The effect of pulsed electromagnetic field exposure on osteoinduction of human mesenchymal stem cells cultured on nano-TiO2 surfaces.

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the repair and regeneration of bone. Considerable efforts have been oriented towards uncovering the best strategy to promote stem cells osteogenic differentiation. In previous studies, hBM-MSCs exposed to physical stimuli such as pulsed electromagnetic fields (PEMFs) or directly seeded on nanostructured titanium surfaces (TiO2) were shown to improve their differentiation to osteoblasts in osteogenic condition. In the present study, the effect of a daily PEMF-exposure on osteogenic differentiation of hBM-MSCs seeded onto nanostructured TiO2 (with clusters under 100 nm of dimension) was investigated. TiO2-seeded cells were exposed to PEMF (magnetic field intensity: 2 mT; intensity of induced electric field: 5 mV; frequency: 75 Hz) and examined in terms of cell physiology modifications and osteogenic differentiation. Results showed that PEMF exposure affected TiO2-seeded cells osteogenesis by interfering with selective calcium-related osteogenic pathways, and greatly enhanced hBM-MSCs osteogenic features such as the expression of early/late osteogenic genes and protein production (e.g., ALP, COL-I, osteocalcin and osteopontin) and ALP activity. Finally, PEMF-treated cells resulted to secrete into conditioned media higher amounts of BMP-2, DCN and COL-I than untreated cell cultures. These findings confirm once more the osteoinductive potential of PEMF, suggesting that its combination with TiO2 nanostructured surface might be a great option in bone tissue engineering applications.

Osteogenic Differentiation of MSC through Calcium Signaling Activation: Transcriptomics and Functional Analysis.

The culture of progenitor mesenchymal stem cells (MSC) onto osteoconductive materials to induce a proper osteogenic differentiation and mineralized matrix regeneration represents a promising and widely diffused experimental approach for tissue-engineering (TE) applications in orthopaedics. Among modern biomaterials, calcium phosphates represent the best bone substitutes, due to their chemical features emulating the mineral phase of bone tissue. Although many studies on stem cells differentiation mechanisms have been performed involving calcium-based scaffolds, results often focus on highlighting production of in vitro bone matrix markers and in vivo tissue ingrowth, while information related to the biomolecular mechanisms involved in the early cellular calcium-mediated differentiation is not well elucidated yet. Genetic programs for osteogenesis have been just partially deciphered, and the description of the different molecules and pathways operative in these differentiations is far from complete, as well as the activity of calcium in this process. The present work aims to shed light on the involvement of extracellular calcium in MSC differentiation: a better understanding of the early stage osteogenic differentiation program of MSC seeded on calcium-based biomaterials is required in order to develop optimal strategies to promote osteogenesis through the use of new generation osteoconductive scaffolds. A wide spectrum of analysis has been performed on time-dependent series: gene expression profiles are obtained from samples (MSC seeded on calcium-based scaffolds), together with related microRNAs expression and in vivo functional validation. On this basis, and relying on literature knowledge, hypotheses are made on the biomolecular players activated by the biomaterial calcium-phosphate component. Interestingly, a key role of miR-138 was highlighted, whose inhibition markedly increases osteogenic differentiation in vitro and enhance ectopic bone formation in vivo. Moreover, there is evidence that Ca-P substrate triggers osteogenic differentiation through genes (SMAD and RAS family) that are typically regulated during dexamethasone (DEX) induced differentiation.

Electro-magnetic field promotes osteogenic differentiation of BM-hMSCs through a selective action on Ca(2+)-related mechanisms.

Exposure to Pulsed Electromagnetic Field (PEMF) has been shown to affect proliferation and differentiation of human mesenchymal stem cells derived from bone marrow stroma (BM-hMSC). These cells offer considerable promise in the field of regenerative medicine, but their clinical application is hampered by major limitations such as poor availability and the time required to differentiate up to a stage suitable for implantation. For this reason, several research efforts are focusing on identifying strategies to speed up the differentiation process. In this work we investigated the in vitro effect of PEMF on Ca(2+)-related mechanisms promoting the osteogenic differentiation of BM-hMSC. Cells were daily exposed to PEMF while subjected to osteogenic differentiation and various Ca(2+)-related mechanisms were monitored using multiple approaches for identifying functional and structural modifications related to this process. The results indicate that PEMF exposure promotes chemically induced osteogenesis by mechanisms that mainly interfere with some of the calcium-related osteogenic pathways, such as permeation and regulation of cytosolic concentration, leaving others, such as extracellular deposition, unaffected. The PEMF effect is primarily associated to early enhancement of intracellular calcium concentration, which is proposed here as a reliable hallmark of the osteogenic developmental stage.

