Fibrocytes: Bringing New Insights Into Mechanisms of Inflammation and Fibrosis

Abstract

Regeneration and fibrosis are integral parts of the recovery process following tissue injury, and impaired regulation of these mechanisms is a hallmark of many chronic diseases. A population of bone marrow-derived mesenchymal progenitor cells known as fibrocytes, play an important role in tissue remodeling and fibrosis in both physiologic and pathologic settings. In this review we summarize the key concepts regarding the pathophysiology of wound healing and fibrosis, and present data to support the contention that circulating fibrocytes are important in both normal repair process and aberrant healing and fibrotic damage associated with a diverse set of disease states.

Introduction

Although the etiology of tissue damage may vary, the reparative process that ensues is remarkably similar (Kisseleva and Brenner, 2008a). Fibrosis is the end result of a series of events that occur after mechanical damage to the epithelium and/or endothelium (Kisseleva and Brenner, 2008b). Initially, inflammatory mediators are released that trigger platelet aggregation, clot formation and the formation of a provisional extracellular matrix. Subsequently, growth factors, cytokines and chemokines are produced and stimulate the proliferation and recruitment of leukocytes that work to remove dead tissue and promote new blood vessel formation. Activated fibroblasts transform into α-smooth muscle actin-expressing myofibroblasts that promote wound contraction. Lastly, epithelial and/or endothelial cells divide and migrate to regenerate the damaged tissue (Wynn, 2008). If this process becomes dysregulated, an excessive accumulation of extracellular matrix components occurs, and the synthesis of new collagen by fibroblasts exceeds the rate at which it is degraded. Over time fibrosis ensues which leads to permanent scarring and organ dysfunction (Kisseleva and Brenner, 2008b, Wynn, 2008).

In the context of physiologic and pathologic fibrosis, tissue fibroblasts and myofibroblasts are classically thought to derive from resident fibroblasts that, in response to tissue injury, proliferate and express constituents of the extracellular matrix. However, it is now thought that fibroblasts can also be derived from two additional sources: (1) from epithelial cells, in a process known as epithelial-mesenchymal transition (Iwano et al., 2002, Kalluri and Neilson, 2003), and (2) from bone marrow-derived circulating progenitor cells (fibrocytes), the focus of this review (Abe et al., 2001, Bucala et al., 1994, Metz, 2003).

Fibrocytes

Original description

Fibrocytes were originally described in 1994 (Bucala et al., 1994): in an experimental model of wound repair, 10% of the cells in the wound chamber were spindle-shaped and expressed collagen, procollagen, and CD34 within one day following injury. Since their appearance was much faster than would be expected by entry of fibroblasts from the surrounding skin, it was thought that they must have originated from the circulation (Bucala, 2008). These cells were named fibrocytes (a term combining fibroblast with leukocyte). Fibrocytes are a unique CD45+ cell population that are distinct from monocytes, dendritic cells, T lymphocytes, fibroblasts, epithelial cells, endothelial cells, and Langerhans cells. They exhibit prominent cell surface projections on scanning electron microscopy making them morphologically distinct from leukocytes (Bucala et al., 1994). Fibrocytes comprise 0.1–1% of the nucleated cells in the peripheral blood in healthy hosts (Metz, 2003, Phillips et al., 2004, Quan et al., 2004).

Bone marrow origin

There is substantial information available supporting the hypothesis that fibrocytes are derived from the bone marrow. Fibrocytes are characterized by the expression of collagen I, collagen III, fibronectin, major histocompatibility complex II, CD11b, CD13, CD34, and CD45 but they do not express CD14, CD3, or CD10 (Abe et al., 2001, Aiba and Tagami, 1997, Bucala et al., 1994, Ebihara et al., 2006, Phillips et al., 2004, Pilling et al., 2003, Pilling et al., 2006, Quan et al., 2004, Yang et al., 2002). The co-expression of collagen and the other hematologic markers (such as CD45) are used to identify fibrocytes: early in culture, fibrocytes express CD34, CD45, collagen I, and vimentin. Fibrocytes also express a number of chemokine receptors on their surface including CCR3, CCR5, CCR7, and CXCR4, and can migrate to wound sites in response to specific chemokine gradients (Abe et al., 2001). They do not, however, express T cell markers (CD3, CD4, and CD8), B cell markers (CD19), the interleukin (IL)-2 receptor chain CD25, the low affinity Fc gamma receptor III (CD16), and myeloid markers (CD14 and nonspecific esterase) (Abe et al., 2001, Bucala et al., 1994, Metz, 2003, Phillips et al., 2004, Quan et al., 2004, Schmidt et al., 2003).

