Tissue Repair in Asthma: The Origin of Airway Subepithelial Fibroblasts and Myofibroblasts

Mattoli S.

Publication Details

Asthma is characterized by functional and structural alterations of the bronchial epithelium, chronic airway inflammation and remodeling of the normal bronchial architecture. Bronchial myofibroblasts are thought to play a crucial role in the pathogenesis of subepithelial fibrosis, a prominent aspect of the remodeling process. The results of the studies reviewed in this report indicate that circulating fibrocytes contribute to the bronchial myofibroblast population and may be responsible for the excessive collagen deposition below the epithelial basement membrane in asthma. More information on the mechanisms regulating the migration and differentiation of these cells in the asthmatic airways may help identify novel targets for therapeutic intervention.

Introduction

Asthma is characterized by structural and functional abnormalities of the bronchial epithelium, accumulation of inflammatory cells in the bronchial mucosa, and remodeling of the airway tissue structure.1-9 The remodeling process has been suspected to contribute to the irreversible airflow obstruction and permanently impaired pulmonary function observed in patients with chronic asthma,10,11 and has become a major target for the development of new anti-asthma drugs.12

Disruption of the epithelium integrity, accumulation of myofibroblasts below the epithelial basement membrane and subepithelial fibrosis are peculiar aspects of airway tissue remodeling in asthma.11,13-16 In allergic asthma, these alterations may be due to an increased susceptibility of the bronchial epithelium to allergen-induced injury or to ineffective repair mechanisms during chronic allergen exposure. Tissue response is similar to that observed in chronic wounds and pathological scaring:17 the massive apoptosis of inflammatory cells and myofibroblasts normally observed when epithelialization has been completed does not occur, as epithelial integrity is not restored, and failure to resolve the inflammatory and structural changes leads to excessive extracellular matrix deposition and deformation of the normal tissue.

In asthma, the thickened lamina reticularis beneath the airway epithelium contains abnormally high amounts of collagens I, III and V; fibronectin and tenascin.14,18 Like in chronic wounds and other conditions where excessive extracellular matrix deposition occurs as a consequence of impaired wound healing, most of these proteins are thought to be produced by the numerous myofibroblasts present in the subepithelial zone,11,14 particularly collagen I, collagen III and fibronectin. In order to find a suitable target to prevent or inhibit the remodeling process in asthma, the origin of bronchial myofibroblasts and the mechanisms involved in the accumulation of those cells need to be elucidated.

The main objective of this report is to review the data indicating that circulating fibrocytes contribute to the myofibroblast population in injured tissues, may function as precursors of bronchial myofibroblasts in asthma and may play a crucial role in the genesis of subepithelial fibrosis.

Phenotypic and Functional Characteristics of Circulating Fibrocytes

Mature fibrocytes represent a unique population of cells that express fibroblast products, such as collagen I, in conjunction with the hematopoietic stem cell antigen CD34, the leukocyte common antigen CD45 and the marker of the myeloid lineage cells CD13.19-23 They originate from CD13+/CD45+/CD34+/collagen I- precursors present in the CD14+ fraction of peripheral blood mononuclear cells, which become CD14-/collagen I+ during the maturation process.19,20 The phenotypic characteristics of these precursors have been poorly investigated, but their differentiation into mature fibrocytes may be up-regulated by interaction with activated T lymphocytes.19,20

Mature fibrocytes normally comprise less than 1% of the circulating pool of nonred cells. They do not constitutively express the myofibroblast marker α-smooth muscle actin (SMA),19,22 but acquire the myofibroblast phenotype under in vitro stimulation with fibrogenic cytokines that are produced in exaggerated quantities in the airways of asthmatic patients, such as TGF-β1 24 and endothelin-1.25 When cultured in presence of these cytokines, fibrocytes develop bundles of actin microfilaments indicative of a contractile phenotype and show the other ultrastructural characteristics of fibroblasts undergoing differentiation into myofibroblasts.22 Exposure to TGF-β1 also increases collagen I immunoreactivity in cultured fibrocytes19 and markedly enhances the release of collagen I, collagen III and fibronectin from these cells.22 The differentiation of circulating fibrocytes into fibroblasts and myofibroblasts in vitro is associated with a down-regulation of the expression of the surface antigens CD34,22,23,26 CD4523,26 and CD13.23 Human and murine circulating fibrocytes are quite similar in terms of phenotypic characteristics and response to TGF-β1 in vitro.22,23

Phenotypic Characteristics and Bone Marrow Origin of Tissue Fibrocytes

A recent study23 was addressed to investigate the phenotypic characteristics and bone marrow origin of fibrocytes in the wounded skin of female BALB/c mice that had received a male whole bone marrow transplant after total body irradiation (sex-mismatched bone marrow chimera mice). At 4 and 7 days post-wounding, numerous fibrocytes could be isolated from digested fragments of wounded tissue by Percoll density gradient centrifugation and immunomagnetic depletion of contaminating cells. More than 95% of the isolated cells expressed CD13 in conjunction with collagen I, as assessed by double-staining with fluorescent antibodies against CD13 and collagen I and analysis of stained cells by fluorescence-activated cell sorting. At an early stage of the healing process, a substantial proportion of these cells (mean % ± SE: 33.8% ± 8.5% from 4 to 5 experiments) also expressed the myofibroblast marker α-SMA, further increasing (to 58.7% ± 9.4%) at day 7 post-wounding. These data indicated that circulating fibrocytes rapidly enter a phase of differentiation into myofibroblasts once they have migrated at the site of tissue injury. During the differentiation process, the proportion of fibrocytes expressing CD45 and CD34 progressively decreased: on the average, more than 20% of the isolated fibrocytes lose CD45 and CD34 expression between day 4 and day 7 post-wounding.

