Cannabidiol improves lung function and inflammation in mice submitted to LPS-induced acute lung injury.

We have previously shown that the prophylactic treatment with cannabidiol (CBD) reduces inflammation in a model of acute lung injury (ALI). In this work we analyzed the effects of the therapeutic treatment with CBD in mice subjected to the model of lipopolysaccharide (LPS)-induced ALI on pulmonary mechanics and inflammation. CBD (20 and 80 mg/kg) was administered (i.p.) to mice 6 h after LPS-induced lung inflammation. One day (24 h) after the induction of inflammation the assessment of pulmonary mechanics and inflammation were analyzed. The results show that CBD decreased total lung resistance and elastance, leukocyte migration into the lungs, myeloperoxidase activity in the lung tissue, protein concentration and production of pro-inflammatory cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2) in the bronchoalveolar lavage supernatant. Thus, we conclude that CBD administered therapeutically, i.e. during an ongoing inflammatory process, has a potent anti-inflammatory effect and also improves the lung function in mice submitted to LPS-induced ALI. Therefore the present and previous data suggest that in the future cannabidiol might become a useful therapeutic tool for the attenuation and treatment of inflammatory lung diseases.

Protective effect of estradiol on acute lung inflammation induced by an intestinal ischemic insult is dependent on nitric oxide.

INTRODUCTION: It has been shown that the innate immune system mediates acute lung inflammation triggered by intestinal trauma. Sexual dimorphism modulates the profile of TH1 and TH2 lymphocytes, and accordingly sex hormones may modulate acute lung inflammation by intestinal ischemia/reperfusion (I/R). Studies indicate that female rats are relatively resistant to organ injury caused by hemorrhagic shock and that the gut of female is more resistant than that of the male to deleterious effects of ischemic injury. At the present study, we investigated the effect of estradiol (E(2)) on the lung inflammation after intestinal I/R and its interaction with the nitric oxide (NO) pathway. METHODS: Anesthetized female rats submitted or not to 7 days ovariectomy (OVx) were subjected to occlusion of the superior mesenteric artery during 45 min, followed by 2 h of reperfusion. Groups of rats were treated with E(2) (17ß-estradiol, 280 µg/kg, s.c.) 24 h before ischemia and/or with the nonselective NO synthase inhibitor L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride) (5 mg/kg, i.v.). In a parallel set of experiments, the selective NO synthase inhibitor, aminoguanidine (50 mg/kg i.v.), was given 1 h before ischemia. In all groups, lung vascular permeability (LVP) was assessed using the Evans blue dye extravasation method, neutrophil recruitment to the tissues by the standard myeloperoxidase (MPO) method, and endothelial NO synthase (eNOS) protein expression by Western blot. RESULTS: In OVx rats, LVP and MPO were increased after intestinal I/R as compared with intact controls. Estradiol reverted the LVP, but not MPO. Aminoguanidine reduced LVP in OVx rats. The E(2) protective effect on LVP was abolished by L-NAME; moreover, an increase in LVP even when compared with OVx rats treated only with L-NAME was observed. In addition, lung eNOS protein expression was reduced in OVx-I/R rats in comparison to intact controls and the E(2) inhibited this effect. CONCLUSIONS: Estradiol treatment is able to reduce lung inflammation due to intestinal I/R, but with the concomitant blockade of NOS activity, this effect is abolished. Nitric oxide probably reduces the vascular deleterious effects of intestinal I/R, and E(2) pretreatment reduces lung inflammation after intestinal I/R and exerts these effects by modulating eNOS protein expression in the lungs.

Enhancement of methacholine-evoked tracheal contraction induced by bacterial lipopolysaccharides depends on epithelium and tumor necrosis factor.