Some studies suggest that fibrocytes can differentiate from CD14+ peripheral blood monocytes that express the receptors for the Fc portion of IgG, CD64, and CD32 (Pilling et al., 2003, Pilling et al., 2006, Varcoe et al., 2006, Yang et al., 2002). Circulating fibrocytes may be present in a subset of CD14+ CD16−monocytes that carry the chemokine receptor, CCR2, on their surface (Gordon and Taylor, 2005, Tacke and Randolph, 2006). At the time of tissue injury this monocyte subset is released from the bone marrow into the peripheral blood and migrates to inflamed sites via a CCR2-mediated pathway (Gordon and Taylor, 2005, Tacke and Randolph, 2006). Human fibrocytes may represent an intermediate stage of differentiation of this monocyte subset into mature fibroblasts and myofibroblasts in tissue (Bellini and Mattoli, 2007). While fibrocytes are most likely of myeloid lineage, more information is needed to determine whether they are derived from a CD14+ progenitor cell.

Differentiation and trafficking

The differentiation of fibrocytes into myofibroblasts is augmented in the presence of transforming growth factor (TGF)-β or endothelin-1, and results in cells that produce fibronectin and collagen, and express the myofibroblast marker α-smooth muscle actin (Abe et al., 2001, Metz, 2003, Phillips et al., 2004, Quan et al., 2004, Schmidt et al., 2003). Fibrocytes spontaneously gain expression of α-smooth muscle actin in culture, and gradually loose the expression of CD34 and CD45 over time depending on the inflammatory milieu (Bucala, 2008): this response can be augmented by exposure of the fibrocytes to TGF-β or endothelin, resulting in differentiation into myofibroblast-type cells. It has been shown that the pro-fibrotic cytokines IL-4 and IL-13 promote fibrocyte differentiation from CD14+ peripheral blood monocytes without inducing proliferation, whereas the anti-fibrotic cytokines IL-12 and interferon (IFN)-γ inhibit fibrocyte differentiation (Shao et al., 2008): IL-4, IL-13 and IFN-γ were found to regulate fibrocyte differentiation through a direct effect on monocytes, whereas IL-12 was found to have an indirect effect possibly through CD16+ NK cells. Fibrocyte differentiation appears to be influenced by a complex profile of cytokines, chemokines and plasma proteins within the area of tissue injury.

Human fibrocytes express several chemokine receptors, including CCR3, CCR5, CCR7, and CXCR4; in contrast, mouse fibrocytes express CXCR4, CCR7, and CCR2 (Abe et al., 2001, Phillips et al., 2004, Mehrad et al., 2009, Moore et al., 2005, Quan et al., 2004). Thus, in interpreting animal studies, it is important to note that the pattern of chemokine receptor expression is not identical between mouse and human fibrocytes. According to the disease process and organ involved, fibrocytes can use different chemokine ligand-receptor pairs for tissue homing. The CXCR4-CXCL12 axis plays an important role in the homing of bone marrow-derived progenitor cells (Murdoch, 2000): CXCR4 is an important chemokine receptor in stem cell trafficking, and the differential expression of CXCL12 in tissues creates the gradient required for trafficking of CXCR4+ cells. Although an early study reported little chemotaxis of fibrocytes to CXCL12 (Abe et al., 2001), our group detected substantial in vitro and in vivo chemotaxis of these cells to CXCL12 (Phillips et al., 2004), and consider the earlier results to be due to methodological differences between the experiments, especially since hypoxia plays an important role for CXCR4 expression and chemotaxis (Mehrad et al., 2009).

Wound healing

Fibrocytes contribute to normal wound healing by several important mechanisms including acting as antigen-presenting cells (Chesney et al., 1997), serving as the contractile force of wound closure via α-smooth muscle actin expression (Abe et al., 2001, Metz, 2003), promoting angiogenesis (Hartlapp et al., 2001), producing cytokines, chemokines, and growth factors that induce fibroblast hyperplasia (Chesney et al., 1998), and secreting components of extracellular matrix (Abe et al., 2001, Bucala et al., 1994). Fibrocytes have been implicated in a wide range of aberrant fibrotic processes involving the lung, heart, and vasculature (Keeley et al., 2009), as well as the skin, kidney, bladder, liver, gallbladder, colon, pancreas, eye, and in infectious disease and tumor metastases.

