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Published ahead of print on June 26, 2008, doi:10.1164/rccm.200710-1557OC
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American Journal of Respiratory and Critical Care Medicine Vol 178. pp. 583-591, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200710-1557OC

Original Article

Increased Circulating Fibrocytes in Asthma with Chronic Airflow Obstruction

Chun-Hua Wang1,2,*, Chien-Da Huang1,3,*, Horng-Chyuan Lin1,2, Kang-Yun Lee1,3, Shu-Min Lin1, Chien-Ying Liu1,3, Kuo-Hsiung Huang1, Yu-Shien Ko4, Kian Fan Chung5 and Han-Pin Kuo1,3

1 Department of Thoracic Medicine, Chang Gung Memorial Hospital, Taipei, Taiwan; 2 Department of Chinese Medicine and 3 Department of Medicine, Chang Gung University, Taoyuan, Taiwan; 4 First Cardiovascular Division, Chang Gung Memorial Hospital, Taipei, Taiwan; and 5 National Heart and Lung Institute, Imperial College London, London, United Kingdom

Correspondence and requests for reprints should be addressed to Han-Pin Kuo, M.D., Ph.D., Department of Thoracic Medicine, Chang Gung Memorial Hospital, 199 Tun-Hwa North Road, Taipei, Taiwan. E-mail: q8828{at}ms11.hinet.net


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: A proportion of patients with asthma present with chronic airflow obstruction (CAO). We hypothesized that this effect may result from increased activity of circulating fibroblast-like progenitor cells (fibrocytes) that could home to the airway mucosal wall.

Objectives: To compare the proportion, proliferation, and differentiation of circulating fibrocytes from patients with asthma with CAO or no airflow obstruction (NOA) and control subjects.

Methods: We investigated circulating fibrocytes in 11 patients with asthma with CAO and a rapid decline in FEV1, 9 patients with asthma with NOA, and 10 nonasthmatic control subjects. Blood nonadherent non-T (NANT) cells were incubated with fetal calf serum or each patient's own serum and fibrocytes expressing CD34, CD45, and collagen I with {alpha}-smooth muscle actin were identified by flow cytometry.

Measurements and Main Results: A higher percentage of circulating fibrocytes in NANT cells was found in patients with CAO when compared with patients with NOA and control subjects. In CAO, the slope of the yearly decline in FEV1 correlated with circulating fibrocytes (r = –0.756, n = 11, P < 0.01). When NANT cells from patients with CAO were cultured in the patients' own sera, more fibrocytes were detected than when cultured in sera from patients with NOA or from normal subjects. An anti–transforming growth factor (TGF)-β1–neutralizing antibody inhibited {alpha}-smooth muscle actin–positive fibrocyte transformation from NANT cells of patients with CAO. Serum TGF-β1 levels were higher in patients with CAO than in patients with NOA or in normal subjects.

Conclusions: Circulating fibrocytes are increased in patients with asthma with CAO and can be transformed by TGF-β1 to myofibroblasts. Fibrocytes may contribute to airway obstruction in asthma.

Key Words: asthma • fibrocytes • myofibroblasts • transforming growth factor-β • airway remodeling



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Blood-borne fibrocytes characterized by expression of collagen I and CD45 and/or CD34 have been identified in asthma and are increased in the airways of patients with asthma after allergen challenge.

What This Study Adds to the Field
Circulating fibrocytes in the nonadherent non–T-cell fraction of peripheral blood mononuclear cells are increased in patients with asthma presenting with airflow obstruction and are correlated with the yearly decline in lung function; they can be transformed by TGF-β1 into myofibroblasts.

 
Chronic asthma is characterized by persistent airway inflammation and structural remodeling of the airways (1–4). The structural remodeling of the asthmatic airway consists of subepithelial fibrosis, submucosal gland hyperplasia, hyperplasia and hypertrophy of airway smooth muscle, and fragility of airway epithelial cells (5, 6). Subepithelial fibrosis is characterized by extensive deposition of extracellular matrix and connective tissue components such as collagens, tenascin, fibronectin, and proteoglycans (7–9), which may lead to increased loss of lung function and work disability (10) and the development of progressive airflow obstruction (11). By virtue of their capacity to produce these constituents of the extracellular matrix, fibroblasts/myofibroblasts may play a major role in the pathogenesis of airway remodeling, and a rapid increase in these cells has been identified in the airway mucosa after allergen challenge of subjects with mild allergic asthma (12).