In the same study, the bone marrow origin of fibrocytes isolated from the wounded tissue of female mice recipients of male bone marrow grafts was also examined by fluorescence in situ hybridization with a mouse Y chromosome paint probe. The Y chromosome could be identified in 94.3% or more of the fibrocytes present in the wounded tissue at day 4 post-wounding, indicating that the vast majority of these cells originated from a bone marrow-derived precursor.

Previous studies in which the origin of fibrocytes was evaluated,21,26 provided conflicting and inconclusive data. Bucala and colleagues21 were unable to demonstrate the bone marrow origin of fibrocytes in sex-mismatched bone marrow chimera mice, while another group could develop fibrocyte-like cells from cultured bone marrow cells of C57Bl/6 mice.26 However, the first study was conducted in female mice that had received a male bone marrow transplant following only 800 rads of total body irradiation, which may not allow a successful ablation of the bone marrow cell population in the female recipients and effective transplantation of the bone marrow cells from male donors.

Identification of Circulating Fibrocytes as Precursors of Bronchial Myofibroblasts in Asthma

Schmidt and colleagues22 identified cells expressing both CD34 and procollagen I mRNA in the bronchial mucosa of patients with chronic allergic asthma. The number of these cells markedly increased during an exacerbation of the disease induced by inhalation of the allergen to which patients were sensitized. At 24 hours after allergen exposure, a substantial proportion of the CD34+/procollagen I mRNA+ cells also expressed α-SMA and localized to areas of collagen deposition below the epithelial basement membrane, in a zone where myofibroblasts accumulate in chronic asthma.11,14 The phenotype of these cells strongly suggested that they were fibrocytes migrated from the peripheral blood, which were differentiating into collagen-producing myofibroblasts at the tissue site.

This possibility was explored by using an animal model of allergic asthma that recapitulates most of the inflammatory and structural alterations of the human disease, including the accumulation of eosinophils within and below the airway epithelium and the thickening of the subepithelial zone, with deposition of collagen and other extracellular matrix proteins.22 In this model, systemically immunized BALB/c mice were challenged with an aerosolized solution of 2.5% ovalbumin (OVA) in phosphate buffered saline (PBS) in a whole body inhalation chamber for 20 minutes, 3 times a week, at intervals of 24 hours, over a period of 8 weeks. Control mice were exposed to the OVA vehicle alone (PBS). During repeated OVA exposures, sensitized mice showed a progressive increase in the number of CD34+/procollagen I mRNA+ cells in the airway wall in comparison with control animals exposed to phosphate buffered saline (PBS). In the airway wall of animals exposed to the allergen for 6 to 8 weeks there were also numerous cells expressing the CD34 antigen in conjunction with α-SMA. By comparison with adjacent tissue sections double-labeled with antibodies against CD34 and procollagen I, it was possible to estimate that, on the average, 44.9% of the CD34+/procollagen I+ cells also expressed α-SMA at 8 weeks of repeated exposure to OVA. At the same time point, the CD34+/ α-SMA+ cells represented about 31% of all cells expressing α-SMA (excluding vessels and smooth muscle cells).

Figure 1, shows the time-course of fibrocyte accumulation in the airway wall of mice chronically exposed to OVA in relation to the kinetics of two key events: the production of TGF-β1 by airway resident cells (fig. 1A) and the deposition of collagen I below the epithelial basement membrane (fig. 1B). In the epithelium and subepithelial area of mice chronically exposed to OVA there was a marked increase in the number of cells showing TGF-β1 immunoreactivity in comparison with mice chronically exposed to PBS for similar periods of time (fig. 1A). The peak of TGF-β1 immunoreactivity was observed between 6 and 8 weeks of chronic exposure to OVA (fig. 1A), when many of the CD34+/procollagen I+ cells also expressed α-SMA (fig. 1C and D). These findings suggested that the CD34+/procollagen I+/α-SMA+ cells were fibrocytes undergoing differentiation into myofibroblasts under the effect of the TGF-β1 produced in excess by epithelial cells and inflammatory cells. Interestingly, in mice chronically exposed to OVA both the increase in TGF-β1 immunoreactivity and the increase in the number of CD34+/procollagen I+/α-SMA+ cells occurred in concomitance with the excessive deposition of collagen I below the epithelial basement membrane (fig. 1A-C). In view of this correlation, and considering that TGF-β1 markedly enhances collagen release in cultured CD34+/collagen I+ fibrocytes acquiring α-SMA expression,22 it is reasonable to think that the fibrocytes undergoing differentiation into myofibroblasts in the subepithelial area were an important source of that collagen and contributed to the development of subepithelial fibrosis.