Fibrotic diseases

Lung

Several lines of evidence support a role for fibrocytes in the development of lung fibrosis (Gomperts and Strieter, 2007). Fibrotic lung diseases are a large group of disorders characterized by varying degrees of inflammation and fibrosis of the lung parenchyma (2002). The clinical course is one of progressive replacement of lung tissue with scar tissue, and clinical deterioration. Among fibrotic lung diseases idiopathic pulmonary fibrosis is the most common.

Idiopathic pulmonary fibrosis

In the context of a mouse model of bleomycin-induced pulmonary fibrosis, fibrocytes have been shown to home to the lungs and contribute to fibrosis (Phillips et al., 2004). In this study, human fibrocytes that were administered intravenously to SCID mice (previously treated with either bleomycin or saline) preferentially homed to the lungs in animals treated with bleomycin. Similarly, in immunocompetent bleomycin-treated mice, the magnitude of lung pro-collagen I and III upregulation correlated with the number of CD45+ collagen I+ CXCR4+ fibrocytes in the bone marrow, in the blood, and in the lung (Phillips et al., 2004). In addition, CXCL12 was significantly increased in the lungs of mice that were treated with bleomycin, supporting the notion that a CXCL12 gradient between the lungs and the plasma promoted the recruitment of the CD45+ collagen I+ CXCR4+ fibrocytes to the lung. The administration of neutralizing anti-CXCL12 antibodies to bleomycin-treated mice resulted in significantly reduced fibrocyte extravasation into the lung, reduced collagen deposition in the lungs, and reduced immunohistochemical expression of α-smooth muscle actin, but did not affect the numbers of other leukocyte populations in the lungs (Phillips et al., 2004).

Several groups have since corroborated these findings in the context of mouse models of lung fibrosis (Ortiz et al., 2003, Rojas et al., 2005). Similar findings have been reported by other investigators using different models of lung fibrosis: (1) in a model of radiation-induced fibrosis, the degree of fibrosis correlated with fibrocyte recruitment to the lung (Epperly et al., 2003); (2) in a endotracheal bleomycin injection model, collagen-producing lung fibroblasts were shown to be derived from bone marrow progenitor cells (Hashimoto et al., 2004); and (3) in a intrapulmonary fluorescein isothiocyanate (FITC)-induced fibrosis model, fibrocytes isolated from the bronchoalveolar lavage fluid and whole lung samples were found to express CCR2, CCR5, CCR7, and CXCR4 (Moore et al., 2005). In the latter study, fibrocytes isolated from the lung expressed CCR2, migrated toward CCL2 and CCL12 ligands, and lost expression of CCR2 when cultured in vitro to a differentiated fibroblast. In CCR2-deficient mice challenged with intrapulmonary FITC, fibrocyte recruitment to the lungs was reduced. Moreover, wildtype mice that received CCR2 −/− bone marrow had reduced recruitment of fibrocytes to the lung and a reduction in pulmonary fibrosis. Transplantation of bone marrow cells from the wildtype mice into irradiated CCR2 −/− mice once again restored the ability to grow fibrocytes from whole lung homogenates and the susceptibility to FITC-induced lung fibrosis (Moore et al., 2005). Additional work from the same investigators suggest that CCR2 ligands play a key role in the accumulation of fibrocytes triggered by intrapulmonary administration of FITC (Moore et al., 2006), and may be involved in the accumulation of fibrocytes in human diseases since the recruitment of human fibrocyte precursors (CD14+ CD16− monocytes) into areas of inflammation is dependent on CCR2 (Tacke and Randolph, 2006).

Lastly, data from several human studies suggest fibrocytes play a pivotal role in the pathogenesis of lung fibrosis. In one study, the numbers of CD45+, collagen I+, CXCR4+ circulating fibrocytes were markedly higher in patients with fibrotic interstitial lung disease than in healthy controls (Mehrad et al., 2007); the CXCL12 ligand expression was also found to be markedly elevated in the lung and plasma of patients with lung fibrosis. In another study, fibrocytes were identified in tissue from 8 out of 9 fibrotic lungs in patients with idiopathic pulmonary fibrosis. While there was a positive correlation between the abundance of fibroblastic foci and the amount of lung fibrocytes (r=0.79; p<0.02), no fibrocytes were identified in normal lungs (Andersson-Sjoland et al., 2008). Moreover, CXCL12 was increased in the plasma of the patients with idiopathic pulmonary fibrosis (and present in about half of their bronchoalveolar samples); and the chemokine level directly correlated with disease severity (higher CXCL12 levels were associated with worse gas exchange) (Andersson-Sjoland et al., 2008). In another study, the proportion of peripheral blood fibrocytes was shown to be increased in patients with acute exacerbations of interstitial pulmonary fibrosis as compared to stable interstitial pulmonary fibrosis, and was an independent predictor of death (Moeller et al., 2009).