Fibrocytes are a distinct population of blood-borne cells that coexpress collagen I (Col-I) and CD45 and/or CD34, and fibroblast products as well as the hematopoietic stem cell and myeloid markers. They may enter sites of tissue injury, and localize to areas of extracellular matrix deposition (13, 14). Fibrocytes also express several chemokine receptors, particularly CXCR4 (15, 16), which has been shown to mediate the effect of CXCL12/SDF-1 (stromal cell–derived factor-1) in causing migration of fibrocytes in pulmonary fibrosis (17). Fibrocytes have been shown to play an important role in the generation of fibrosis in several in vivo models, and inhibition of fibrocyte recruitment leads to a decrease in fibrosis (17–20). Transforming growth factor (TGF)-β1 serves as a key inducer of fibrosis by stimulating the release of growth factors and by inducing the differentiation of fibrocytes into myofibroblast-like cells that produce high levels of extracellular matrix components (15, 21).

Fibrocyte-like cells have been reported to be increased in the airways of patients with asthma after allergen challenge and to differentiate into collagen-producing myofibroblasts (22). In a mouse model of ovalbumin-induced asthma, CD34+Col-I+ cells were present in the circulation and were shown to home into the airway mucosa on exposure to allergen; whereas in mucosal tissue, they lose CD34 expression but continue to express {alpha}-smooth muscle actin ({alpha}-SMA), which are features of myofibroblasts (22). In steroid-naive patients with mild asthma, an increased number of fibrocytes has been observed in the bronchial mucosa as well as in the bronchoalveolar lavage fluid; the number of submucosal fibrocytes has been correlated with the thickness of the sub-basement membrane (23).

We hypothesized that in patients with asthma with chronic airflow obstruction, there could be an increased number of circulating fibrocytes compared with patients with asthma with no loss of lung function, and that these fibrocytes could be myofibroblast precursors. We therefore studied the number and activity of circulating fibrocytes in the peripheral blood of patients with asthma with chronic airflow obstruction, and examined the potential contribution of TGF-β1 to the transformation of myofibroblasts from fibrocytes.


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Population
Twenty nonsmoking patients with asthma with normal lung function (FEV1 > 80% predicted; asthma with no obstruction) and with persistently impaired lung function (post-bronchodilator FEV1 < 60% predicted; chronic obstructive asthma) were recruited (Table 1), according to American Thoracic Society criteria (24). Patients with chronic obstructive asthma had a rapid decline in FEV1, as measured over the previous 5 years, compared with patients with asthma with FEV1 greater than 80% predicted (see the online supplement). Ten healthy volunteers with normal lung function and PC20 (provocative concentration of methacholine needed to reduce FEV1 by 20%; >16 mg/ml), and normal serum IgE levels were recruited. This study was approved by the Chang Gung Memorial Hospital (Taipei, Taiwan) Ethics Committee and subjects gave informed consent.


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  		TABLE 1. CLINICAL CHARACTERISTICS OF SUBJECTS

 
Circulating Progenitor Fibrocytes
Peripheral blood mononuclear cells were separated from whole blood by Ficoll-Hypaque density gradient centrifugation (25, 26). Mononuclear cells at the interface were harvested, washed twice, and resuspended in Iscove's modified Dulbecco's medium (IMDM). The nonadherent mononuclear cell fraction was separated by centrifugation, resuspended in IMDM, and mixed with sheep red blood cells, and depleted of E-rosette–forming cells by a second Ficoll-Hypaque centrifugation. T cells were further depleted with anti-CD3 monoclonal antibody; T cells represented less than 1% of nonadherent non-T (NANT) cells.