Smoke exposure, wheezing, and asthma development: a systematic review and meta-analysis in unselected birth cohorts.

BACKGROUND: The understanding of the role of smoking exposure in the induction of wheezing and asthma in children is important for prevention. METHODS: A systematic review of literature and a meta-analysis were conducted to identify studies on unselected prospective birth cohorts. The effect of exposure to maternal/parental smoking on the induction of current wheezing or asthma was evaluated in children aged 6 months, <6 years, and ≥6 years. Pooled odds ratios (OR) with 95% confidence intervals (CI) were estimated. RESULTS: We identified 43 papers. Exposure to maternal prenatal smoking was associated with an increased risk of wheezing in <6-year-olds (OR 1.36; 95% CI: 1.19-1.55) and of wheezing or asthma in ≥6-year-olds (OR: 1.22, 95% CI: 1.03-1.44). A positive association (OR: 1.24, 95% CI: 1.11-1.38) was also found in the only three studies that evaluated exposure to maternal prenatal smoking alone. Postnatal exposures to maternal/parental smoking were associated with wheezing in <6-year-olds (OR: 1.21; 95% CI: 1.13-1.31 and OR: 1.30; 95% CI: 1.13-1.51), although it was often impossible to separate the role of postnatal from that of prenatal exposure; data in schoolchildren are limited and this precluded a meta-analysis. No clear association was found between exclusive postnatal exposure and wheezing or asthma. CONCLUSIONS: We confirmed an important role of prenatal exposure to maternal smoking on the induction of wheezing and asthma in offspring, particularly in the first years of life. More studies with a consistent number of subjects only exposed to smoke postnatally are needed to better investigate the harmful effects on the induction of wheezing or asthma, particularly in schoolchildren.

Corticosteroids may favor proliferation of thoracic inflammatory myofibroblastic tumors.

Inflammatory myofibroblastic tumor (IMT) was thought to represent a benign post-infectious or post-inflammatory process cured by surgical resection. However, reports of cases with an aggressive clinical course suggest the need for caution about the prognosis. The treatment of choice is a complete surgical resection, while medical treatment options are limited. Corticosteroid therapy has been used with some success in unresectable lesion. However, rapid progression of lung IMT after prednisone treatment has been reported, raising the hypothesis that corticosteroids may favor a tumultuous proliferation of this lesion, possibly through immunosuppression. We here report a similar observation and suggest that other mechanisms may be involved. A 5-year and 6-month-old boy presented with a 72 hr history of breathlessness, initially responsive to albuterol and prednisone. He represented 15 days later with increasing symptoms despite further prednisone treatment. CT chest scan showed a mass lesion in the tracheal lumen, which on biopsy was found to be an IMT. The possibility that prednisone may have an enhancing effect on IMT cell proliferation is demonstrated through IMT cell culture and discussed.

Expression of vascular remodelling markers in relation to bradykinin receptors in asthma and COPD.

BACKGROUND: Vascular remodelling plays a central role in asthma and chronic obstructive pulmonary disease (COPD). Bradykinin (BK) is a vasoactive proinflammatory peptide mediating acute responses in asthma. We investigated the role of angiogenic factors in relation to BK receptors in asthma and COPD. METHODS: Bronchial biopsies from 33 patients with COPD, 24 old (≥50 years) patients with (≥50 years) asthma, 18 old control smokers, 11 old control non-smokers, 15 young (≤40yrs) patients with (≤40 years) asthma and 10 young control non-smokers were immunostained for CD31, vascular endothelial growth factor-A (VEGF-A), angiogenin and BK receptors (B2R and B1R). Fibroblast and endothelial co-localisation of relevant molecules were performed by immunofluorescence. BK-induced VEGF-A and angiogenin release was studied (ELISA) in bronchial fibroblasts from subjects with asthma and COPD. RESULTS: In bronchial lamina propria of old patients with asthma, CD31 and VEGF-A(+) cell numbers were higher than old control non-smokers (p<0.05). Angiogenin(+), B2R(+) and B1R(+) cell numbers in old patients with asthma were higher than in old control non-smokers, control smokers and patients with COPD (p<0.01). Angiogenin(+) cell numbers were higher in patients with COPD than both old control groups (p<0.05). In all patients with asthma the number of B2R(+) cells was positively related to the numbers of B1R(+) (rs=0.43), angiogenin(+) (rs=0.42) and CD31 cells (rs=0.46) (p<0.01). Angiogenin(+) cell numbers were negatively related to forced expiratory volume in 1 s (rs=-0.415, p=0.008). Double immunofluorescence revealed that CD31 cells of capillary vessels coexpressed B2R and that fibroblasts coexpressed B2R, VEGF-A and angiogenin. BK (10(-6)M) induced significant angiogenin release in fibroblasts from asthma and to a lesser extent in COPD. CONCLUSIONS: Unlike COPD, this study suggests the involvement of BK receptors in bronchial vascular remodelling in asthma.