Asthma

Chronic asthma is characterized by persistent airway inflammation and structural aberrant remodeling of the airways (Davies et al., 2003). The bronchial mucosa of asthmatic patients shows mixed degrees of both on-going inflammation and repair. The remodeling process leads to thickening of the airway wall and can lead to irreversible decline in lung function. The subepithelial fibrosis in asthma is a result of extensive deposition of extracellular matrix and connective tissue components. In one study, following inhalation of allergen, fibrocytes expressing CD34 and pro-collagen I mRNA were found in the airways of patients and a substantial proportion of these fibrocytes also expressed α-smooth muscle actin (Schmidt et al., 2003). Similarly, in a different study, a rapid increase in myofibroblasts was seen in the airway mucosa after allergen challenge in subjects with mild asthma (Gizycki et al., 1997). Others have shown that in bronchial biopsies from patients with mild asthma, cells expressing CD34, CD45 and α-smooth muscle actin consistent with fibrocytes, differentiated into myofibroblasts and resulted in increased thickness of the lamina reticularis (Nihlberg et al., 2006). These fibrocytes appeared in clusters close the epithelial basement membrane and their density correlated with the thickness of the lamina reticularis. The expression of CCR7 in the myofibroblasts in the bronchial mucosa in asthma patients was shown in another study and provides further evidence that these cells may be of fibrocyte origin (Kaur et al., 2006). These investigators also observed an increased production of CCL19 and its receptor CCR7, suggesting that the CCL19/CCR7 axis may be important in fibrocyte recruitment in asthma (Kaur et al., 2006).

Increased number of circulating fibrocytes has also been shown in patients with asthma. In one study, an increased number of CD34+ CD45+ Col I+ fibrocytes were found in the peripheral blood of patients with chronic persistent obstructive asthma, but not in patients with normal lung function (Wang et al., 2008). These fibrocytes had higher proliferation potential and increased differentiability into α-smooth muscle actin myofibroblasts when exposed to TGF-β1. Moreover, there was a significant correlation between the percentage of fibrocytes in the peripheral blood of patients with chronic obstructive asthma and the average slope of the yearly decline in FEV1 (Wang et al., 2008). In a separate study, investigators found increased numbers of fibrocytes in the peripheral blood of patients with severe refractory asthma compared to normal controls (1.4 × 10⁴/mL versus 0.4 × 10⁴/ml, p=0.002). Moreover, they also found increased numbers of fibrocytes in the lamina propria from bronchial biopsy specimens in patients with severe refractory asthma compared to normal controls (1.9/mm² versus 0/mm², p<0.0001) (Saunders et al., 2009). Lastly, since fibrocytes are potent antigen-presenting cells, they may amplify the allergic reaction by capturing antigens that cross the epithelial barrier (Chesney et al., 1997).

Pulmonary hypertension

Pulmonary hypertension is characterized by elevated pulmonary arterial pressures, increased pulmonary vascular resistance, and right ventricular failure (McLaughlin et al., 2009). The vascular remodeling seen in pulmonary hypertension is characterized by marked fibroproliferative changes in the pulmonary artery adventitia (Farber and Loscalzo, 2004). In a set of experiments using two different animal models of pulmonary hypertension (rats and calves), investigators showed a significant accumulation of fibrocytes in the adventitia and media of the remodeled pulmonary arteries (Frid et al., 2006). Moreover, the contribution of circulating fibrocytes to the vascular remodeling process was confirmed using depletion studies: reduction of fibrocytes in the circulation of chronically hypoxic rats led to a marked attenuation of adventitial thickening and decreased fibrosis (Frid et al., 2006). Others have also suggested a role for circulating bone marrow-derived cells in chronic hypoxia-induced remodeling. These investigators transplanted the bone marrow of enhanced green fluorescent protein (GFP)-transgenic mice to lethally irradiated syngeneic mice, and exposed the chimera mice to hypoxia. Marked vascular remodeling was observed and a large number of GFP+ cells were observed in the adventitia of the pulmonary artery in the mice exposed to hypoxia compared to controls. These cells expressed α-smooth muscle actin and contributed to the pulmonary vascular remodeling process (Hayashida et al., 2005).

