Effects of fluticasone propionate and budesonide on the expression of immune defense genes in bronchial epithelial cells.

BACKGROUND: COPD patients have increased risk of pneumonia when treated with fluticasone propionate (FP), whereas this is generally not the case with budesonide (BUD) treatment. We hypothesized that BUD and FP differentially affect the expression of immune defense genes. METHODS: Human bronchial epithelial 16HBE cells and air-liquid interface (ALI)-cultured primary bronchial epithelial cells (PBECs) were pre-treated with clinically equipotent concentrations of BUD or FP (0.16-16 nM BUD and 0.1-10 nM FP), and the expression of immune defense genes was studied at baseline and after exposure to rhinovirus (RV16). RESULTS: Using microfluidic cards, we observed that both BUD and FP significantly suppressed CXCL8, IFNB1 and S100A8 mRNA expression in unstimulated 16HBE cells. Interestingly, BUD, but not FP, significantly increased lactotransferrin (LTF) expression. The difference between the effect of BUD and FP on LTF expression was statistically significant and confirmed by qPCR and at the protein level by western blotting. RV16 infection of ALI-cultured PBECs significantly increased the expression of CCL20, IFNB1 and S100A8, but not of LTF or CAMP/LL-37. In these RV16-exposed cells, LTF expression was again significantly higher upon pre-treatment with BUD than with FP. The same was observed for S100A8, but not for CCL20, IFNB1 or CAMP/LL-37 expression. CONCLUSIONS: Treatment of human bronchial epithelial cells with BUD results in significantly higher expression of specific immune defense genes than treatment with FP. The differential regulation of these immune defense genes may help to explain the clinical observation that BUD and FP treatment differ with respect to the risk of developing pneumonia in COPD.

The ß2-subtype of adrenoceptors mediates inhibition of pro-fibrotic events in human lung fibroblasts.

Fibrosis is part of airway remodelling observed in bronchial asthma and COPD. Pro-fibrotic activity of lung fibroblasts may be suppressed by ß-adrenoceptor activation. We aimed, first, to characterise the expression pattern of ß-adrenoceptor subtypes in human lung fibroblasts and, second, to probe ß-adrenoceptor signalling with an emphasis on anti-fibrotic actions. Using reverse transcription PCR, messenger RNA (mRNA) encoding ß(2)-adrenoceptors was detected in MRC-5, HEL-299 and primary human lung fibroblasts, whereas transcripts for ß(1)- and ß(3)-adrenoceptors were not found. Real-time measurement of dynamic mass redistribution in MRC-5 cells revealed ß-agonist-induced G(s)-signalling. Proliferation of MRC-5 cells (determined by [(3)H]-thymidine incorporation) was significantly inhibited by ß-agonists including the ß(2)-selective agonist formoterol (-logIC(50), 10.2) and olodaterol (-logIC(50), 10.6). Formoterol's effect was insensitive to ß(1)-antagonism (GCP 20712, 3 µM), but sensitive to ß(2)-antagonism (ICI 118,551; apparent, pA (2), 9.6). Collagen synthesis in MRC-5 cells (determined by [(3)H]-proline incorporation) was inhibited by ß-agonists including formoterol (-logIC(50), 10.0) and olodaterol (-logIC(50), 10.3) in a ß(2)-blocker-sensitive manner. α-Smooth muscle actin, a marker of myo-fibroblast differentiation, was down-regulated at the mRNA and the protein level by about 50% following 24 and 48 h exposure to 1 nM formoterol, a maximally active concentration. In conclusion, human lung fibroblasts exclusively express ß(2)-adrenoceptors and these mediate inhibition of various markers of pro-fibrotic cellular activity. Under clinical conditions, anti-fibrotic actions may accompany the therapeutic effect of long-term ß(2)-agonist treatment of bronchial asthma and COPD.

Anti-inflammatory activity of beta2-agonists in primary lung epithelial cells is independent of glucocorticoid receptor.

