Additive anti-inflammatory effect of formoterol and budesonide on human lung fibroblasts.

BACKGROUND: It has been shown that treatment with a long acting beta2 agonist in addition to a glucocorticoid is beneficial in the treatment of asthma. In asthma inflammatory cells, particularly eosinophils, migrate into the pulmonary tissue and airway lumen by means of adhesion molecules expressed on resident tissue cells--that is, fibroblasts--and become activated by cytokines and adhesive interactions. A study was undertaken to determine whether an interaction exists between the long acting beta2 agonist formoterol and the glucocorticoid budesonide on inhibition of adhesion molecule expression, as well as chemo/cytokine production by human lung fibroblasts. METHODS: Lung fibroblasts were preincubated with therapeutically relevant drug concentrations of 10(-8) M to 10(-10) M. Cells were stimulated with interleukin (IL)-1beta (1 or 10 U/ml) for 8 hours and supernatants were collected for measurement of GM-CSF and IL-8 concentrations. The cells were fixed and subjected to a cell surface ELISA technique to measure the expression of ICAM-1 and VCAM-1. RESULTS: Formoterol exerted an additive effect on the inhibition of IL-1beta stimulated ICAM-1 and VCAM-1 upregulation and GM-CSF production by budesonide in concentrations of 10(-9) M and above (p<0.05). IL-8 production was not influenced by formoterol. CONCLUSION: Formoterol exerts an additive effect on the anti-inflammatory properties of budesonide. In vitro data support the finding that the combination of budesonide and formoterol in asthma treatment strengthens the beneficial effect of either drug alone.

Effects of budesonide and formoterol on eosinophil activation induced by human lung fibroblasts.

Budesonide and formoterol are extensively used in current asthma therapy. Budesonide is known as potent antiinflammatory agent and formoterol also appears to have some antiinflammatory properties. We investigated inhibitory effects of these drugs on eosinophil activation in vitro as induced by fibroblast-conditioned medium (FCM). We measured the modulation of expression of clonal designator (CD)11b and L-selectin with flow cytometry after 4 h or 16 h of culture of eosinophils when budesonide or formoterol was applied either directly to the eosinophils while they were stimulated with FCM (direct method) or when each drug was applied to lung fibroblasts from which conditioned medium was then administered to eosinophils (indirect method). In the direct method, budesonide (10(-)(8) M) inhibited the modulation of CD11b (44 [25th to 75th percentiles: 26 to 66]% of control) and L-selectin (30 [-13 to 48]% of control) only after 16 h, and not after 4 h. Formoterol did not directly inhibit the modulation of eosinophil CD11b and L-selectin expression. In the indirect method, both budesonide and formoterol inhibited lung fibroblast activation, resulting in diminished eosinophil activation after 4 h. Budesonide or formoterol at 10(-)(8) M inhibited upregulation of CD11b to 26 [15 to 40]% and 38 [23 to 46]%, respectively, and inhibited L-selectin shedding to 14 [-3 to 50]% and 27 [2 to 62]%, respectively, of control values. These results show that budesonide inhibits eosinophil activation primarily through effects on lung fibroblasts, presumably by inhibiting production of granulocyte-macrophage colony-stimulating factor. After longer incubation periods, budesonide also directly inhibits eosinophil activation. In contrast, formoterol can inhibit eosinophil activation only via inhibitory effects on lung fibroblasts. We did not observe an additional effect of formoterol, beyond the effects induced by budesonide under any circumstance studied. Lung fibroblasts, in addition to eosinophils, may serve as important target cells for antiinflammatory treatment in asthma.

Budesonide and formoterol inhibit ICAM-1 and VCAM-1 expression of human lung fibroblasts.

