**4. Fibrocytes participation in repair processes**

Wound repair is a complex process that results from the coordinated release of cytokines, chemokines, and growth factors, leading successively to the recruitment and activation of different cells into the injured site from the very initial phases of repair (Gurtner GC et al., 2008). Fibrocytes have been postulated as important players of the tissue repair process since they have the ability to rapidly home to sites of tissue together with the infiltrating inflammatory cells that act to prevent infection and degrade damaged connective tissue components (Bucala et al., 1994).

Fibrocytes secrete proinammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin (IL)-6, IL-8, IL-10, macrophage inammatory protein-1α/β (MIP-1α/β) CCchemokine ligands (CCL) -3 and -4 in response to IL-1β which is an important mediator of wound healing response (Chesney et al., 1998). The fibrocyte products MIP-1α , MIP-1β, and monocyte chemotactic protein-1 (MCP-1) are potent T cell chemoattractants and may act to specifically recruit CD4+ T cells into the tissue repair microenvironment; moreover, the fibrocytes increase the cell surface expression of leukocyte adhesion molecules, such as intercellular adhesion molecule 1 (ICAM1), which would enhance leukocyte trafficking (Chesney et al., 1998). Interestingly, in addition to these functions, fibrocytes may play an early and important role in the initiation of antigen-specific immunity. Thus, it has been demonstrated that peripheral blood fibrocytes: express the surface proteins required for antigen presentation, including class II major histocompatability complex molecules: HLA-

Hematopoietic Derived Fibrocytes: Emerging Effector Cells in Fibrotic Disorders 325

which raises the question if fibrocytes main contribution to the process of tissue repair is only a direct participation in the production of ECM components. In this context, it is important to emphasize that fibrocytes secrete paracrine growth factors such as connective tissue growth factor (CTGF), PDGF, FGF and TGF-β1 that induce proliferation, migration and differentiation of fibroblasts to myofibroblasts in culture (Chesney et al., 1998, Wang et al., 2007). These findings suggest that the predominant role of brocytes in scarring could be

Constitutive

Constitutive; ↑ with TGF-β1 or IL-1β

the regulation of the functions of local broblasts.

Platelet-Derived Growth Factor A (PDGF-α)

Fibroblast Growth Factor basic (bFGF) Granulocyte-Monocyte Colony Stimulating

Vascular Endothelial Cell Growth Factor

Transforming Growth Factor-beta1 (TGF-β1) Connective Tissue Growth Factor (CTGF)

Insulin Growth Factor 1 (IGF1)

Hepatocyte Growth Factor (HGF)

**Growth factors** 

Factor (GM-CSF)

(VEGF)

**Cytokines** 

**Chemokines** 

IL-8

GROα MIP-1α MIP-1β MCP-1 **MMPs**  MMP-2

MMP-7

**Proteins secreted by fibrocytes Pattern of Expression** 

Tumoral Necrosis Factor-alpha (TNF-α) Induced by IL-1β stimulation

MMP-9 Constitutive; ↑ with TGF-β<sup>1</sup>

MMP-8 Constitutive; ↓ with TGF-β<sup>1</sup>

Table 2. Fibrocytes pattern of expression for diverse proteins.

IL-1α Constitutive

IL-6 Induced by IL-1β or TNF-α stimulation IL-10 Induced by IL-1β or TNF-α stimulation

DP, -DQ, and –DR; the costimulatory molecules CD80 and CD86, and the adhesion molecules CD11a, CD54, and CD58. Fibrocytes are potent stimulators of antigen-specific T cells in vitro, and migrate to lymph nodes and sensitize naïve T cells in situ (Chesney et al., 1997). Likewise, brocytes may also participate in the development of the innate immune response; in porcine models, specic in vitro stimulation of fibrocytes for TLR 2, 4, 7 or TLR3 leads rapidly to the translocation of the NF-kB transcription factor and the production of high levels of IL-6 (Balmelli et al., 2007); on the other hand, exposure to innate immune stimulation in the form of TLR agonists induces an increased expression of MHC class I and II molecules and of the co-stimulatory proteins CD80 and CD86 on fibrocytes, which enables these cells to function as antigen-presenting cells for the activation of cytotoxic CD8+ T cells. All these findings indicate that brocytes may recognize a large variety of pathogens such as viruses or bacteria and could be part of the initiation of innate immune responses (Balmelli et al., 2005 and 2007).