In patients with asthma and chronic obstructive pulmonary disease, the addition of long-acting beta(2)-agonists (LABA) to glucocorticosteroids (GCS) results in better control than increasing the dose of GCS alone. In smooth muscle cells and fibroblasts, one apparent underlying mechanism involves the ability of LABAs to activate the glucocorticoid receptor (GR). The present study investigates the effects of formoterol (FORM), salmeterol (SALM) and budesonide (BUD) on GR activation in bronchial epithelial cells via tumour necrosis factor-alpha-stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) release, GR nuclear translocation and GR-regulated reporter gene activity. Both BUD and FORM inhibited GM-CSF release by < or = 50%. The combination of these two drugs, in clinically relevant concentrations, inhibited GM-CSF release by 85% down to unstimulated levels. A similar inhibition was obtained when combining BUD and SALM. The ability of FORM to inhibit GM-CSF synthesis was not altered by small interfering RNA-mediated depletion of GR and FORM nor SALM-induced GR translocation into the cell nucleus. In addition, FORM did not activate GR-regulated reporter gene activity (SALM was not tested), in contrast to the clear effect of BUD. It was concluded that in bronchial epithelial cells, inhibition of granulocyte-macrophage colony-stimulating factor synthesis by formoterol and salmeterol does not act via previously demonstrated glucocorticoid receptor-related mechanisms, suggesting an alternative pathway in these cells.

Advances in asthma and COPD treatment: combination therapy with inhaled corticosteroids and long-acting beta 2-agonists.

Asthma treatment guidelines advocate the use of long-acting beta2-agonists (LABA) in addition to inhaled corticosteroids (ICS) in patients whose asthma is uncontrolled by ICS alone, thereby addressing two processes fundamental to asthma: bronchoconstriction and inflammation. Superior control--including a reduction in severe exacerbations--of asthma and COPD by ICS/LABA combination therapy has been demonstrated. Results from clinical studies suggest additive and potentially synergistic effects when the two agents are used in combination. No new safety-related issues have been identified with ICS/LABA compared with the monocomponents. The exact mechanisms for the enhanced efficacy of ICS/LABA combinations are under investigation but likely include drug interactions at the receptor level and interwoven signalling pathways, which may result in improved function of 2- adrenoceptors and steroid receptors. Data from preclinical studies provide evidence of additive, compensatory, complementary and synergistic effects of ICS and LABA in the control of inflammation and airway and lung remodelling. These effects may contribute to the improved efficacy seen when treating asthma and COPD with ICS/LABA combinations in clinical studies. Two ICS/LABA combination products are available: budesonide/formoterol (Symbicort) and salmeterol/fluticasone propionate (SeretideTM). An ICS/LABA combination in a single inhaler represent safe, effective and convenient treatment options for the management of patients with asthma and COPD. Clinical results also suggest that adjustable dosing with budesonide/formoterol provides better asthma control than fixed dosing. Further elucidation of the underlying mechanisms responsible for this superior disease control is needed.

Gut mucosal uptake and retention characteristics contribute to the high intestinal selectivity of budesonide compared with prednisolone in the rat.

BACKGROUND: Budesonide and prednisolone are both effective for the treatment of inflammatory bowel disease, but budesonide produces fewer adverse systemic effects. High first-pass hepatic inactivation of budesonide partially explains its favourable ratio between topical and systemic activity, but it is probable that its uptake and retention in intestinal target tissues are also contributory. AIM: To compare the uptake and retention of radio-labelled budesonide and prednisolone in rat ileal mucosa in vivo. METHODS: 3H-Budesonide and 3H-prednisolone were applied for 10 min directly to a perfused segment of rat ileum in vivo, followed by saline lavage every 10 min. Steroid uptake into the mucosa and submucosa was assessed at 20 min and 4 h. The uptake of budesonide was also measured in allergen-challenged animals vs. saline-challenged controls to assess whether inflammation of the mucosa with ongoing plasma exudation would impair its uptake. RESULTS: Budesonide was better absorbed into ileal tissue (15-fold at 20 min) than prednisolone and better retained (50-fold at 4 h) after topical administration. The uptake of budesonide was not impaired by exudation processes following allergen challenge. CONCLUSIONS: The higher uptake and retention characteristics of budesonide in gut mucosa should contribute to its greater intestinal selectivity compared with that of prednisolone.