Identification and Quantitation of Circulating Fibrocytes
Fibrocytes were detected by flow cytometry, using both phycoerythrin (PE)-conjugated anti-CD34 and anti–Col-I antibodies (22). Freshly isolated peripheral blood NANT cells (1 x 105 cells/ml) or NANT cells cultured for a given period of time were immersed in permeabilizing solution and incubated with mouse anti-human collagen I antibodies, followed by fluorescein isothiocyanate (FITC)–conjugated anti-mouse antibodies. Next, the cell pellet was incubated with PE-conjugated anti-CD34 and peridinin chlorophyll protein (PerCP)–conjugated anti-CD45 antibodies. The cell suspension was analyzed with a BD FACScan flow cytometer (BD Biosciences, San Jose, CA) equipped with an argon ion laser. Offline analysis was performed with CellQuest software (see the online supplement).

Culture of Fibrocytes
NANT cells were cultured in the presence of 30% fetal calf serum (FCS), or 30% patient's own serum, or 30% serum from normal subjects. The proportion of fibrocytes was determined by triple staining with PE-conjugated anti-CD34 antibodies, PerCP-conjugated anti-CD45 antibodies, and mouse anti–human Col-I antibodies followed by FITC-conjugated anti-mouse antibody. Cytocentrifuge-prepared smears of cultured cells were made for morphologic identification and cell counts.

To study the role of TGF-β1, NANT cells were incubated with 30% serum and anti–TGF-β1 antibodies (0.1, 1, and 3 µg/ml) for 14 days. Cells were incubated with mouse anti–human {alpha}-SMA antibodies, followed by PE-conjugated anti-mouse antibodies. Single-color analysis of the stained cells was done with the BD FACScan flow cytometer.

Immunofluorescence Labeling
NANT cells were incubated with primary monoclonal mouse anti-human antibodies: PE-conjugated anti-CD34 and anti–Col-I. Cells were fixed in 4% paraformaldehyde and 0.5% Triton X-100 in phosphate-buffered saline as a permeabilization agent before collagen I staining, followed by FITC-conjugated anti-mouse antibody. Nuclear staining was performed (see the online supplement). The fluorescence-labeled slides were examined with a Leica TCS 4D confocal laser scanning microscopy system (Leica Microsystems Heidelberg, Mannheim, Germany) (27).

Serum TGF-β1 and IL-13
Serum TGF-β1 and IL-13 were measured by quantitative sandwich-type enzyme-linked immunoassay (28).

Data Analysis
Nonparametric Kruskal-Wallis analysis was used, with subsequent Mann-Whitney U testing (two-tailed) to assess significance. Preplanned comparisons were made and significant values were confirmed by Newman-Keuls analysis. Data are represented as means ± SEM. The null hypothesis was rejected at P < 0.05.


   RESULTS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fibrocytes in Peripheral Blood
Using multiparametric flow cytometry (Figure 1A), the percentage of bone marrow–derived fibrocytes (CD34+CD45+Col-I+ cells) in the peripheral blood was higher in patients with chronic obstructive asthma (27.6 ± 3.2% of NANT cells, n = 11; P < 0.001) compared with patients with asthma with unimpaired lung function (6.4 ± 1.1%) and in normal subjects (6.5 ± 1.0%) (Figure 1B). Similarly, when expressed as absolute counts per milliliter of blood, fibrocytes were also increased in patients with chronic obstructive asthma (6.4 [±2.1] x 104 cells/ml, n = 11; P < 0.05) compared with patients with asthma with normal lung function (0.8 [±0.2] x 104 cells/ml, n = 9).


Figure 1
Figure 1
Figure 1
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  		Figure 1. (A) Flow cytometric analysis of nonadherent non-T (NANT) cells by immunostaining of CD34, CD45, and collagen I (Col-I). Horizontal and vertical lines mark fluorescence intensity greater than background observed with irrelevant phycoerythrin (PE)-, fluorescein isothiocyanate (FITC)–, and peridinin chlorophyll protein (PerCP)–conjugated anti-CD34, anti–Col-I, and anti-CD45, respectively, in isotype-matched control subjects. In NANT cells, some cells coexpressed CD45 and CD34 (top right) or CD45 and Col-I (bottom left). Circulating fibrocytes showed CD45+ cells that also expressed CD34 and Col-I (bottom right). (B) Percentage of CD34+CD45+Col-I+ circulating fibrocytes in the peripheral blood of normal subjects (n = 10), patients with asthma without airflow obstruction (asthma no obstruction, n = 9), and patients with chronic obstructive asthma (n = 11). The horizontal lines indicate mean values. (C) Relationship between the slope of the yearly decline in FEV1 by linear regression and the percentage of circulating fibrocytes in nonadherent mononuclear cells with T-cell depletion in patients with chronic obstructive asthma.