The glucocorticoid budesonide and the long-acting beta2-adrenoceptor agonist formoterol are used in asthma therapy for their anti-inflammatory and bronchodilating effects, respectively. Since expression of adhesion molecules on resident cells in the lung plays an important role in asthmatic inflammatory responses, the effects of these drugs on the cytokine-induced intercellular adhesion molecule-1 (ICAM)-1 and vascular cell adhesion molecule-1 (VCAM)-1 expression of human lung fibroblasts were investigated. Budesonide and formoterol were added in the absence or presence of interleukin (IL)-1beta, tumour necrosis factor-alpha (TNF-alpha), interferon gamma (IFN-gamma) or IL-4 to human lung fibroblasts; ICAM-1 and VCAM-1 expression were measured after 8 h using a cell surface enzyme linked immunosorbent assay (ELISA). It was found that both budesonide and formoterol significantly inhibited (p<0.05) the increased expression of ICAM-1 and VCAM-1 after stimulation with IL-1beta (maximal inhibition (median (25-75% percentiles) 50 (48-52) and 61% (42-69), respectively, with budesonide and 55 (50-73) and 86% (64-94), respectively, with formoterol (10(-7) M)), TNF-alpha (maximal inhibition 49 (46-57) and 57% (44-68), respectively, with budesonide and 44 (40-75) and 62% (52-83) respectively, with formoterol), IFN-gamma (maximal inhibition 64% (41-67) with budesonide and 39% (29-49) with formoterol for ICAM-1) and IL-4 (maximal inhibition 82% (69-92) with budesonide and 43% (33-67) with formoterol for VCAM-1) in a dose-dependent manner. The results show that budesonide, as well as formoterol, in probably clinically relevant concentrations inhibits cytokine-induced adhesion molecule expression on human lung fibroblasts from a concentration of 10(-9) M. This inhibitory effect on resident cells may have implications for the infiltration of inflammatory cells into pulmonary tissue during therapy with these drugs in asthma.

Interferon-gamma and interleukin-4 differentially regulate ICAM-1 and VCAM-1 expression on human lung fibroblasts.

The expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and more specifically vascular adhesion molecule-1 (VCAM-1) on lung fibroblasts may be important for migration of inflammatory cells through the submucosa to the airway lumen in the asthmatic inflammatory response. This study aimed to assess which cytokines are regulating ICAM-1 and VCAM-1 expression on human lung fibroblasts. For this purpose, confluent fibroblast cultures (derived from lung tissue from a nonasthmatic donor) were stimulated for 4 h with interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha, interferon (IFN)gamma, IL-4, IL-5 or transforming growth factor (TGF)beta. IL-1beta (optimal concentration (OC) 1 U x mL(-1)) and TNFalpha (OC 100 U x mL(-1)) both increased ICAM-1 and VCAM-1 expression. IFNgamma (OC 2 U x mL(-1)) increased only ICAM-1 expression and IL-4 (OC 5 ng x mL(-1)) increased only VCAM-1 expression, whereas IL-5 (20 ng x mL(-1)) and TGFbeta (10 ng x mL(-1)) did not influence ICAM-1 or VCAM-1 expression. ICAM-1 expression reached a plateau at 8-12 h after cytokine stimulation and remained constant for at least 24 h. VCAM-1 showed a transient increased expression within 24 h after IL-1beta and TNFalpha stimulation. In contrast, VCAM-1 expression did not decrease after maximal expression at 4 h upon IL-4 stimulation. It is concluded that the Helper-1T-cell, type cytokine interferon gamma and the Helper-2 T-cell type cytokine interleukin-4 differentially regulate intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression on human lung fibroblasts. The proinflammatory cytokines interleukin-1beta and tumour necrosis factor alpha increase both intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, without differential regulation of the expression of these adhesion molecules.

Eosinophil derived neurotoxin (EDN) levels in commercial human urinary preparations of glycoprotein hormones.