Blood vessel formation during normal physiological processes, such as wound healing, is highly regulated by a delicate balance between pro- and antiangiogenic factors. As mentioned, circulating brocytes have been shown to migrate to early wound sites where angiogenesis occurs, fibrocytes produce and secrete active matrix metalloproteinase 9 and 2 (MMP-9: gelatinase B; MMP-2: gelatinase A) (Hartlapp I et al., 2001, García-de-Alba et al., 2010), which are implicated in the proteolysis of the basement membrane early during the invasion stage of angiogenesis. In addition, cultured brocytes constitutively secrete vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), insulin growth factor (IGF-I) and hematopoietic factors as granulocyte monocyte-colony stimulating factor (GM-CSF) that induce endothelial cell migration, proliferation, and alignment of endothelial cells into tubular-like structures in vitro. In like manner cultured brocytes (and brocyte-conditioned media) showed the ability to promote angiogenesis in vivo using a Matrigel implant model, (Hartlapp I et al., 2001).

Interestingly, it has been reported that Th2 cytokines (IL-4 and IL-13) induce, whereas Th1 cytokines (IFN-γ and IL-12) inhibit the CD14+ monocyte to brocyte differentiation. When added together the probrocyte activities of IL-4 and IL-13 and the brocyte-inhibitory activities of IFN-γ and IL-12 counteract each other in a concentration-dependent form. By contrast, the brocyte-inhibitory activity of the plasma protein serum amyloid P (SAP) dominates over the probrocyte activities of IL-4 and IL-13. These results might indicate that the complex mix of cytokines and plasma proteins present in inammatory lesions, wounds, and brosis will inuence brocyte differentiation (Shao et al., 2008). Consistent with this data, it was recently reported that CD14+ monocytes can differentiate in vitro into two different subtypes of fibrocytes depending on the presence or absence of serum in the culture media, which could resemble the changes in serum protein concentrations that occur during tissue repair, inflammation and its resolution (Curnow et al., 2010).

Fibrocytes also contribute to normal wound healing by serving as the contractile force of wound closure via α-smooth muscle actin expression (Abe et al., 2001; Metz, 2003), and secreting components of the extracellular matrix (collagen I, collagen III, fibronectin) (Abe et al., 2001; Bucala et al., 1994). Interestingly, it has been reported that the capacity to produce collagen of fibrocytes from normal subjects or from burn patients is less than that of fibroblasts (dermal and lung fibroblasts) (Wang et al., 2007; García-de-Alba et al., 2010),

DP, -DQ, and –DR; the costimulatory molecules CD80 and CD86, and the adhesion molecules CD11a, CD54, and CD58. Fibrocytes are potent stimulators of antigen-specific T cells in vitro, and migrate to lymph nodes and sensitize naïve T cells in situ (Chesney et al., 1997). Likewise, brocytes may also participate in the development of the innate immune response; in porcine models, specic in vitro stimulation of fibrocytes for TLR 2, 4, 7 or TLR3 leads rapidly to the translocation of the NF-kB transcription factor and the production of high levels of IL-6 (Balmelli et al., 2007); on the other hand, exposure to innate immune stimulation in the form of TLR agonists induces an increased expression of MHC class I and II molecules and of the co-stimulatory proteins CD80 and CD86 on fibrocytes, which enables these cells to function as antigen-presenting cells for the activation of cytotoxic CD8+ T cells. All these findings indicate that brocytes may recognize a large variety of pathogens such as viruses or bacteria and could be part of the initiation of innate immune responses (Balmelli

Blood vessel formation during normal physiological processes, such as wound healing, is highly regulated by a delicate balance between pro- and antiangiogenic factors. As mentioned, circulating brocytes have been shown to migrate to early wound sites where angiogenesis occurs, fibrocytes produce and secrete active matrix metalloproteinase 9 and 2 (MMP-9: gelatinase B; MMP-2: gelatinase A) (Hartlapp I et al., 2001, García-de-Alba et al., 2010), which are implicated in the proteolysis of the basement membrane early during the invasion stage of angiogenesis. In addition, cultured brocytes constitutively secrete vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), insulin growth factor (IGF-I) and hematopoietic factors as granulocyte monocyte-colony stimulating factor (GM-CSF) that induce endothelial cell migration, proliferation, and alignment of endothelial cells into tubular-like structures in vitro. In like manner cultured brocytes (and brocyte-conditioned media) showed the ability to promote angiogenesis in vivo using a Matrigel implant model, (Hartlapp I et al.,

