Flow Cytometric Identification of Fibrocytes in the Human Circulation

Abstract

Because the incidence of organ fibrosis increases with age, various fibrosing disorders are projected to account for significant increases in morbidity, mortality, and healthcare costs in the years to come. Treatments for these diseases are scarce and better understanding of the immunopathogenesis of fibrosis and its relationship to aging are sorely needed. One area of interest in this field is the role that fibrocytes might play in the development of tissue remodeling and fibrosis. Fibrocytes are mesenchymal progenitor cells presumed to be of monocyte origin that possess the tissue remodeling properties of tissue resident fibroblasts such as extracellular matrix production and α-SMA-related contractile properties, as well as the immunologic functions typically attributed to macrophages including production of cytokines and chemokines, antigen presentation, regulation of leukocyte trafficking, and modulation of angiogenesis. Fibrocytes could participate in the development of age-related fibrosing disorders through any or all of these functions. This chapter presents methods that have been developed for the study of circulating human fibrocytes. Protocols for the quantification of fibrocytes in the human circulation will be presented along with discussion of the technical challenges that are frequently encountered in this field. It is hoped that this information will facilitate further investigation of the relationship between fibrocytes, aging, and fibrosis, and perhaps uncover new areas of study in these difficult-to-treat and deadly diseases.

Fibrocytes, Wound Healing, and Corneal Fibrosis

“…8 Fibrocytes also express the pan-myeloid antigen marker CD13, reinforcing the theory that hematopoietic progenitor cells (likely myeloid progenitor cells) are the source of fibrocytes rather than MSCs. 24,25 Varcoe et al 16 show that circulating labeled leukocytes infiltrate tissue injured in vivo and progressively acquire the fibrocyte phenotype (CD34 + CD45 + vimentin + ), which further supports the hypothesis that fibrocytes arise from hematopoietic cells.…”

“…Also, unlike monocytes, fibrocytes produce collagen type I and collagen type III and express CD34. 24,27,35 Fibrocytes can also be distinguished from dendritic cells because they do not express typical surface proteins of dendritic cells or their precursors (such as CD1a, CD10 and CD83). 27 In addition, fibrocytes do not express the B-cell marker CD19 or T-cell markers CD3, CD4, CD8, CD25 or CD56.…”

“…27 Fibrocytes also express the chemokine receptors CCR2, CCR3, CCR5, CCR7, CXCR4, CXCR6, and CX3CR1, host defense Fc receptors for immunoglobulin G (CD32 and CD64), antigen presentation and costimulatory molecules of lymphocyte activation (major histocompatibility complex [MHC] class I and II/CD80 and CD86), and cell surface enzymes (CD10 and CD13). 24,25,27,[36][37][38] The complete list of fibrocytes markers is presented in Table 1. 8,24,25,39 In the majority of studies reported, fibrocytes were identified by a combination of expressed markers, for example, the simultaneous expression of CD34 or CD45 or CD11b and collagen type I or collagen type III.…”

Fibrocyte measurement in peripheral blood correlates with number of cultured mature fibrocytes in vitro and is a potential biomarker for interstitial lung disease in Rheumatoid Arthritis

“…The traditional method for measuring circulating fibrocytes is to first isolate PBMCs and hereafter perform permeabilization and fixation for intracellular staining [22]. This method involves several washing steps, is time consuming, costly and requires large volumes of venous blood, which makes it difficult to use in daily clinical practice (see Table 3 for comparison of the two methods).…”

“…collagen type 1/pro-collagen type 1/vimentin) [22]. However, this methodology is time consuming and expensive.…”