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Originally published online as doi:10.1189/jlb.0906587 on June 5, 2007

Published online before print June 5, 2007
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(Journal of Leukocyte Biology. 2007;82:449-456.)
© 2007 by Society for Leukocyte Biology

Fibrocytes in lung disease

Brigitte N. Gomperts* and Robert M. Strieter{dagger},1

* Mattel Children’s Hospital, Department of Pediatrics, Division of Pediatric Hematology Oncology, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, USA; and
{dagger} Department of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia, USA

1 Correspondence: Department of Medicine, University of Virginia School of Medicine, P.O. Box 800466, Charlottesville, VA 22908-0466, USA. E-mail: strieter{at}virginia.edu


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ABSTRACT
 
Fibrocytes were first described over a decade ago as a population of cells in circulation with fibroblast-like properties, which were involved in tissue repair. Since that time, we have learned a significant amount about these bone marrow-derived cells, which contribute to wound healing and fibrosis. Fibrocytes express leukocyte markers such as CD34, CD45, and CD13 and also express mesenchymal markers such as pro-collagens I and III, vimentin, and fibronectin. In addition, they have been shown to express the chemokine receptors CXCR4 and CCR7, which appear to be important in cellular trafficking from the vascular to the extravascular compartment. Fibrocytes have been shown to contribute to a number of fibrotic disorders, and here, we review their involvement in lung diseases including pulmonary fibrosis, asthma, and vascular remodeling.

Key Words: chemokines • chemokine receptors • stem cells • progenitor cells • pulmonary fibrosis


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INTRODUCTION
 
The fibrocyte is a unique cell population that has been implicated in wound repair
The fibroblast/myofibroblast is critical for the generation of the extracellular matrix (ECM), which serves as the foundation for re-establishment of tissue integrity and provides tissue support. In 1994, Bucala and associates [1 ] discovered a unique, blood-borne cell with fibroblast-like properties. This cell was found not to be a leukocyte, and its presence in wounds was not a result of local infiltration of surrounding, connective tissue fibroblasts (see Fig. 2 ) [1 ]. The term "fibrocyte" has subsequently been assigned to this novel and distinct population of blood-borne cells with fibroblast-like features [1 ]. By scanning electron microscopy, fibrocytes are morphologically distinct from leukocytes and display prominent cell surface projections, which are intermediate in size between microvilli and pseudopodia [1 ]. Differentiated fibrocytes from peripheral blood are characterized as spindle-shaped, negative for CD14 and nonspecific esterase stain (i.e., not monocytes or macrophages) as well as for cell surface markers for epithelial and endothelial cells [1 , 2 ]. Abe and colleagues [3 ], however, were able to demonstrate that peripheral blood fibrocytes could be cultured from a CD14+ cell population, and therefore, more than one population of fibrocytes may exist in circulation.


Figure 2
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  		Figure 2. Some of the remodeling processes in which fibrocytes are involved. Fibrocytes are circulating cells, which have now been shown to play a role in pulmonary fibrosis, adipogenesis, vascular wall remodeling with pulmonary hypertension, and wound healing. IPF, Idiopathic pulmonary fibrosis.

Fibrocytes, as defined by collagen 1+CD45+ and/or CD34+ in expression, only comprise 0.1–0.5% of the nucleated cells in peripheral blood [2 , 4 , 5 ]. In culture (i.e., presence of serum), fibrocytes begin to express {alpha}-smooth muscle actin ({alpha}SMA) spontaneously. The presence of serum in the culture medium inhibits the development of fibrocytes from PBMCs in vitro, and serum amyloid P and immune complexes appear to be factors in inhibiting fibrocyte differentiation [6 ]. The use of enriched, serum-containing media accelerates and favors the differentiation of fibrocytes into fibroblasts and myofibroblasts [6 , 7 ]. Addition of TGF-ß or endothelin increases the levels of {alpha}SMA expression markedly, which corresponds with differentiation of these cells into myofibroblasts [2 3 4 5 , 8 ]. Fibrocytes express the fibroblast markers, such as vimentin, collagens I and III, and fibronectin [1 , 2 , 5 ], and are negative for CD3, CD4, CD8, CD16, CD19, CD25, and CD54. In addition, fibrocytes express the adhesion molecules CD11b and CD18 (Table 1 ) [1 , 3 4 5 ]. Moreover, fibrocytes express the common leukocyte antigen (CD45RO), the pan-myeloid antigen (CD13), HLA-DR, and the hematopoietic stem cell antigen CD34 (Table 1) [1 2 3 4 5 , 8 ].