 
Because fibrocytes have previously been described only in the adherent cell fraction, we determined the proportion of fibrocytes in the adherent and nonadherent cell fractions; we studied another group of three patients with chronic obstructive asthma and three patients with asthma with normal lung function. In the NANT fraction, fibrocytes were also significantly higher in patients with chronic obstructive asthma (25.5 ± 0.6%, n = 3) compared with patients with asthma with normal lung function (6.1 ± 0.4%, n = 3; P < 0.05). The percentage of circulating CD45+CD34+Col-I+ fibrocytes in the 2-hour adherent cell fraction was 7.3 ± 3.1% of total fibrocytes in patients with chronic obstructive asthma and 11.0 ± 3.7% in patients with asthma with normal lung function. Therefore, most fibrocytes were found in the nonadherent fraction.

To further characterize circulating fibrocytes, we analyzed freshly isolated NANT cells triple-stained for Col-I, CD45, and CXCR4 or CCR7 by flow cytometry. We found that the majority of CD45+Col-I+ cells expressed CXCR4 (83.0 ± 2.0%, n = 7) whereas the proportion of CCR7 expression in CD45+Col-I+ cells was lower (32.8 ± 2.8%, n = 7; P < 0.01) compared with that of CXCR4 expression (data not shown).

We have calculated the decline in FEV1 on the basis of yearly FEV1 data points measured during the 5 years of observation, using regression analysis. There was a significant correlation between the percentage of fibrocytes of NANT cells in the peripheral blood of patients with chronic obstructive asthma and the "average" slope of the yearly decline in FEV1 (Figure 1C). There was no correlation with FEV1 (percent predicted). There was no significant difference in the percentage of circulating fibrocytes between patients with atopic and nonatopic asthma with either chronic obstruction or normal lung function. Of the NANT cells expressing CD34, 86% also expressed collagen I as confirmed by immunocytochemistry (Figure 2).


Figure 2
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  		Figure 2. Simultaneous immunocytochemistry for CD34 surface marker (red), intracellular collagen I (green), and nuclei (blue), respectively, on circulating fibrocytes in peripheral blood nonadherent mononuclear cells with T-cell depletion. The confocal images of (A) to (D) are with IgG control and those from (E) to (H) are with the respective antibodies. The merged picture shows double immunostaining of CD34+Col-I+ cells (yellow).

 
Proliferation of Circulating Fibrocytes
To correct for the different percentages of fibrocytes in NANT cells from patients with asthma and normal subjects, the proliferative capacity of CD34+CD45+Col-I+ fibrocytes in NANT cells after culture for 7 and 14 days was expressed as the ratio of fibrocytes grown after culture compared with fibrocytes in NANT cells at the start of culture. After 7 days, there was a greater number of fibrocytes grown from NANT cells cultured in 30% patients' own serum for patients with chronic obstructive asthma (4.7 ± 1.5-fold increase, n = 11; P < 0.05) than in normal subjects (1.5 ± 0.4-fold increase, n = 10) or in patients with asthma with normal lung function (1.7 ± 0.8-fold increase, n = 9). Such an effect was not found when cells were cultured with 30% FCS (Figure 3A). At 14 days, fibrocytes grown from NANT cells cultured with either 30% FCS or 30% patients' own serum increased in patients with chronic obstructive asthma by 4.4- and 7.4-fold, respectively. At 7 and 14 days, no increase was found in fibrocytes from normal subjects (1.0 ± 0.4-fold and 1.0 ± 0.3-fold, respectively; n = 10) and from patients with asthma with normal lung function (1.3 ± 0.3-fold and 1.6 ± 0.3-fold, respectively; n = 9) (Figure 3B).