Figure 1. Kinetics of increased TGF-β1 expression (A) and subepithelial collagen I deposition (B) in relation to the time-course of fibrocyte accumulation (C) and differentiation into myofibroblasts (D) in the airway wall of mice chronically exposed to OVA or PBS.

Figure 1

Kinetics of increased TGF-β1 expression (A) and subepithelial collagen I deposition (B) in relation to the time-course of fibrocyte accumulation (C) and differentiation into myofibroblasts (D) in the airway wall of mice chronically exposed to (more...)

Mechanisms Potentially Involved in the Recruitment of Fibrocytes into the Airways in Asthma

By tracking labeled circulating fibrocytes in the mouse model of allergic asthma, Schmidt and colleagues22 provided direct evidence that in mice chronically exposed to OVA fibrocytes are recruited into the airway wall from the peripheral blood and localize to areas of ongoing collagen deposition below the airway epithelium. The labeled fibrocytes that accumulated in the airways had a phenotype different from the phenotype of labeled fibrocytes present in the peripheral blood. They expressed α-SMA while labeled fibrocytes present in the peripheral blood did not, and showed increased collagen I immunoreactivity. The expression of the CD34 antigen on labeled fibrocytes isolated from the airway wall was reduced in comparison with the labeled cells recovered from the peripheral blood. Taken together, these results indicated that the labeled fibrocytes recruited from the circulation into the airways had entered into a phase of differentiation into myofibroblasts at the tissue site.

The same investigators also evaluated some of the mechanisms potentially involved in the recruitment of circulating fibrocytes into the airways. The secondary lymphoid cytokine (SLC), also known as CCL21 in mice, has been shown to attract human and murine fibrocytes via the CCR7 receptor.19 In the airway wall of mice chronically exposed to OVA for 5 to 6 weeks, there was an increased expression of SLC/CCL21 compared with the airway wall of mice exposed to PBS for the same periods of time (fig. 2A). The sources of this chemokine were predominantly endothelial cells (fig. 2B), but some SLC/CCL21 immunoreactivity was also observed in the epithelium and in the inflammatory infiltrate below the epithelium. The intravenous administration of a 30-μg dose of a neutralizing antibody against mouse SLC/CCL21 1 hour before the last exposure to OVA at 5 weeks significantly reduced the number of CD34+/ procollagen I+ cells present in the airway wall 24 hours following that OVA exposure, while the administration of a control antibody had no significant effect (fig.2C). However, the number of CD34+/procollagen I+ fibrocytes was still significantly higher in the airway wall of OVA-challenged mice than in the airway wall of control mice (fig. 2C). The administration of higher doses of the antibody against SLC/CCL21 did not change the results. These findings indicated that SLC/CCL21 may represent one of the factors contributing to the recruitment of circulating fibrocytes into the airways during subepithelial fibrogenesis elicited by chronic allergen exposure in BALB/c mice and that it is not the only factor involved. Another possible candidate could be the CXC chemokine stromal cell-derived factor-1 (SDF-1, also known as CXCL12), as murine and human fibrocytes seem to express the CXCR4 receptor for this chemoattractant.19,26 However, there are conflicting data about the ability of fibrocytes to migrate in response to this chemokine in vitro and in vivo,19,26 and more studies are needed to elucidate the role of SDF-1/CXCL12 in the recruitment of fibrocytes at the tissue sites during wound healing and in airway remodeling.

Figure 2. A) Increased expression of SLC/CCL21 in the airway wall of mice exposed to OVA for 5 and 6 weeks in comparison with control mice exposed to PBS for the same periods of time.

Figure 2

A) Increased expression of SLC/CCL21 in the airway wall of mice exposed to OVA for 5 and 6 weeks in comparison with control mice exposed to PBS for the same periods of time. Tissue sections were stained with an antibody against CCL21. Stained cells were (more...)

Conclusions

The data reviewed in this report indicate that circulating fibrocytes contribute to the bronchial myofibroblast population in asthma and may be involved in the genesis of subepithelial fibrosis through the release of excessive amounts of collagen and other extracellular matrix proteins below the bronchial epithelium. One of the chemokines that may induce the migration of fibrocytes from the circulation into the airways is SLC/CCL21. Once migrated at the tissue site, circulating fibrocytes may differentiate into myofibroblasts under the effect of fibrogenic cytokines produced by epithelial cells and inflammatory cells, particularly TGF-β1.

It should be noted that the data discussed above do not exclude the possibility that other, TGF-β1 insensitive, bone-marrow derived mesenchymal cell progenitors27 or epithelial cells undergoing epithelial-mesenchymal transition as a result of epithelial injury28 might also be involved in the development of subepithelial fibrosis in asthma. Thus, future studies should be addressed to investigate whether multiple cell types contribute to subepithelial fibrogenesis in this disease. Nonetheless, on the basis of the data currently available, it is reasonable to anticipate that a better understanding of the factors regulating the migration and differentiation of fibrocytes in asthmatic airways may help identify suitable targets for the development of new anti-asthma drugs.

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