Urinary tract

Regardless of the specific etiology, renal fibrosis characterized by glomerulosclerosis and interstitial fibrosis, is the most common final pathway of renal failure (Sakai et al., 2006). Moreover, the extent of renal fibrosis within the kidney is an independent predictor of clinical outcomes (Nath, 1998). Histologically, renal fibrosis is defined by tubular atrophy and dilation, interstitial leukocyte infiltration, and increased interstitial matrix deposition. It has been estimated that up to 15% of the myofibroblasts contributing to renal fibrosis are of bone marrow origin (Kisseleva and Brenner, 2008a). The renin-angiotensin system and angiotensin II play significant roles in renal fibrosis: the renin-angiotensin system promotes inflammation by increased expression of cytokines, chemokines, growth factors, and reactive oxygen species, while angiotensin II induces endothelial dysfunction, and up-regulates adhesion molecules (Ruiz-Ortega et al., 2006). Moreover, in a murine model of renal fibrosis, it has been suggested that fibrocytes might contribute to fibrosis by an angiotensin II dependent pathway (Sakai et al., 2008). In this study, using two models of renal fibrosis (unilateral ureteral obstruction and chronic angiotensin II infusion) angiotensin II type 2 receptor-deficient mice had increased renal fibrosis and fibrocyte infiltration and a concomitant upregulation of procollagen type I compared with wild-type mice. In addition, the number of fibrocytes in the bone marrow of the angiotensin II type 2 receptor-deficient mice were increased. Pharmacologic inhibition of angiotensin II type 1 receptor reduced the degree of renal fibrosis and the number of fibrocytes in the kidney and in the bone marrow.

These same investigators examined whether the contribution of fibrocytes to renal fibrosis was dependent on their trafficking to the kidney via CCL21/CCR7 signaling (Sakai et al., 2006). In a murine model of renal fibrosis, they found that circulating CD45+ Col I+ fibrocytes infiltrated the diseased kidneys, and that these fibrocytes expressed CCR7. The inhibition of the CCL21/CCR7 signaling using a neutralizing antibody reduced the severity of the fibrocyte infiltration, the degree of kidney fibrosis (by almost 50%), and the renal expression of TGF-β1 and Col I; thus suggesting that fibrocytes contribute to renal fibrosis by the production of Col I and that this process requires CCl21/CCR7 signaling. Moreover, CD34 + spindle shaped cells have also been detected in tubulointerstitial lesions in patients with glomerulonephritis and closely correlated with interstitial volume (Okon et al., 2003).

Lastly, in a pathologic study of chronic cystitis and invasive urothelial carcinomas of the bladder, CD34+ fibrocytes were found in the lamina propria and tunica muscularis in specimens obtained from patients with chronic cystitis, while there was complete loss of CD34+ fibrocytes in specimens obtained from patients with invasive urothelial carcinoma (Nimphius et al., 2007).

Multi-organ fibrotic diseases

Nephrogenic systemic fibrosis

Nephrogenic systemic fibrosis is a disease that occurs in patients with renal insufficiency, especially those who are dialysis-dependent, and is strongly associated with the use of gadolinium-based contrast agents (Galan et al., 2006, Morcos and Thomsen, 2008): The onset of the disease varies from several days to several months following exposure to gadolinium-based contrast. It is a debilitating disease that is characterized by the development of discolored plaques on the skin of the extremities and trunk. Over time, contractures develop and complete loss of range of motion can occur. Although initially thought to affect only the skin (hence, the original name was nephrogenic fibrosing dermopathy), involvement of the lungs, heart and liver can occur (Mendoza et al., 2006) and is associated with increased mortality (Cowper et al., 2008, Marckmann et al., 2008). Histologically, fibrocytes defined as CD34+ Col I+ procollagen I+ cells populate the lesions of nephrogenic systemic fibrosis (Cowper and Bucala, 2003). Many questions remain about the pathogenesis of this disorder, the role of gadolinium-based contrast agents, and whether it is a disease of aberrant fibrocyte activation, trafficking or function (Bucala, 2008). Lastly, continued research into the pathophysiology of nephrogenic systemic fibrosis may shed light on other systemic fibrosing disorders such as systemic sclerosis and scleroderma.