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  		Table 1. Classic Fibrocyte Expression Patterns

Although early time-points of fibrocyte culture are associated with the expression of CD34+, CD45+, Col I+, and vimentin+, differentiation to myofibroblast-like cells after exposure to TGF-ß or endothelin results in the subsequent gain in expression of {alpha}SMA and loss of expression of CD34 [2 , 5 , 8 , 9 ] and CD45 [2 ]. These cellular changes support the notion that these cells lose their "stem" and common "leukocyte" markers as they differentiate. Therefore, the classic markers for these cells in circulation are CD45+, CD34+, Col I+, and vimentin+ (Table 1) [1 , 2 , 5 ]. When isolated initially, fibrocytes do express CD34, and CD34 expression is classically considered characteristic of hematopoietic stem cells, but it is not yet clear whether fibrocytes are actually derived from hematopoietic or mesenchymal progenitor/stem cells [1 , 4 , 5 ].

Fibrocytes have been found to have a number of functions other than promoting fibrosis. They are potent APCs and can elicit the recruitment and activation of T cells [10 ]. Fibrocytes can also induce an angiogenic phenotype in cultured endothelial cells and are involved with angiogenesis in vivo [11 ]. They can also secrete chemokines, cytokines, and growth factors, which are relevant in mediating fibroproliferation [4 , 12 ]. Fibrocytes have been found in a variety of tissues under normal and pathologic conditions [4 ].

Fibrocyte trafficking
Trafficking of leukocytes involves a complicated array of adhesion molecules, chemoattractants, and chemoattractant receptors, which allow the leukocytes to exit the bone marrow and extravasate to a specific region within tissue [13 ]. Classic cell trafficking has been well-described for leukocytes but is still being worked out for fibrocytes. Work to date suggests that there is significant overlap between the mechanisms of trafficking between leukocytes and fibrocytes.

The chemokine biological system is made up of chemoattractant ligands and their seven transmembrane receptors, and chemokines appear to be involved in fibrocyte trafficking [13 ]. Human fibrocytes have been shown to express the chemokine receptors CCR3, CCR5, CCR7, and CXCR4 (Table 1 ; gene chip and superarray data of fibrocyte chemokine gene expression, unpublished data) [3 , 5 ]. In contrast, mouse fibrocytes have been shown to express CCR7, CXCR4, and CCR2 (Table 1) [3 , 5 , 14 ]. CXCR4 is a pivotal chemokine receptor in hematopoietic and nonhematopoietic stem cell homing, and the differential expression of CXCL12 in tissues creates a gradient, which is essential for trafficking of CXCR4+ cells [15 ]. Fibrocytes have been shown to express CXCR4, consistent with their progenitor/stem cell phenotype and to migrate in response to CXCL12 in vitro (Fig. 1 ) [2 ]. Moreover, the biological axis of CXCL12/CXCR4 has been demonstrated to play a major role in mediating the contribution of fibrocytes to pulmonary fibrosis [2 ].


Figure 1
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  		Figure 1. The CXCR4/CXCL12 biological axis in fibrocyte trafficking. Lung injury results in high levels of CXCL12 expression. This creates a chemokine gradient for CXCR4+ fibrocytes to be released from the bone marrow and recruited from the circulation to the lungs. DAD, Diffuse alveolar damage.

Fibrocytes also express CCR7, which is a chemokine receptor that is important in dendritic cell and T cell migration. Phillips et al. [2 ] identified a population of fibrocytes that expressed CCR7, which were distinct from the CXCR4-expressing fibrocytes in bleomycin-induced, pulmonary fibrosis. They noted that the intrapulmonary recruitment of CD45+Col I+CXCR4+ fibrocytes was greater than CD45+Col I+CCR7+ fibrocytes, which correlated with collagen deposition in the lungs of bleomycin-exposed mice [2 ]. In mice, CXCR4, CCR7, and potentially, CCR2 appear to mediate recruitment of fibrocytes to the lung [2 , 14 ].

