Review
Circulating fibrocytes: collagen-secreting cells of the peripheral blood

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Abstract

Since the original description of circulating fibrocytes in 1994, our knowledge of this unique cell population has grown steadily. While initially described in the context of wound repair, fibrocytes have since been found to participate in granuloma formation, antigen presentation, and various fibrosing disorders. Fibrocytes produce matrix proteins such as vimentin, collagens I and III, and they participate in the remodeling response by secreting matrix metalloproteinases. Fibrocytes also are a rich source of inflammatory cytokines, growth factors, and chemokines that provide important intercellular signals within the context of the local tissue environment. Moreover, fibrocytes express the immunological markers typical of an antigen-presenting cell, and they are fully functional for the presentation of antigen to naı̈ve T cells. Fibrocytes can further differentiate, and they may represent a systemic source of the contractile myofibroblast that appears in many fibrotic lesions. Clinically, there is evidence that patients with hypertrophic scars such as keloids, and those affected by scleroderma and other fibrosing disorders have fibrocytes in their lesions. Recently, a new disease entity called nephrogenic fibrosing dermopathy (NFD) has been described, and the fibrocyte may play an important etiopathogenic role in disease development. Nephrogenic fibrosing dermopathy occurs in patients with renal insufficiency and leads to thickening and hardening of the skin, especially of the extremities. Ongoing research is focusing on the molecular signals that influence fibrocyte migration, proliferation, and function in the context of normal physiology and pathology.

Introduction

Circulating fibrocytes were first identified in 1994 as a result of studies in a model system of wound repair, and defined by their growth characteristics and unique surface phenotype (Bucala et al., 1994). Fibrocytes produce collagen and other matrix proteins, but they also circulate in the peripheral blood and express cell surface markers indicative of a hematopoetic origin. This new leukocyte sub-population hence was termed “fibrocytes”, which combines the Greek “kytos” referring to cell, and “fibro”, which is from the Latin denoting fiber (Fig. 1, Fig. 2). This designation is not unique however, and may lead to some overlap as the term “fibrocyte” also appears in histopathologic literature as a synonym for “mature” fibroblasts. In a separate context, a cell called a “fibrocyte” is also a constituent of the inner ear. Perhaps a more specific term for the circulating fibrocyte should be considered, such as the “alitofibrocyte”, which incorporates “alitis” from the Greek for wanderer, thereby emphasizing the important blood-borne nature of these cells.

Cells of mesenchymal origin are the precursors to numerous constituents of structural, supportive, blood, cardiac and bone tissue. In structural and supportive tissue, these cells typically have an irregular star (stellate), or spindle (fusiform) shape and delicate branching cytoplasmic extensions that form an interlacing network throughout the tissue. It is generally considered that mesenchymal cells can mature into tissue fibroblasts (Burkitt & Heath, 1997). Fibroblasts play a major role in wound repair, yet by historical definition they are not of hematopoietic origin. For a number of decades, there was an active debate regarding the extent to which connective tissue scar was the result of an ingrowth of adjacent mesenchymal or fibroblast-like elements versus the hematogenous entry of circulating, fibroblast precursors (Dunphy, 1963).

In older literature, references to so-called “blood-borne fibroblasts” and “fibroblast-like cells” exist and likely represent the first observations of cells with the current, molecular-defined features of circulating fibrocytes. Indeed, the potential derivation of matrix-producing cells from the peripheral blood was discussed for almost 150 years beginning with the work of Paget in 1863, Conheim in 1867, Fischer in 1925, and Maximov in 1928 (Paget, 1863, Stirling & Kakkar, 1969). It is likely that in experimental studies of wound repair that go back to at least the 1940s, the cells in the circulating blood that appeared capable of producing connective tissue were indeed “fibrocytes” (Stirling & Kakkar, 1969). In one such study, Stirling and Kakkar used careful cannulation and diffusion chamber techniques to demonstrate that collagen-producing cells were not contaminants dislodged from the blood vessel wall, but indeed were derived from circulating blood elements (Stirling & Kakkar, 1969). Fibrocytes are structural, marrow derived (to be discussed later in this review), and found in the peripheral circulation, typical features found in tissues of mesenchymal origin.