Inhaled bacterial lipopolysaccharides (LPSs) induce an acute tumour necrosis factor-alpha (TNF-α-) dependent inflammatory response in the murine airways mediated by Toll-like receptor 4 (TLR4) via the myeloid differentiation MyD88 adaptor protein pathway. However, the contractile response of the bronchial smooth muscle and the role of endogenous TNFα in this process have been elusive. We determined the in vivo respiratory pattern of C57BL/6 mice after intranasal LPS administration with or without the presence of increasing doses of methacholine (MCh). We found that LPS administration altered the basal and MCh-evoked respiratory pattern that peaked at 90 min and decreased thereafter in the next 48 h, reaching basal levels 7 days later. We investigated in controlled ex vivo condition the isometric contraction of isolated tracheal rings in response to MCh cholinergic stimulation. We observed that preincubation of the tracheal rings with LPS for 90 min enhanced the subsequent MCh-induced contractile response (hyperreactivity), which was prevented by prior neutralization of TNFα with a specific antibody. Furthermore, hyperreactivity induced by LPS depended on an intact epithelium, whereas hyperreactivity induced by TNFα was well maintained in the absence of epithelium. Finally, the enhanced contractile response to MCh induced by LPS when compared with control mice was not observed in tracheal rings from TLR4- or TNF- or TNF-receptor-deficient mice. We conclude that bacterial endotoxin-mediated hyperreactivity of isolated tracheal rings to MCh depends upon TLR4 integrity that signals the activation of epithelium, which release endogenous TNFα.

Effects of single or repeated amphetamine treatment and withdrawal on lung allergic inflammation in rats.

The effects of single or repeated amphetamine (AMPH) treatment and those of AMPH withdrawals on immune-mediated lung inflammatory response were studied in rats. Two experiments were done. In the first, rats egg-albumin (OVA) sensitized were singularly or repeatedly (21 days, once daily) treated with AMPH (1.0 mg/kg) or with a similar number and volume of 0.9% NaCl. The OVA aerosol challenge was performed 12 h after the single or last repeated AMPH treatment and also 72 and 120 h after AMPH withdrawal. In the second experiment, the effects of reserpine (1.0 mg/kg/day for 5 consecutive days) on single AMPH actions on lung allergic response of rats were analyzed. Single and repeated AMPH treatment induced opposite actions on Bronchoalveolar lavage fluid (BAL) cellularity of allergic rats: single treatment decreased and repeated treatment increased the total number of cells as well as those of macrophages, neutrophils and eosinophils. Our data also showed that single but not repeated AMPH treatment decreased the number of neutrophils, monocytes and lymphocytes in the peripheral blood, and increased the total number of bone marrow cells in rats sensitized and challenged with OVA. Furthermore, it was shown that reserpine treatment precluded the effects of single AMPH treatment on cellular migration to the lung of OVA-sensitized and challenged rats. It was concluded that AMPH effects on lung inflammatory response and cell recruitment to the lung in allergic rats rely at least partially on corticosterone serum levels. The possible involvement of vesicular monoamine transporter type 2 (VMAT2) with these observed effects was discussed.

Interference of methysergide, a specific 5-hydroxytryptamine receptor antagonist, with airway chronic allergic inflammation and remodelling in a murine model of asthma.

BACKGROUND: Airway remodelling encompasses the structural changes observed in asthmatic airways. Mast cells, through the release of histamine and 5-hydroxytryptamine (serotonin), are implicated in early asthmatic reactions, bronchoconstriction and mucosal oedema, and in the development of bronchial hyperresponsiveness. However, the association between serotonin and remodelling processes in murine model of airways inflammation remains to be elucidated. OBJECTIVE: As serotonin is released by murine mast cells upon antigen challenge, we tested the hypothesis of its involvement in the development of inflammatory and remodelling processes in a murine model of chronic airway inflammation following prolonged allergen challenge. Methods BALB/c mice were exposed to aerosolized ovalbumin for 20 min 2 days a week, for 4 consecutive weeks. Two hours before each challenge, they were treated with methysergide (intranasally, 40 microg/kg). Forty-eight hours after the last aerosol challenge, bronchoalveolar lavage (BAL) and lung tissue were collected for analysis. RESULTS: Methysergide inhibited the allergen-induced increase in airway eosinophilia, reduced T helper type 2 (Th2) cytokines in lung, spleen or thoracic lymph nodes, and specific IgE levels. The extravasation of plasma and fibronectin production in the lung, and collagen deposition in the lung were also inhibited after methysergide treatment. Although methysergide treatment induced an increase in IFN-gamma levels, experiments with neutralizing antibody suggest that this is not responsible for inhibition. In addition, instillation of serotonin to immunized mice induced eosinophil recruitment to BAL, Th2 cytokine production and fibronectin release in lung as well as collagen deposition. CONCLUSION: Serotonin may contribute to the development and maintenance of remodelling through the release of cytokines and of fibrogenic mediators. Serotonin should therefore be considered as relevant for the development and maintenance of airway remodelling.