Eosinophil derived neurotoxin (EDN) is a ubiquitous human ribonuclease, occurring not only in eosinophils, but also in many tissues and body fluids. It may be a contaminant of commercial human urinary preparations of chorionic gonadotropin (hCG) and other glycoprotein hormones. Here we describe the use of a fast commercial assay to quantify this contaminant and demonstrate that the content varies much between different commercial glycoprotein hormone preparations. As this ribonuclease may have a cytotoxic activity on certain cells, it is useful to be able to determine its quantity in a fast and reliable way in these preparations.

Eosinophil and mast cell parameters in children with stable moderate asthma.

UNLABELLED: Mast cells and eosinophils are important cells that contribute to the process of inflammation in asthma either by activating other cells or by secreting products which are potentially toxic to the respiratory epithelium. The influx of these cells in the airways and the secretion of toxic products by these cells is abrogated by inhaled corticosteroids. METHODS: In a double blind randomised, placebo controlled, study in children with stable moderate asthma (N = 34, 15 children received fluticasone propionate (FP), an inhaled corticosteroid, and 19 children used a placebo), we investigated the influence of treatment with FP 100 microg b.d. on various parameters of inflammation: number of eosinophils, secretory products of eosinophils i.e. ECP and EDN (in serum and urine) and a secretory product of mast cells, histamine, which is determined as the compound to which histamine is converted and excreted by the human body: NT-methyl-histamine. RESULTS: Previously we reported that lung function increased and bronchial hyperresponsiveness decreased in the 30 children that completed the study during treatment with FP. In these children we found that none of the laboratory parameters of inflammation changed significantly during treatment with either FP or placebo. However, the decrease in urinary EDN almost reached significance (P = 0.07). CONCLUSIONS: Our results indicate that the number of eosinophils, serum ECP and EDN and urinary EDN as well as urinary NT-methyl-histamine do not reflect asthma disease activity in children with stable moderate asthma. Our data on urinary EDN warrant further study of the use of this parameter to monitor asthma in children.

Changes in CD11b and L-selectin expression on eosinophils are mediated by human lung fibroblasts in vitro.

Eosinophilic airway infiltration is a central feature in asthma. Eosinophils recovered from bronchoalveolar fluid show an activated phenotype, e.g., increased CD11b and decreased L-selectin expression. We investigated whether lung fibroblasts are able to activate eosinophils in vitro, and if so, which activating factor is most important. CD11b and L-selectin expression of isolated peripheral blood eosinophils were measured by flow cytometry after coculture with normal lung fibroblasts or their conditioned medium. We found that eosinophil CD11b expression increased (154% and 210%, p < 0.05) and L-selectin expression decreased (59% and 35.5%, p < 0.05) on eosinophils compared with baseline (100%) after 4 and 24 h of coculture with interleukin-1-beta (IL-1beta)-stimulated fibroblasts, respectively. Conditioned medium of stimulated fibroblasts also increased CD11b expression, but to a smaller extent (p < 0.05). L-selectin expression of eosinophils in cocultures was not different from that of eosinophils in conditioned medium. Only anti-granulocyte/macrophage colony-stimulating factor (anti-GM-CSF) reduced the activation of eosinophils in conditioned medium to almost basal levels (p < 0.05). An increase in CD11b expression is mediated by cytokines as well as direct cell contact, whereas a decrease in L-selectin expression is only mediated by cytokines. GM-CSF released by fibroblasts is an important factor in the modulation of both CD11b and L-selectin expression. These results show that lung fibroblasts can activate eosinophils by both adhesive interactions and by soluble factors.

Prevalence of sensitization to the storage mites Acarus siro, Tyrophagus putrescentiae, and Lepidoglyphus destructor in allergic patients with different degrees of sensitization to the house-dust mite Dermatophagoides pteronyssinus.