Figure 3
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  		Figure 3. Proliferative capacity of fibrocyte-like progenitor cells cultured for 7 days (A) or 14 days (B) in the presence of 30% fetal calf serum (FCS) or 30% patient's own serum and stem cell factor, and number of {alpha}-smooth muscle actin–positive ({alpha}-SMA+) myofibroblast cells cultured from nonadherent non-T cells for 14 days in the presence of 30% FCS or 30% patient's own serum and stem cell factor (C) from normal subjects (n = 10), patients with asthma with normal pulmonary function test (asthma no obstruction, n = 9), or patients with chronic obstructive asthma (n = 11). Horizontal lines indicate mean values.

 
Differentiation of Circulating Fibrocytes
The expression of {alpha}-SMA as a marker of differentiation for myofibroblasts was determined by labeling cells grown from NANT cells with an anti–{alpha}-SMA monoclonal antibody. After 14 days, there were higher numbers of cells that had differentiated into myofibroblasts with either 30% FCS or 30% patients' own serum in patients with chronic obstructive asthma (32.7 [±6.7] and 53.7 [±12.1] x 104 cells/ml, respectively; n = 11) than in normal subjects (3.8 [±0.6] and 3.2 [±0.4] x 104 cells/ml, respectively; n = 10; P < 0.001) or in patients with asthma with normal lung function (3.8 [±0.8] and 3.2 [±0.8] x 104 cells/ml, respectively; n = 9; P < 0.01) (Figure 3C). There was no difference in {alpha}-SMA+ myofibroblast-like cells grown from NANT cells cultured with 30% FCS or 30% patients' own serum between normal subjects and patients with asthma with normal lung function (Figure 3C).

TGF-β1 in Myofibroblast Differentiation
To determine the role of TGF-β1 in the differentiation of fibrocytes, NANT cells were cultured in IMDM with 30% patient's own serum in the presence of an anti–TGF-β1 neutralizing antibody. After 14 days, spindle-shaped myofibroblasts were observed, but this differentiation was inhibited by the anti–TGF-β1 antibody (Figures 4A–4E). The myofibroblast phenotype was confirmed by positive staining with a PE-conjugated anti–{alpha}-SMA antibody, as analyzed by flow cytometry. The anti–TGF-β1 antibody dose-dependently reduced the numbers of {alpha}-SMA+ myofibroblasts grown from NANT cells in patients with chronic obstructive asthma but had no effect on myofibroblasts grown from NANT cells of normal subjects or patients with asthma with normal lung function (Figure 4F).


Figure 4
Figure 4
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  		Figure 4. Effect of anti–transforming growth factor (TGF)-β1 antibody on the numbers of {alpha}-smooth muscle actin–positive ({alpha}-SMA+) myofibroblast cells cultured from nonadherent non-T (NANT) cells for 14 days in normal subjects (n = 7) or patients with asthma with no airflow obstruction (n = 9) or patients with chronic obstructive asthma (n = 11). Pictures of spindle-shaped myofibroblasts grown from NANT cells of patients with chronic obstructive asthma show inhibition by anti–TGF-β1–neutralizing antibodies (A–E). (A) Serum; (B) serum + IgG; (C) serum + anti-TGF-β1 Ab (0.1 µg/ml); (D)serum + anti-TGF-β1 Ab (1 µg/ml); (E) serum + anti-TGF-β1 Ab (3 µg/ml); (F) Mean data of myofibroblasts in the presence of anti–TGF-β1–neutralizing antibody. **P < 0.01 compared with corresponding group in presence of serum alone.

 
Serum TGF-β1 and IL-13
Serum levels of TGF-β1 were significantly higher in patients with chronic obstructive asthma (952.3 ± 50.0 pg/ml) than in normal subjects (631.3 ± 61.4 pg/ml, P = 0.0006) and patients with asthma with normal lung function (737.4 ± 62.8 pg/ml, n = 9; P = 0.014) (Figure 5A). For comparison, the level of TGF-β1 in the 30% FCS culture medium was 92.9 ± 2.2 pg/ml for 11 replicates. In contrast, there was no difference in serum IL-13 levels (Figure 5B).