Role of sex hormones in allergic inflammation in mice.

BACKGROUND: A number of clinical studies have documented both a pro- and anti-inflammatory role for sex hormones in the context of lung inflammation and worsening of asthma. OBJECTIVE: To determine the role of sex hormones in a murine model of allergic inflammation and airway hyper-responsiveness (AHR) induced by ovalbumin (OVA). METHODS: Female BALB/c were sensitized to OVA on days 0 and 7 and subsequently challenged on day 14 over a 3-day period. Mice had their ovaries removed either 7 days before or 8 days after the first OVA injection on day 0. Pulmonary eosinophilia and AHR were measured 24 h following the last antigen challenge. In other experiments, ovariectomized mice (Ovx) were pre-treated with oestradiol benzoate. In further studies, the effect of the oestradiol antagonist tamoxifen on allergic inflammation in intact mice was evaluated. Spleens from all groups were collected for proliferation assays and measurement of cytokine release. RESULTS: Removal of the ovaries 7 days before sensitization to OVA significantly inhibited lung eosinophilia and IL-5 levels in lung lavage. Furthermore, airway reactivity (maximum response) but not sensitivity (PC100) to methacholine were significantly reduced in these mice. Proliferation of spleen cells and release of IL-5 collected from Ovx mice was significantly attenuated compared with spleen cells obtained from non-Ovx mice. Ovx mice treated with oestradiol benzoate presented partially restored levels of eosinophils and IL-5 in sensitized mice. Moreover, pharmacological antagonism of the effect of endogenous oestrogen with tamoxifen significantly reduced the number of eosinophils in the lung of intact sensitized mice, reproducing the effect of ovariectomy, and suggested a role for oestrogen in the process of antigen sensitization in female mice. In contrast, removal of ovaries 8 days after the first OVA injection failed to alter significantly pulmonary eosinophilia or AHR to methacholine in comparison with non-Ovx mice. Moreover, removal of the ovaries 8 days after the sensitization period induced a significant increase in levels of IL-5 in lung fluid. Spleen cells collected from these mice also had a significantly higher proliferation index and production of IL-5 in response to OVA than non-Ovx mice. Treatment with oestradiol benzoate partially reduced levels of eosinophils present in the lung of Ovx mice, supporting an anti-inflammatory role of sex hormones during the effector phase of the response to inhaled antigen. CONCLUSION: Sex hormones play a dual role in regulating allergic lung inflammation in mice.

Effects of amphetamine on immune-mediated lung inflammatory response in rats.

OBJECTIVE: The present study analyzed the effects of acute amphetamine (AMPH) treatment on immune-mediated lung inflammatory response in rats. METHODS: There were four experiments. In the first and second experiments, rats were treated with AMPH (1 mg/kg) or 0.9% NaCl, and locomotor activity (experiment 1) and serum AMPH concentrations (experiment 2) were measured 1 or 12 h after treatment. In the third experiment, rats which were immunized with ovalbumin (OVA) were treated 14 days later with 0.9% NaCl or AMPH (1 mg/kg). Twelve hours after these treatments, all animals were submitted to challenge by 1% OVA inhalation being analyzed afterwards for bronchoalveolar lavage fluid (BAL), peripheral blood and bone marrow cellularity. In the fourth and final experiment, rats were treated and studied as for experiment 3, except that half of the animals within each group were previously treated with metyrapone prior to the OVA challenge. RESULTS: In the non-immunized rats, AMPH treatment induced an increase in locomotor activity synchronized to high serum AMPH concentrations 1 h after, but not 12 h after treatment. In OVA-challenged rats, AMPH treatment decreased the total number of inflammatory cells, recovered in both BAL and peripheral blood and increased the total number of bone marrow cells. These effects, observed 1 day after OVA challenge, were abrogated by previous metyrapone treatment. CONCLUSION: AMPH treatment changed HPA-axis responsiveness to the stress condition imposed by the OVA challenge decreasing lung and blood leukocytes cellularity most probably via corticosterone actions on bone marrow activity.