Fibrocyte plasticity
Fibrocytes have been shown to transition to {alpha}SMA+ myofibroblasts in the presence of serum or high concentrations of TGF-ß [2 3 4 5 , 8 ]. In addition, fibrocytes can differentiate into adipocytes and behave in a similar manner as preadipocytes from visceral or s.c. adipose tissue [16 ] (Fig. 2 ). It is interesting that specific ECM molecules (i.e., type I collagen) and TGF-ß can inhibit the differentiation of a fibrocyte to an adipocyte by down-regulating peroxisome proliferator-activated receptor {gamma} [16 ]. cDNA microarray analysis of the differentiation of a fibrocyte to an adipocyte revealed gene expression clusters, which were similar to visceral or s.c. preadipocyte-to-adipocyte differentiation [16 ]. Moreover, fibrocytes, which were undergoing adipogenesis, were engrafted into SCID mice and formed human adipose tissue [16 ]. Furthermore, fibrocytes exposed to appropriate culturing conditions in vitro have been found to differentiate to chondrocytes and osteoblasts (R. M. Strieter, unpublished data). Thus, fibrocytes may best be described as circulating mesenchymal progenitor cells (CMPC), as they demonstrate the ability to differentiate into multiple mesenchymal cell types. Therefore, in the context of fibroproliferative disorders, developing strategies to attenuate the recruitment of CMPC into tissue will impact on whether these cells integrate into tissue and contribute to fibrosis or are novel cells, which can contribute to obesity associated with metabolic syndrome.


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FIBROCYTES IN PULMONARY FIBROSIS
 
Pulmonary fibrosis
Idiopathic interstitial pneumonias (IIPs) are a heterogeneous group of diffuse, parenchymal lung disorders resulting from injury to the lung parenchyma and associated with varying degrees of inflammation and fibrosis [17 , 18 ]. IIPs can be classified into seven distinct entities based on clinical manifestations, pathology, and radiologic features [18 ]. One of these entities, IPF, is a progressive and fatal disease. IPF is defined as a chronic fibrosing form of IIP, which is limited to the lungs and associated with biopsy-proven pathology, showing a histologic pattern of usual interstitial pneumonia (UIP) [17 , 18 ], which is not unique to IPF and has been reported in other diseases associated with interstitial lung disease, such as scleroderma, rheumatoid arthritis, and end-stage hypersensitivity pneumonitis [19 ].

Neither the natural history nor the exact pathogenesis of UIP is currently known. UIP is diagnosed on the basis of temporally heterogeneous areas of normal lung, active fibrosis, and end-stage honeycomb fibrosis [17 ]. This histopathology suggests that the pathological events are occurring at different points in time and ultimately lead to dysregulated repair with aberrant vascular remodeling and marked deposition of ECM. IIPs usually occur as a continuum with overlapping features of chronic inflammation, which lead to end-stage fibrosis. Significant histopathological variability is found in surgical lung biopsies from patients with IIP [20 ]. Usually, there is inter- and intralobar, histopathologic variability of IIP with components of chronic inflammation [i.e., nonspecific interstitial pneumonia (NSIP)] with more fibrosis (i.e., UIP). The presence of UIP or discordant UIP with NSIP on biopsy appears to dictate a worse prognosis, as compared with concordant NSIP [21 ]. These data support the fact that evolving pulmonary fibrosis equates to a poorer response to conventional, immunosuppressive agents and a worse prognosis. As pulmonary fibrosis appears to be a dynamic process, the opportunity to target cells, which promote fibrosis (i.e., fibroblasts/myofibroblasts), would be a novel strategy to attenuate the development of pulmonary fibrosis.