Bradykinin-induced asthmatic fibroblast/myofibroblast activities via bradykinin B2 receptor and different MAPK pathways.

Bradykinin drives normal lung fibroblasts into myofibroblasts, induces fibroblast proliferation and activates mitogen activated protein kinase pathways (MAPK) but its effects on bronchial fibroblasts from asthmatics (HBAFb) have not been yet studied. We studied bradykinin-induced fibroblast proliferation and differentiation and the related intracellular mechanisms in HBAFb compared to normal bronchial fibroblasts (HNBFb). Bradykinin-stimulated HBAFb and HNBFb were used to assess: bradykinin B2 receptor expression by Western blot analysis; cell proliferation by [(3)H] thymidine incorporation; α-smooth muscle actin (SMA) expression/polymerization by Western blot and immunofluorescence; epidermal growth factor (EGF) receptor, extracellular-regulated kinase (ERK) 1/2 and p38 MAPK activation by immunoprecipitation and Western blot, respectively. Constitutive bradykinin B2 receptor and α-SMA expression was higher in HBAFb as compared to HNBFb. Bradykinin increased bradykinin B2 receptor expression in HBAFb. Bradykinin, via bradykinin B2 receptor, significantly increased fibroblast proliferation at lower concentration (10(-11)M) and α-SMA expression/polymerization at higher concentration (10(-6)M) in both cells. Bradykinin increased ERK1/2 and p38 phosphorylation via bradykinin B2 receptor; EGF receptor inhibitor AG1478 and panmetalloproteinase inhibitor GM6001 blocked bradykinin-induced ERK1/2 activation but not p38 phosphorylation. Bradykinin, via bradykinin B2 receptor, induced EGF receptor phosphorylation that was suppressed by AG1478. In HBAFb AG1478, GM6001, the ERK1/2-inhibitor U0126 and the p38 inhibitor SB203580 suppressed bradykinin-induced cell proliferation, but only SB203580 reduced myofibroblast differentiation. These data indicate that bradykinin is actively involved in asthmatic bronchial fibroblast proliferation and differentiation, through MAPK pathways and EGF receptor transactivation, by which bradykinin may contribute to airway remodeling in asthma, opening new horizons for potential therapeutic implications in asthmatic patients.

Cytokines induce tight junction disassembly in airway cells via an EGFR-dependent MAPK/ERK1/2-pathway.