Melatonin modulates allergic lung inflammation.

Asthma is an inflammatory lung disease characterized by cell migration, bronchoconstriction and hyperresponsiveness, and can be induced, as an experimental model, by ovalbumin sensitization followed by a challenge. In addition to the well-known immunostimulatory effects of melatonin, research has identified some of its anti-inflammatory properties. In this study, we evaluated the influence of pinealectomy and melatonin administration on cell migration in an experimental model of allergic airway inflammation. We evaluated, in pinealectomized rats treated or not with melatonin, cell migration into the bronchoalveolar fluid, the number of cells and their proliferative activity in the bone marrow, and plasma corticosterone levels. Pinealectomy reduces, 24 hr after the challenge, the total cell number count in the lung and bone marrow cell proliferation, without changing the number of cells in the bone marrow or in the peripheral blood. This fact suggests that melatonin is important in the control of cell recruitment from the bone marrow and the migration of those cells to the lung. Melatonin administration to pinealectomized rats seems to restore the ability of cells to migrate from the bone marrow to the bronchoalveolar fluid. So, the development of specific inhibitors of melatonin would benefit patients with asthma.

Pharmacological modulation of allergic inflammation in the rat airways and association with mast cell heterogeneity.

Administration of ovalbumin by aerosol to sensitised rats produced a rapid (15 min) protein exudation in different airway tissues, as determined by Evans blue staining. This was associated with marked mast cell degranulation determined by histological examination, with there being no difference between mucosal and connective tissue mast cells. A 5-day administration regimen with compound 48/80 selectively depleted connective tissue mast cell (positive to berberine staining) without modifying ovalbumin-induced plasma protein extravasation. Treatment of rats with dexamethasone (1 mg/kg, -12 h) or nor-dihydroguaiaretic acid (30 mg/kg i.p., -30 min) significantly reduced ovalbumin-induced protein extravasation and preserved mucosal mast cell morphology. Indomethacin (4 mg/kg i.v., -30 min) exerted no effect on either parameter. In conclusion, we propose the mucosal mast cell as a target cell responsible at least partly for the inhibitory actions of known anti-inflammatory drugs. We suggest an involvement of endogenous leukotriene(s), but not prostanoid(s), in mucosal mast cell activation/degranulation.

Modulation of hypersensitivity reaction by lipids given orally.

The effect of lipids administration by gavage (0.4% body weight) given daily during four weeks on the hypersensitivity reaction in trachea, upper and lower bronchi, liver, kidney, mesentery, and pancreas was investigated in male rats. The plasma exudation was assessed by using Evans blue (EB) dye extravasation method. There was a significant difference in the permeability of the organs in nonimmunized rats. The immunization increased the vascular permeability and the response with the organs varied greatly. The effect of lipids on anaphylactic reaction was compared to those of untreated rats (control group). The EB extravasation was significantly increased in the trachea obtained from rats treated with cocoa butter and soybean oil. In the upper bronchi of rats treated with soybean oil, the EB extravasation was increased. However, in the lower bronchi, none of the treatments with lipids changed the extravasation of EB. The same was observed in the liver and kidney. The animals treated with lipids by gavage did not present differences in EB extravasation in the mesentery. However, in the pancreas and duodenum, the treatment with fish and soybean oils and cocoa butter markedly lowered EB extravasation.

Nitric oxide modulates Na+, K+-ATPase activity through cyclic GMP pathway in proximal rat trachea.

