Northern analysis, macrophage inflammatory protein (MIP)-2 mRNA in BAL cells increased markedly 1 h after NaVO3 instillation and reduced a little bit at 4 h, whereas MIP-1alpha mRNA in BAL cells was expressed relatively high 1 h after NaVO3 instillation, although a basal expression was detected in control group, and returned rapidly nearly to control level at 4 h. Since MIP-2 is a potent PMN chemoattractant and MIP-1alpha is a potent macrophage/monocyte chemoattractant has been well known. The facts that PMN influx was preceded by increased MIP-2 mRNA expression, suggesting that MIP-2 is involved in the development of NaVO3-induced pulmonary inflammation, whereas increased MIP-1alpha mRNA expression was followed by decreased AMs in BAL cells, suggesting AMs might be activated by MIP-1alpha, adherent to the lining surface of the airways and then resistant to be washed out. To delineate the mechanisms of transcriptional activation, we recently cloned the 5'-flanking region of the MIP-2 gene. The promotor region contains consensus binding sites for transcription factor nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1). Using electrophoretic mobility shift assay, increased nuclear NF-kappaB, not AP-1, binding activity was detected 1 h after NaVO3 instillation, which correlated with the induction of MIP-2 mRNA. p65 (Rel A) and p50 protein appears to be involved in MIP-2 NF-kappaB binding. Taken together, our studies suggest that MIP-2 is an important mediator of NaVO3-induced pulmonary inflammation in the rat model. In addition, elevated MIP-2 mRNA levels are accompanied by increased NF-kappaB binding activity in BAL cells, suggesting possible MIP-2 transcriptional regulation through NF-kappaB.

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.

Distinct functions of interferon-gamma for chemokine expression in models of acute lung inflammation.

Challenge of the immune system with bacterial superantigens or endotoxin induces the systemic release of cytokines followed by lethal septic shock. The lung is particularly susceptible to systemic toxin exposure resulting in acute leucocyte infiltration and vascular damage. In the present study, the functions of interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) for chemokine regulation during acute lung inflammation were examined. Following administration of the superantigen, staphylococcal enterotoxin B (SEB), lung mRNA levels of the chemokines cytokine-induced neutrophil chemo-attractant (KC), lipopolysaccharide-induced CXC chemokine (LIX), macrophage chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1alpha and MIP-2 were increased to a similar extent both in controls and in mice deficient for the IFN-gamma or 55 000 MW TNF receptors. In contrast, interferon-inducible protein-10 (IP-10) and monokine induced by IFN-gamma (Mig) mRNA expression was markedly reduced in mice deficient for IFN-gamma or IFN-gamma receptor, but not in 55 000 MW TNF receptor knockout mice. In situ hybridization experiments demonstrated that IP-10 was highly expressed in lung interstitial macrophages of C57BL/6, but not of IFN-gamma receptor-deficient mice. In contrast to SEB administration, treatment with lipopolysaccharide resulted in a strong induction of IP-10 and Mig in IFN-gamma receptor-deficient mice. Together, these results establish a critical function of IFN-gamma for chemokine induction in acute lung inflammation that is dependent on the nature of the inflammatory stimulus.

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

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

Pulmonary Cytokine and Chemokine mRNA Levels after Inhalation of Lipopolysaccharide in C57BL/6 Mice

Inhaled endotoxin (lipopolysaccharide, LPS) can induce acute lung injury and at high doses may lead to respiratory distress syndrome. Using a mouse model of acute lung inflammation induced by inhalation of low doses of LPS we examined the kinetics of chemokine, proinflammatory cytokine, and metallothionein. Eight-week-old C57BL/6 mice were dosed for 10 min with LPS, resulting in an estimated alveolar dose of < 10 ng LPS/mouse, and euthanized 2,6, or 24 h postexposure. Analysis of bronchoalveolar lavage fluid demonstrated increased polymorphonuclear neutro-phils (PMNs) of 6.94, 32.7, and 38.8% after 2, 6, and 24 h, respectively. Examination of proinflammatory cytokine, chemokine, and Mt mRNA in the lung revealed increases for messages encoding IL-1α, IL-1β3, IL-6, IFN-γ, TNFα, Eotaxin, MIP-1α, MIP-1β3, MIP-2, Mt, and IP-10, while messages encoding IL-12, IL-10, IFN-β, Ltn, MCP-1, TGFβ1+2, and RANTES were unchanged from those of sham-exposed mice 2 h postexposure. By 6 h most messages had returned to near control levels. Comparison to 5 mg/kg body weight intraperitoneal injection and 5 μg/mouse intratracheal instillation 2 h postexposure demonstrated similar message responses. Our results demonstrate that low levels of LPS exposure by inhalation induce a strong PMN response and a selective cytokine response in the lung, supporting the hypothesis that PMNs may regulate inflammatory processes via cytokine and Chemokine response.