The origin of the fibroblasts/myofibroblasts in pulmonary fibrosis
The classic concept of the origin of fibroblasts/myofibroblasts in lung tissue during the pathogenesis of pulmonary fibrosis is that tissue injury activates the resident interstitial fibroblasts to differentiate into myofibroblasts. These resident cells then migrate into the intra-alveolar space, proliferate, and express constituents of the ECM, leading to fibrosis in the alveolae and interstitium [22 23 24 ]. Another contemporary concept suggests that lung injury and changes in the microenvironment of the epithelium induce epithelial cells to transition to a mesenchymal phenotype and become fibroblasts/myofibroblasts and that these cells subsequently contribute to fibroproliferation [1 , 3 , 24 , 25 ]. A third concept implicates circulating fibrocytes (CMPC) in pulmonary fibrosis. Here, the CMPC may be derived from bone marrow precursor cells and traffic and home via specific chemoattractants into sites of tissue injury, where they could proliferate, differentiate to myofibroblasts, and contribute to the generation of ECM, relevant to pulmonary fibrosis [1 , 3 , 4 , 24 ].

Evidence to support the role of fibrocytes in the development of pulmonary fibrosis
The role of fibrocytes in the pathogenesis of human pulmonary fibroses is controversial; however, there is evidence that they may play a role. Phillips et al. [2 ] identified a population of CD45+Col I+CXCR4+ human fibrocytes and observed significant chemotaxis of these cells in vitro in response to the CXCR4 ligand CXCL12 [2 ]. A murine model of bleomycin-induced pulmonary fibrosis was used to examine the kinetics and magnitude of migration of these fibrocytes in vivo [2 ]. Purified human fibrocytes were injected intravascularly into SCID mice, which had already been exposed to bleomycin or saline for 4 days [2 ]. After a further 4 days, the mice were killed, and the lungs were analyzed for the presence of infiltrating human fibrocytes [2 ]. Significantly greater numbers of human CD45+Col I+CXCR4+ fibrocytes were observed in bleomycin-treated lungs in SCID mice, compared with those lungs that received saline alone [2 ]. Using the bleomycin mouse model in immunocompetent C57Bl/6 mice, they also found that transcription of pro-collagen I and pro-collagen III was up-regulated dramatically in mice exposed to bleomycin as compared with mice treated with saline alone, and there was also an increase in total collagen protein by Sircol assay [2 ].

Once they had characterized the kinetics of collagen deposition in C57Bl/6 mice in response to bleomycin, they performed a kinetic analysis of fibrocyte infiltration in the lungs of bleomycin-exposed mice [2 ]. The CD45+Col I+CXCR4+ cells began to appear in the lung 2 days after bleomycin treatment, became maximal at Day 8, and remained elevated at Days 16 and 20 [2 ]. Expression of CD45+Col I+CXCR4+ cells in the lung of saline-treated mice also increased initially, before returning to the levels observed in naive mice by Days 16 and 20 [2 ]. The most likely explanation for the latter phenomenon was that intratracheal instillation of saline itself can promote an inflammatory response. The number of CD45+Col I+CXCR4+ fibrocytes found in the bone marrow of bleomycin-challenged animals was markedly greater than the cells found in normal saline control mice [2 ]. Recently, we have found that fibrocytes can be mobilized into the circulation when mice are exposed to G-CSF, M-CSF, and GM-CSF (R. M. Strieter, unpublished data; Fig. 1 ). These data suggest that the bone marrow is at least one potential source of CD45+Col I+CXCR4+-circulating fibrocytes.

The CXCR4 ligand, CXCL12, was significantly higher in the lungs of animals exposed to bleomycin for 8 days than the comparable saline control or the naive control [2 ]. Thus, these data support the notion that a CXCL12 gradient existed between the lungs and plasma of bleomycin-treated mice, which could promote the recruitment of CD45+Col I+CXCR4+ fibrocytes to the fibrotic lung (Fig. 1) . A similar chemokine gradient between the lungs and plasma of bleomycin-treated mice existed for CCL21 (6Ckine) but not for CCL19 (ELC), the putative ligands for CCR7 [2 ].