Scleroderma

Scleroderma is a connective tissue disease of unknown etiology that is characterized by small vessel vasculopathy and irreversible fibrosis effecting the skin and other organs (LeRoy et al., 1988). Adenosine and one of its receptors (the adenosine A_2A receptor) have been shown to play important roles in the development of diffuse dermal fibrosis following bleomycin treatment in a murine model of scleroderma (Chan et al., 2006). In a separate study using the same model, investigators stained skin samples from normal mice, bleomycin-treated wild type A_2A receptor knock-out mice, and A_2A antagonist-treated mice for procollagen α2 Type I and CD34 (fibrocytes) (Katebi et al., 2008): the investigators found more fibrocytes in the dermis of the bleomycin-treated mice compared to the normal mice, and the increase in fibrocyte number was abrogated by blockade of the adenosine A_2A receptor, suggesting that recruitment of fibrocytes into tissue plays an important role in scleroderma. While a small case–control study of 8 patients with limited scleroderma, showed no difference between cases and controls in the expression of circulating monocyte surface molecules associated with fibrocytes (Russo et al., 2007), the study had major limitations including a small sample size and the enrollment of patients with limited scleroderma only.

It has previously been shown that patients with scleroderma have low levels of serum and tissue amyloid P compared to healthy controls (Pilling et al., 2003), and that serum levels of amyloid P play a key role in fibrocyte differentiation. In a study using specimens from scleroderma patients, investigators found very few CD34+ cells in the affected areas of skin compared to healthy controls and patients with other collagen vascular diseases (Aiba et al., 1994): a finding that is consistent with the observation that CD34 expression, which is increased during the early active inflammatory phase of wound healing, is down-regulated over time (Aiba and Tagami, 1997, Yang et al., 2005).

Skin

Fibrocytes play an integral role in the delicate balance between normal repair of skin wounds and pathologic fibrosis by regulating the production of extracellular matrix components as well as extracellular matrix-modifying enzymes (Metz, 2003). During the healing process of extensive burn wounds if excess deposition of extracellular matrix occurs, two different forms of aberrant wound healing (keloid and hypertrophic scar formation) may result (Ehrlich et al., 1994).

Keloid formation and hypertrophic scars

While keloids and hypertrophic scars are morphologically different, they share one important feature: over time the expression of CD34 on the fibrocytes within these wounds decreases, whereas the expression of proline-4-hydroxylase (an enzyme involved in collagen synthesis) increases (Aiba and Tagami, 1997), a finding that has been documented by others (Abe et al., 2001, Phillips et al., 2004). Moreover, in burn patients the number of circulating fibrocytes is significantly increased (up to 10% of peripheral blood mononuclear cells) compared to that of normal individuals (<0.5%) (Yang et al., 2002). In hypertrophic scars, spindle-shaped cells expressing leukocyte-specific protein-1 and pro-collagen I (fibrocytes) are abundant (Yang et al., 2005). In a separate experimental model of wound healing, investigators found that cells that expressed α-smooth muscle actin were significantly increased in the wound compared to normal skin: these cells also expressed CD45, and CD34 thus consistent with fibrocytes (Mori et al., 2005). Similar experiments in female mice that received a male bone marrow transplant following total body irradiation, the Y chromosome was identified in the nuclei of the fibrocytes isolated from the wound site, supporting the role of fibrocytes in would healing and their bone marrow origin (Mori et al., 2005). Lastly, it has been suggested that the predominant role of fibrocytes in hypertrophic scarring secondary to burn injury may be to regulate the function of local fibroblasts (Wang et al., 2007).

Gastrointestinal

Fibrocytes have also been implicated in the fibrosis of the liver, gallbladder, and pancreas. Regardless of the etiology, chronic liver disease can eventually lead to the excess accumulation of type I collagen and fibrosis. In a murine model of bile duct ligation-induced liver fibrosis, investigators found bone marrow-derived collagen-expressing GFP+ cells in the liver of chimeric mice (Kisseleva et al., 2006). The majority of these bone marrow-derived cells co-expressed collagen-GFP+ and CD45+, suggesting that collagen-producing fibrocytes were recruited from the bone marrow to the damaged liver (Kisseleva et al., 2006): it has been estimated that bone marrow-derived fibrocytes comprise 4–6% of all collagen-producing cells in the liver (Kisseleva and Brenner, 2008a).