Interestingly, it has been reported that Th2 cytokines (IL-4 and IL-13) induce, whereas Th1 cytokines (IFN-γ and IL-12) inhibit the CD14+ monocyte to brocyte differentiation. When added together the probrocyte activities of IL-4 and IL-13 and the brocyte-inhibitory activities of IFN-γ and IL-12 counteract each other in a concentration-dependent form. By contrast, the brocyte-inhibitory activity of the plasma protein serum amyloid P (SAP) dominates over the probrocyte activities of IL-4 and IL-13. These results might indicate that the complex mix of cytokines and plasma proteins present in inammatory lesions, wounds, and brosis will inuence brocyte differentiation (Shao et al., 2008). Consistent with this data, it was recently reported that CD14+ monocytes can differentiate in vitro into two different subtypes of fibrocytes depending on the presence or absence of serum in the culture media, which could resemble the changes in serum protein concentrations that occur

Fibrocytes also contribute to normal wound healing by serving as the contractile force of wound closure via α-smooth muscle actin expression (Abe et al., 2001; Metz, 2003), and secreting components of the extracellular matrix (collagen I, collagen III, fibronectin) (Abe et al., 2001; Bucala et al., 1994). Interestingly, it has been reported that the capacity to produce collagen of fibrocytes from normal subjects or from burn patients is less than that of fibroblasts (dermal and lung fibroblasts) (Wang et al., 2007; García-de-Alba et al., 2010),

during tissue repair, inflammation and its resolution (Curnow et al., 2010).

et al., 2005 and 2007).

2001).

which raises the question if fibrocytes main contribution to the process of tissue repair is only a direct participation in the production of ECM components. In this context, it is important to emphasize that fibrocytes secrete paracrine growth factors such as connective tissue growth factor (CTGF), PDGF, FGF and TGF-β1 that induce proliferation, migration and differentiation of fibroblasts to myofibroblasts in culture (Chesney et al., 1998, Wang et al., 2007). These findings suggest that the predominant role of brocytes in scarring could be the regulation of the functions of local broblasts.


Table 2. Fibrocytes pattern of expression for diverse proteins.

Hematopoietic Derived Fibrocytes: Emerging Effector Cells in Fibrotic Disorders 327

**TGF-β1** (Smad2/3 and SAPK/JNK MAPK pathways)

**Osteogenic media** (Dexamethasone, ascorbate, β-Glycerophosphate)

Fig. 2. Schematic summary of the mediators and inhibitors of CD14+monocyte to fibrocyte

differentiation, and fibrocytes differentiation to other mesenchymal cells. TGZ: trogliotazone. A crosstalk between PPARγ and TGF-β1 exists, where they can strongly inhibit each other signaling, making clear that a complex and critical balance exists between both of them. It is noteworthy that the expression of hematopoietic markers decrease as fibrocytes differentiate into other mesenchymal cells, while specific markers for that given

DAPI COL I MMP-8 MERGE

Fig. 3. Fluorescent immunocytochemistry showing a group of fibrocytes positive for

**Chondrogenic media** (Dexamethasone, ascorbate, β-Glycerophosphate + TGF-β3)

> **Leptin α- SMA Collagen II Osteonectin**

**(PPARγ) agonist TGZ** ↑aP2

**Fibrocytes**

**Monocyte**

**+ + +**

TGF-β1 ET1

Th2 cytokines (IL4, IL13)

**---**

SAP

TLR2

collagen I and MMP-8 staining.

Th1 cytokines (IFNγ, TNF, IL12)

> **CD34 CD45**

cell increase their expression during differentiation.

**Myofibroblast**

**Chondrocyte**

**Osteoblast**

**Adipocyte**

#### **4.1 Migration and homing**

Fibrocytes trafficking from the bone marrow and circulation to the organs or site of lesion is given through several chemokines. Human brocytes express diverse chemokine receptors, including CCR3, CCR5, CCR7, and CXCR4; whereas mouse brocytes express CXCR4, CCR7, and CCR2 (Abe et al., 2001; Phillips et al., 2004; Moore et al., 2005; Mehrad et al., 2009). Secondary lymphoid tissue chemokine (SLC/CCL21) and its receptor CCR7 was the first chemokine-chemokine receptor system described to induce the recruitment of brocytes as a mechanism of migration to wound sites (Abe et al., 2001). In humans as in mice CCL21 is constitutively abundant in lymphoid tissues, particularly in the lymph nodes and spleen but it is also expressed at lower levels in some non-lymphoid tissues, including the kidneys and lungs (Gunn et al., 1998; Abe et al., 2001; Sakai N et al., 2006).

CXCR4 is an important chemokine receptor for stem and immune cell migration, high levels of CXCL12, which is the only known ligand for CXCR4, were found in the lungs and plasma of patients with IPF and these levels correlated with circulating brocyte concentrations (Mehrad B et al., 2007; Andersson-Sjöland A et al., 2008).

