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.

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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 stratum griseum superficiale (SGS) of the superior colliculus contains a high concentration of the recently described GABA C receptor. In a previous study, it was postulated that activation of these receptors on inhibitory interneurons functions to disinhibit projection cells that relay visual information to the thalamus and brainstem. To test this model, we used in vitro whole-cell patch-clamp methods to measure effects of GABA and muscimol on EPSCs and IPSCs evoked in rat SGS by electrical optic layer stimulation. The neurons were filled with biocytin for later morphological characterization. As expected, bath applications of GABA and muscimol always strongly depressed evoked PSCs at concentrations of Ͼ100 and Ͼ1 M, respectively. However, at lower agonist concentrations, which most likely activate GABA C but not GABA A receptors, effects were not uniform. Evoked responses were suppressed by both agonists in 48% of the neurons, whereas the remaining cells exhibited enhanced responses with increased evoked EPSCs, decreased evoked IPSCs, or both types of change. Most morphologically identified cells with suppressed responses (14 of 17 cells) had morphological characteristics of putative GABAergic interneurons, whereas almost all cells with enhanced responses (8 of 10 cells) had morphological characteristics of projection cells. Finally, all effects of GABA and muscimol at low concentrations were blocked by (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid, a specific GABA C receptor antagonist, but not by the specific GABA A receptor antagonist bicuculline. Taken together, these results indicate that in SGS, GABA C receptors are predominantly expressed by GABAergic neurons and that activation of these receptors leads to disinhibition of SGS projection cells.

The C-C motif chemokine ligand 5 (CCL5), CCL11, and CCL24 are involved in the pathogenesis of asthma, and their function is mainly associated with the airway recruitment of eosinophils. This study tested their ability to induce the migration of circulating fibrocytes, which may contribute to the development of irreversible airflow obstruction in severe asthma. The sputum fluid phase (SFP) from patients with severe/treatment-refractory asthma (PwSA) contained elevated concentrations of CCL5, CCL11, and CCL24 in comparison with the SFP from patients with non-severe/treatment-responsive asthma (PwNSA). The circulating fibrocytes from PwSA expressed the receptors for these chemokines at increased levels and migrated in response to recombinant CCL5, CCL11, and CCL24. The SFP from PwSA induced the migration of autologous fibrocytes, and its activity was significantly attenuated by neutralization of endogenous CCL5, CCL11, and CCL24. These findings suggest that CCL5, CCL11, and CCL24 may contribute to the airway recruitment of fibrocytes in severe asthma.

The fibrocytes are thought to serve as a source of newly deposited collagens I and III during reparative processes and in certain fibrotic disorders, but their matrix remodelling properties are incompletely understood. We evaluated their ability to produce several extracellular matrix (ECM) components, in comparison with fibroblasts, and to participate in collagen turnover. The collagen gene expression profile of fibrocytes differed from that of fibroblasts because fibrocytes constitutively expressed relatively high levels of the mRNA encoding collagen VI and significantly lower levels of the mRNA encoding collagens I, III and V. The proteoglycan (PG) gene expression profile was also different in fibrocytes and fibroblasts because fibrocytes constitutively expressed the mRNA encoding perlecan and versican at relatively high levels and the mRNA encoding biglycan and decorin at low and very low levels, respectively. Moreover, fibrocytes expressed the mRNA for hyaluronan synthase 2 at higher level than fibroblasts. Significant differences between the two cell populations were also demonstrated by metabolic labelling and analysis of the secreted collagenous proteins, PGs and hyaluronan. Fibrocytes constitutively expressed the scavenger receptors CD163 and CD204 as well as the mannose receptors CD206 and Endo180, and internalized and degraded collagen fragments through an Endo180-mediated mechanism. The results of this study demonstrate that human fibrocytes exhibit ECM remodelling properties previously unexplored, including the ability to participate in collagen turnover. The observed differences in collagen and PG expression profile between fibrocytes and fibroblasts suggest that fibrocytes may predominantly have a matrix-stabilizing function.

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