Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow
Introduction
Myofibroblasts are transiently found at the site of tissue injury and are believed to play a pivotal role in the healing process [1]. By secreting the extracellular matrix proteins involved in tissue repair and by promoting the contraction of the granulation tissue through the expression of the contractile protein α-smooth muscle actin (α-SMA), these cells are essential for wound closure [1], [2], [3], [4].
Recent studies have provided evidence that myofibroblasts may not derive from tissue fibroblasts, as previously postulated, but may originate from a bone-marrow-derived precursor [5], [6]. The phenotype of this precursor is still unknown, but it has been hypothesized that this cell may develop from circulating fibrocytes [6], [7].
Fibrocytes are unique circulating cells that express hematopoietic surface markers and myeloid antigens together with fibroblast products [8]. They have been isolated from murine and human peripheral blood mononuclear cells as CD14-negative cells that express collagen (Col) I in conjunction with CD34, CD11b, CD45, and CD13 [8]. Results from our own laboratory [7] and other groups [9], [10], [11] have indicated that circulating fibrocytes may be involved in tissue repair during wound healing [9], [10] in the airway subepithelial fibrosis and airway remodeling of patients with allergic asthma [7], and in the pulmonary fibrosis induced by bleomycin in mice [11]. Although it has been found that fibrocytes differentiate into myofibroblasts in vitro when stimulated with fibrogenic cytokines involved in tissue repair, such as transforming growth factor-β1 (TGF-β1) [7], [10], only one study [7] has provided evidence that myofibroblasts may originate from fibrocytes in vivo. In addition, there are conflicting and inconclusive data about the origin of fibrocytes from the bone marrow [9], [11]. Therefore, it is presently unknown whether these cells may contribute to the population of bone-marrow-derived myofibroblasts previously identified in wounded skin and other tissues [5], [6].
In this study, we evaluated the phenotype of fibrocytes in the wounded skin of BALB/c mice and investigated whether these cells differentiate into myofibroblasts in vivo and in vitro. We also examined their origin using the wounded tissue of sex-mismatched bone marrow chimera mice.
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Antibodies and reagents
The primary antibodies used for the immunostaining of tissue sections were an anti-CD13 (either the clone R3-242, BD Biosciences-Pharmingen, San Diego, CA, USA, or the rat anti-mouse CD13 from Serotec Inc., Raleigh, NC, USA), rat anti-mouse CD45 (Serotec) and an anti α-SMA (Clone B-4, NeoMarkers, Lab Vision Corporation, Fremont, CA, USA). The cyanine (Cy)2- and Rhodamine Red-X-conjugated donkey anti-rat or goat anti-mouse secondary antibodies for immunofluorescence were from Jackson
Characterization of myofibroblasts and fibrocytes in wounded tissue
The immunohistochemical analysis of sections of wounded tissue showed a time-dependent increase in the number of α-SMA-positive cells in comparison with sections of unwounded skin (excluding vessels) (Figs. 1A–C). A substantial proportion of these myofibroblasts co-expressed CD13 (56.4 ± 6.2% at day 4 and 51.6 ± 5.4% at day 7 post-wounding) (Figs. 1D,E). The proportion of α-SMA+ cells that also expressed CD45 was 46.7 ± 4.8% at day 4 and 39.8 ± 3.5% at day 7 post-wounding (Figs. 1D,F).
To
Discussion
This study provides direct evidence that circulating fibrocytes contribute to the myofibroblast population in wounded skin and that they originate from the bone marrow.
In the wounded skin, a substantial proportion of myofibroblasts (α-SMA positive cells, excluding vessels) expressed the surface markers CD13 and CD45, which they would not express if they were differentiating from tissue fibroblasts [14]. Indeed, the results of our preliminary experiments indicated that tissue fibroblasts do not
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