Given the importance of the CXCR4/CXCL12 biological axis in fibrocyte recruitment, a strategy to deplete CXCL12 was used in the bleomycin-lung mouse model. Pulmonary collagen deposition was reduced significantly under conditions of CXCL12 depletion in bleomycin-exposed mice in comparison with those mice exposed to bleomycin and treated with control antibodies [2 ]. The addition of neutralizing CXCL12 antibodies to bleomycin-treated lungs did not, however, completely attenuate collagen deposition to the level of the saline control [2 ]. Taken together, these data indicate that inhibition of the CXCR4/CXCL12 chemotactic axis reduces intrapulmonary recruitment of CD45+Col I+CXCR4+ fibrocytes and abrogates lung fibrosis significantly in bleomycin-exposed mice.

To exclude that the effect of CXCL12 depletion influenced the infiltration of conventional leukocyte populations, FACS analysis of CD4 and CD8 T cells, NK cells, neutrophils, and monocytes/macrophages was performed from single cell suspensions of bleomycin-exposed lungs in animals treated with anti-CXCL12 or control antibodies [2 ]. There was no statistical difference in the numbers of CD4 and CD8 T cells, NK cells, neutrophils, and monocytes/macrophages trafficking to the lungs of bleomycin-exposed mice treated with anti-CXCL12 or control antibodies. Although CXCL12 clearly does mediate recruitment of CXCR4+ leukocytes to the lungs, the fact that neutralizing anti-CXCL12 antibodies did not block migration of leukocytes here can be explained by the observation that these PBMCs express chemokine receptors other than CXCR4. It is possible therefore, that inhibition of a single chemokine receptor/ligand combination (i.e., CXCR4/CXCL12) does not prevent these conventional leukocytes from intrapulmonary infiltration in response to chemokines other than CXCL12. Furthermore, these findings support the notion that blocking the CXCR4/CXCL12 biological axis under conditions of bleomycin-induced pulmonary fibrosis affected only the infiltration of fibrocytes and perhaps other nonleukocyte progenitor cells into the lung.

To verify further that systemic addition of anti-CXCL12 antibodies selectively reduced intrapulmonary infiltration of CD45+Col I+CXCR4+ fibrocytes and attenuated bleomycin-induced pulmonary fibrosis, H&E staining and morphometric analysis with a collagen-specific dye (picosirus red) were performed and showed that depletion of CXCL12 reduced bleomycin-induced pulmonary fibrosis significantly [2 ]. In addition, blocking the recruitment of CD45+Col I+CXCR4+ fibrocytes to bleomycin-exposed lungs also reduced expression of immunohistochemical expression of {alpha}SMA [2 ]. This suggests that CD45+Col I+CXCR4+ fibrocytes recruited from the peripheral circulation may ultimately develop an {alpha}SMA+ phenotype in fibrotic lungs, which is compatible with the in vitro findings for human fibrocytes.

The role for fibrocytes in the development of pulmonary fibrosis is supported by the following: The kinetics of fibrocyte extravasation into the lung paralleled maximal pulmonary fibrosis; blocking CXCL12 resulted in marked attenuation of fibrocyte extravasation into the lung, which correlated directly with a marked reduction in pulmonary fibrosis; and blocking CXCL12 inhibited fibrocyte extravasation into the lungs but did not attenuate other leukocyte populations. However, the exact origin of the fibrotic cell types in human pulmonary fibroses has not been established yet. One reason for this is that the known markers of fibrocytes are changing constantly as the cell traffics and engrafts, and so, immunohistochemical studies, such as that by Choi et al. [26 ], are unable to dynamically track the cell and follow it from its origin. Mehrad and associates [27 ] examined the numbers of CD45+Col1+CXCR4+ in patients with fibrotic interstitial lung disease and found an order of magnitude higher number of fibrocytes in patients compared with healthy volunteers. The CXCL12 ligand expression was also found to be elevated markedly in the lung and plasma of patients with lung fibrosis [27 ]. Therefore, it appears that the CXCR4/CXCL12 biological axis is important in the trafficking of human fibrocytes in the setting of pulmonary fibrosis.

