Inducible Nitric Oxide Synthase—Knockout Mice Exhibit Resistance to Pleurisy and Lung Injury Caused by Carrageenan (original) (raw)
Related papers
Life Sciences, 2003
In the present study, by comparing the responses in wild-type mice (iNOSWT) and mice lacking (iNOSKO) the inducible (or type 2) nitric oxide synthase (iNOS), we investigated the correlation between endogenous nitric oxide (NO) and prostaglandin (PG) generation in carrageenan-induced pleurisy. The inflammatory response in iNOSKO mice was significantly reduced in respect to iNOSWT animals, as demonstrated by the exudate volume ( À 63%) and numbers of infiltrating cells ( À 62%). The levels of NOx in the pleural exudate from carrageenan-treated mice were significantly (p < 0.01) decreased in iNOSKO mice (16 F 7.6 nmoles/mice) compared to iNOSWT animals (133 F 9 nmoles/mice). Similarly, the amounts of PGE 2 in the pleural exudates of carrageenan-treated animals were significantly (p < 0.01) lower in iNOSKO compared to iNOSWT mice (120 F 20 pg/mice vs. 308 F 51 pg/mice). Also the amounts of 6-keto-PGF 1a produced by lungs from carrageenan-treated iNOSKO mice (1.01 F 0.10 ng/tissue mg) were significantly (p < 0.01) reduced compared to iNOSWT carrageenan-treated mice (2.1 F 0.09 ng/tissue mg). In conclusion our results confirm, by the use of iNOSKO mice that in carrageenan-induced pleurisy NO positively modulates PG biosynthesis. D
Regulatory Effects of iNOS on Acute Lung Inflammatory Responses in Mice
The American Journal of Pathology, 2003
The role of endogenous NO in the regulation of acute lung injury is not well defined. We investigated the effects of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) on the acute inflammatory response in mouse lungs. Acute lung injury was induced by intratracheal instillation of bacterial lipopolysaccharide (LPS) into wild-type (WT) mice and mice deficient in iNOS (iNOS ؊/؊ ) or eNOS (eNOS ؊/؊ ). Endpoints of inflammatory injury were myeloperoxidase (MPO) content and leak of albumin into lung. Inflammatory injury was similar in WT and eNOS ؊/؊ mice but was substantially increased in iNOS ؊/؊ mice. Bronchoalveolar lavage (BAL) fluids of iNOS ؊/؊ and WT mice showed similar levels of CXC chemokines (MIP-2, KC) but enhanced levels of CC chemokines (MCP-1, MCP-3). Increased lung content of MPO in iNOS ؊/؊ mice was reduced by anti-MCP-1 to values found in WT mice.
Role of IL-6 in the pleurisy and lung injury caused by carrageenan
Journal of immunology (Baltimore, Md. : 1950), 1999
In the present study we used IL-6 knockout mice (IL-6KO) to evaluate the role of IL-6 in the inflammatory response caused by injection of carrageenan into the pleural space. Compared with carrageenan-treated IL-6 wild-type (IL-6WT) mice, carrageenan-treated IL-6KO mice exhibited a reduced degree of pleural exudation and polymorphonuclear cell migration. Lung myeloperoxidase activity and lipid peroxidation were significantly reduced in IL-6KO mice compared with those in IL-6WT mice treated with carrageenan. Immunohistochemical analysis for nitrotyrosine and poly(A)DP-ribose polymerase revealed a positive staining in lungs from carrageenan-treated IL-6WT mice. No positive staining for nitrotyrosine or PARS was found in the lungs of the carrageenan-treated IL-6KO mice. Staining of lung tissue sections obtained from carrageenan-treated IL-6WT mice with an anti-cyclo-oxygenase-2 Ab showed a diffuse staining of the inflamed tissue. Furthermore, expression of inducible nitric oxide synthas...
Naunyn-Schmiedeberg's Archives of Pharmacology, 1999
We studied the involvement of nuclear factor-κB (NF-κB) in the regulation of inducible nitric oxide synthase expression in carrageenin-induced rat pleurisy. Injection of 0.2 ml of 1% λ-carrageenin into the pleural cavity of male Wistar rats caused after 6 h: (a) exudate formation and leukocyte migration into the pleural cavity; (b) inducible NO synthase protein expression and accumulation of NO 2plus NO 3in pleural exudate; (c) increase in p50/p65 nuclear level as well as ΝF-κB/DNA binding activity. Treatment of rats with pyrrolidine dithiocarbamate (10, 30, and 100 mg/kg) and N-α-p-tosyl-L-lysine chloromethylketone (30 mg/kg), two inhibitors of NF-κB activation, given subcutaneously concomitantly with carrageenin, caused a significant inhibition of all the parameters assayed. These results suggest that in carrageenin-induced rat pleurisy the activation of ΝF-κB plays a key role in inducible NO synthase protein expression and in the development of inflammatory response.