Balancing act with chemokines in angiogenesis &amp; inflammation

Two functional types of chemokines have been identified. Researchers believe that any upset in the balance of these 2 groups can lead to the onset of certain pathological processes, such as tumour development and inflammation. But correction of the imbalance may well lead to disease improvement. Progress in the balancing act was reported at the IBC USA conference on Chemotactic Cytokines [Philadelphia, US; October 1995]. Studies looking specifically at the effects of interleukin-8 (IL-8) blockade in models of acute lung inflammation were also presented.

Pentoxifylline and Lung Ischemia—Reperfusion Injury

Pentoxifylline (PTX) attenuates neutrophil-mediated lung injury in several models of acute lung inflammation. Because pulmonary neutrophil sequestration is the main determinant of ischemia-reperfusion (IR) injury in lung transplantation, we sought to determine whether or not PTX prevented IR injury in isolated perfused rat and rabbit lungs submitted to IR, and in pigs after left lung allotransplantation. In rat lungs after IR, the coefficient of lung endothelial permeability (K fc ) increased by 112 ± 12% in controls and by 27 ± 8% (p < 0.001) in PTX-treated lungs. After IR, lung myeloperoxidase and blood neutrophil count decrease were lower with PTX than in controls, and the changes in K fc were correlated with the percentage decrease in blood neutrophils during reperfusion. In rabbit lungs, endothelium-dependent relaxation in isolated pulmonary arterial rings was decreased in the control group and normal in the PTX group. In pigs ventilated with pure oxygen, the PaO 2 was greater in the PTX group than in the control group (423 ± 49 vs. 265 ± 43 mm Hg ; p < 0.05), whereas the total pulmonary vascular resistance was lower (15 ± 1 vs. 30 ± 9 mm Hg/L/min ; p < 0.02). After reperfusion, the decrease in circulating leukocyte count fell by 35 ± 3% in the control group and remained unchanged in the PTX group, and the leukocyte count per microscopic field in the transplanted lung was lower in the PTX group than in the control group (p < 0.02). In conclusion, PTX prevented IR lung endothelium injury and improved post-IR lung function by decreasing neutrophil lung sequestration, and this agent might be useful in clinical lung transplantation.

Cytokine expression by neutrophils and macrophages in vivo: endotoxin induces tumor necrosis factor-alpha, macrophage inflammatory protein-2, interleukin-1 beta, and interleukin-6 but not RANTES or transforming growth factor-beta 1 mRNA expression in acute lung inflammation.

Using a rat model of acute lung inflammation induced by intratracheal instillation of lipopolysaccharide (LPS), we investigated the kinetics of mRNA expression and the potential cellular sources of tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2), interleukin (IL)-1 beta, IL-6, RANTES, and transforming growth factor-beta 1 (TGF-beta 1). By Northern blot analysis, TNF-alpha and MIP-2 mRNAs in total lung tissue increased markedly by 30 min and peaked by 1 h after LPS exposure, whereas expression of IL-1 beta and IL-6 was not detected until 1 h and peaked within 6 h. In contrast, neither RANTES nor TGF-beta 1 mRNA was induced by LPS throughout 72 h, although a basal expression was detected in both saline- and LPS-treated lung tissues. At 1 h after LPS, the bronchoalveolar lavage (BAL) fluid contained about 98% alveolar macrophages (AM), whereas by 6 or 12 h, 88% of BAL cells were polymorphonuclear neutrophils (PMN). Upon extraction of total RNA after separation of AM from PMN in BAL, Northern analysis showed that at 1 h, AM expressed pronounced signals for TNF-alpha, MIP-2, IL-1 beta, and IL-6. At 6 and 12 h, however, while cytokine transcripts decreased in AM, PMN exhibited strong signals for these cytokines. A low basal noninducible signal for TGF-beta 1 but not RANTES was detected in both AM and PMN. Finally, by in situ hybridization techniques, PMN in the lung tissue, particularly those located in the vicinity of the bronchiole and vasculature, were demonstrated to localize MIP-2 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)