The presence and distribution of CD34+ fibrocytes have been analyzed in neoplastic and inflammatory lesions of both the pancreas (Barth et al., 2002a, Kuroda et al., 2004), and the bladder (Nimphius et al., 2007). In one pathologic study, the stroma of normal pancreatic tissue had very few, diffusely scattered CD34+ fibrocytes, while tissue from patients with chronic pancreatitis had not only an increased number of CD34+ fibrocytes, but also an increased number of α-smooth muscle actin myofibroblasts (Barth et al., 2002a): Tissue obtained from pancreatic ductal adenocarcinoma, however, was devoid of CD34+ fibrocytes. Similar findings were reported in a separate study, and these investigators suggested that the CD34+ fibrocytes may play a role in the regulation of tumor growth in the pancreas (Kuroda et al., 2004).

In a pathologic study of normal gallbladder and chronic cholecystitis, myofibroblasts were absent from the wall of the normal gallbladder, but CD34+ cells were found in the stroma (Kuroda et al., 2005): In chronic cholecystitis, however, CD34+ cells were either absent or very limited in number and location. Lastly, investigators using in a murine dextran sodium sulfate (DSS) colitis model showed that compared to normal mucosa without inflammation, there was a significant accumulation of immature oval-shaped CD45+ Col I+ fibrocytes in the submucosal layer of the colon on day 7 following the oral administration of 3% DSS, and by day 14 they had differentiated into the classically described spindle-shaped fibrocytes (Uehara et al., 2009). They also showed that the accumulation of CD45+ Col I+ fibrocytes preceeded the appearance of α-smooth muscle actin + myofibroblasts. Regarding the shape of the cells, the investigators hypothesized that the oval-shaped fibrocytes seen during the early phase of colitis may be functioning as antigen-presenting cells, and that their differentiation into spindle-shaped cells may play an important role in initiating the healing process.

Stromal remodeling and tumor metastases

Stromal remodeling precipitated by invasive carcinomas is characterized by a loss of CD34+ expression and a gain of α-smooth muscle actin expression resulting in a phenotype change from CD34+ fibrocytes to α-smooth muscle actin myofibroblasts (Barth and Westhoff, 2007). This process is similar for many different tumors and may play a role in local invasion as well as systemic dissemination. It is thought that in the face of decreased antigen presentation by CD34+ fibrocytes, the hosts’ immune control directed against invasive carcinoma cells may become dysregulated. The disappearance of CD34+ fibrocytes and the appearance of α-smooth muscle actin myofibroblasts associated with the transformation into an invasive tumor have been associated with tumors of the breast (Barth et al., 2002b, Ebrahimsade et al., 2007, Ramaswamy et al., 2003), cervix (Barth et al., 2002c), oral cavity/larynx/pharynx (Barth et al., 2004, Kojc et al., 2005), malignant melanoma (Wessel et al., 2008), anus (Sakai and Matsukuma, 2002), and renal pelvis and ureter (Kuroda et al., 2006).

Infectious disease

Lyme disease

Fibrocytes have been implicated in the pathogenesis of Lyme disease (Grab et al., 1999, Grab et al., 2002). In one study (Grab et al., 1999), fibrocytes from Rhesus monkeys were isolated and characterized by flow cytometry. Borrelia Burgdoferi were incubated with human or monkey fibrocyte cultures in vitro. The investigators found that B. burgdorferi bound to both human and monkey fibrocytes in vitro, suggesting that the interaction between B. burgdorferi and peripheral blood fibrocytes may contribute to the inflammatory arthritis that is associated with Lyme disease.

Eye

Proliferative vitreoretinopathy is characterized by the development of fibrocellular membranes on the retina and is the most common cause of failure of retinal reattachment surgery. In an experiment using 16 epiretinal membranes from 16 eyes undergoing vitreoretinal surgery for the treatment of retinal detachment complicated by proliferative vitreoretinopathy investigators showed that cells expressing α-smooth muscle actin were present on all membranes (Abu El-Asrar et al., 2008). Moreover, using immunohistochemical techniques, there were cells that co-expressed both CD34 and α-smooth muscle actin and CD45 and α-smooth muscle actin. The investigators also demonstrated that the presence of CCR7+ α-smooth muscle actin+ and CXCR4+ α-smooth muscle actin+ cells and demonstrated that CCL21 and CXCL12 proteins were localized in myofibroblasts.