The methods that currently are employed for the isolation and growth of peripheral blood fibrocytes are similar to the ones described in older literature (Chesney & Bucala, 2001). The present techniques rely on the entry of “fibroblast-like” cells into wound chambers, or the derivation of “fibroblast-like” cells from the buffy coat of peripheral blood obtained from different mammalian species. A simple procedure is to draw a blood sample into heparinized tubes, dilute 2:1 with saline, and separate the buffy coat from the red cells by Ficoll Hypaque density-gradient centrifugation. The leukocyte-rich buffy coat is then removed, washed, and resuspended in DMEM supplemented with 10%, heat-inactivated fetal bovine serum. The resulting mononuclear cells then are plated onto tissue culture ware and grown in DMEM supplemented with 10% heat-inactivated fetal calf serum. Fibronectin pre-coating of the culture surfaces increases the yield and better sustains the growth of fibrocytes. After 2 days, the non-adherent cells (largely T cells) are aspirated off, and the remaining adherent cells cultivated for 14 days. Over time, the contaminating monocytes die off, and fibrocytes appear as clusters of stellate, elongated or spindle-shaped cells that show long cellular processes. After a prolonged period of culture, fibrocytes then begin contracting into a fusiform shape.

Flow cytometric studies confirmed that peripheral blood fibrocytes express the CD34 cell surface antigen. CD34 is a 110 kD integral membrane glycoprotein that was initially believed to be expressed exclusively on hematopoietic stem cells, including various myeloid and lymphoid progenitor cells (Bucala et al., 1994; Brown, Greaves & Molgaard, 1991). We now know that embryonic fibroblasts, endothelial cells, and bone marrow stromal cells also express CD34 (Brown et al., 1991, Fina et al., 1990). Studies in different laboratories have affirmed the utility of CD34 as a marker for identifying fibrocytes, although it has become apparent that fibrocyte expression of CD34 decreases over time, both in culture, and under certain conditions in vivo (Aiba & Tagami, 1997, Aiba et al., 1994, Bucala et al., 1994, Barth et al., 2002a, Barth et al., 2002b, Hirohata et al., 2001). Additional cell surface markers that are indicative of the hematologic origin of fibrocytes include CD11b, CD45, HLA-DR, CD71, CD80, and CD86 (Table 1). It is likely that the in situ environment influences the durability of fibrocyte CD34 expression. For example, fibrocytes derived from wound chambers, which likely represent an inflammatory milieu, maintain CD34+ expression over time (Bucala et al., 1994). More recent studies have utilized additional markers to identify fibrocytes, such as Type I pro-collagen (Schmidt et al., 2003). The minimum criteria of the co-expression of collagen production and uniquely hematologic markers is likely sufficient to describe fibrocytes in different pathologic lesions. Other commonly used markers for fibrocytes include the pan-leukocyte antigen CD45, and HLA-DR, which reflects the antigen presenting ability of these cells (Bucala et al., 1994; Chesney, Bacher, Bender & Bucala, 1997). Other markers of connective tissue matrix production, such as vimentin, and prolyl 4-hydroxylase, also have been employed to identify fibrocytes in hypertrophic scars and keloids (Aiba & Tagami, 1997).

Section snippets

Function

In the classic descriptions of wound repair, the host response is initiated immediately upon the traumatic disruption of tissue. Chemoattractants induced by injury to endothelium and other tissue elements lead to the successive recruitment of diverse cells into the injured site (Robson & Smith, 1992). In the first stage of wound repair, termed the inflammatory phase, neutrophils and monocytes enter the wound and remove clot, cell debris and invading bacteria. Monocytes mature into macrophages

Tissue origin

Fibrocytes are unusual, if not unique, in that they are matrix-producing cells of the peripheral blood. Initial studies in sex-mismatched, bone marrow-transplanted mice showed that after the re-constitution of female hosts with male bone marrow, the fibrocytes cultured from the peripheral blood originated from the host and not the donor cells. Based on this result, it was suggested that fibrocytes may have their origin from bone marrow stroma, which is a radio-resistant, meshwork of

Clinical role

The clinical relevance of fibrocytes centered initially on their potential role in wound repair and in inflammatory fibrosis, as typified by granuloma formation (Bucala et al., 1994). More recently, evidence for a role for fibrocytes in tumors, hypertrophic scars or keloids, scleroderma and related disorders, and pulmonary fibrosis has begun to emerge.

Future directions

Studies performed over the last 10 years have provided strong support for the fibrocyte as a collagen-producing cell of the peripheral blood. The accumulated evidence also indicates that circulating fibrocytes play an important role in the inflammatory and proliferative phases of wound repair. This is evidenced by the early migration of fibrocytes into wounds, and by their regulated production of both matrix proteins and growth regulating cytokines. Fibrocytes also have been identified in

Acknowledgements

These studies were supported by the Scleroderma Foundation (RB).

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