Figure 5
Figure 5
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  		Figure 5. Serum levels of (A) TGF-β1 and (B) IL-13 in normal subjects (n = 10), patients with asthma with no airflow obstruction (n = 9), and patients with chronic obstructive asthma (n = 11). Horizontal lines indicate mean values.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We found increased numbers of CD34+CD45+Col-I+ circulating fibrocytes in the peripheral blood of patients with chronic persistent obstructive asthma, but not in that of patients with asthma with normal lung function. The yearly decline in FEV1 was significantly correlated with the percentage of circulating fibrocytes in patients with chronic obstructive asthma. When cultured in the presence of FCS or in patients' own serum, the number of CD34+CD45+Col-I+ circulating fibrocytes of patients with chronic obstructive asthma was higher compared with those from patients with asthma with normal lung function. Fibrocyte-like progenitor cells in the peripheral blood of patients with chronic obstructive asthma therefore possessed enhanced growth potential and were committed to preferential development into {alpha}-SMA+ myofibroblasts, an effect that can be mediated by TGF-β1. The increased number of circulating fibrocytes, and the enhanced proliferative capacity and differentiation into fibroblasts/myofibroblasts, support a potential role for these circulating progenitor cells in the persistence and progression of airway obstruction in chronic asthma.

We observed increased numbers of fibrocytes, among patients with asthma with chronic airflow obstruction, in the nonadherent non–T-cell population of peripheral blood mononuclear cells. Fibrocytes were first described in the adherent cells of peripheral blood mononuclear cells after 7–14 days of culture (13). We now demonstrate that fibrocytes are also present in the nonadherent (after 2 h only) and non–T-cell portion of the peripheral blood mononuclear cells, and they represent about 80 to 90% of the fibrocyte population as measured by CD34+CD45+Col-I+ cells. The fibrocyte counts isolated from these fractions were 64,000 cells per milliliter of blood from patients with chronic obstructive asthma, representing an eightfold increase relative to patients with asthma with no airflow obstruction.

TGF-β1 may play an important role in the differentiation of fibrocyte-like progenitor cells from patients with chronic persistent obstructive asthma but not from patients with asthma without airflow obstruction into myofibroblasts, as defined by the expression of {alpha}-SMA. We also showed that there were increased levels of TGF-β1 in the serum of patients with chronic obstructive asthma compared with patients with asthma with normal lung function and normal subjects. This finding is not surprising because TGF-β1 is known to enhance the differentiation of pluripotential CD45+CD34+Col-I+ fibrocytes into fibroblasts/myofibroblasts with increased production of Col-I (22). However, our data indicate that this specific response to TGF-β1 is observed only in fibrocytes from patients with asthma with airflow obstruction. Addition of TGF-β1 markedly increases the level of {alpha}-SMA expression, which corresponds with differentiation of fibrocytes into myofibroblasts (14–17, 22). TGF-β1 drives fibrocyte-to-myofibroblast differentiation through the activation of Smad2/3 and SAPK (stress-activated protein kinase)/JNK (c-jun N-terminal kinase) MAPK (mitogen-activated protein kinase) pathways (29), which in turn stimulates {alpha}-SMA expression and also contributes to the production of collagen and other extracellular matrix proteins that promote airway remodeling. TGF-β1 is overexpressed in the airways of patients with asthma (30, 31). The proliferative capacity and differentiation into {alpha}-SMA myofibroblasts of CD45+CD34+Col-I+ progenitor cells in the peripheral blood of patients with chronic obstructive asthma were increased in the presence of patients' own serum compared with those of normal subjects or patients with asthma with normal lung function. Neutralizing anti–TGF-β1 antibodies had a nearly 90% inhibitory effect on the differentiation of circulating fibrocytes into myofibroblasts. Our results are also similar to a report (32) showing that higher levels of TGF-β1 are produced by burn patient fibrocytes. In addition, a TGF-β1–neutralizing antibody reduced the effect of media from burn patient fibrocytes on dermal fibroblast proliferation, migration, and collagen lattice contraction. These findings support the concept that TGF-β1 can prime circulating fibrocyte progenitor cells to proliferate and enhance their subsequent ability to differentiate into myofibroblasts in patients with asthma with persistent airflow obstruction. Whether TGF-β1 has effects on apoptosis of the cells needs further exploration.

