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Notes: Background In bronchial mucosa, T cells are in close association with fibroblasts. This cell contact raises the possibility of cross-talk between the two cell types through cytokines, such as interleukin-4 (IL-4).Objective We postulated that IL-4 may modulate collagen synthesis and degradation in the fibroblasts of asthmatics.Methods Bronchial fibroblasts from asthmatics (BAF) and normal controls (BNF) were stimulated with IL-4. Procollagen I gene expression and protein production were measured by real-time PCR, RT-PCR, and radioimmunoassay. The effect of IL-4 on the regulation of procollagen I (α1) promoter was studied through transient cell transfections. The implication of Sp1 and AP-1 in regulating IL-4-induced procollagen I (α1) production was determined. The effect of IL-4 on metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) production and gene expression was evaluated.Results Following IL-4 stimulation, there was a significant increase in the expression of mRNA of procollagen I (α1) by human bronchial fibroblasts of asthmatics and controls. IL-4 has a dose–response effect on mRNA, with a maximal effect at 5 ng/mL, as determined by real-time PCR. The maximal increase in procollagen I (α1) was observed at 6 h after IL-4 stimulation in both BNF and BAF. BAFs have a greater increase in the procollagen I (α1)/β2 microglobulin ratio after 6 h of IL-4 stimulation (4.1×10−2±0.03 to 20.8×10−2±0.1) compared with BNF (2.9×10−2±0.006 to 9.2×10−2±0.08) (P=0.001). In transient transfection experiments, IL-4 increased promoter activity by threefold in BAF and BNF. Sp1 was up-regulated after IL-4 stimulation and AP-1 was down-regulated as shown by electrophoretic mobility shift assay. IL-4 decreased MMP-2 protein and mRNA levels, and did not alter TIMP-2 production.Conclusions IL-4 positively regulates procollagen I (α1) transcription by direct promoter activation and increases the TIMP-2/MMP-2 ratio, thereby supporting the profibrotic effect of this cytokine. Thus, this study emphasizes that IL-4 may be considered as a link between inflammation and collagen deposition observed in asthmatic airways.

Notes: Background Pathophysiology of corticosteroid (CS)-resistant asthma remains incompletely understood.Objective To determine if failure of asthma to clinically improve with CS is due to a defective response of airway bronchial inflammation to these drugs.Methods Twenty-one asthmatics having a decreased baseline FEV1 that improved ≥ 30% with inhaled β2 agonist got bronchial biopsies before and at the end of an oral CS treatment (methylprednisolone 40 mg daily for 14 days). They were arbitrarily divided into two groups according to baseline FEV1 improvement following this treatment: ≥ 23% designated as CS-sensitive (CSS) (n = 10) and 〈 15% as CS-resistant (CSR) (n = 11).Results Before oral CS, counts of bronchial mucosa inflammatory cells identified by immunohistochemistry (CD3, MBP, tryptase, CD68, neutrophil elastase and CD25 for lymphocytes, eosinophils, mast cells, macrophages, neutrophils and IL-2 receptors, respectively) were similar in CSS and CSR subjects. Oral CS decreased CD3+ cell counts (medians: 60–20 cells/mm2; P = 0.014) and MBP+ cell counts (medians: 19–4 cells/mm2; P = 0.03) in CSS asthmatics, but only tryptase+ cell counts in CSR asthmatics (medians: 30–18 cells/mm2; P = 0.05). Few bronchial neutrophil elastase+ cells were observed and their counts were similar in the two groups of asthmatics before and when on oral CS (all medians: = 2 cells/mm2).Conclusions These data show that, in these subjects with moderate to severe asthma, lymphocytes and eosinophils constitute most of the inflammatory cells infiltrating the bronchial mucosa. They also demonstrated that clinical impaired response to CS is associated with a persistent bronchial mucosa cellular infiltrate despite oral CS treatment. Additional studies are required to determine the role of this CS-resistant bronchial inflammation in the impaired asthma clinical response to these drugs.