Topical and oral anti-inflammatory activity of budesonide compared with oral prednisolone in an animal model using allergen-induced gut mucosal exudation of plasma as a marker.

BACKGROUND: Development of topically active glucocorticosteroids with minimal systemic effects is paramount in improving therapy in inflammatory bowel disease. Our experimental model in the rat has proved useful for assessing topical versus systemic anti-inflammatory potency of glucocorticosteroids on the inflamed gut. METHODS: Experiments were performed on allergen-sensitized perfused rat ileum in vivo. Mucosal exudation of plasma, induced by local allergen perfusion, was measured as the appearance of circulating 125I-labelled albumin in the gut lumen. Experiments compared the anti-exudative effects of oral budesonide (0.1 mg/kg) with oral prednisolone (1, 3.3 or 10 mg/kg) and saline, given by oral gavage 24 h prior to allergen challenge, and of topical budesonide (3 x 10(-5) mol/L) with saline, administered in the perfusate 4 h prior to allergen challenge. Systemic glucocorticosteroid activity was assessed by weighing thymus glands after sacrifice. RESULTS: Allergen-induced plasma exudation was significantly reduced by oral budesonide, oral prednisolone (dose-dependently) and topically applied budesonide; topical budesonide was effective within 4 h. While prednisolone significantly reduced the relative thymus weight at both 3.3 and 10 mg/kg, budesonide given orally, 0.1 mg/kg, or topically, 3 x 10(-5) mol/L, had no significant effect. CONCLUSION: Budesonide, administered orally or topically, shows higher selectivity for the gut mucosa than prednisolone and produces local anti-inflammatory responses comparable to prednisolone, without the accompanying systemic effects.

Prolonged airway activity and improved selectivity of budesonide possibly due to esterification.

We addressed the question of whether the prolonged local retention of the glucocorticoid (GC) budesonide (BUD) within airway tissue, due to reversible fatty acid esterification, is associated with protracted topical anti-inflammatory activity and improved airway selectivity, when compared with fluticasone propionate (FP). BUD or FP at 25 nmol/kg was administered intratracheally or subcutaneously to adrenalectomized rats, followed by lipopolysaccharide (LPS) intratracheal instillation. The trachea and main bronchi were lavaged 6 h after LPS, and tumor necrosis factor-alpha (TNF-alpha) concentration and cell number in the lavage fluid were measured. Instilled 1 h before LPS, both GCs reduced TNF-alpha by 70% (p < 0.05) and mononuclear cells by 55% (p < 0.01), with no reduction in neutrophils. Instilled 6 h before LPS, a significant reduction of TNF-alpha (59%, p < 0.02) and mononuclear cells (47%, p < 0.05) was achieved only with BUD. After subcutaneous administration, no significant effects were observed. BUD did not exert higher systemic activity than FP, measured as plasma corticosterone suppression. In conclusion, BUD exerted a more prolonged topical anti-inflammatory activity, and a higher airway selectivity than FP, possibly because of its reversible fatty acid esterification within airway tissue. This may contribute to the high efficacy and safety of BUD in asthma, even with once-daily inhalation.

Adrenalectomy permits a late, local TNF-alpha release in LPS-challenged rat airways.