Epithelial barrier permeability is altered in inflammatory respiratory disorders by a variety of noxious agents through modifications of the epithelial cell structure that possibly involve tight junction (TJ) organization. To evaluate in vitro whether pro-inflammatory cytokines involved in the pathogenesis of respiratory disorders could alter TJ organization and epithelial barrier integrity, and to characterize the signal transduction pathway involved Calu-3 airway epithelial cells were exposed to TNF-a, IL-4 and IFN-g to assess changes in: (a) TJ assembly, that is, occludin and zonula occludens (ZO)-1 expression and localization, evaluated by confocal microscopy; (b) apoptotic activity, quantified using terminal transferase deoxyuridine triphosphate nick-end labeling staining; (c) epithelial barrier integrity, detected as transmembrane electrical resistance and expressed as G(T) values; (d) epidermal growth factor receptor (EGFR)-dependent mitogenactivated protein (MAP) kinase (MAPK)/extracellular signal-regulated kinases (ERK)1/2 phosphorylation, assessed by western blotting. Exposure to cytokines for 48 h induced a noticeable downregulation of the TJ transmembrane proteins. The degree ZO-1 and occludin colocalization was 62±2% in control cultures and significantly decreased in the presence of TNF-a (47±3%), IL-4 (43±1%) and INF-g (35±3%). Although no apoptosis induction was detected following exposure to cytokines, changes in the epithelial barrier integrity were observed, with a significant enhancement in paracellular conductance. G(T) values were, respectively, 1.030±0.0, 1.300±0.04, 1.260±0.020 and 2.220±0.015 (mS/cm²)1000 in control cultures and in those exposed to TNF-a, IFN-g and IL-4. The involvement of EGFR-dependent MAPK/ERK1/2 signaling pathway in cytokine-induced damage was demonstrated by a significant increase in threonine/tyrosine phosphorylation of ERK1/2, already detectable after 5 min incubation. All these cytokine-induced changes were markedly prevented when Calu-3 cells were cultured in the presence of an EGFR inhibitor (AG1478, 1 µM) or a MAP kinase inhibitor (U0126, 25 µM). In conclusion, cytokine-induced epithelial injury includes TJ disassembly and epithelial barrier permeability alteration and involves the EGFR-dependent MAPK/ERK1/2 signaling pathway.

Bronchial airway epithelial cell damage following exposure to cigarette smoke includes disassembly of tight junction components mediated by the extracellular signal-regulated kinase 1/2 pathway.

BACKGROUND: Through a variety of biochemical mechanisms, cigarette smoke (CS) may damage airway epithelium, altering its normal structure and function. Injury to epithelium may include changes in tight junction (TJ) integrity with impairment of epithelial barrier function. METHODS AND RESULTS: To study the effect of the exposure to CS condensate (CSC) on TJ integrity, two human bronchial epithelial cell lines (HBECs), BEAS-2B and 16HBE14o-, were used. Exposure of the two HBECs to CSC resulted in a time-dependent and concentration-dependent disassembly of TJs, which were already detectable at 24 h at all the CSC concentrations tested (5%, 10%, and 20%), associated with changes in cell shape, suggesting cell damage. However, a significant inhibition of cell growth and an increase in DNA fragmentation were detected only at the highest CSC concentration tested (20%) at 48 and 72 h, respectively. The involvement of epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase (ERK) 1/2 cascade in CSC-induced damage was shown by the observation that exposure to CSC (5%) induced a marked phosphorylation of ERK1/2, already detectable after 5-min incubation and confirmed by the demonstration that not only ERK1/2 phosphorylation but also CSC-induced TJ disassembly and DNA fragmentation were partially inhibited by a mitogen-activated protein kinase kinase inhibitor (U0126) and completely blocked by a EGFR inhibitor (AG1478). CONCLUSION: CSC-induced damage to airway epithelium includes disassembly of TJs, modulated through the EGFR-ERK1/2 signaling pathway.

Modulation of human lung fibroblast functions by ciclesonide: evidence for its conversion into the active metabolite desisobutyryl-ciclesonide.

BACKGROUND: Ciclesonide, an inhaled corticosteroid administered as inactive compound with almost no binding affinity for the glucocorticoid receptor, is clinically effective in asthma being converted by airway epithelial cells into its active metabolite desisobutyryl-(des)-ciclesonide. AIM: To evaluate whether ciclesonide could directly modulate in vitro bronchial fibroblast functions being converted into des-ciclesonide by these pluripotent cells involved in the regulation of airway inflammation and remodelling. METHODS: Ciclesonide (0.09-9.0 microM) was added to a human adult lung fibroblast cell line (CCL-202), seeded in medium in the presence of the following cytokines and growth factors: (a) basic fibroblast growth factor (bFGF) for cell proliferation, measured by tritiated thymidine ([3H]TdR) incorporation; (b) tumour necrosis factor (TNF)-alpha, to stimulate intercellular adhesion molecule (ICAM)-1 expression and monocyte chemoattractant protein-1 (MCP-1) and eotaxin release, evaluated by flow cytometry and ELISA, respectively; (c) transforming growth factor (TGF)-beta1, for induction of alpha smooth muscle actin (alpha-SMA) protein expression and modification of the organization of alpha-SMA stress fibres, evaluated by Western blot analysis and fluorescence microscopy. RESULTS: The presence of ciclesonide in cell cultures induced a significant downregulation of: (a) bFGF-induced fibroblast proliferation and TNF-alpha-induced ICAM-1 expression, at the 0.3-9.0 microM concentrations (p<0.05); (b) TNF-alpha-induced MCP-1 release, at all the concentrations tested (p<0.05); (c) TNF-alpha-induced eotaxin release, at the three highest concentrations (0.9-9.0 microM) (p<0.05); (d) TGF-beta1-induced of alpha-SMA protein expression at the 0.3-3.0 microM concentrations, associated with a reduction in the organization of alpha-SMA stress fibres. CONCLUSIONS: These data show at cellular level an effective anti-inflammatory activity of ciclesonide on human lung fibroblasts and support the hypothesis that also these cells, in addition to airway epithelial cells, may be involved in converting the parental compound into its active metabolite in the airways.