Notes: Background Remodelling of the asthmatic airway includes increased deposition of proteoglycan (PG) molecules. One of the stimuli driving airway remodelling may be excessive mechanical stimulation.Objective We hypothesized that fibroblasts from asthmatic patients would respond to excessive mechanical strain with up-regulation of message for PGs.Methods We obtained fibroblasts from asthmatic patients (AF) and normal volunteers (NF) using endobronchial biopsy. Cells were maintained in culture until the fifth passage and then grown on a flexible collagen-coated membrane. Using the Flexercell device, cells were then subjected to cyclic stretch at 30% amplitude at 1 Hz for 24 h. Control cells were unstrained. Total RNA was extracted from the cell layer and quantitative RT-PCR performed for decorin, lumican and versican mRNA.Results In unstrained cells, the expression of decorin mRNA was greater in AF than NF. With strain, NF showed increased expression of versican mRNA and AF showed increased expression of versican and decorin mRNA. The relative increase in versican mRNA expression with strain was greater in AF than NF.Conclusions These data support the hypothesis that proteoglycan message is increased in asthmatic fibroblasts subject to mechanical strain. This finding has implications for the mechanisms governing airway wall remodelling in asthma.

Notes: Background Tissue eosinophils express more membrane receptors and release more mediators than blood eosinophils, suggesting that migration from blood to tissue modulates eosinophil phenotype and functions.Objective We postulated that eosinophil passage through endothelial basement membrane, an important step of eosinophil migration into tissue, may be responsible for some of these changes.Method We previously showed that 5-oxo-6, 8, 11, 14-eicosatetraenoic acid (5-oxo-ETE) in combination with IL-5 promotes eosinophil migration through Matrigel®, a mouse tumour cell-derived basement membrane. Using this model, we evaluated the effect of trans-Matrigel migration on purified human blood eosinophil expressions of CD44, CD69 and HLA-DR that either increase or appear on activated eosinophils, and releases of peroxidase (EPO), leukotriene (LT) C4 and granulocyte-monocyte colony stimulating factor (GM-CSF).Results IL-5, but not 5-oxo-ETE, increased eosinophil expression of CD44 and CD69. Migration of eosinophils through Matrigel significantly increased CD44 expression level over the one induced by IL-5 (P = 0.0001). Migration through Matrigel did not modify CD69 expression compared with the one obtained in the presence of IL-5 alone; however, incubation of eosinophils on Matrigel decreased IL-5-induced CD69 (P = 0.0001). Trans-Matrigel migration did not modify HLA-DR expression, nor EPO, LTC4 and GM-CSF releases.Conclusion These data show that in vitro trans-Matrigel migration and Matrigel contact modulate eosinophil membrane receptor expression. Consequently, they suggest that migration through basement membrane mediates changes in cell-surface phenotype observed on activated eosinophils and probably prepares them for interactions with tissue components and cells.

Notes: A previous study suggested that the long-acting β2-adrenergic agonist salmeterol (SM) had inhibitory effects on bronchial mucosal inflammation 6 hours after allergen exposure.To further evaluate the influence of SM on allergen-induced airway inflammation.We studied, in a randomized, double-blind, cross-over trial, 16 mild asthmatic patients who had a dual asthmatic response to allergen inhalation. Subjects received 50 µg of SM or placebo (P), twice daily for 1 week each, separated by a 2-week wash-out period. At the end of each treatment period, after withholding SM for 24 h, they had a methacholine inhalation test (medication was resumed after the test), followed 24 h later by an AC with the concentration of allergen that had induced a LAR at baseline. Airway inflammation was assessed 24 h after the AC by bronchoalveolar lavage (BAL) (n = 16) and bronchial biopsies (n = 13).As expected, SM improved baseline FEV1 and PC20 (P ≤ 0.009) and decreased the allergen-induced early bronchoconstrictive response. There were no significant differences in BAL cell counts after the two treatments. On bronchial biopsies, numbers (median, mm2) of submucosal CD45 (P: 43 ± 23; SM: 161 ± 43, P = 0.031), CD45Ro (P: 37 ± 19; SM: 126 ± 41, P = 0.047) and AA1 positive cells (P: 38 ± 6, SM: 65 ± 17, P = 0.006) were significantly higher after SM than P treatment. The numbers of CD4 (P: 11 ± 10; SM: 32 ± 7, P = 0.085), HLA-DR (P: 65 ± 30; SM: 116 ± 36, P = 0.079) and EG2 positive cells (P: 25 ± 15; SM: 38 ± 26, P = 0.09) tended to increase with SM treatment.In summary, compared to placebo, 1 week of regular use of SM is associated with an increase in bronchial inflammatory cells 24 h after AC. This is in keeping with the recommendation that salmeterol should only be used with an anti-inflammatory agent, particularly in the context of significant allergen exposure.