European journal of …, 2000
Carrageenan causes enhanced formation of reactive oxygen species, which contribute to the pathophysiology of inflammation. We have investigated the effects of tempol, a membrane-permeable radical scavenger, in rats subjected to carrageenan-induced pleurisy. Ž. Treatment of rats with tempol 10, 30, or 100 mgrkg 15 min prior to carrageenan attenuated the pleural exudation and the migration of Ž. polymorphonuclear cells caused by carrageenan dose dependently. Tempol also attenuated the lung injury histology as well as the increase in the tissue levels of myeloperoxidase and malondialdehyde caused by carrageenan in the lung. However, tempol did not inhibit the activity of inducible nitric oxide synthase in the lungs. Immunohistochemical analysis for nitrotyrosine revealed positive staining in Ž. lungs from carrageenan-treated rats. Lung tissue sections from carrageenan-treated rats also showed positive staining for poly-ADP-ribose Ž. synthetase PARS. The degree of staining for nitrotyrosine and PARS was markedly reduced in tissue sections obtained from Ž. Ž. carrageenan-treated rats, which had received tempol 100 mgrkg. Furthermore, treatment of rats with tempol significantly reduced i the Ž. Ž. Ž. formation of peroxynitrite, ii the DNA damage, iii the impairment in mitochondrial respiration, and iv the fall in the cellular level of NAD q observed in macrophages harvested from the pleural cavity of rats treated with carrageenan. Tempol also attenuated the cell injury Ž. caused by hydrogen peroxide 1 mM in cultured human endothelial cells. This study provides the first evidence that tempol, a small molecule which permeates biological membranes and scavenges ROS, attenuates the degree of inflammation and tissue damage associated with carageenan-induced pleurisy in the rat. The mechanisms of the anti-inflammatory effect of tempol are discussed.
Lung sources and cytokine requirements for in vivo expression of inducible nitric oxide synthase
American Journal of Respiratory Cell and Molecular Biology, 1995
Products of inducible nitric oxide synthase (iNOS) are known to be involved in lung injury following intrapulmonary deposition of immunoglobulin G immune complexes (IgG-ICx). In the current studies rat alveolar macrophages stimulated in vitro with murine interferon')' (IFN-')'), tumor necrosis factor a, interleukin Io , (IL-la) , lipopolysaccharide (LPS), or IgG-ICx immunostained for iNOS and produced nitrite/nitrate-(N02-INO)-) in a dose-and time-dependent manner requiring availability of L-arginine. Under the same conditions, IL-4 and IL-I0 reduced N0 2-INO}-generation. Type II alveolar epithelial cells, which were obtained from normal rat lungs and stimulated in vitro with IgG-ICx, LPS, or IFN-')', also immunostained for iNOS and generated N0 2-INO}-. Special techniques of bronchoalveolar lavage (BAL) were used to retrieve alveolar macrophages and type II alveolar epithelial cells. Under these conditions, intrapulmonary deposition of LPS yielded BAL fluids containing increased amounts of N0 2-I NO}-and macrophages that spontaneously released N0 2-INO}-and stained for iNOS. After intrapulmonary deposition of IgG both macrophages as well as type II cells (retrieved by BAL) spontaneously produced N0 2-INO}-and both cell types immunostained for iNOS (approximately 20 % of all type II cells and 35 %of all alveolar macrophages). Using dual fluorescence staining for cell identification, frozen sections of lung tissue after IgG immune complex deposition revealed iNOS in both alveolar macrophages and type II cells. Finally, in the immune complex model of alveolitis, the appearance of iNOS in macrophages as well as macrophage production in vitro of N0 2-INO}-was dependent on the in vivo availability of tumor necrosis factor a, ILl , and IFN-')'. These studies suggest a dual cell source for nitric oxide in inflamed lungs and the requirements for iNOS of several cytokines. Although there is abundant evidence that the pathogenesis of immunoglobulin G immune complex (IgG-ICx)-induced tissue injury requires the role of complement, the participation of phagocytic cells (neutrophils and macrophages) and products from L-arginine oxidation produced by nitric oxide synthase (NOS), precise events that can be linked to the causation of injury, are not clearly defined. It has been postulated that the influx and activation of phagocytic cells result in the local generation of toxic oxygen products (0 2-, H 202 , hydroxyl radical, and hypochlorous acid), toxic products of
The protective role of endogenous glutathione in carrageenan-induced pleurisy in the rat
European Journal of Pharmacology, 1999
Peroxynitrite, a potent cytotoxic oxidant formed by the reaction of nitric oxide (NO) with the superoxide anion, was recently proposed to play a major pathogenic role in the inflammatory process. Here we have investigated the effects of endogenous melatonin, a known scavenger of peroxynitrite, in rats subjected to carrageenan-induced pleurisy. Endogenous melatonin was depleted in rats maintained on 24 h light cycle for 1 wk. In vivo depletion of endogenous melatonin enhanced the carrageenan-induced degree of pleural exudation and polymorphonuclear leukocyte migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase activity and lipid peroxidation were significantly increased in melatonin-deprived rats. However, the inducible NO synthase in lung samples was unaffected by melatonin depletion. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in lungs from carrageenan-treated rats that was markedly enhanced in melatonin-deprived rats. Furthermore, melatonin depletion significantly increased peroxynitrite formation as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, enhanced DNA damage and the decrease in mitochondrial respiration and reduced the cellular levels of NAD؉ in macrophages harvested from the pleural cavity of rats subjected to carrageenaninduced pleurisy. In vivo treatment with exogenous melatonin (15 mg/kg intraperitoneal) significantly reversed the effects of melatonin depletion. Thus, endogenous melatonin plays an important protective role against carrageenan-induced local inflammation.-Cuzzocrea, S., Tan, D.-X., Costantino, G., Mazzon, E., Caputi, A. P., Reiter, R. J. The protective role of endogenous melatonin in carrageenan-induced pleurisy in the rat. FASEB J. 13, 1930 -1938 (1999) 1930 0892-6638/99/0013-1930/$02.25 © FASEB * P Ͻ 0.01 vs. MEL ϩ/ϩ.