In other studies that support the role of fibrocytes in the pathogenesis of pulmonary fibrosis, Hashimoto and associates [28 ] performed bone marrow transplantation of wild-type mice with GFP-labeled bone marrow. They had durable engraftment with >92% of CD45+ cells in the bone marrow being GFP-positive. These mice were then exposed to endotracheal bleomycin injury, and analysis of the lungs from these mice showed large clusters of bone marrow-derived, GFP-positive cells in areas of active fibrosis related to bleomycin exposure. More than 80% of the collagen I-expressing cells in the bleomycin-injured lungs were GFP+ and therefore, of bone marrow origin. However, dual-staining of GFP-positive cells in the lung showed almost no staining for {alpha}SMA, implying that in this model and at the time-point assessed, the lung myofibroblasts did not appear to arise from bone marrow-derived cells [28 ]. These findings were in contrast with the findings of Philips et al. [2 ] and another GFP bone marrow chimeric mouse model in BALB/c mice, where analysis of GFP+ bone marrow-derived fibrocytes in skin wounds demonstrated clear evidence for the expression of {alpha}SMA in GFP+ fibrocytes at Day 4 postwounding [29 ]. As further confirmation that fibrocytes are bone marrow-derived, we have transplanted lethally irradiated, wild-type mice with GFP+ bone marrow and demonstrated a marked reduction in GFP+ fibrocytes in the lungs of bleomycin-exposed animals under conditions of CXCL12 depletion (R. M. Strieter, unpublished data).

Bone marrow-derived myofibroblasts have also been found in pulmonary-organizing alveolitis/fibrosis after lung irradiation in mice [30 ]. For these studies, GFP+ male bone marrow was transplanted into wild-type female mice, and the mice were then exposed to total lung irradiation. A significant number of GFP+ bone marrow-derived cells were seen in the fibrotic areas and were also vimentin-positive, although less than 1% of the endothelial cells in the areas of fibrosis was GFP+ [30 ]. Bromodeoxyuridine labeling of developing fibrotic areas showed that dividing cells were GFP+, Y-chromosome+, and vimentin+ predominantly. These data suggest that CMPC from the bone marrow are involved in the fibrotic response of the lung to radiation injury and that once recruited, these CMPC proliferate at sites of injury and fibrosis. Mice, which were treated subsequently with an intratracheal injection of a radio-protectant 24 h before total lung irradiation, demonstrated a significant decrease in GFP+, vimentin+ cells in the injured lung [30 ].

In another study, Moore and colleagues [14 ] examined the contribution of fibrocytes to fibrosis in a FITC-induced, lung-injury model. In this study, fibrocytes were obtained by bronchoalveolar lavage (BAL) from a population of cells, which had transmigrated to the airway lumen. In addition, they assessed fibrocytes from minced lung specimens. The BAL cells or minced lungs were cultured and analyzed for the dual expression of collagen I and CD45. Continued culture of these fibrocytes resulted in loss of CD45 expression but a persistence of collagen I expression. They found that populations of fibrocytes from B6/129F2 and C57Bl/6 strains of mice expressed CXCR4, CCR5, CCR7, and CCR2 [14 ]. This is in contrast to human fibrocytes, which express low levels of CCR2 after isolation [16 ]. Fibrocytes isolated from the mouse lungs expressed CCR2, migrated to CCL2 and CCL12 ligands, and lost expression of CCR2 when cultured in vitro to a differentiated fibroblast [14 ].

They next treated wild-type and CCR2–/– mice on the C57BL/6 or B6/129F2 backgrounds with intratracheal FITC and found that the absolute number of lung fibrocytes present in cultures from the BAL of wild-type mice was significantly greater than noted in cultures from CCR2–/– mice [14 ]. The absolute number of lung fibrocytes in C57BL/6 mice was higher than in B6/129F2 mice, which correlated with the differences observed previously in the magnitude of the fibrotic response to FITC between these two mouse strains. They explained the differences in fibrocyte accumulation in the BAL from wild-type compared with CCR2–/– mice treated with FITC as reflecting differences in recruitment, as there was no difference in proliferation of these cells in culture [14 ].

In this same study, they next performed bone marrow transplants with wild-type bone marrow in lethally irradiated CCR2–/– mice. They showed that recruitment of lung fibrocytes in response to FITC injury was restored in the mice that received CCR2+/+ bone marrow. Lung fibrocytes cultured from the CCR2+/+ bone marrow transplant mice were positive for CCR2, indicating that they were of donor origin [14 ]. Conversely, when CCR2–/– mice received a CCR2–/– bone marrow transplant, the mice were protected from FITC-induced fibrosis [14 ]. They then added CCL2, TGF-ß1, and IL-13 to the fibrocyte cultures to determine their effect on collagen production. TGF-ß1 and CCL2 increased collagen I production from fibrocytes, but IL-13 did not. In contrast, IL-13 and TGF-ß1 stimulated the production of collagen I by fibroblasts, but CCL2 had no effect [14 ].

