The mechanisms contributing to airway wall remodeling in asthma are under investigation to identify appropriate therapeutic targets. Bronchial myofibroblasts would represent an important target because they play a crucial role in the genesis of subepithelial fibrosis, a characteristic feature of the remodeling process, but their origin is poorly understood. We hypothesized that they originate from fibrocytes, circulating cells with the unique characteristic of expressing the hemopoietic stem cell Ag CD34 and collagen I. In this study we show that allergen exposure induces the accumulation of fibrocyte-like cells in the bronchial mucosa of patients with allergic asthma. These cells are CD34-positive; express collagen I and α-smooth muscle actin, a marker of myofibroblasts; and localize to areas of collagen deposition below the epithelium. By tracking labeled circulating fibrocytes in a mouse model of allergic asthma, we provide evidence that fibrocytes are indeed recruited into the bronchial tissue following allergen exposure and differentiate into myofibroblasts. We also show that human circulating fibrocytes acquire the myofibroblast phenotype under in vitro stimulation with fibrogenic cytokines that are produced in exaggerated quantities in asthmatic airways. These results indicate that circulating fibrocytes may function as myofibroblast precursors and may contribute to the genesis of subepithelial fibrosis in asthma.

Human fibrocytes are mesenchymal progenitors that exhibit mixed morphological and molecular characteristics of hematopoietic stem cells, monocytes and fibroblasts. They likely represent the obligate intermediate stage of differentiation into mature mesenchymal cells of a bone marrow-derived precursor of the monocyte lineage under permissive conditions. On in vitro stimulation with pro-fibrotic cytokines and growth factors, human fibrocytes produce large quantities of extracellular matrix components and further differentiate into cells identical to the contractile myofibroblasts that emerge at the tissue sites during repair processes and in some fibrotic lesions. Studies in various animal models of wound healing or fibrotic diseases have confirmed the ability of fibrocytes to differentiate into mature mesenchymal cells in vivo and have suggested a causal link between fibrocyte accumulation and ongoing tissue fibrogenesis or vascular remodeling in response to tissue damage or hypoxia. Fibrocytes synthesizing new collagen or acquiring myofibroblast markers have been detected in human hypertrophic scars, in the skin of patients affected by nephrogenic systemic fibrosis, in human atherosclerotic lesions, and in pulmonary diseases characterized by repeated cycles of inflammation and repair, like asthma. The presence of fibrocyte-like cells has been reported in human chronic pancreatitis and chronic cystitis. Similar cells also populate the stroma surrounding human benign tumors. The available data indicate that human fibrocytes serve as a source of mature mesenchymal cells during reparative processes and in fibrotic disorders or stromal reactions predominantly associated with a persistent inflammatory infiltrate or with the selective recruitment of monocytes induced by ischemic changes and tumor development. A deeper understanding of the mechanisms involved in fibrocyte differentiation in these pathological conditions may lead to the development of novel therapies for preventing detrimental tissue or vascular remodeling and metastatic progression of invasive tumors.

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The respiratory epithelium represents the first barrier encountered by airborne Ags. Two major dust mite Ags, Der p3 and Der p9, are serine proteases that may activate lung epithelial cells by interaction with the protease-activated receptor 2 (PAR-2). In this study both Der p3 and Der p9 cleaved the peptide corresponding to the N terminus of PAR-2 at the activation site. Both Ags sequentially stimulated phosphoinositide hydrolysis, transient cytosolic Ca2+ mobilization, and release of GM-CSF and eotaxin in human pulmonary epithelial cells. These responses were similar to those observed with trypsin and a specific PAR-2 agonist and were related to the serine protease activity of Der p3 and Der p9. Cell exposure to the Ags resulted in a refractory period, indicating that a PAR had been cleaved. Partial desensitization to Der p3 and Der p9 by the PAR-2 agonist suggested that PAR-2 was one target of the Ags. However, PAR-2 was not the only target, because the PAR-2 agonist caused less desensitization to Der p3 and Der p9 than did trypsin. A phospholipase C inhibitor prevented the cytokine-releasing effect of the PAR-2 agonist and abolished or reduced (>70%) the cytokine-releasing effects of Der p3 and Der p9. Our results suggest that Der p 3 and Der p9 may induce a nonallergic inflammatory response in the airways through the release of proinflammatory cytokines from the bronchial epithelium and that this effect is at least in part mediated by PAR-2.

Fibrocytes contribute to the fibrotic changes most frequently observed in forms of asthma where inflammation is driven by T helper type 2 (Th2) cells. The mechanisms that regulate the profibrotic function of asthmatic fibrocytes are largely unknown. We isolated circulating fibrocytes from patients with allergen-exacerbated asthma, who showed the presence of fibrocytes, together with elevated concentrations of interleukin (IL)-4 and IL-13 and slightly increased concentrations of the Th17 cell-derived IL-17A, in induced sputum. Fibrocytes stimulated with IL-4 and IL-13 produced high levels of collagenous and non-collagenous matrix components and low levels of proinflammatory cytokines. Conversely, fibrocytes stimulated with IL-17A proliferated and released proinflammatory factors that may promote neutrophil recruitment and airway hyperresponsiveness. IL-17A also indirectly increased α-smooth muscle actin but not collagen expression in fibrocytes. Thus, fibrocytes may proliferate and express a predominant profibrotic or proinflammatory phenotype in asthmatic airways depending on the local concentrations of Th2- and Th17-derived cytokines.

The reproducibility of sputum cell counts was examined and the cell counts in patients with asthma were compared with those in patients with chronic bronchitis. Three groups of subjects were studied. Sputum from eight patients with chronic asthma and with sputum production were studied to determine the reproducibility of sputum cell counts. The findings in 10 non-smokers with asthma uncomplicated by other airway disease examined at the time of an exacerbation with sputum (group 2) were compared with those from eight smokers with chronic cough and sputum but no features of asthma (group 3). Sputum plugs were selected by microscopy to ensure their origin from the lower respiratory tract. A total cell count was performed on a trypsinised suspension, and differential and metachromatic cell counts were performed on undiluted plugs. The within specimen and test-retest reproducibility of these measurements was high (reliability coefficient, R, = 0.99 and 0.89). The sputum of the asthmatic patients was characterised by eosinophilia (69%, range 46-92%) and the presence of formaldehyde blockable metachromatic cells (1.5%, range 0.6-2.8%). In comparison, the sputum of the patients with chronic bronchitis had few eosinophils (0.5%) or metachromatic cells (0.14%); the dominant cell type was the macrophage (83%). It is concluded that sputum cell counts are reproducible in the short term, the inflammation of asthma is characterised by eosinophilia and metachromatic cells in sputum, and sputum may provide a useful source of cells for investigating the cellular characteristics of airway inflammation.

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