The normal rise of circulating endogenous glucocorticosteroids (GCS), in response to immunological stimuli, can be impaired in patients with inflammatory diseases. The aim of this study was to investigate whether abolition of the endogenous GCS response promotes local production of the pro-inflammatory cytokine, tumour necrosis factor (TNF)-alpha, in challenged airways and affects the cellular response in rats. In adrenalectomized or sham operated rats, the trachea and main bronchi were lavaged at various times after intratracheal instillation of low dose lipopolysaccharide (LPS). TNF-alpha in lavage fluid and plasma corticosterone were measured, and cells were differentiated. In adrenalectomized rats, LPS-induced in the airways a biphasic TNF-alpha release peaking at 2 and 6 h, whereas in sham operated rats the second peak was absent; probably inhibited by the strong rise of plasma corticosterone. The second peak was abolished in adrenalectomized rats by pretreatment with exogenous GCS. The LPS-induced neutrophil influx and a decrease in mononuclear cells were prolonged in adrenalectomized rats. In conclusion, abolition of the endogenous glucocorticosteroid response promotes the late release of tumour necrosis factor-alpha in the airways and prolongs the cellular response. This suggests that a normal rise of endogenous glucocorticosteroid after an immunological trigger contributes to a dampening of the late inflammatory activity.

Reversible fatty acid conjugation of budesonide. Novel mechanism for prolonged retention of topically applied steroid in airway tissue.

A high airway concentration might be required for the antiasthmatic efficacy of inhaled glucocorticosteroids (GCS). The topical uptake and retention of GCS in airway tissue were compared for GCS of the inhaled type [budesonide (BUD), fluticasone propionate (FP), and beclomethasone dipropionate (BDP)] and of the noninhaled type (dexamethasone and hydrocortisone). 3H-labeled GCS solutions were administered into rat airways by either perfusion of trachea in vivo, intratracheal instillation, or inhalation. Radioactivity was determined in the airway tissue, lung parenchyma, and plasma 20 min to 24 hr after exposure. Ethanol extracts of exposed tracheas were analyzed by HPLC. Exposed tracheas were also incubated in vitro in buffer, and the released radioactivity was analyzed by HPLC. BUD, FP, and BDP were equally well taken up into the airway tissue; their uptake was 25-130 times greater than that of dexamethasone and hydrocortisone. BUD was shown to form very lipophilic intracellular fatty acid esters (at carbon 21) in the airway and lung tissue after topical application. In large airways 20 min after administration, approximately 70-80% of retained BUD was conjugated. BUD stored in esterified form in the tissue was retained in large airways for a prolonged time, compared with FP and BDP, which do not form such conjugates. The fatty acid conjugation of BUD is reversible in vivo; BUD conjugates are gradually hydrolyzed and free BUD is regenerated. This reversible conjugation may improve airway selectivity, as well as prolong the local anti-inflammatory action of BUD in the airways and might be one explanation for why BUD is efficacious in the treatment of mild asthma when inhaled once daily.

Inhibition of nitric oxide synthase reduces Sephadex-induced oedema formation in the rat lung: dependence on intact adrenal function.

In the present study we have investigated the effect of L-nitro arginine mono methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase on Sephadex induced inflammation in the rat lung. Instillation of Sephadex into the airways induced an inflammatory reaction characterized by a long-lasting interstitial oedema, measured as an increase in lung weight, and an influx of inflammatory cells into the airways. L-NAME given s.c. prevented the increase in lung weight following Sephadex instillation. The inactive enantiomer D-NAME had no effect, nor did aminoguanidine which indicates that this effect of L-NAME was mediated by inhibition of the constitutive form of NOS. Treatment with L-NAME did not reduce an established oedema. In contrast, L-NAME tended to enhance the influx of oesinophils into the airways of Sephadex-instilled animals. L-NAME did not have any effect on the development of oedema in adrenalectomized rats or in animals where formation of glucocorticosteroids (GCS) was inhibited with metyrapone. L-NAME did not however, increase plasma levels of corticosterone. The present results indicate that, in this model, inhibition of NO-synthesis has marked anti-inflammatory effects. The underlying mechanism is complex but seems not to involve prevention of overproduction of NO.

Attenuation of bradykinin-induced mucosal inflammation by topical budesonide in rat trachea.