In another study by the same group, fibrocytes were recruited to FITC-injured lung in CCL2 knockout mice, unlike CCR2 knockout mice, implying that CCL2 was not essential for fibrocyte trafficking to the FITC-injured lung. These findings were demonstrated further by blocking CCL2 activity, which did not prevent fibrosis significantly [31 ]. However, neutralization of the CCR2 ligand, CCL12, reduced fibrosis significantly in the FITC mouse model of lung injury [31 ]. The authors suggested that CCL12 is the CCR2 ligand that promotes fibrosis in the mouse lung. However, this may be more relevant to mouse biology, as human fibrocytes have been found to express low levels of CCR2, which increases markedly when the cells undergo adipogenesis [16 ].


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FIBROCYTES IN ASTHMA
 
Repair and remodeling of the airway in asthma
Inflammation and airway remodeling are the hallmarks of asthma, and both contribute to disease persistence and progression [32 34 35 ]. The remodeling of asthmatic airways results in subepithelial fibrosis, submucosal gland hyperplasia and hypertrophy, hyperplasia of myofibroblasts, hyperplasia and hypertrophy of myocytes, and hypertrophy of airway epithelial cells [34 , 35 ]. The mechanisms of remodeling are not well understood, but a number of cytokines and inflammatory mediators have been implicated [34 ]. Identifying and characterizing the defects in proliferation and differentiation of the progenitor epithelial cells involved in the regeneration of the pulmonary epithelium in asthma have important therapeutic implications, as modifying the abnormal regeneration of these cells could provide novel treatments for asthma. In this regard, furthering our understanding of the potential contribution of CMPC to these processes may aid in the development of specific therapeutic interventions.

Fibrocytes in airway remodeling in asthma
Schmidt and associates [8 ] showed that fibrocyte-like cells exist in the airway of asthmatic patients, increase in number after antigen challenge, and appear to differentiate into collagen-producing myofibroblasts. They showed that there are significant numbers of CD34+Col I+ cells as well as a few CD34+{alpha}SMA+ cells below the basement membrane in the bronchial mucosa of asthmatic patients and that these cells increase dramatically 24 h after exposure to an allergen [8 ]. They used systematic sensitization of BALB/c mice with OVA as a mouse model of chronic asthma, which has been shown to result in airway remodeling, characterized by thickening of the lamina propria by subepithelial deposition of fibronectin and collagen [8 ]. In this mouse model, CD34+Col I+ cells were also seen, as were CD34+{alpha}SMA+ cells. They then isolated CD34+Col I+ cells from mouse peripheral blood and stained the cells with intravital PKH-26 fluorescent dye. These labeled cells were injected into the tail vein of the chronically OVA-exposed BALB/c mice, and flow cytometric analysis of single cell suspensions from the airway tissue of these mice showed a significant increase in the number of fibrocytes in the airway submucosa compared with PBS-injected controls. These PKH-26-labeled cells in the airway submucosa also showed loss of CD34 expression and an increase in {alpha}SMA expression [8 ]. Finally, they showed that cultures of human CD34+Col I+ cells in serum-free medium constitutively expressed {alpha}SMA, and the addition of endothelin-1 and TGF-ß1 resulted in an increase in fibronectin and collagen III in the culture medium [8 ].