Recently (Mehrad et al., 2009) reported that most (but not all) freshly isolated human brocytes expressed CXCR4, whereas 46% expressed CCR2 and 9% expressed CCR7. Approximately 30% were CCR2/CXCR4+ and most CCR7+ cells also expressed CCR2, but there was no overlap between CXCR4+ and CCR7+ receptors.

It has been reported an association between serum concentration of MCP-1 and high levels of CD45/pro-Col-I+ brocytes in the circulation of scleroderma patients with interstitial lung disease (ILD) or in healthy aging subjects, suggesting that MCP-1 may be also involved in mobilization of brocytes into the peripheral blood. (S. Mathai et al., as cited in Herzog & Bucala , 2010),

Thus, fibrocytes can use different chemokine–chemokine receptor axis for tissue homing and this might be related to the type of process (acute or chronic) or to the organs involved; however, the mechanisms implicated in the migration through basement membranes and extracellular matrix and subsequent tissue homing remain unclear. In this context, it was recently reported that fibrocytes express several MMP's (MMP- 2, 7, 8 and 9) (Fig 3) that may facilitate the process of migration of fibrocytes from the circulation to the tissues in response to chemokine gradients (Garcia-de-Alba et al., 2010). In this work it was showed that fibrocytes transmigration towards CXCL12 or PDGF through collagen I coated migration chambers, was highly associated with the collagenase MMP8, while migration through a combination of proteins of basal membrane was facilitated by gelatinases MMP2 and MMP9. Thus, these MMPs may ease cell migration by breaking down matrix barriers or affecting the state of cell-matrix interactions and also may play an important role in the remodelling of ECM. Interestingly, PDGF showed to be a more potent chemotactic agent when migration was given through collagen I coated chambers, possibly indicating that when fibrocytes have arrived to lung interstitium, PDGF plays an important role as a chemoattractant through lung parenchyma.

Fibrocytes trafficking from the bone marrow and circulation to the organs or site of lesion is given through several chemokines. Human brocytes express diverse chemokine receptors, including CCR3, CCR5, CCR7, and CXCR4; whereas mouse brocytes express CXCR4, CCR7, and CCR2 (Abe et al., 2001; Phillips et al., 2004; Moore et al., 2005; Mehrad et al., 2009). Secondary lymphoid tissue chemokine (SLC/CCL21) and its receptor CCR7 was the first chemokine-chemokine receptor system described to induce the recruitment of brocytes as a mechanism of migration to wound sites (Abe et al., 2001). In humans as in mice CCL21 is constitutively abundant in lymphoid tissues, particularly in the lymph nodes and spleen but it is also expressed at lower levels in some non-lymphoid tissues, including the kidneys and lungs (Gunn et al., 1998; Abe et

CXCR4 is an important chemokine receptor for stem and immune cell migration, high levels of CXCL12, which is the only known ligand for CXCR4, were found in the lungs and plasma of patients with IPF and these levels correlated with circulating brocyte concentrations

Recently (Mehrad et al., 2009) reported that most (but not all) freshly isolated human brocytes expressed CXCR4, whereas 46% expressed CCR2 and 9% expressed CCR7. Approximately 30% were CCR2/CXCR4+ and most CCR7+ cells also expressed CCR2, but

It has been reported an association between serum concentration of MCP-1 and high levels of CD45/pro-Col-I+ brocytes in the circulation of scleroderma patients with interstitial lung disease (ILD) or in healthy aging subjects, suggesting that MCP-1 may be also involved in mobilization of brocytes into the peripheral blood. (S. Mathai et al., as cited in Herzog &

Thus, fibrocytes can use different chemokine–chemokine receptor axis for tissue homing and this might be related to the type of process (acute or chronic) or to the organs involved; however, the mechanisms implicated in the migration through basement membranes and extracellular matrix and subsequent tissue homing remain unclear. In this context, it was recently reported that fibrocytes express several MMP's (MMP- 2, 7, 8 and 9) (Fig 3) that may facilitate the process of migration of fibrocytes from the circulation to the tissues in response to chemokine gradients (Garcia-de-Alba et al., 2010). In this work it was showed that fibrocytes transmigration towards CXCL12 or PDGF through collagen I coated migration chambers, was highly associated with the collagenase MMP8, while migration through a combination of proteins of basal membrane was facilitated by gelatinases MMP2 and MMP9. Thus, these MMPs may ease cell migration by breaking down matrix barriers or affecting the state of cell-matrix interactions and also may play an important role in the remodelling of ECM. Interestingly, PDGF showed to be a more potent chemotactic agent when migration was given through collagen I coated chambers, possibly indicating that when fibrocytes have arrived to lung interstitium, PDGF plays an important role as a chemoattractant through

**4.1 Migration and homing** 

al., 2001; Sakai N et al., 2006).