The extravasation of plasma proteins and formation of interendothelial gaps in submucosal microvessels by mucosally-applied bradykinin (BK), were studied in the rat trachea. The effects of topical and systemic (s.c.) glucocorticoid budesonide (BUD) were investigated in the presence or absence of inhibitors of BK-degradtive enzymes (captopril and thiorphan 10 microM to inhibit angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP), respectively). Inhibition of these enzymes markedly increased the inflammatory responses to BK. Topical BUD (3 microM, 10 min contact, 90 min before BK) significantly decreased the volume of plasma in the tracheal lumen, both in the absence and presence of the enzyme inhibitors. Thus, the main anti-transudation mechanism of topical BUD is not related to modulation of BK-breakdown. However, this may be the mechanism for systemic BUD. Neither topical nor systemic BUD prevented interendothelial gap formation.

Native fluorescence of extravasated proteins. A new method for quantitation of macromolecular vascular leakage.

Macromolecular vascular leakage into rat tracheal lumen is quantified by a new method that does not require the administration of an exogenous macromolecular tracer or dye to the animals. The magnitude of vascular leakage is determined by the concentration of extravasated plasma proteins measured by their native fluorescence and assayed against diluted plasma. Native fluorescence of proteins (PNF), when excited at 295 nm and measured at 340 nm, is almost exclusively due to tryptophan fluorescence. The PNF assay can be used in the range of protein concentration of 0.1-120 micrograms/mL. The results obtained in the PNF assay were found to be equivalent to the results from two other conventional assays where fluorescent or radioactive albumin was used as the external macromolecular tracer.

An in situ preparation of rat trachea for studying the effects of mucosally applied anti-asthma drugs on mediator-induced plasma exudation.

An animal model is described for testing the effects of anti-asthma drugs on mediator-induced airway inflammation. An in situ segment of trachea is slowly perfused with normal saline in spontaneously breathing, anaesthetised rats. Both the anti-asthma drug and the inflammatory mediator can be applied directly to the airway mucosal surface at the same and/or different times. The amount of plasma in samples of perfusate is monitored. Application of a mediator, such as bradykinin, results in an increase in the amount of plasma in the tracheal lumen (and hence in the perfusate). Plasma in the perfusate can be estimated without injecting a labelled tracer for plasma exudation by using a protein native fluorescence (PNF) method as described by Miller-Larsson & Brattsand (3).

A method of description of single muscle fibre action potential by an analytical function V(t, r).

The paper describes a new method of analytical description of single muscle fibre action potential suitable for computer simulation. The description introduced is a product of quadratic and gaussian functions. The coefficients of the function are determined on the basis of dependences of SFAP parameters on electrode-to-fibre distance combined from the transformed results of electrophysiological experiments and modelling. The description is being used for computer simulation of motor unit action potential which results will be described in forthcoming papers.

Motor unit action potential field--modelling results.

The theoretical field of a motor unit (MU) action potential (MUP) was considered. It has been proved that in normal muscles the shape of a smooth threephasic MUP curve is determined mostly by the spatial distribution of MU muscle fibres. Phenomena called "time dispersion" are of prime importance in pathology, where they complicate normal threephasic MUP. Amplitudes and time parameters of model threephasic MUP were analyzed as a function of the radial distance R from the geometrical centre of the motor unit territory (MUT) and approximated by mathematical expressions. It appeared that analysis of radial variability of MUP curve allows conclusions to be made about the MUT size and the spatial distribution of MU muscle fibres. These anatomical features of a MU are often changed in pathological muscles, thus the proposed methods of their evaluation could be helpful in diagnosis of neuromuscular diseases.

An analysis of extracellular single muscle fibre action potential field--modelling results.

The radial variability of extracellular single muscle fibre action potential (SFAP) field was studied on the basis of an SFAP theoretical model originally constructed by Rosenfalck (1969). Amplitude and time parameters of the SFAP as a function of the radial distance r less than or equal to 10 mm from the fibre were described by simple mathematical expressions. The results obtained were compared with existing experimental data from different authors and discussed in detail.