The histamine-induced enhanced expression of vascular cell adhesion molecule-1 by nasal polyp-derived fibroblasts is inhibited by levocetirizine.

BACKGROUND: Histamine, a key chemical mediator in allergic reaction, exhibits an array of pro-inflammatory effects that include the activation of fibroblasts. The aim of this study was to evaluate whether histamine could stimulate nasal polyp-derived fibroblasts to express vascular cell adhesion molecule (VCAM)-1, a surface molecule involved in structural-inflammatory cell interaction and whether levocetirizine could inhibit this induction. METHODS: Primary nasal polyp tissue-derived fibroblasts were stimulated with histamine (10-1000 microM) or interleukin (IL)-4 plus tumor necrosis factor (TNF)-alpha (0.5-5 ng/mL) and VCAM-1 expression was evaluated by flow cytometry analysis. The inhibitory effect of the selective H1-antagonist levocetirizine (0.01-10.0 microM) on VCAM-1 expression was also tested. RESULTS: Compared with unstimulated cultures, histamine or IL-4 + TNF-alpha, at the highest concentrations tested, significantly increase VCAM-1 expression (p < 0.05). To evaluate the ability of levocetirizine to downregulate VCAM-1 expression, fibroblasts were stimulated with histamine (1000 microM) or IL-4 + TNF-alpha (5 ng/mL), in the presence of the drug (0.01-10.0 microM). The histamine-induced VCAM-1 expression was effectively inhibited by levocetirizine (0.1-10.0 microM) (p < 0.05). No effect of the drug on IL-4 + TNF-alpha-induced VCAM-1 expression was observed. CONCLUSIONS: Histamine upregulates VCAM-1 expression on nasal polyp-derived fibroblasts and this phenomenon, relevant to allergic late-phase inflammation, is effectively inhibited by levocetirizine.

The effect of transforming growth factor (TGF)-beta1 and (TGF)-beta2 on nasal polyp fibroblast activities involved upper airway remodeling: modulation by fluticasone propionate.

Transforming growth factor (TGF)-beta may play a significant role in nasal polyposis pathogenesis, possibly through fibroblast activation. We studied the effects of two TGF-beta isoforms (TGF-beta1 and TGF-beta2) on nasal polyposis fibroblasts by evaluating cell proliferation and differentiation into myofibroblasts. In addition, the inhibitory activity of different concentrations of fluticasone propionate (F.P.) was tested in this in vitro system. Primary nasal polyp tissue-derived fibroblasts were stimulated with different concentrations (1, 10 and 20 ng/ml) of TGF-beta1 and TGF-beta2 for different incubation periods (24, 48 and 72 h) and cell proliferation [3H thymidine ([3H]TdR) incorporation] and alpha-smooth muscle actin (alpha-SMA) expression (immunocytochemistry) was evaluated. The lowest concentration of TGF-beta1 (1 ng/ml) induced a significant increase in [3H]TdR incorporation at 48 and 72 h (p<0.05, each comparison), while in the presence of TGF-beta (10 ng/ml) and TGF-beta2 (1 ng/ml) the enhancement in cell proliferation was significant only after 48 h (p<0.05, each comparison with the unstimulated cells). In contrast, a significant increase in alpha-SMA expression was observed in the presence of the two highest concentration of both TGF-beta isoforms, at 48 and 72 h for TGF-beta1 (p<0.05, each comparison), but only at 72 h for TGF-beta2 (<0.05, each comparison). Finally, at all concentrations tested, F.P. significantly inhibited the TGF-beta1 and TGF-beta2-induced 3HTdR incorporation (p<0.01, each comparison) and the alpha-SMA expression (p<0.05, each comparison). Thus, in vitro different concentrations of TGF-beta1 and TGF-beta2 appear to sequentially stimulate primary nasal polyp tissue-derived fibroblast proliferation and myofibroblast differentiation. These activities are effectively inhibited by F.P.