Heart and vasculature

Intimal hyperplasia

Intimal hyperplasia, the thickening of the tunica intima of blood vessels, is a universal response to vessel injury, and is an important cause of bypass graft failure and arterial restenosis following percutaneous revascularization of coronary and peripheral arteries. In an ovine model of carotid artery intimal hyperplasia, a population of labeled circulating leukocytes that infiltrated the intima in vivo expressed CD45, CD34 and vimentin, and showed α-smooth muscle actin immunoreactivity during the remodeling process (Varcoe et al., 2006). Since this unique combination of surface markers is consistent with the surface markers of fibrocytes, the investigators suggested that intimal hyperplasia is, at least in part, caused by the migration of these cells (Varcoe et al., 2006).

Atherosclerosis

In a rabbit model of atherosclerosis, compared to controls, the atherosclerotic plaque in rabbits fed a high cholesterol diet contained cells that stained positive for CD34, CD45, α-smooth muscle actin, and prolyl-4 hydroxylase on immunohistochemistry, suggesting a hematopoietic origin and fibroblast/myofibroblast-like phenotype (Zulli et al., 2005). The fibrous cap plays an integral role in the stability of the atherosclerotic plaque: plaques with thin fibrous caps are more vulnerable to disruption and thrombotic vessel occlusion than those with thick fibrous caps. Recently, fibrocytes have been identified within the fibrous cap of human carotid endarterectomy specimens (Medbury et al., 2008). These investigators examined atherosclerotic specimens for fibrocytes by immunohistochemistry staining for CD34, procollagen I and leukocyte specific protein-1/procollagen I, and found fibrocytes in areas of plaque growth and tissue repair, suggesting that fibrocytes contribute to the formation and strength of the fibrous cap. Moreover, they found that these cells co-localized with TGF-β.

Ischemic cardiomyopathy

In a closed chest mouse model of ischemic cardiomyopathy, repetitive ischemia/reperfusion episodes (consisting of multiple, daily 15-minute vessel occlusions, not associated with myocardial necrosis) resulted in a fibrotic cardiomyopathy and global left ventricular dysfunction (Dewald et al., 2003). The same investigators subsequently showed that these repetitive ischemia/reperfusion episodes were associated with a markedly prolonged induction of CCL2; and that the resulting left ventricular dysfunction could be prevented by either genetic deletion of CCL2, or injection of a neutralizing anti-CCL2 antibody (Dewald et al., 2005). In a separate study using the same model of ischemic cardiomyopathy, in addition to prolonged induction of CCL2, increased numbers of small spindle-shaped cells in the myocardium that expressed collagen I, α-smooth muscle actin, CD34, and CD45, consistent with fibrocytes, were seen (Haudek et al., 2006). Inhibition of circulating monocyte differentiation with serum amyloid P, a member of the pentraxin family of autocoids that binds to FcγR and modifies the phenotype and pathophysiological functions of monocytes (Mold et al., 2002), resulted in reduced fibrosis and improved global and regional ventricular function. Moreover, after treatment with serum amyloid P, isolated fibroblasts lacked the small spindle-shaped morphology characteristic of fibrocytes, and were identical to fibroblasts isolated from sham hearts; the subpopulation of CD34+/CD45+ cells were no longer detected (Haudek et al., 2006). The hematopoietic origin of the fibrocyte was confirmed by using a chimeric mouse that expressed β-galactosidase in bone-marrow originating cells (Haudek et al., 2006): after ischemia/reperfusion a subpopulation of isolated cardiac fibroblasts demonstrated the distinctive morphology of fibrocytes (small spindle-shaped cells), and was positive for lacZ expression, as well as CD34 and collagen I.

Myxomatous mitral valve degeneration

Fibrocytes have been associated with myxomatous mitral valve degeneration the most common cause of isolated mitral valve insufficiency. In one study, myxomatous mitral valves removed from patients with mitral regurgitation, stained positive for vimentin (a characteristic of myofibroblasts) (Rabkin et al., 2001). In a separate pathologic study, investigators found that CD34+ fibrocytes made up the majority of mitral valve stromal cells, suggesting that CD34+ fibrocytes are involved in the pathogenesis of myxomatous mitral valve degeneration (Barth et al., 2005).

Conclusion

The bone marrow-derived fibrocyte is a unique circulating cell that plays a crucial role in both normal and aberrant healing processes. They maintain a pivotal position in balancing between normal healing and excess fibrosis that can lead to organ damage and ultimate failure. Further research is needed in order to more fully understand their role in health and disease.

Abbreviations

IL

interleukin

IFN

interferon

DSS

dextran sodium sulfate

FITZ

fluorescein isothiocyanate

TGF

transforming growth factor