Thickening of the airway epithelial basement membrane from fibrosis is often seen in asthmatics, but the origins of this fibrosis are not known. Nihlberg and colleagues [36 ] cultured BAL fluid from mild steroid, naive asthmatics with definite bronchial hyper-responsiveness and divided the patients into two groups, depending on whether they could culture fibrocytes from their BAL fluid. They used these two patient groups and normal control subjects for their subsequent studies about the number of fibrocytes and thickness of the basement membrane from airway biopsy specimens. They first used confocal microscopy of the airway tissue and found that there was a 14-fold increase in the number of CD34+{alpha}SMA+CD45RO+ cells and a 16-fold increase in the number of CD34+Col I+ cells in the proximity of the basement membrane in patients from whom they could culture fibrocytes, compared with control subjects. They also saw a twofold increase in CD34+{alpha}SMA+CD45RO+ cells and CD34+Col I+ cells in patients from whom they could not culture fibrocytes when compared with control samples. They found that the fibrocytes were located in close proximity to the basement membrane in patients with mild asthma, that the thickness of the basement membrane was greatest in patients from whom fibrocytes in BAL fluid could be cultured, and that the basement membrane thickness correlated with the number of fibrocytes in the airway [36 ].


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FIBROCYTES IN PULMONARY VASCULAR REMODELING
 
Chronic pulmonary hypertension is characterized by adventitial cell proliferation, vascular wall ECM deposition, and expansion of myofibroblasts in the large and small pulmonary arteries. This process has now been shown to occur, not only from resident fibroblasts but also from circulating, mesenchymal, progenitor cells (Fig. 2) [37 ]. However, there is some controversy as to whether the circulating cells described by Frid and colleagues [37 ] are true fibrocytes, as they describe cells that are monocyte/macrophage-like with CD11b+CD13+CD14+ surface markers, whereas classical fibrocytes are CD11b+CD13+CD14– (Table 1) [1 , 2 , 37 ]. In chronically hypoxic, neonatal rat and calf models, they found that cells in the adventitia coexpressed CD45, Col I, CD14, CD11b, and collagen-prolyl-hydroxylase-{alpha} [37 ]. Cells in the adventitia also coexpressed CD45 and {alpha}SMA, as well as CD68 and {alpha}SMA [37 ]. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-5,5' (DiI) liposomes were injected into the circulation of rats, taken up by phagocytosis and then incorporated into the cell membrane of monocytes [37 ]. DiI-labeled cells were found in the pulmonary artery adventitia of chronically hypoxic rats and not in control, normoxic rats [37 ]. The majority of these labeled cells also expressed CD11b and ED1 and ED2, and many coexpressed Col I.

Varcoe and colleagues [38 ] isolated CD14+-nucleated cells from the peripheral blood of healthy donors and cultured them on fibronectin in the presence of platelets isolated from autologous, platelet-rich plasma. They demonstrated these cells to be CD14+CD34+Col I+{alpha}SMA+ by Day 14 of culture. They also used an in vivo ovine model to demonstrate the contribution of circulating fibrocytes to intimal hyperplasia. They labeled peripheral blood leukocytes with CFSE and then injected the CFSE-labeled leukocytes through an internal jugular catheter. Twenty-four hours later, the animals underwent surgery to receive a carotid artery synthetic patch graft. The animals were killed 1, 2, and 4 weeks postsurgery, and the graft was examined for CFSE fluorescence. At 2–4 weeks postsurgery, they found that 50% of the cells of the intima of the graft were fluorescent, indicating that these cells arose from the circulating, CFSE-labeled leukocytes [38 ].

These studies highlight the potential importance of circulating fibrocytes or fibrocyte-like cells in vascular adventitial thickening in pulmonary hypertension, as resident adventitial fibroblasts played a much smaller role in the changes seen in the vascular adventitia in pulmonary hypertension. These findings also support that targeting these fibrocyte-like cells may have therapeutic implications in the treatment of pulmonary hypertension.


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CONCLUSIONS
 
The discovery of fibrocytes and their role in promoting fibrosis have changed our thinking about many disease processes, which are associated with fibrosis. Many questions remain unanswered, such as: What are the signals involved in the recruitment of fibrocytes? Are these signals different in humans and mice? What are the factors involved in their plasticity/differentiation? What is the role of the microniche in promoting fibrocyte progression to a differentiated myofibroblast? All of these questions are critical to our understanding of fibrosis and need to be addressed to design therapeutic strategies to attenuate fibrocyte biology and prevent their contribution to fibrotic disorders in organs such as the lungs.

Received September 22, 2006; revised March 26, 2007; accepted April 9, 2007.


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REFERENCES
 
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