The prevalence of sensitization to the storage mites Acarus siro (AS), Tyrophagus putrescentiae (TP), and Lepidoglyphus destructor (LD) was studied in 250 sera of patients with different degrees of sensitization to the house-dust mite Dermatophagoides pteronyssinus (DP) by measuring IgE binding to extracts of the storage mites. Additionally, allergenic cross-reactivity between DP and the storage mite species was studied by RAST inhibition with five individual sera (and a pool of these sera) with moderate IgE levels to all three storage mites and to DP. Increased serum IgE to storage mites was found in 46% of the 200 patients sensitized to DP. Increased prevalence rates of IgE titers to storage mites were associated with higher IgE levels to DP. In 50 sera without sensitization to DP, only five sera showed increased IgE to one of the storage mites. Extracts of TP almost completely inhibited the IgE binding to AS, and vice versa. DP inhibited IgE binding to all storage mites up to 60%, whereas IgE binding to DP was only minimally inhibited by extracts of storage mites. In conclusion, cosensitization to storage mites is a frequent finding in patients sensitized to DP. Although this is largely the result of cross-reactivity between different mite species, it may nevertheless be of clinical significance in patients exposed to storage mites.

Eosinophils and eosinophil-derived proteins in children with moderate asthma.

Laboratory parameters can contribute to the diagnosis of asthma, which is often a difficult procedure in paediatric patients. The aim of this study was to investigate the value of eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) in the diagnosis of paediatric asthma. The number of eosinophils, serum ECP and EDN, and urinary EDN were determined in 22 children with stable, allergic asthma, aged 4-14 yrs, and in 17 age-matched healthy controls. Symptoms were monitored, the peak expiratory flow rate (PEFR) was recorded in the younger children, and lung function tests (forced expiratory volume in one second (FEV1) and the provocative concentration of histamine causing a 20% fall in FEV1 (PC20)) were performed in the older children. None of the asthmatic children had respiratory symptoms. PEFR was not significantly different in asthmatic children compared to controls. The FEV1 % predicted was significantly lower compared to controls. The number of eosinophils, serum ECP and EDN, and urinary EDN were significantly higher in asthmatic children compared with controls. After correction of serum ECP and EDN, and urinary EDN for the number of eosinophils, the differences between patients and controls disappeared. The nocturnal PEFR and the FEV1 were significantly related to urinary EDN. The results suggest that serum and urinary concentration of eosinophil-derived proteins can be determined instead of the number of eosinophils to support the diagnosis of asthma in childhood. The urinary concentration of eosinophil-derived neurotoxin can be especially valuable in young children, because in this age group quantification of lung function cannot be performed and blood sampling can be difficulty.

Standardization of house dust mite extracts. Liberation of allergenic and antigenic components in relation to extraction conditions.

The spontaneous release of house dust mite components from cultures of Dermatophagoides pteronyssinus into slightly buffered water was studied against time, using both continuous and discontinuous extraction procedures. It was shown that proteins, carbohydrates, IgE binding components and precipitating antigenic components were rapidly released from the house dust mite cultures, reaching a maximal liberation within 1 h of extraction. Repeated extractions of house dust mite cultures (discontinuous extraction) showed an additional release of IgE components but the IgE binding potency declined after successive extractions, while showing increasing release of immunological inactive components. IgE binding to antigens immobilized to polystyrene surfaces (IgE-ELISA) appeared to be less sensitive compared with cyanogen-bromide activated discs (IgE-RAST). It was concluded that extraction procedures of house dust mite cultures with short incubation time of 1 h or less are to be preferred.

Standardization of allergenic extracts of Aspergillus fumigatus. Liberation of IgE-binding components during cultivation.

During cultivation of Aspergillus fumigatus a rapid liberation of IgE-binding components was found reaching maximum values during the logarithmic phase of growth (phase I). After a fall in IgE-binding titers during phase II, appearance of additional IgE-binding components was noted during the period of lysis of the microorganism (phase III). These latter allergenic components are different from the phase I IgE-binding components, as was shown by crossed-inhibition studies. The number of precipitating antigenic components was not related with the corresponding IgE-binding titers and showed an increase during all phases of growth. The rapid changes in both IgE- and IgG-binding properties and the discrepancies between precipitating properties and IgE binding are discussed in relation to standardization and quality control of aspergillus extracts.