The present work demonstrated that nitric oxide (NO) modulates Na+, K+-ATPase activity in the proximal rat trachea. Sodium nitroprusside induced concentration-dependent (10-100 microM) stimulation in proximal trachea Na+, K+-ATPase activity. The effect was specific for Na+, K+-ATPase since Mg-ATPase activity was unaffected. This NO-donor changed neither Na+, K+-ATPase nor Mg-ATPase activity in the distal segment. The modulatory action on Na+, K+-ATPase induced by sodium nitroprusside was linked to an increase in nitrates/nitrites and cyclic GMP levels in proximal segments. Modulation of proximal Na+, K+-ATPase activity by sodium nitroprusside was mimicked by S-nitroso-N-acetylpenicillamine (100 microM) and 8-bromo-cyclic GMP (100 microM). Both sodium nitroprusside and 8-bromo-cyclic GMP effects on Na+, K+-ATPase activity of proximal segments of trachea were blocked by 2 microM of KT 5823 (a cyclic GMP-dependent protein kinase inhibitor), but not by 0.5 microM of KT 5720 (a cyclic AMP-dependent protein kinase inhibitor). Both kinase inhibitors decreased proximal Na+, K+-ATPase activity, but did not change Mg-ATPase activity. Okadaic acid (1 microM), a phosphatase-1 inhibitor, increased proximal Na+, K+-ATPase but not Mg-ATPase activity. The effect of okadaic acid was non-additive with that of 8-bromo-cGMP on Na+, K+-ATPase activity. Our results suggest that NO modulates proximal rat trachea Na+, K+-ATPase activity through cyclic GMP and cyclic GMP-dependent protein kinase.

Modulation by lipid mediators of immune complex-induced lung inflammation in mice.

The present study characterized a murine model of immune complex-induced pneumonitis and investigated the role of platelet-activating factor (PAF) and eicosanoids as mediators of lung neutrophil infiltration and hemorrhagic lesions. Rabbit antibodies to bovine serum albumin were injected into the airways and bovine serum albumin was injected intravenously into C3H/HePas and BALB/c mice. After 24 h, a significant increase in neutrophil infiltration and hemoglobin concentration in the bronchoalveolar lavage fluid and lung parenchyma was observed in both strains despite the C3H/HePas strain being 10 times more sensitive to PAF. Neutrophil influx and vascular lesions were not affected by pre-treatment of the mice with the PAF receptor antagonist, WEB 2170 (5-(2-chlorphenyl)carbonyl)-3,4-dihydro- 10-methyl-3-((4-morpholinyl)-2H,7H-cyclopenta(4,5)thieno(3,2-f)(1,2,4)-t riazolo-(4,3-a)(1,4)-diazepine). In contrast, neutrophil influx and vascular lesions were increased by the cyclo-oxygenase inhibitor, indomethacin, and reduced by the inhibitor of leukotriene synthesis, MK 886 (3-[1-(4-chlorobenzyl-3-t-butyl-thio-t-isopropyl-indol-2y-1]-2-2-+ ++dimethylpropanoic acid) and by the leukotriene B4 receptor antagonist, RO 0254094 (2-[(5-carboxypentyl)-6-[6-[3,4-dihidro-4-oxo-8-propyl-2H-1-benzop yran-7-yl)hexyl] benzenepropanoic acid). Increased levels of leukotriene B4, leukotriene C4/D4, thromboxane B2 were found in bronchoalveolar lavage fluid 4 h after induction of the reaction. There is also a tendency to increased prostaglandins E2 levels. Neutrophil infiltration and vascular lesions in immune complex-induced pneumonitis in mice are mediated by leukotriene B4.

Lipid mediators, tumor necrosis factor and nitric oxide and their interactions in immune-complex-induced lung injury.