Mediators of Inflammation, 2001
Background: Although myeloperoxidase (MPO) and adenosine-deaminase (ADA) levels are markers of activated leukocytes, both enzymes have not been currently addressed in inflammation models. Aims: This study evaluates whether the concentrations of these enzymes are significantly correlated with the content of leukocytes in a pleurisy model. Methods: The pleurisy was induced by carrageenan (1%) in mice, and the parameters analyzed 4 and 48 h after. Results: After the induction of inflammation (4 h), MPO and ADA levels peaked in parallel to neutrophils (p < 0.01). Regarding the second phase of pleurisy (48 h), the highest concentrations of ADA were detected in parallel to the highest levels of mononuclears (p < 0.01). At this time, MPO levels and neutrophils remained elevated, although at lower levels than those found at 4 h. A significant positive correlation was found among neutrophils and MPO, and mononuclears and ADA (p < 0.01). Conclusions: These findings support the evidence that both enzymes are markers of the inflammatory process, and provide new tools for a better understanding of the immunoregulatory pathways that occur in inflammation.
Frontiers in Pharmacology
The ecto-5'-nucleotidase (ecto-5'NT/CD73) represents a crucial enzyme for endogenous adenosine generation. Several findings have shown that CD73 plays an important role in regulating vascular permeability and immune cell function. Adenosine 5'-(α,βmethylene)diphosphate (APCP) is a CD73 inhibitor, widely used as pharmacological tool to investigate the role of CD73/adenosine pathway in several in vitro and in vivo models, although it has been also shown to inhibit other ectoenzymes involved in adenosinergic pathway. Here, we evaluated the effect of APCP in the development of inflammation in carrageenan-induced pleurisy model. We found that treatment with APCP (400 μg/ rat) significantly increased cell accumulation, exudate formation, and pro-inflammatory cytokine content into the pleural cavity in the acute phase (4 h) of inflammation, with no differences in the sub-acute phase (72 h) except for the regulation of monocyte chemotactic protein-1 levels. In addition, cells collected by pleural lavage fluids of APCPtreated rats, 4 h following carrageenan injection, showed increased ability to migrate in vitro, both in presence and in absence of N-formyl-L-methionyl-L-leucyl-L-phenylalanine as chemotactic stimulus, compared to cells obtained by control rats. Our results demonstrate that APCP exacerbates the early phase of carrageenan-induced pleurisy by controlling pleural effusion and polymorphonuclear migration in vivo and ex vivo. This effect is likely dependent upon CD73 inhibition, although an inhibitory effect of other ectoenzymes cannot be ruled out.
Molecular Medicine
Background: We have recently demonstrated that 17estradiol (E2) inhibits the increase of inducible nitric oxide synthetase (iNOS) activity in selected model systems such as macrophages, microglia, smooth muscle cells, and proposed that this effect might be associated with an antiinflammatory activity of this hormone. Here we investigate the effects of endogenous estrogens in rats subjected to carrageenan-induced pleurisy. Materials and Methods: Adult female rats were ovariectomized 3 weeks before the experiments to deplete circulating estrogens. Selected inflammatory markers, landmarks of the delayed phase of carrageenan-induced pleurisy, were measured in intact (N-OVX), and ovariectomized (OVX) female rats. In addition, the effect of hormone replacement was evaluated in ovariectomized rats with intraperitoneal injection of 17-estradiol (E2; 50 g/kg) 1 hr before carrageenan treatment (OVX ϩ E2). Results: Ovariectomy enhanced the carrageenan-induced degree of pleural exudation and polymorphonuclear leukocyte migration in rats subjected to carrageenaninduced pleurisy. Lung myeloperoxidase (MPO) activity