Bucala , 2010),

lung parenchyma.

(Mehrad B et al., 2007; Andersson-Sjöland A et al., 2008).

there was no overlap between CXCR4+ and CCR7+ receptors.

Fig. 2. Schematic summary of the mediators and inhibitors of CD14+monocyte to fibrocyte differentiation, and fibrocytes differentiation to other mesenchymal cells. TGZ: trogliotazone. A crosstalk between PPARγ and TGF-β1 exists, where they can strongly inhibit each other signaling, making clear that a complex and critical balance exists between both of them. It is noteworthy that the expression of hematopoietic markers decrease as fibrocytes differentiate into other mesenchymal cells, while specific markers for that given cell increase their expression during differentiation.

Fig. 3. Fluorescent immunocytochemistry showing a group of fibrocytes positive for collagen I and MMP-8 staining.

Hematopoietic Derived Fibrocytes: Emerging Effector Cells in Fibrotic Disorders 329

Fibrocyte recruitment to damaged lungs has been proved to be mediated by several chemokine/chemokine receptor interactions. Thus, in a model of fluorescein isothiocyanate (FITC)-induced lung fibrosis, it was demonstrated that significantly higher numbers of fibrocytes are present in the airspaces of fluorescein isothiocyanate-injured CCR2+/+ mice

expressed CCR2 and migrated toward CCL2 and CCL12 ligands. Interestingly, CCL2 stimulated collagen secretion by lung fibrocytes, which differentiated towards a myofibroblast phenotype, transition that was associated with loss of CCR2 expression

Importantly, interruption of the chemokine axis attenuated both brocyte accumulation and pulmonary brosis (Phillips et al., 2004; Moore et al., 2006), strengthening the notion that these chemokine/chemokine receptor axis are the main responsible of fibrocytes trafficking to the lungs; however, under which biological/pathological conditions one or other chemokine/chemokine receptor system is activated, or if they represent redundant

Recently several independent research groups have identied brocytes in different forms of brotic human lung disease. In an initial study, it was reported that circulating brocytes expressing CXCR4 and both lung and plasma levels of CXCL12 were elevated in IPF patients (Mehrad 2007). CXCL12 levels showed a positive correlation with higher number of circulating brocytes in the peripheral blood of these patients. Later, Andersson-Sjöland et al., evaluated the presence of brocytes in the lung of patients with idiopathic pulmonary brosis by immunouorescence and confocal microscopy. Fibrocytes were identied with different combinations of markers in most brotic lungs; interestingly, no brocytes were identied in normal lungs. They also found a positive correlation between the abundance of broblastic foci and the amount of lung brocytes and a negative correlation between plasma levels of CXCL12 with lung function tests (lung diffusing capacity for carbon monoxide and oxygen saturation on exercise) (Andersson-Sjöland et al., 2008). These ndings indicate that circulating brocytes may contribute to the expansion of the

On the other hand, as mentioned earlier in this chapter, fibrocytes constitutively synthesize and release to the medium important amounts of MMP-2, MMP-7, MMP-8, and MMP-9

MMPs consist of a large family of zinc endoproteases, collectively capable of degrading all ECM components (Pardo et al., 2006). However, ECM represents only a fraction of their proteolytic targets, and moreover, a given MMP can act on various proteins and, in turn, affect a variety of processes. Gelatinases (MMP-2 and MMP-9) have been found upregulated in human pulmonary brosis and animal models of lung brosis (Swiderski et al., 1998; Selman et al., 2000; Oikonomidi et al., 2009). The overexpression of MMP-2 and MMP-9 has been mainly associated with their capacity to provoke disruption of alveolar epithelial basement membrane and enhanced broblast invasion into the alveolar spaces (Ruiz et al., 2003; Pardo et al., 2006). In the case of fibrocytes, these MMPs may facilitate the process of migration from the circulation to the interstitial and alveolar spaces in response to SDF-1/CXCL12 synthesized by alveolar epithelial cells (Andersson-Sjöland et al., 2008; Garcíade-Alba et al., 2010). TGF-β1–stimulated brocytes signicantly increase gene and protein

broblast/myobroblast population in idiopathic pulmonary brosis.

/- mice (Moore et al., 2005; 2006). Fibrocytes isolated from the lung

compared to CCR2-

(Moore et al 2005).

mechanisms, yet remains to be elucidated.

(García-de-Alba et al., 2010).