We investigated the contribution of eicosanoids, platelet-activating factor, tumor necrosis factor and nitric oxide to the neutrophil influx and development of pulmonary haemorrhagic lesions following immune-complex-induced pneumonitis in rats and possible interactions between these mediators. Increased levels of leukotriene B4 and tumor necrosis factor, measured by enzyme immunoassay and L-929 cytotoxicity assay, were found in the bronchoalveolar lavage 1 and 4 h after induction of the reaction, respectively, and their release was dependent on the previous generation of platelet activating factor. Antagonism of leukotriene B4 receptors by RO-0254094 (2-[(5-carboxypentyl])oxy]-6-[6-[3,4-dihydro-4-oxo-8-propyl-2H-1-benzopy ran-7-yl)oxy]hexyl] benzenepropanoic acid), inhibition of nitric oxide synthesis by L-NAME (Nw-nitro-L-arginine methyl ester) and antagonism of PAF-receptors by WEB-2170 (5-(2-chlorphenyl)-3-4-dihydro-10-methyl-3-((4-morpholinyl)carbony l)-2 H,7H-cyclopenta (4,5)thieno(3,2-f)(1,2,4)-triazolo-4,3,a)91,4)diazepine), significantly inhibited the intensity of haemorrhage, evaluated by the increased levels of extravascular hemoglobin in homogenates of lung tissues. Little evidence support the role of tumor necrosis factor in these lesions. The infiltration of neutrophils, evaluated by measuring myeloperoxidase in homogenates of lungs, was reduced by compounds L-663,536 (3-[1-(4 chlorobenzyl)-3-t-butyl thio-5-isopropylindol-2-yl]-2-2-dimethylpropanoic acid), WEB-2170 and L-NAME. These results indicate that neutrophil infiltration and haemorrhagic lesions in immune-complex-induced lung inflammation are mediated by platelet activating factor, leukotriene B4 and nitric oxide and point out to interesting interactions between these mediators.

Impairment in connective tissue mast cells degranulation in spontaneously hypertensive rats: stimulus dependent resistance.

1. Microvascular permeability in the mesentery and consequent leakage of protein into the peritoneum of spontaneously hypertensive rats (SHR) and normotensive rats (NTR) was measured in vivo by the extravasation of Evans blue dye. 2. In sensitized NTR, challenge with antigen produced extensive increases in dye extravasation in the mesentery and in peritoneal lavage fluid within 10 min. 3. In sensitized SHR there was no increase in the permeability of the mesentery and a very weak increase in dye extravasation in the peritoneal cavity following challenge. 4. The glucocorticoid antagonist RU38486 did not change the permeability response induced by antigen in sensitized NTR and SHR. 5. However, compound 48/80 was equally effective in either NTR or SHR in causing increased vasopermeability. 6. Mesenteric mast cells in the NTR were degranulated after immunological challenge, whereas those in the SHR were resistant, as measured histologically. 7. Similarly, challenge ex vivo of mesentery from sensitized NTR induced contraction of guinea-pig ileum in co-incubation experiments, whereas SHR mesentery was unresponsive. 8. Plasma levels of antigen-specific IgE and IgG2a in sensitized NTR and SHR were identical. 9. Immune serum from SHR was unable to induce a passive cutaneous anaphylaxis (PCA) reaction in the skin of NTR and SHR did not develop a PCA reaction upon passive sensitization with NTR immune serum. 10. We conclude that the mast cells of SHR are resistant to degranulation following immunological challenge, although the relevant antibodies are present.

Involvement of eicosanoids and PAF in immune-complex alveolitis.

An immune-complex-mediated hypersensitivity reaction induced in the rat lung was followed by release of the eicosanoids thromboxane, prostaglandin E2 and leukotriene B4 into the bronchoalveolar space. Concomitantly, there was a decrease in the number of circulating platelets. The thrombocytopenia was inhibited by a cyclo-oxygenase inhibitor (indomethacin), a platelet activating factor (PAF) antagonist (BN-52021) and an inhibitor of thromboxane (econazole), but was not affected by a lipoxygenase inhibitor (NDGA). These results suggest the involvement of eicosanoids and PAF in the immune complex hypersensitivity reaction in the rat lung and indicate the occurrence of interactions between PAF and thromboxane.