In our short-term culture of fibroblasts we also used stem cell factor, which is not essential for differentiation into myofibroblasts or fibroblasts. In preliminary experiments, we showed that stem cell factor led to a small increase in the number of fibrocytes (approximately 28%), which may represent the acquisition of collagen I expression by CD34+ cells in this cell fraction, and therefore differentiation of these CD34+ cells into fibrocytes.

Migration of circulating fibrocytes into the airway wall requires some induced signal in the lung that is capable of recruiting these extrapulmonary cells. Of the factors that may be involved in the recruitment of fibrocytes into the injured tissue, the chemokine receptors CXCR4 and CCR7 appear to be pivotal for their homing (15–17), and the expression of CXCL12, a chemokine ligand for CXCR4, in tissues may create a gradient needed for trafficking of CXCR4+ fibrocytes (33). Increased immunoreactivity for CXCL12 has been reported in the airways of patients with asthma, with localization to endothelial cells, macrophages, and T cells (34). In a mouse bleomycin model, circulating fibrocytes proliferated and contributed to lung fibrosis after homing in response to the chemokine CXCL12 (17). We confirmed the presence of CXCR4 and CCR7 receptors on freshly isolated fibrocytes (15), although not all fibrocytes expressed these receptors, with CXCR4 expressed on a higher proportion of fibrocytes than CCR7. In addition, the presence of these chemokine receptors provided further support that these CD45+Col-I+ cells isolated from nonadherent and non–T-cell fraction are fibrocytes.

Chronic airway inflammation and airway remodeling are the hallmarks of asthma and may contribute to disease persistence and progression (3, 5, 6). Chronic structural changes of airway remodeling include subepithelial fibrosis with deposition of extracellular matrix, hyperplasia and hypertrophy of smooth muscle and submucosal glands, and an increase in fibroblasts/myofibroblasts (5, 6). Reports strongly suggest that circulating fibrocytes may function as precursors of bronchial myofibroblasts after allergen challenge (22) and can localize in tissue within the proximity of a thicker sub-basement membrane observed in patients with asthma (23). The mechanisms by which remodeling may link to the progressive decline in lung function leading to chronic airflow obstruction seen in some patients with asthma (11, 35), as in the patients with chronic airflow obstruction we studied, are unclear. Circulating fibrocytes that possess hematopoietic as well as fibroblast-like properties can migrate from the bone marrow into the sites of tissue injury and mediate tissue repair (14, 15, 18). Fibrocytes, at sites of tissue injury, secrete inflammatory cytokines and extracellular matrix proteins, and promote angiogenesis and wound contraction (15, 36). Circulating fibrocytes from our patients with asthma with chronic airflow obstruction and a more rapid decline in lung function show higher proliferative capacity and a greater degree of differentiability into {alpha}-SMA+ myofibroblasts, which are key mesenchymal cells implicated in changing the composition of the airway wall matrix (35).

In conclusion, we have shown that fibrocytes in the peripheral blood of patients with asthma with progressive airway obstruction possess higher proliferation potential, and increased differentiability into {alpha}-SMA+ myofibroblasts, under the actions of TGF-β1. These cells may play a crucial role in the remodeling of the airways that could contribute to chronic airflow obstruction.


   FOOTNOTES
 
* These authors contributed equally to this work. Back

Supported by a grant from the National Science Council, Taiwan (NSC-96-2628-B-182-022-my3).

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200710-1557OC on June 26, 2008

Conflict of Interest Statement: C.H.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.D.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.C.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.Y.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.M.L does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.Y.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.H.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Y.S.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.F.C. has participated in scientific advisory boards for Novartis, Merck, GlaxoSmithKline, Mundipharma, and Chiesi Farmaceutici. H.P.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form October 22, 2007; accepted in final form June 24, 2008


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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