Human bronchial fibroblasts exhibit a mesenchymal stem cell phenotype and multilineage differentiating potentialities.

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate along different pathways including chondrogenic, osteogenic and adipogenic lineages. MSCs with a fibroblast-like morphology have been identified in human fetal lung. However, their frequency and characterization in human adult lung have not been yet evaluated. Therefore, we analyzed the mesenchymal phenotype and differentiation ability of cultured human adult bronchial fibroblast-like cells (Br) in comparison with those of mesenchymal cell progenitors isolated from fetal lung (ICIG7) and adult bone marrow (BM212) tissues. Surface immunophenotyping by flow cytometry revealed a similar expression pattern of antigens characteristic of marrow-derived MSCs, including CD34 (-), CD45 (-), CD90/Thy-1 (+), CD73/SH3, SH4 (+), CD105/SH2 (+) and CD166/ALCAM (+) in Br, ICIG7 and BM212 cells. There was one exception, STRO-1 antigen, which was only weakly expressed in Br cells. Analysis of cytoskeleton and matrix composition by immunostaining showed that lung and marrow-derived cells homogeneously expressed vimentin and nestin proteins in intermediate filaments while they were all devoid of epithelial cytokeratins. Additionally, alpha-smooth muscle actin was also present in microfilaments of a low number of cells. All cell types predominantly produced collagen and fibronectin extracellular matrix as evidenced by staining with the monoclonal antibodies to collagen prolyl 4-hydroxylase and fibronectin isoforms containing the extradomain (ED)-A together with ED-B in ICIG7 cells. Br cells similarly to fetal lung and marrow fibroblasts were able to differentiate along the three adipogenic, osteogenic and chondrogenic mesenchymal pathways when cultured under appropriate inducible conditions. Altogether, these data indicate that MSCs are present in human adult lung. They may be actively involved in lung tissue repair under physiological and pathological circumstances.

Concentration-dependent activity of mometasone furoate and dexamethasone on blood eosinophils isolated from atopic children: modulation of Mac-1 expression and chemotaxis.

Treatment of asthma with corticosteroids results in downregulation of eosinophilic airway inflammation. We evaluated in vitro the activity of an "inhaled" corticosteroid, mometasone furoate (MF), and of a "systemic" corticosteroid, dexamethasone (DEX), on eosinophil functions, i.e. adhesion molecule expression and cell chemotaxis. Partially purified blood eosinophils were obtained from 18 asthmatic subjects sensitized to house dust mites. The expression of the macrophage antigen (Mac)-1 (CD11b/CD18) was measured by specific monoclonal antibody (mAb) staining and flow cytometry analysis at baseline or after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or with recombinant human (rh) granulocyte macrophage-colony stimulating factor (GM-CSF) plus a mAb anti-human (ah) IgE low affinity receptor [FcepsilonRII or CD23]. Cell chemotaxis toward the complement fragment 5a (C5a) or rh interleukin (IL)-5 was evaluated in Boyden microchambers by light microscopy. Eosinophils showed a significant increase in Mac-1 expression after activation with fMLP or with rh GM-CSF plus ah CD23 mAbs (p<0.05, each comparison) and a remarkable chemotactic response to both C5a or rh IL-5 (p<0.001, each comparison). To test the inhibitory activity of MF and DEX on eosinophil functions, the cells were preincubated for 3 h with four concentrations (0.1, 1, 10 and 100 nM) of each of the two drugs, before being activated by fMLP or by rh GM-CSF plus ah CD23 mAbs or tested with C5a or with rh IL-5. Independently of the stimulus used, both Mac-1 expression and eosinophil migration were effectively downregulated by preincubation with MF or DEX at 1, 10 and 100 nM (p<0.05). The inhibitory activity on cell chemotaxis in response to both C5a or with rh IL-5 was higher for MF than DEX, but only at the highest concentration tested (p<0.05, each comparison). These data demonstrate that concentrations of MF similar to those obtained in vivo are highly effective in inhibiting eosinophil functions involved in airway inflammation.